The interplay of non-specific binding, target-mediated clearance and FcRn interactions on the pharmacokinetics of humanized antibodies

PubMed Central

Datta-Mannan, Amita; Lu, Jirong; Witcher, Derrick R; Leung, Donmienne; Tang, Ying; Wroblewski, Victor J

2015-01-01

The application of protein engineering technologies toward successfully improving antibody pharmacokinetics has been challenging due to the multiplicity of biochemical factors that influence monoclonal antibody (mAb) disposition in vivo. Physiological factors including interactions with the neonatal Fc receptor (FcRn) and specific antigen binding properties of mAbs, along with biophysical properties of the mAbs themselves play a critical role. It has become evident that applying an integrated approach to understand the relative contribution of these factors is critical to rationally guide and apply engineering strategies to optimize mAb pharmacokinetics. The study presented here evaluated the influence of unintended non-specific interactions on the disposition of mAbs whose clearance rates are governed predominantly by either non-specific (FcRn) or target-mediated processes. The pharmacokinetics of 8 mAbs representing a diverse range of these properties was evaluated in cynomolgus monkeys. Results revealed complementarity-determining region (CDR) charge patch engineering to decrease charge-related non-specific binding can have a significant impact on improving the clearance. In contrast, the influence of enhanced in vitro FcRn binding was mixed, and related to both the strength of charge interaction and the general mechanism predominant in governing the clearance of the particular mAb. Overall, improved pharmacokinetics through enhanced FcRn interactions were apparent for a CDR charge-patch normalized mAb which was affected by non-specific clearance. The findings in this report are an important demonstration that mAb pharmacokinetics requires optimization on a case-by-case basis to improve the design of molecules with increased therapeutic application. PMID:26337808

Minimized virus binding for tests of barrier materials.

PubMed Central

Lytle, C D; Routson, L B

1995-01-01

Viruses are used to test the barrier properties of materials. Binding of virus particles during passage through holes in the material may yield misleading test results. The choices of challenge virus and suspending medium may be important for minimizing confounding effects that might arise from such binding. In this study, different surrogate viruses, as well as different support media, were evaluated to determine optimal test parameters. Two membranes with high-binding properties (nitrocellulose and cationic polysulfone) were used as filters to compare binding activities of different surrogate challenge viruses (MS2, phi X174, T7, PRD1, and phi 6) in different media. The media consisted of buffered saline with surfactants, serum, or culture broth as additives. In addition, elution rates of viruses that bound to the membranes were determined. The results suggest that viruses can bind by hydrophobic and electrostatic interactions, with phi X174 displaying the lowest level of binding by either process. The nonionic detergents Triton X-100 and Tween 80 (0.1%) equally minimized hydrophobic interactions. Neither anionic nor cationic surfactants were as effective at nontoxic levels. Serum was effective at reducing both hydrophobic and electrostatic binding, with 2% being sufficient for eliminating binding under our test conditions. Thus, phi X174 remains the best choice as a surrogate virus to test barrier materials, and Triton X-100 (0.1%) remains a good choice for reducing hydrophobic binding. In addition, binding of viruses by barrier materials is unlikely to prevent passage of blood-borne pathogens. PMID:7574603

Simulation optimization of spherical non-polar guest recognition by deep-cavity cavitands

PubMed Central

Wanjari, Piyush P.; Gibb, Bruce C.; Ashbaugh, Henry S.

2013-01-01

Biomimetic deep-cavity cavitand hosts possess unique recognition and encapsulation properties that make them capable of selectively binding a range of non-polar guests within their hydrophobic pocket. Adamantane based derivatives which snuggly fit within the pocket of octa-acid deep cavity cavitands exhibit some of the strongest host binding. Here we explore the roles of guest size and attractiveness on optimizing guest binding to form 1:1 complexes with octa-acid cavitands in water. Specifically we simulate the water-mediated interactions of the cavitand with adamantane and a range of simple Lennard-Jones guests of varying diameter and attractive well-depth. Initial simulations performed with methane indicate hydrated methanes preferentially reside within the host pocket, although these guests frequently trade places with water and other methanes in bulk solution. The interaction strength of hydrophobic guests increases with increasing size from sizes slightly smaller than methane to Lennard-Jones guests comparable in size to adamantane. Over this guest size range the preferential guest binding location migrates from the bottom of the host pocket upwards. For guests larger than adamantane, however, binding becomes less favorable as the minimum in the potential-of-mean force shifts to the cavitand face around the portal. For a fixed guest diameter, the Lennard-Jones well-depth is found to systematically shift the guest-host potential-of-mean force to lower free energies, however, the optimal guest size is found to be insensitive to increasing well-depth. Ultimately our simulations show that adamantane lies within the optimal range of guest sizes with significant attractive interactions to match the most tightly bound Lennard-Jones guests studied. PMID:24359375

Insights into an original pocket-ligand pair classification: a promising tool for ligand profile prediction.

PubMed

Pérot, Stéphanie; Regad, Leslie; Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A; Villoutreix, Bruno O; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket properties for ligand binding.

Insights into an Original Pocket-Ligand Pair Classification: A Promising Tool for Ligand Profile Prediction

PubMed Central

Reynès, Christelle; Spérandio, Olivier; Miteva, Maria A.; Villoutreix, Bruno O.; Camproux, Anne-Claude

2013-01-01

Pockets are today at the cornerstones of modern drug discovery projects and at the crossroad of several research fields, from structural biology to mathematical modeling. Being able to predict if a small molecule could bind to one or more protein targets or if a protein could bind to some given ligands is very useful for drug discovery endeavors, anticipation of binding to off- and anti-targets. To date, several studies explore such questions from chemogenomic approach to reverse docking methods. Most of these studies have been performed either from the viewpoint of ligands or targets. However it seems valuable to use information from both ligands and target binding pockets. Hence, we present a multivariate approach relating ligand properties with protein pocket properties from the analysis of known ligand-protein interactions. We explored and optimized the pocket-ligand pair space by combining pocket and ligand descriptors using Principal Component Analysis and developed a classification engine on this paired space, revealing five main clusters of pocket-ligand pairs sharing specific and similar structural or physico-chemical properties. These pocket-ligand pair clusters highlight correspondences between pocket and ligand topological and physico-chemical properties and capture relevant information with respect to protein-ligand interactions. Based on these pocket-ligand correspondences, a protocol of prediction of clusters sharing similarity in terms of recognition characteristics is developed for a given pocket-ligand complex and gives high performances. It is then extended to cluster prediction for a given pocket in order to acquire knowledge about its expected ligand profile or to cluster prediction for a given ligand in order to acquire knowledge about its expected pocket profile. This prediction approach shows promising results and could contribute to predict some ligand properties critical for binding to a given pocket, and conversely, some key pocket properties for ligand binding. PMID:23840299

Functional assay for T4 lysozyme-engineered G protein-coupled receptors with an ion channel reporter.

PubMed

Niescierowicz, Katarzyna; Caro, Lydia; Cherezov, Vadim; Vivaudou, Michel; Moreau, Christophe J

2014-01-07

Structural studies of G protein-coupled receptors (GPCRs) extensively use the insertion of globular soluble protein domains to facilitate their crystallization. However, when inserted in the third intracellular loop (i3 loop), the soluble protein domain disrupts their coupling to G proteins and impedes the GPCRs functional characterization by standard G protein-based assays. Therefore, activity tests of crystallization-optimized GPCRs are essentially limited to their ligand binding properties using radioligand binding assays. Functional characterization of additional thermostabilizing mutations requires the insertion of similar mutations in the wild-type receptor to allow G protein-activation tests. We demonstrate that ion channel-coupled receptor technology is a complementary approach for a comprehensive functional characterization of crystallization-optimized GPCRs and potentially of any engineered GPCR. Ligand-induced conformational changes of the GPCRs are translated into electrical signal and detected by simple current recordings, even though binding of G proteins is sterically blocked by the added soluble protein domain. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes.

PubMed

Srinivasulu, Yerukala Sathipati; Wang, Jyun-Rong; Hsu, Kai-Ti; Tsai, Ming-Ju; Charoenkwan, Phasit; Huang, Wen-Lin; Huang, Hui-Ling; Ho, Shinn-Ying

2015-01-01

Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes.

Characterizing informative sequence descriptors and predicting binding affinities of heterodimeric protein complexes

PubMed Central

2015-01-01

Background Protein-protein interactions (PPIs) are involved in various biological processes, and underlying mechanism of the interactions plays a crucial role in therapeutics and protein engineering. Most machine learning approaches have been developed for predicting the binding affinity of protein-protein complexes based on structure and functional information. This work aims to predict the binding affinity of heterodimeric protein complexes from sequences only. Results This work proposes a support vector machine (SVM) based binding affinity classifier, called SVM-BAC, to classify heterodimeric protein complexes based on the prediction of their binding affinity. SVM-BAC identified 14 of 580 sequence descriptors (physicochemical, energetic and conformational properties of the 20 amino acids) to classify 216 heterodimeric protein complexes into low and high binding affinity. SVM-BAC yielded the training accuracy, sensitivity, specificity, AUC and test accuracy of 85.80%, 0.89, 0.83, 0.86 and 83.33%, respectively, better than existing machine learning algorithms. The 14 features and support vector regression were further used to estimate the binding affinities (Pkd) of 200 heterodimeric protein complexes. Prediction performance of a Jackknife test was the correlation coefficient of 0.34 and mean absolute error of 1.4. We further analyze three informative physicochemical properties according to their contribution to prediction performance. Results reveal that the following properties are effective in predicting the binding affinity of heterodimeric protein complexes: apparent partition energy based on buried molar fractions, relations between chemical structure and biological activity in principal component analysis IV, and normalized frequency of beta turn. Conclusions The proposed sequence-based prediction method SVM-BAC uses an optimal feature selection method to identify 14 informative features to classify and predict binding affinity of heterodimeric protein complexes. The characterization analysis revealed that the average numbers of beta turns and hydrogen bonds at protein-protein interfaces in high binding affinity complexes are more than those in low binding affinity complexes. PMID:26681483

Key structural features of nonsteroidal ligands for binding and activation of the androgen receptor.

PubMed

Yin, Donghua; He, Yali; Perera, Minoli A; Hong, Seoung Soo; Marhefka, Craig; Stourman, Nina; Kirkovsky, Leonid; Miller, Duane D; Dalton, James T

2003-01-01

The purposes of the present studies were to examine the androgen receptor (AR) binding ability and in vitro functional activity of multiple series of nonsteroidal compounds derived from known antiandrogen pharmacophores and to investigate the structure-activity relationships (SARs) of these nonsteroidal compounds. The AR binding properties of sixty-five nonsteroidal compounds were assessed by a radioligand competitive binding assay with the use of cytosolic AR prepared from rat prostates. The AR agonist and antagonist activities of high-affinity ligands were determined by the ability of the ligand to regulate AR-mediated transcriptional activation in cultured CV-1 cells, using a cotransfection assay. Nonsteroidal compounds with diverse structural features demonstrated a wide range of binding affinity for the AR. Ten compounds, mainly from the bicalutamide-related series, showed a binding affinity superior to the structural pharmacophore from which they were derived. Several SARs regarding nonsteroidal AR binding were revealed from the binding data, including stereoisomeric conformation, steric effect, and electronic effect. The functional activity of high-affinity ligands ranged from antagonist to full agonist for the AR. Several structural features were found to be determinative of agonist and antagonist activities. The nonsteroidal AR agonists identified from the present studies provided a pool of candidates for further development of selective androgen receptor modulators (SARMs) for androgen therapy. Also, these studies uncovered or confirmed numerous important SARs governing AR binding and functional properties by nonsteroidal molecules, which would be valuable in the future structural optimization of SARMs.

A DFT study for the structural and electronic properties of Zn m Se n nanoclusters

NASA Astrophysics Data System (ADS)

Yadav, Phool Singh; Pandey, Dheeraj Kumar

2012-09-01

An ab initio study has been performed for the stability, structural and electronic properties of 19 small zinc selenide Zn m Se n ( m + n = 2-4) nanoclusters. Out of these nanoclusters, one nanocluster is found to be unstable due to its imaginary vibrational frequency. A B3LYP-DFT/6-311G(3df) method is used in the optimization of the geometries of the nanoclusters. We have calculated the zero point energy (ZPE), which is ignored by the other workers. The binding energies (BE), HOMO-LUMO gaps and bond lengths have been obtained for all the optimized nanoclusters. For the same value of ` m' and ` n', we designate the most stable structure the one, which has maximum final binding energy (FBE) per atom. The adiabatic and vertical ionization potentials (IP) and electron affinities (EA), dipole moments and charge on atoms have been investigated for the most stable nanoclusters. For the same value of ` m' and ` n', the nanocluster containing maximum number of Se atoms is found to be most stable.

Non-additivity of functional group contributions in protein-ligand binding: a comprehensive study by crystallography and isothermal titration calorimetry.

PubMed

Baum, Bernhard; Muley, Laveena; Smolinski, Michael; Heine, Andreas; Hangauer, David; Klebe, Gerhard

2010-04-09

Additivity of functional group contributions to protein-ligand binding is a very popular concept in medicinal chemistry as the basis of rational design and optimized lead structures. Most of the currently applied scoring functions for docking build on such additivity models. Even though the limitation of this concept is well known, case studies examining in detail why additivity fails at the molecular level are still very scarce. The present study shows, by use of crystal structure analysis and isothermal titration calorimetry for a congeneric series of thrombin inhibitors, that extensive cooperative effects between hydrophobic contacts and hydrogen bond formation are intimately coupled via dynamic properties of the formed complexes. The formation of optimal lipophilic contacts with the surface of the thrombin S3 pocket and the full desolvation of this pocket can conflict with the formation of an optimal hydrogen bond between ligand and protein. The mutual contributions of the competing interactions depend on the size of the ligand hydrophobic substituent and influence the residual mobility of ligand portions at the binding site. Analysis of the individual crystal structures and factorizing the free energy into enthalpy and entropy demonstrates that binding affinity of the ligands results from a mixture of enthalpic contributions from hydrogen bonding and hydrophobic contacts, and entropic considerations involving an increasing loss of residual mobility of the bound ligands. This complex picture of mutually competing and partially compensating enthalpic and entropic effects determines the non-additivity of free energy contributions to ligand binding at the molecular level. (c) 2010 Elsevier Ltd. All rights reserved.

Computational optimization of AG18051 inhibitor for amyloid-beta binding alcohol dehydrogenase enzyme

NASA Astrophysics Data System (ADS)

Marques, Alexandra T.; Antunes, Agostinho; Fernandes, Pedro A.; Ramos, Maria J.

Amyloid-beta (Abeta) binding alcohol dehydrogenase (ABAD) is a multifunctional enzyme involved in maintaining the homeostasis. The enzyme can also mediate some diseases, including genetic diseases, Alzheimer's disease, and possibly some prostate cancers. Potent inhibitors of ABAD might facilitate a better clarification of the functions of the enzyme under normal and pathogenic conditions and might also be used for therapeutic intervention in disease conditions mediated by the enzyme. The AG18051 is the only presently available inhibitor of ABAD. It binds in the active-site cavity of the enzyme and reacts with the NAD+ cofactor to form a covalent adduct. In this work, we use computational methods to perform a rational optimization of the AG18051 inhibitor, through the introduction of chemical substitutions directed to improve the affinity of the inhibitor to the enzyme. The molecular mechanics-Poisson-Boltzmann surface area methodology was used to predict the relative free binding energy of the different modified inhibitor-NAD-enzyme complexes. We show that it is possible to increase significantly the affinity of the inhibitor to the enzyme with small modifications, without changing the overall structure and ADME (absorption, distribution, metabolism, and excretion) properties of the original inhibitor.

Mechanistic and quantitative insight into cell surface targeted molecular imaging agent design.

PubMed

Zhang, Liang; Bhatnagar, Sumit; Deschenes, Emily; Thurber, Greg M

2016-05-05

Molecular imaging agent design involves simultaneously optimizing multiple probe properties. While several desired characteristics are straightforward, including high affinity and low non-specific background signal, in practice there are quantitative trade-offs between these properties. These include plasma clearance, where fast clearance lowers background signal but can reduce target uptake, and binding, where high affinity compounds sometimes suffer from lower stability or increased non-specific interactions. Further complicating probe development, many of the optimal parameters vary depending on both target tissue and imaging agent properties, making empirical approaches or previous experience difficult to translate. Here, we focus on low molecular weight compounds targeting extracellular receptors, which have some of the highest contrast values for imaging agents. We use a mechanistic approach to provide a quantitative framework for weighing trade-offs between molecules. Our results show that specific target uptake is well-described by quantitative simulations for a variety of targeting agents, whereas non-specific background signal is more difficult to predict. Two in vitro experimental methods for estimating background signal in vivo are compared - non-specific cellular uptake and plasma protein binding. Together, these data provide a quantitative method to guide probe design and focus animal work for more cost-effective and time-efficient development of molecular imaging agents.

Cofactor-binding sites in proteins of deviating sequence: comparative analysis and clustering in torsion angle, cavity, and fold space.

PubMed

Stegemann, Björn; Klebe, Gerhard

2012-02-01

Small molecules are recognized in protein-binding pockets through surface-exposed physicochemical properties. To optimize binding, they have to adopt a conformation corresponding to a local energy minimum within the formed protein-ligand complex. However, their conformational flexibility makes them competent to bind not only to homologous proteins of the same family but also to proteins of remote similarity with respect to the shape of the binding pockets and folding pattern. Considering drug action, such observations can give rise to unexpected and undesired cross reactivity. In this study, datasets of six different cofactors (ADP, ATP, NAD(P)(H), FAD, and acetyl CoA, sharing an adenosine diphosphate moiety as common substructure), observed in multiple crystal structures of protein-cofactor complexes exhibiting sequence identity below 25%, have been analyzed for the conformational properties of the bound ligands, the distribution of physicochemical properties in the accommodating protein-binding pockets, and the local folding patterns next to the cofactor-binding site. State-of-the-art clustering techniques have been applied to group the different protein-cofactor complexes in the different spaces. Interestingly, clustering in cavity (Cavbase) and fold space (DALI) reveals virtually the same data structuring. Remarkable relationships can be found among the different spaces. They provide information on how conformations are conserved across the host proteins and which distinct local cavity and fold motifs recognize the different portions of the cofactors. In those cases, where different cofactors are found to be accommodated in a similar fashion to the same fold motifs, only a commonly shared substructure of the cofactors is used for the recognition process. Copyright © 2011 Wiley Periodicals, Inc.

Three-dimensional structure-activity relationship modeling of cocaine binding to two monoclonal antibodies by comparative molecular field analysis.

PubMed

Paula, Stefan; Tabet, Michael R; Keenan, Susan M; Welsh, William J; Ball, W James

2003-01-17

Successful immunotherapy of cocaine addiction and overdoses requires cocaine-binding antibodies with specific properties, such as high affinity and selectivity for cocaine. We have determined the affinities of two cocaine-binding murine monoclonal antibodies (mAb: clones 3P1A6 and MM0240PA) for cocaine and its metabolites by [3H]-radioligand binding assays. mAb 3P1A6 (K(d) = 0.22 nM) displayed a 50-fold higher affinity for cocaine than mAb MM0240PA (K(d) = 11 nM) and also had a greater specificity for cocaine. For the systematic exploration of both antibodies' binding specificities, we used a set of approximately 35 cocaine analogues as structural probes by determining their relative binding affinities (RBAs) using an enzyme-linked immunosorbent competition assay. Three-dimensional quantitative structure-activity relationship (3D-QSAR) models on the basis of comparative molecular field analysis (CoMFA) techniques correlated the binding data with structural features of the ligands. The analysis indicated that despite the mAbs' differing specificities for cocaine, the relative contributions of the steric (approximately 80%) and electrostatic (approximately 20%) field interactions to ligand-binding were similar. Generated three-dimensional CoMFA contour plots then located the specific regions about cocaine where the ligand/receptor interactions occurred. While the overall binding patterns of the two mAbs had many features in common, distinct differences were observed about the phenyl ring and the methylester group of cocaine. Furthermore, using previously published data, a 3D-QSAR model was developed for cocaine binding to the dopamine reuptake transporter (DAT) that was compared to the mAb models. Although the relative steric and electrostatic field contributions were similar to those of the mAbs, the DAT cocaine-binding site showed a preference for negatively charged ligands. Besides establishing molecular level insight into the interactions that govern cocaine binding specificity by biopolymers, the three-dimensional images obtained reflect the properties of the mAbs binding pockets and provide the initial information needed for the possible design of novel antibodies with properties optimized for immunotherapy. Copyright 2003 Elsevier Science Ltd.

The Kinetic Mechanism for Cytochrome P450 Metabolism of Type II Binding Compounds: Evidence Supporting Direct Reduction

PubMed Central

Pearson, Joshua; Dahal, Upendra P.; Rock, Daniel; Peng, Chi-Chi; Schenk, James O.; Joswig-Jones, Carolyn; Jones, Jeffrey P.

2011-01-01

The metabolic stability of a drug is an important property that should be optimized during drug design and development. Nitrogen incorporation is hypothesized to increase the stability by coordination of nitrogen to the heme iron of cytochrome P450, a binding mode that is referred to as type II binding. However, we noticed that the type II binding compound 1 has less metabolic stability at subsaturating conditions than a closely related type I binding compound 3. Three kinetic models will be presented for type II binder metabolism; 1) Dead-end type II binding, 2) a rapid equilibrium between type I and II binding modes before reduction, and 3) a direct reduction of the type II coordinated heme. Data will be presented on reduction rates of iron, the off rates of substrate (using surface plasmon resonance) and the catalytic rate constants. These data argue against the dead-end, and rapid equilibrium models, leaving the direct reduction kinetic mechanism for metabolism of the type II binding compound 1. PMID:21530484

The Solution Structure, Binding Properties, and Dynamics of the Bacterial Siderophore-binding Protein FepB*

PubMed Central

Chu, Byron C. H.; Otten, Renee; Krewulak, Karla D.; Mulder, Frans A. A.; Vogel, Hans J.

2014-01-01

The periplasmic binding protein (PBP) FepB plays a key role in transporting the catecholate siderophore ferric enterobactin from the outer to the inner membrane in Gram-negative bacteria. The solution structures of the 34-kDa apo- and holo-FepB from Escherichia coli, solved by NMR, represent the first solution structures determined for the type III class of PBPs. Unlike type I and II PBPs, which undergo large “Venus flytrap” conformational changes upon ligand binding, both forms of FepB maintain similar overall folds; however, binding of the ligand is accompanied by significant loop movements. Reverse methyl cross-saturation experiments corroborated chemical shift perturbation results and uniquely defined the binding pocket for gallium enterobactin (GaEnt). NMR relaxation experiments indicated that a flexible loop (residues 225–250) adopted a more rigid and extended conformation upon ligand binding, which positioned residues for optimal interactions with the ligand and the cytoplasmic membrane ABC transporter (FepCD), respectively. In conclusion, this work highlights the pivotal role that structural dynamics plays in ligand binding and transporter interactions in type III PBPs. PMID:25173704

Identification of DNA-binding proteins using multi-features fusion and binary firefly optimization algorithm.

PubMed

Zhang, Jian; Gao, Bo; Chai, Haiting; Ma, Zhiqiang; Yang, Guifu

2016-08-26

DNA-binding proteins (DBPs) play fundamental roles in many biological processes. Therefore, the developing of effective computational tools for identifying DBPs is becoming highly desirable. In this study, we proposed an accurate method for the prediction of DBPs. Firstly, we focused on the challenge of improving DBP prediction accuracy with information solely from the sequence. Secondly, we used multiple informative features to encode the protein. These features included evolutionary conservation profile, secondary structure motifs, and physicochemical properties. Thirdly, we introduced a novel improved Binary Firefly Algorithm (BFA) to remove redundant or noisy features as well as select optimal parameters for the classifier. The experimental results of our predictor on two benchmark datasets outperformed many state-of-the-art predictors, which revealed the effectiveness of our method. The promising prediction performance on a new-compiled independent testing dataset from PDB and a large-scale dataset from UniProt proved the good generalization ability of our method. In addition, the BFA forged in this research would be of great potential in practical applications in optimization fields, especially in feature selection problems. A highly accurate method was proposed for the identification of DBPs. A user-friendly web-server named iDbP (identification of DNA-binding Proteins) was constructed and provided for academic use.

Uncertainty analysis of the nonideal competitive adsorption-donnan model: effects of dissolved organic matter variability on predicted metal speciation in soil solution.

PubMed

Groenenberg, Jan E; Koopmans, Gerwin F; Comans, Rob N J

2010-02-15

Ion binding models such as the nonideal competitive adsorption-Donnan model (NICA-Donnan) and model VI successfully describe laboratory data of proton and metal binding to purified humic substances (HS). In this study model performance was tested in more complex natural systems. The speciation predicted with the NICA-Donnan model and the associated uncertainty were compared with independent measurements in soil solution extracts, including the free metal ion activity and fulvic (FA) and humic acid (HA) fractions of dissolved organic matter (DOM). Potentially important sources of uncertainty are the DOM composition and the variation in binding properties of HS. HS fractions of DOM in soil solution extracts varied between 14 and 63% and consisted mainly of FA. Moreover, binding parameters optimized for individual FA samples show substantial variation. Monte Carlo simulations show that uncertainties in predicted metal speciation, for metals with a high affinity for FA (Cu, Pb), are largely due to the natural variation in binding properties (i.e., the affinity) of FA. Predictions for metals with a lower affinity (Cd) are more prone to uncertainties in the fraction FA in DOM and the maximum site density (i.e., the capacity) of the FA. Based on these findings, suggestions are provided to reduce uncertainties in model predictions.

Theoretical Studies for Dendrimer-Based Drug Delivery.

PubMed

Bello, Martiniano; Fragoso-Vázquez, Jonathan; Correa-Basurto, José

2017-01-01

Numerous theoretical studies have been performed to iteratively optimize the physicochemical properties such as dendrimer size and surface constituents in solution, as well as their molecular recognition properties for drugs, lipid membranes, nucleic acids and proteins, etc. Molecular modeling approaches such as docking and molecular dynamic (MD) simulations have supported experimental efforts by providing important insights into the structural properties of dendrimers in solution and possible binding properties of drugs at the atomic level. We review the utilization of molecular modelling tools to obtain insight into the study and design of dendrimers, with particular importance placed on the improvement of binding properties of dendrimers for their use as drug nanocarriers and to increase the water solubility properties and drug delivery. The modeling studies discussed in this review have provided substantial insight into the physicochemical properties of dendrimers in solution, including solvent pH and counterion distribution, at the atomic level, as well as the elucidation of some of the key interactions in solution of unmodified and modified dendrimers with some drugs of pharmaceutics interest and biological systems such as nucleic acids, proteins and lipid membranes. the described studies illustrate that whether simulations will be run at the all-atom or coarse-grained level, physicochemical conditions such as the type of force field, the treatment of electrostatics effects, counterion distribution, protonation state of dendrimers, and dendrimer concentrations which have been probed to play a crucial role in the structural behavior and binding properties must be prudently incorporated in the simulations. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Inhibition of HIV-1 by a peptide ligand of the genomic RNA packaging signal Psi.

PubMed

Dietz, Julia; Koch, Joachim; Kaur, Ajit; Raja, Chinnappan; Stein, Stefan; Grez, Manuel; Pustowka, Anette; Mensch, Sarah; Ferner, Jan; Möller, Lars; Bannert, Norbert; Tampé, Robert; Divita, Gilles; Mély, Yves; Schwalbe, Harald; Dietrich, Ursula

2008-05-01

The interaction of the nucleocapsid NCp7 of the human immunodeficiency virus type 1 (HIV-1) Gag polyprotein with the RNA packaging signal Psi ensures specific encapsidation of the dimeric full length viral genome into nascent virus particles. Being an essential step in the HIV-1 replication cycle, specific genome encapsidation represents a promising target for therapeutic intervention. We previously selected peptides binding to HIV-1 Psi-RNA or stem loops (SL) thereof by phage display. Herein, we describe synthesis of peptide variants of the consensus HWWPWW motif on membrane supports to optimize Psi-RNA binding. The optimized peptide, psi-pepB, was characterized in detail with respect to its conformation and binding properties for the SL3 of the Psi packaging signal by NMR and tryptophan fluorescence quenching. Functional analysis revealed that psi-pepB caused a strong reduction of virus release by infected cells as monitored by reduced transduction efficiencies, capsid p24 antigen levels, and electron microscopy. Thus, this peptide shows antiviral activity and could serve as a lead compound to develop new drugs targeting HIV-1.

AutoSite: an automated approach for pseudo-ligands prediction—from ligand-binding sites identification to predicting key ligand atoms

PubMed Central

Ravindranath, Pradeep Anand; Sanner, Michel F.

2016-01-01

Motivation: The identification of ligand-binding sites from a protein structure facilitates computational drug design and optimization, and protein function assignment. We introduce AutoSite: an efficient software tool for identifying ligand-binding sites and predicting pseudo ligand corresponding to each binding site identified. Binding sites are reported as clusters of 3D points called fills in which every point is labelled as hydrophobic or as hydrogen bond donor or acceptor. From these fills AutoSite derives feature points: a set of putative positions of hydrophobic-, and hydrogen-bond forming ligand atoms. Results: We show that AutoSite identifies ligand-binding sites with higher accuracy than other leading methods, and produces fills that better matches the ligand shape and properties, than the fills obtained with a software program with similar capabilities, AutoLigand. In addition, we demonstrate that for the Astex Diverse Set, the feature points identify 79% of hydrophobic ligand atoms, and 81% and 62% of the hydrogen acceptor and donor hydrogen ligand atoms interacting with the receptor, and predict 81.2% of water molecules mediating interactions between ligand and receptor. Finally, we illustrate potential uses of the predicted feature points in the context of lead optimization in drug discovery projects. Availability and Implementation: http://adfr.scripps.edu/AutoDockFR/autosite.html Contact: sanner@scripps.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:27354702

Directed evolution of glutathione transferases towards a selective glutathione-binding site and improved oxidative stability.

PubMed

Axarli, Irine; Muleta, Abdi W; Chronopoulou, Evangelia G; Papageorgiou, Anastassios C; Labrou, Nikolaos E

2017-01-01

Glutathione transferases (GSTs) are a family of detoxification enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic compounds. A library of alpha class GSTs was constructed by DNA shuffling using the DNA encoding the human glutathione transferase A1-1 (hGSTA1-1) and the rat glutathione transferase A1-1 (rGSTA1-1). Activity screening of the library allowed the selection of a chimeric enzyme variant (GSTD4) that displayed high affinity towards GSH and GSH-Sepharose affinity adsorbent, higher k cat /K m and improved thermal stability, compared to the parent enzymes. The crystal structures of the GSTD4 enzyme in free form and in complex with GSH were determined to 1.6Šand 2.3Šresolution, respectively. Analysis of the GSTD4 structure showed subtle conformational changes in the GSH-binding site and in electron-sharing network that may contribute to the increased GSH affinity. The shuffled variant GSTD4 was further optimized for improved oxidative stability employing site-saturation mutagenesis. The Cys112Ser mutation confers optimal oxidative stability and kinetic properties in the GSTD4 enzyme. DNA shuffling allowed the creation of a chimeric enzyme variant with improved properties, compared to the parent enzymes. X-ray crystallography shed light on how recombination of a specific segment from homologous GSTA1-1 together with point mutations gives rise to a new functionally competent enzyme with improved binding, catalytic properties and stability. Such an engineered GST would be useful in biotechnology as affinity tool in affinity chromatography as well as a biocatalytic matrix for the construction of biochips or enzyme biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

Interpreting linear support vector machine models with heat map molecule coloring

PubMed Central

2011-01-01

Background Model-based virtual screening plays an important role in the early drug discovery stage. The outcomes of high-throughput screenings are a valuable source for machine learning algorithms to infer such models. Besides a strong performance, the interpretability of a machine learning model is a desired property to guide the optimization of a compound in later drug discovery stages. Linear support vector machines showed to have a convincing performance on large-scale data sets. The goal of this study is to present a heat map molecule coloring technique to interpret linear support vector machine models. Based on the weights of a linear model, the visualization approach colors each atom and bond of a compound according to its importance for activity. Results We evaluated our approach on a toxicity data set, a chromosome aberration data set, and the maximum unbiased validation data sets. The experiments show that our method sensibly visualizes structure-property and structure-activity relationships of a linear support vector machine model. The coloring of ligands in the binding pocket of several crystal structures of a maximum unbiased validation data set target indicates that our approach assists to determine the correct ligand orientation in the binding pocket. Additionally, the heat map coloring enables the identification of substructures important for the binding of an inhibitor. Conclusions In combination with heat map coloring, linear support vector machine models can help to guide the modification of a compound in later stages of drug discovery. Particularly substructures identified as important by our method might be a starting point for optimization of a lead compound. The heat map coloring should be considered as complementary to structure based modeling approaches. As such, it helps to get a better understanding of the binding mode of an inhibitor. PMID:21439031

Mathematical modeling of vesicle drug delivery systems 2: targeted vesicle interactions with cells, tumors, and the body.

PubMed

Ying, Chong T; Wang, Juntian; Lamm, Robert J; Kamei, Daniel T

2013-02-01

Vesicles have been studied for several years in their ability to deliver drugs. Mathematical models have much potential in reducing time and resources required to engineer optimal vesicles, and this review article summarizes these models that aid in understanding the ability of targeted vesicles to bind and internalize into cancer cells, diffuse into tumors, and distribute in the body. With regard to binding and internalization, radiolabeling and surface plasmon resonance experiments can be performed to determine optimal vesicle size and the number and type of ligands conjugated. Binding and internalization properties are also inputs into a mathematical model of vesicle diffusion into tumor spheroids, which highlights the importance of the vesicle diffusion coefficient and the binding affinity of the targeting ligand. Biodistribution of vesicles in the body, along with their half-life, can be predicted with compartmental models for pharmacokinetics that include the effect of targeting ligands, and these predictions can be used in conjunction with in vivo models to aid in the design of drug carriers. Mathematical models can prove to be very useful in drug carrier design, and our hope is that this review will encourage more investigators to combine modeling with quantitative experimentation in the field of vesicle-based drug delivery.

Isolation, Characterization and Lipid-Binding Properties of the Recalcitrant FtsA Division Protein from Escherichia coli

PubMed Central

Zorrilla, Silvia; Reija, Belén; Alfonso, Carlos; Mingorance, Jesús; Rivas, Germán; Jiménez, Mercedes

2012-01-01

We have obtained milligram amounts of highly pure Escherichia coli division protein FtsA from inclusion bodies with an optimized purification method that, by overcoming the reluctance of FtsA to be purified, surmounts a bottleneck for the analysis of the molecular basis of FtsA function. Purified FtsA is folded, mostly monomeric and interacts with lipids. The apparent affinity of FtsA binding to the inner membrane is ten-fold higher than to phospholipids, suggesting that inner membrane proteins could modulate FtsA-membrane interactions. Binding of FtsA to lipids and membranes is insensitive to ionic strength, indicating that a net contribution of hydrophobic interactions is involved in the association of FtsA to lipid/membrane structures. PMID:22761913

In silico optimization of pharmacokinetic properties and receptor binding affinity simultaneously: a 'parallel progression approach to drug design' applied to β-blockers.

PubMed

Advani, Poonam; Joseph, Blessy; Ambre, Premlata; Pissurlenkar, Raghuvir; Khedkar, Vijay; Iyer, Krishna; Gabhe, Satish; Iyer, Radhakrishnan P; Coutinho, Evans

2016-01-01

The present work exploits the potential of in silico approaches for minimizing attrition of leads in the later stages of drug development. We propose a theoretical approach, wherein 'parallel' information is generated to simultaneously optimize the pharmacokinetics (PK) and pharmacodynamics (PD) of lead candidates. β-blockers, though in use for many years, have suboptimal PKs; hence are an ideal test series for the 'parallel progression approach'. This approach utilizes molecular modeling tools viz. hologram quantitative structure activity relationships, homology modeling, docking, predictive metabolism, and toxicity models. Validated models have been developed for PK parameters such as volume of distribution (log Vd) and clearance (log Cl), which together influence the half-life (t1/2) of a drug. Simultaneously, models for PD in terms of inhibition constant pKi have been developed. Thus, PK and PD properties of β-blockers were concurrently analyzed and after iterative cycling, modifications were proposed that lead to compounds with optimized PK and PD. We report some of the resultant re-engineered β-blockers with improved half-lives and pKi values comparable with marketed β-blockers. These were further analyzed by the docking studies to evaluate their binding poses. Finally, metabolic and toxicological assessment of these molecules was done through in silico methods. The strategy proposed herein has potential universal applicability, and can be used in any drug discovery scenario; provided that the data used is consistent in terms of experimental conditions, endpoints, and methods employed. Thus the 'parallel progression approach' helps to simultaneously fine-tune various properties of the drug and would be an invaluable tool during the drug development process.

Capture of Pb2+ and Cu2+ Metal Cations by Neisseria meningitidis-type Capsular Polysaccharides.

PubMed

Ghimire, Sujan; McCarthy, Pumtiwitt C

2018-05-05

Heavy metal pollution of water is a significant environmental and public health concern. Current biological strategies for heavy metal removal from water are performed using microbial biopolymers, including polysaccharides, that are already fully formed. This creates limitations in adapting polysaccharides to increase binding affinity for specific metals. We propose that altering the specificity of polysaccharide-producing enzymes could be beneficial to improving metal capture by modified polysaccharides. We assess binding of Cu 2+ and Pb 2+ metal cations to Neisseria meningitidis -type polysaccharides. All concentrations of metal cations tested were able to completely bind to colominic acid. This polymer is equivalent to the capsular polysaccharide of N. meningitidis serogroup B comprised of a homopolymer of negatively charged sialic acid. There was slightly less binding observed with N. meningitidis serogroup W, which contains repeating units of the neutral sugar galactose and sialic acid. Our work represents the first assessment of the metal-binding properties of these capsular polysaccharides. Future work will seek to optimize metal-binding with Neisseria meningitidis serogroup W polysaccharide.

Fragment Based Optimization of Metabotropic Glutamate Receptor 2 (mGluR2) Positive Allosteric Modulators in the Absence of Structural Information.

PubMed

Szabó, György; Túrós, György I; Kolok, Sándor; Vastag, Mónika; Sánta, Zsuzsanna; Dékány, Miklós; Lévay, György I; Greiner, István; Natsumi, Minami; Tatsuya, Watanabe; Keserű, György M

2018-03-14

Metabotropic glutamate receptor 2 (mGluR2) positive allosteric modulators (PAMs) have been implicated as potential pharmacotherapy for psychiatric conditions. Screening our corporate compound deck, we identified a benzotriazole fragment (4) that was rapidly optimized to a potent and metabolically stable early lead (16). The highly lipophilic character of 16, together with its limited solubility, permeability, and high protein binding, however, did not allow reaching of the proof of concept in vivo. Since further attempts on the optimization of druglike properties were unsuccessful, the original hit 4 has been revisited and was optimized following the principles of fragment based drug discovery (FBDD). Lacking structural information on the receptor-ligand complex, we implemented a group efficiency (GE) based strategy and identified a new fragment like lead (60) with more balanced profile. Significant improvement achieved on the druglike properties nominated the compound for in vivo proof of concept studies that revealed the chemotype being a promising PAM lead targeting mGluR2 receptors.

On optima: the case of myoglobin-facilitated oxygen diffusion.

PubMed

Wittenberg, Jonathan B

2007-08-15

The process of myoglobin/leghemoglobin-facilitated oxygen diffusion is adapted to function in different environments in diverse organisms. We enquire how the functional parameters of the process are optimized in particular organisms. The ligand-binding properties of the proteins, myoglobin and plant symbiotic hemoglobins, we discover, suggest that they have been adapted under genetic selection pressure for optimal performance. Since carrier-mediated oxygen transport has probably evolved independantly many times, adaptation of diverse proteins for a common functionality exemplifies the process of convergent evolution. The progenitor proteins may be built on the myoglobin scaffold or may be very different.

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

PubMed Central

Hu, Liang; Chen, Dong-ying

2009-01-01

Aim: To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions. Methods: A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination. Results: The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R2) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×103(mol/L)-1 and 59.5%, respectively. Conclusion: Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma. PMID:19890364

Spectro Analytical, Computational and In Vitro Biological Studies of Novel Substituted Quinolone Hydrazone and it's Metal Complexes.

PubMed

Nagula, Narsimha; Kunche, Sudeepa; Jaheer, Mohmed; Mudavath, Ravi; Sivan, Sreekanth; Ch, Sarala Devi

2018-01-01

Some novel transition metal [Cu (II), Ni (II) and Co (II)] complexes of nalidixic acid hydrazone have been prepared and characterized by employing spectro-analytical techniques viz: elemental analysis, 1 H-NMR, Mass, UV-Vis, IR, TGA-DTA, SEM-EDX, ESR and Spectrophotometry studies. The HyperChem 7.5 software was used for geometry optimization of title compound in its molecular and ionic forms. Quantum mechanical parameters, contour maps of highest occupied molecular orbitals (HOMO) and lowest unoccupied molecular orbitals (LUMO) and corresponding binding energy values were computed using semi empirical single point PM3 method. The stoichiometric equilibrium studies of metal complexes carried out spectrophotometrically using Job's continuous variation and mole ratio methods inferred formation of 1:2 (ML 2 ) metal complexes in respective systems. The title compound and its metal complexes screened for antibacterial and antifungal properties, exemplified improved activity in metal complexes. The studies of nuclease activity for the cleavage of CT- DNA and MTT assay for in vitro cytotoxic properties involving metal complexes exhibited high activity. In addition, the DNA binding properties of Cu (II), Ni (II) and Co (II) complexes investigated by electronic absorption and fluorescence measurements revealed their good binding ability and commended agreement of K b values obtained from both the techniques. Molecular docking studies were also performed to find the binding affinity of synthesized compounds with DNA (PDB ID: 1N37) and "Thymidine phosphorylase from E.coli" (PDB ID: 4EAF) protein targets.

Targeted therapy using nanotechnology: focus on cancer

PubMed Central

Sanna, Vanna; Pala, Nicolino; Sechi, Mario

2014-01-01

Recent advances in nanotechnology and biotechnology have contributed to the development of engineered nanoscale materials as innovative prototypes to be used for biomedical applications and optimized therapy. Due to their unique features, including a large surface area, structural properties, and a long circulation time in blood compared with small molecules, a plethora of nanomaterials has been developed, with the potential to revolutionize the diagnosis and treatment of several diseases, in particular by improving the sensitivity and recognition ability of imaging contrast agents and by selectively directing bioactive agents to biological targets. Focusing on cancer, promising nanoprototypes have been designed to overcome the lack of specificity of conventional chemotherapeutic agents, as well as for early detection of precancerous and malignant lesions. However, several obstacles, including difficulty in achieving the optimal combination of physicochemical parameters for tumor targeting, evading particle clearance mechanisms, and controlling drug release, prevent the translation of nanomedicines into therapy. In spite of this, recent efforts have been focused on developing functionalized nanoparticles for delivery of therapeutic agents to specific molecular targets overexpressed on different cancer cells. In particular, the combination of targeted and controlled-release polymer nanotechnologies has resulted in a new programmable nanotherapeutic formulation of docetaxel, namely BIND-014, which recently entered Phase II clinical testing for patients with solid tumors. BIND-014 has been developed to overcome the limitations facing delivery of nanoparticles to many neoplasms, and represents a validated example of targeted nanosystems with the optimal biophysicochemical properties needed for successful tumor eradication. PMID:24531078

Unveiling Stability Criteria of DNA-Carbon Nanotubes Constructs by Scanning Tunneling Microscopy and Computational Modeling

DOE PAGES

Kilina, Svetlana; Yarotski, Dzmitry A.; Talin, A. Alec; ...

2011-01-01

We present a combined approach that relies on computational simulations and scanning tunneling microscopy (STM) measurements to reveal morphological properties and stability criteria of carbon nanotube-DNA (CNT-DNA) constructs. Application of STM allows direct observation of very stable CNT-DNA hybrid structures with the well-defined DNA wrapping angle of 63.4 ° and a coiling period of 3.3 nm. Using force field simulations, we determine how the DNA-CNT binding energy depends on the sequence and binding geometry of a single strand DNA. This dependence allows us to quantitatively characterize the stability of a hybrid structure with an optimal π-stacking between DNA nucleotides and themore » tube surface and better interpret STM data. Our simulations clearly demonstrate the existence of a very stable DNA binding geometry for (6,5) CNT as evidenced by the presence of a well-defined minimum in the binding energy as a function of an angle between DNA strand and the nanotube chiral vector. This novel approach demonstrates the feasibility of CNT-DNA geometry studies with subnanometer resolution and paves the way towards complete characterization of the structural and electronic properties of drug-delivering systems based on DNA-CNT hybrids as a function of DNA sequence and a nanotube chirality.« less

Essential role of conformational selection in ligand binding.

PubMed

Vogt, Austin D; Pozzi, Nicola; Chen, Zhiwei; Di Cera, Enrico

2014-02-01

Two competing and mutually exclusive mechanisms of ligand recognition - conformational selection and induced fit - have dominated our interpretation of ligand binding in biological macromolecules for almost six decades. Conformational selection posits the pre-existence of multiple conformations of the macromolecule from which the ligand selects the optimal one. Induced fit, on the other hand, postulates the existence of conformational rearrangements of the original conformation into an optimal one that are induced by binding of the ligand. In the former case, conformational transitions precede the binding event; in the latter, conformational changes follow the binding step. Kineticists have used a facile criterion to distinguish between the two mechanisms based on the dependence of the rate of relaxation to equilibrium, kobs, on the ligand concentration, [L]. A value of kobs decreasing hyperbolically with [L] has been seen as diagnostic of conformational selection, while a value of kobs increasing hyperbolically with [L] has been considered diagnostic of induced fit. However, this simple conclusion is only valid under the rather unrealistic assumption of conformational transitions being much slower than binding and dissociation events. In general, induced fit only produces values of kobs that increase with [L] but conformational selection is more versatile and is associated with values of kobs that increase with, decrease with or are independent of [L]. The richer repertoire of kinetic properties of conformational selection applies to kinetic mechanisms with single or multiple saturable relaxations and explains the behavior of nearly all experimental systems reported in the literature thus far. Conformational selection is always sufficient and often necessary to account for the relaxation kinetics of ligand binding to a biological macromolecule and is therefore an essential component of any binding mechanism. On the other hand, induced fit is never necessary and only sufficient in a few cases. Therefore, the long assumed importance and preponderance of induced fit as a mechanism of ligand binding should be reconsidered. © 2013 Elsevier B.V. All rights reserved.

Thermodynamic Studies for Drug Design and Screening

PubMed Central

Garbett, Nichola C.; Chaires, Jonathan B.

2012-01-01

Introduction A key part of drug design and development is the optimization of molecular interactions between an engineered drug candidate and its binding target. Thermodynamic characterization provides information about the balance of energetic forces driving binding interactions and is essential for understanding and optimizing molecular interactions. Areas covered This review discusses the information that can be obtained from thermodynamic measurements and how this can be applied to the drug development process. Current approaches for the measurement and optimization of thermodynamic parameters are presented, specifically higher throughput and calorimetric methods. Relevant literature for this review was identified in part by bibliographic searches for the period 2004 – 2011 using the Science Citation Index and PUBMED and the keywords listed below. Expert opinion The most effective drug design and development platform comes from an integrated process utilizing all available information from structural, thermodynamic and biological studies. Continuing evolution in our understanding of the energetic basis of molecular interactions and advances in thermodynamic methods for widespread application are essential to realize the goal of thermodynamically-driven drug design. Comprehensive thermodynamic evaluation is vital early in the drug development process to speed drug development towards an optimal energetic interaction profile while retaining good pharmacological properties. Practical thermodynamic approaches, such as enthalpic optimization, thermodynamic optimization plots and the enthalpic efficiency index, have now matured to provide proven utility in design process. Improved throughput in calorimetric methods remains essential for even greater integration of thermodynamics into drug design. PMID:22458502

A look at ligand binding thermodynamics in drug discovery.

PubMed

Claveria-Gimeno, Rafael; Vega, Sonia; Abian, Olga; Velazquez-Campoy, Adrian

2017-04-01

Drug discovery is a challenging endeavor requiring the interplay of many different research areas. Gathering information on ligand binding thermodynamics may help considerably in reducing the risk within a high uncertainty scenario, allowing early rejection of flawed compounds and pushing forward optimal candidates. In particular, the free energy, the enthalpy, and the entropy of binding provide fundamental information on the intermolecular forces driving such interaction. Areas covered: The authors review the current status and recent developments in the application of ligand binding thermodynamics in drug discovery. The thermodynamic binding profile (Gibbs energy, enthalpy, and entropy of binding) can be used for lead selection and optimization (binding enthalpy, selectivity, and adaptability). Expert opinion: Binding thermodynamics provides fundamental information on the forces driving the formation of the drug-target complex. It has been widely accepted that binding thermodynamics may be used as a decision criterion along the ligand optimization process in drug discovery and development. In particular, the binding enthalpy may be used as a guide when selecting and optimizing compounds over a set of potential candidates. However, this has been recently called into question by arguing certain difficulties and in the light of certain experimental examples.

Properties of human brain sodium channel α-subunits expressed in HEK293 cells and their modulation by carbamazepine, phenytoin and lamotrigine

PubMed Central

Qiao, Xin; Sun, Guangchun; Clare, Jeffrey J; Werkman, Taco R; Wadman, Wytse J

2014-01-01

Background and purpose Voltage-activated Na+ channels contain one distinct α-subunit. In the brain NaV1.1, NaV1.2, NaV1.3 and NaV1.6 are the four most abundantly expressed α-subunits. The antiepileptic drugs (AEDs) carbamazepine, phenytoin and lamotrigine have voltage-gated Na+ channels as their primary therapeutic targets. This study provides a systematic comparison of the biophysical properties of these four α-subunits and characterizes their interaction with carbamazepine, phenytoin and lamotrigine. Experimental approach Na+ currents were recorded in voltage-clamp mode in HEK293 cells stably expressing one of the four α-subunits. Key results NaV1.2 and NaV1.3 subunits have a relatively slow recovery from inactivation, compared with the other subunits and NaV1.1 subunits generate the largest window current. Lamotrigine evokes a larger maximal shift of the steady-state inactivation relationship than carbamazepine or phenytoin. Carbamazepine shows the highest binding rate to the α-subunits. Lamotrigine binding to NaV1.1 subunits is faster than to the other α-subunits. Lamotrigine unbinding from the α-subunits is slower than that of carbamazepine and phenytoin. Conclusions and implications The four Na+ channel α-subunits show subtle differences in their biophysical properties, which, in combination with their (sub)cellular expression patterns in the brain, could contribute to differences in neuronal excitability. We also observed differences in the parameters that characterize AED binding to the Na+ channel subunits. Particularly, lamotrigine binding to the four α-subunits suggests a subunit-specific response. Such differences will have consequences for the clinical efficacy of AEDs. Knowledge of the biophysical and binding parameters could be employed to optimize therapeutic strategies and drug development. PMID:24283699

Post-SELEX optimization of aptamers.

PubMed

Gao, Shunxiang; Zheng, Xin; Jiao, Binghua; Wang, Lianghua

2016-07-01

Aptamers are functional single-stranded DNA or RNA oligonucleotides, selected in vitro by SELEX (Systematic Evolution of Ligands by Exponential Enrichment), which can fold into stable unique three-dimensional structures that bind their target ligands with high affinity and specificity. Although aptamers show a number of favorable advantages such as better stability and easier modification when compared with the properties of antibodies, only a handful of aptamers have entered clinical trials and only one, pegaptanib, has received US Food and Drug Administration approval for clinical use. The main reasons that limit the practical application of aptamers are insufficient nuclease stability, bioavailability, thermal stability, or even affinity. Some aptamers obtained from modified libraries show better properties; however, polymerase amplification of nucleic acids containing non-natural bases is currently a primary drawback of the SELEX process. This review focuses on several post-SELEX optimization strategies of aptamers identified in recent years. We describe four common methods in detail: truncation, chemical modification, bivalent or multivalent aptamer construction, and mutagenesis. We believe that these optimization strategies should improve one or more specific properties of aptamers, and the type of feature(s) selected for improvement will be dependent on the application purpose.

Development of a Novel Method to Detect Prostate Cancer Circulating Tumor Cells (CTCs) Based on Epithelial-Mesenchymal Transition Biology

DTIC Science & Technology

2015-12-01

different incubation periods for the cell lines (1 hour and overnight). We found that room temperature incubation provided the optimal temperature ...properties, either directly (through modification of residues in the complementarity determining region (CDR)) or indirectly through allosteric effects ...showed that the reduction in antigen binding affinity is associated with handling the antibody (e.g. temperature , buffer, purification steps) rather

Targeted Lymphoma Cell Death by Novel Signal Transduction Modifications

DTIC Science & Technology

2010-07-14

CD22 -binding peptides that initiate signal transduction and apoptosis in non-Hodgkin’s lymphoma (NHL), 2) optimize CD22 -mediated signal transduction...and lymphomacidal properties of ligand blocking anti- CD22 monoclonal antibodies (mAbs) and peptides with CD22 -specific phosphatase inhibition and 3...correlate mAb-mediated and anti- CD22 peptide-mediated in vivo physiologic changes, efficacy, and tumor targeting using advanced immuno-positron

Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

PubMed

Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

2011-11-15

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that bear them.

The rise of fragment-based drug discovery.

PubMed

Murray, Christopher W; Rees, David C

2009-06-01

The search for new drugs is plagued by high attrition rates at all stages in research and development. Chemists have an opportunity to tackle this problem because attrition can be traced back, in part, to the quality of the chemical leads. Fragment-based drug discovery (FBDD) is a new approach, increasingly used in the pharmaceutical industry, for reducing attrition and providing leads for previously intractable biological targets. FBDD identifies low-molecular-weight ligands (∼150 Da) that bind to biologically important macromolecules. The three-dimensional experimental binding mode of these fragments is determined using X-ray crystallography or NMR spectroscopy, and is used to facilitate their optimization into potent molecules with drug-like properties. Compared with high-throughput-screening, the fragment approach requires fewer compounds to be screened, and, despite the lower initial potency of the screening hits, offers more efficient and fruitful optimization campaigns. Here, we review the rise of FBDD, including its application to discovering clinical candidates against targets for which other chemistry approaches have struggled.

Lead Optimization Studies on FimH Antagonists: Discovery of Potent and Orally Bioavailable Ortho-substituted Biphenyl Mannosides

PubMed Central

Han, Zhenfu; Pinkner, Jerome S.; Ford, Bradley; Chorell, Erik; Crowley, Jan M.; Cusumano, Corinne K.; Campbell, Scott; Henderson, Jeffrey P.; Hultgren, Scott J.; Janetka, James W.

2012-01-01

Herein, we describe the X-ray structure-based design and optimization of biaryl mannoside FimH inhibitors. Diverse modifications to the biaryl ring to improve drug-like physical and pharmacokinetic properties of mannosides were assessed for FimH binding affinity based on their effects on hemagglutination and biofilm formation along with direct FimH binding assays. Substitution on the mannoside phenyl ring ortho to the glycosidic bond results in large potency enhancements of several-fold higher than corresponding unsubstituted matched pairs and can be rationalized from increased hydrophobic interactions with the FimH hydrophobic ridge (Ile13) or “tyrosine gate” (Tyr137 and Tyr48) also lined by Ile52. The lead mannosides have increased metabolic stability and oral bioavailability as determined from in vitro PAMPA predictive model of cellular permeability and in vivo pharmacokinetic studies in mice, thereby representing advanced preclinical candidates with promising potential as novel therapeutics for the clinical treatment and prevention of recurring urinary tract infections. PMID:22449031

Exploiting conformational dynamics in drug discovery: design of C-terminal inhibitors of Hsp90 with improved activities

PubMed Central

Moroni, Elisabetta; Zhao, Huiping; Blagg, Brian S.J.; Colombo, Giorgio

2014-01-01

The interaction that occurs between molecules is a dynamic process that impacts both structural and conformational properties of the ligand and the ligand binding site. Herein, we investigate the dynamic cross-talk between a protein and the ligand as a source for new opportunities in ligand design. Analysis of the formation/disappearance of protein pockets produced in response to a first-generation inhibitor assisted in the identification of functional groups that could be introduced onto scaffolds to facilitate optimal binding, which allowed for increased binding with previously uncharacterized regions. MD simulations were used to elucidate primary changes that occur in the Hsp90 C-terminal binding pocket in the presence of first-generation ligands. This data was then used to design ligands that adapt to these receptor conformations, which provides access to an energy landscape that is not visible in a static model. The newly synthesized compounds demonstrated anti-proliferative activity at ~150 nanomolar concentration. The method identified herein may be used to design chemical probes that provide additional information on structural variations of Hsp90 C-terminal binding site. PMID:24397468

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks

PubMed Central

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long – range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions. PMID:27857564

The role of charged surface residues in the binding ability of small hubs in protein-protein interaction networks.

PubMed

Patil, Ashwini; Nakamura, Haruki

2007-01-01

Hubs are highly connected proteins in a protein-protein interaction network. Previous work has implicated disordered domains and high surface charge as the properties significant in the ability of hubs to bind multiple proteins. While conformational flexibility of disordered domains plays an important role in the binding ability of large hubs, high surface charge is the dominant property in small hubs. In this study, we further investigate the role of the high surface charge in the binding ability of small hubs in the absence of disordered domains. Using multipole expansion, we find that the charges are highly distributed over the hub surfaces. Residue enrichment studies show that the charged residues in hubs are more prevalent on the exposed surface, with the exception of Arg, which is predominantly found at the interface, as compared to non-hubs. This suggests that the charged residues act primarily from the exposed surface rather than the interface to affect the binding ability of small hubs. They do this through (i) enhanced intra-molecular electrostatic interactions to lower the desolvation penalty, (ii) indirect long - range intermolecular interactions with charged residues on the partner proteins for better complementarity and electrostatic steering, and (iii) increased solubility for enhanced diffusion-controlled rate of binding. Along with Arg, we also find a high prevalence of polar residues Tyr, Gln and His and the hydrophobic residue Met at the interfaces of hubs, all of which have the ability to form multiple types of interactions, indicating that the interfaces of hubs are optimized to participate in multiple interactions.

Synthesis, hybridization characteristics, and fluorescence properties of oligonucleotides modified with nucleobase-functionalized locked nucleic acid adenosine and cytidine monomers.

PubMed

Kaura, Mamta; Kumar, Pawan; Hrdlicka, Patrick J

2014-07-03

Conformationally restricted nucleotides such as locked nucleic acid (LNA) are very popular as affinity-, specificity-, and stability-enhancing modifications in oligonucleotide chemistry to produce probes for nucleic acid targeting applications in molecular biology, biotechnology, and medicinal chemistry. Considerable efforts have been devoted in recent years to optimize the biophysical properties of LNA through additional modification of the sugar skeleton. We recently introduced C5-functionalization of LNA uridines as an alternative and synthetically more straightforward approach to improve the biophysical properties of LNA. In the present work, we set out to test the generality of this concept by studying the characteristics of oligonucleotides modified with four different C5-functionalized LNA cytidine and C8-functionalized LNA adenosine monomers. The results strongly suggest that C5-functionalization of LNA pyrimidines is indeed a viable approach for improving the binding affinity, target specificity, and/or enzymatic stability of LNA-modified ONs, whereas C8-functionalization of LNA adenosines is detrimental to binding affinity and specificity. These insights will impact the future design of conformationally restricted nucleotides for nucleic acid targeting applications.

Numerical Optimization of Density Functional Tight Binding Models: Application to Molecules Containing Carbon, Hydrogen, Nitrogen, and Oxygen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Krishnapriyan, A.; Yang, P.; Niklasson, A. M. N.

New parametrizations for semiempirical density functional tight binding (DFTB) theory have been developed by the numerical optimization of adjustable parameters to minimize errors in the atomization energy and interatomic forces with respect to ab initio calculated data. Initial guesses for the radial dependences of the Slater- Koster bond integrals and overlap integrals were obtained from minimum basis density functional theory calculations. The radial dependences of the pair potentials and the bond and overlap integrals were represented by simple analytic functions. The adjustable parameters in these functions were optimized by simulated annealing and steepest descent algorithms to minimize the value ofmore » an objective function that quantifies the error between the DFTB model and ab initio calculated data. The accuracy and transferability of the resulting DFTB models for the C, H, N, and O system were assessed by comparing the predicted atomization energies and equilibrium molecular geometries of small molecules that were not included in the training data from DFTB to ab initio data. The DFTB models provide accurate predictions of the properties of hydrocarbons and more complex molecules containing C, H, N, and O.« less

Numerical Optimization of Density Functional Tight Binding Models: Application to Molecules Containing Carbon, Hydrogen, Nitrogen, and Oxygen

DOE PAGES

Krishnapriyan, A.; Yang, P.; Niklasson, A. M. N.; ...

2017-10-17

New parametrizations for semiempirical density functional tight binding (DFTB) theory have been developed by the numerical optimization of adjustable parameters to minimize errors in the atomization energy and interatomic forces with respect to ab initio calculated data. Initial guesses for the radial dependences of the Slater- Koster bond integrals and overlap integrals were obtained from minimum basis density functional theory calculations. The radial dependences of the pair potentials and the bond and overlap integrals were represented by simple analytic functions. The adjustable parameters in these functions were optimized by simulated annealing and steepest descent algorithms to minimize the value ofmore » an objective function that quantifies the error between the DFTB model and ab initio calculated data. The accuracy and transferability of the resulting DFTB models for the C, H, N, and O system were assessed by comparing the predicted atomization energies and equilibrium molecular geometries of small molecules that were not included in the training data from DFTB to ab initio data. The DFTB models provide accurate predictions of the properties of hydrocarbons and more complex molecules containing C, H, N, and O.« less

The Importance of Being Tyrosine: Lessons in Molecular Recognition from Minimalist Synthetic Binding Proteins

PubMed Central

Koide, Shohei; Sidhu, Sachdev S.

2010-01-01

Summary Combinatorial libraries built with severely restricted chemical diversity have yielded highly functional synthetic binding proteins. Structural analyses of these minimalist binding sites have revealed the dominant role of large tyrosine residues for mediating molecular contacts and of small serine/glycine residues for providing space and flexibility. The concept of using limited residue types to construct optimized binding proteins mirrors findings in the field of small molecule drug development, where it has been proposed that most drugs are built from a limited set of side chains presented by diverse frameworks. The physicochemical properties of tyrosine make it the amino acid that is most effective for mediating molecular recognition, and protein engineers have taken advantage of these characteristics to build tyrosine-rich protein binding sites that outperform natural proteins in terms of affinity and specificity. Knowledge from preceding studies can be used to improve current designs, and thus, synthetic protein libraries will continue to evolve and improve. In the near future, it seems likely that synthetic binding proteins will supersede natural antibodies for most purposes, and moreover, synthetic proteins will enable many new applications beyond the scope of natural proteins. PMID:19298050

Enhanced reaction kinetics in biological cells

NASA Astrophysics Data System (ADS)

Loverdo, C.; Bénichou, O.; Moreau, M.; Voituriez, R.

2008-02-01

The cell cytoskeleton is a striking example of an `active' medium driven out-of-equilibrium by ATP hydrolysis. Such activity has been shown to have a spectacular impact on the mechanical and rheological properties of the cellular medium, as well as on its transport properties: a generic tracer particle freely diffuses as in a standard equilibrium medium, but also intermittently binds with random interaction times to motor proteins, which perform active ballistic excursions along cytoskeletal filaments. Here, we propose an analytical model of transport-limited reactions in active media, and show quantitatively how active transport can enhance reactivity for large enough tracers such as vesicles. We derive analytically the average interaction time with motor proteins that optimizes the reaction rate, and reveal remarkable universal features of the optimal configuration. We discuss why active transport may be beneficial in various biological examples: cell cytoskeleton, membranes and lamellipodia, and tubular structures such as axons.

Probing the Interaction between Cyclic ADTC1 Ac-CADTPPVC-NH2) Peptide with EC1-EC2 domain of E-cadherin using Molecular Docking Approach

NASA Astrophysics Data System (ADS)

Siahaan, P.; Wuning, S.; Manna, A.; Prasasty, V. D.; Hudiyanti, D.

2018-04-01

Deeply understanding that intermolecular interaction between molecules on the paracellular pathway has given insight to its microscopic and macroscopic properties. In the paracellular pathway, synthetic cyclic ADTC1 (Ac-CADTPPVC-NH2) peptide has been studied to modulate EC1-EC2 domain, computationally using molecular docking method. The aim of this research is to probe the effect of amino acid alanine (A) of ADTC1 on its interaction properties. The study carried out in two steps: 1. the optimization using GROMACS v4.6.5 program and; 2. Determination of the interaction properties using AutoDock 4.2 program. The interaction was done for A-J box, and the best position of the binding site and binding energy on the OC and CC ADTC1 peptides against the EC1-EC2 domain of E-cadherin was selected. The result showed that the CC of the F box ADTC1 has the best interaction with binding energy of - 26.36 kJ/mol and its energy was lower than ADTC5 without alanine amino acid. ADTC1 interacted with EC1 of EC1-EC2 on Asp1, Trp2, Val3, Ile4, Ile24, Lys25, Ser26, Asn27, and Met92 residues.

Engineered Metal-Phenolic Capsules Show Tunable Targeted Delivery to Cancer Cells.

PubMed

Ju, Yi; Cui, Jiwei; Sun, Huanli; Müllner, Markus; Dai, Yunlu; Guo, Junling; Bertleff-Zieschang, Nadja; Suma, Tomoya; Richardson, Joseph J; Caruso, Frank

2016-06-13

We engineered metal-phenolic capsules with both high targeting and low nonspecific cell binding properties. The capsules were prepared by coating phenolic-functionalized hyaluronic acid (HA) and poly(ethylene glycol) (PEG) on calcium carbonate templates, followed by cross-linking the phenolic groups with metal ions and removing the templates. The incorporation of HA significantly enhanced binding and association with a CD44 overexpressing (CD44+) cancer cell line, while the incorporation of PEG reduced nonspecific interactions with a CD44 minimal-expressing (CD44-) cell line. Moreover, high specific targeting to CD44+ cells can be balanced with low nonspecific binding to CD44- cells simply by using an optimized feed-ratio of HA and PEG to vary the content of HA and PEG incorporated into the capsules. Loading an anticancer drug (i.e., doxorubicin) into the obtained capsules resulted in significantly higher cytotoxicity to CD44+ cells but lower cytotoxicity to CD44- cells.

Platinum Monolayer Shell on Non-Noble Metal Core Electrocatalysts for the Hydrogen Oxidation Reaction

NASA Astrophysics Data System (ADS)

Teeluck, Krishani Malini

According to the United States Environmental Protection Agency, as of 2015, transportation accounted for 32% of the carbon dioxide emissions in the United States (and all carbon dioxide emissions in the U.S. accounted for 82.2% of all greenhouse gases from human activity). A hydrogen fuel cell is a device that efficiently produces electrical energy directly from a chemical reaction, with zero carbon emissions, and therefore holds great promise in alleviating our dependence on harmful use of energy sources. Due to their clean emissions and high efficiencies, there has been focus on the hydrogen fuel cell for vehicle applications using proton exchange membrane and alkaline fuel cells. Although the proton exchange membrane fuel cell is currently being used in vehicles, their high cost limits their feasibility in the market. This has inspired the development of the alkaline fuel cell whose efficiency and simplicity suggest the possibility of manufacturing high power fuel cell vehicles at a low cost, since the electrocatalysts in the alkaline fuel cell can be made from non-noble metals. Although the hydrogen oxidation reaction is one of the fastest electrochemical reactions in acidic media, it is two orders of magnitude slower in alkaline media, which hinders the overall efficiency of the alkaline fuel cell. Pure platinum is currently the best catalyst for the hydrogen oxidation reaction, but platinum’s high cost and rarity yields economic issues, rendering the technology futile if it cannot be commercialized. Furthermore, platinum’s hydrogen binding energy is slightly stronger than the optimal hydrogen binding energy. As the hydrogen oxidation reaction happens only on the surface of the catalyst, there is no need for platinum content beyond the exterior. Since tungsten and nickel are cheap, as well as abundant, they are ideal elements to replace the core of the catalyst with, while leaving a platinum shell surrounding this core. The activity of the hydrogen oxidation reaction when using a platinum monolayer shell on a nickel tungsten core electrocatalyst is explored, and it was found that the novel catalyst created here exhibits kinetics that rival pure platinum, but at less than half the platinum content, suggesting that nickel and tungsten modify the electronic properties of platinum in a way that enhances its activity for the hydrogen oxidation reaction. Furthermore, the hydrogen binding energy of this novel electrocatalyst was found to be weaker than the optimal binding energy (rather than stronger, as seen in pure platinum), indicating the possibility of modifying the electronic properties of platinum for a more optimal hydrogen binding energy.

The creation of a biomimetic interface between boron-doped diamond and immobilized proteins.

PubMed

Hoffmann, René; Kriele, Armin; Obloh, Harald; Tokuda, Norio; Smirnov, Waldemar; Yang, Nianjun; Nebel, Christoph E

2011-10-01

Immobilization of proteins on a solid electrode is to date done by chemical cross-linking or by addition of an adjustable intermediate. In this paper we introduce a concept where a solid with variable surface properties is optimized to mediate binding of the electron-transfer protein Cytochrome c (Cyt c) by mimicking the natural binding environment. It is shown that, as a carbon-based material, boron-doped diamond can be adjusted by simple electrochemical surface treatments to the specific biochemical requirements of Cyt c. The structure and functionality of passively adsorbed Cyt c on variously terminated diamond surfaces were characterized in detail using a combination of electrochemical techniques and atomic force microscopy with single-molecule resolution. Partially oxidized diamond allowed stable immobilization of Cyt c together with high electron transfer activity, driven by a combination of electrostatic and hydrophobic interactions. This surface mimics the natural binding partner, where coarse orientation is governed by electrostatic interaction of the protein's dipole and hydrophobic interactions assist in formation of the electron transfer complex. The optimized surface mediated electron transfer kinetics around 100 times faster than those reported for other solids and even faster kinetics than on self-assembled monolayers of alkanethiols. Copyright © 2011 Elsevier Ltd. All rights reserved.

GABAA receptor activity modulating piperine analogs: In vitro metabolic stability, metabolite identification, CYP450 reaction phenotyping, and protein binding.

PubMed

Zabela, Volha; Hettich, Timm; Schlotterbeck, Götz; Wimmer, Laurin; Mihovilovic, Marko D; Guillet, Fabrice; Bouaita, Belkacem; Shevchenko, Bénédicte; Hamburger, Matthias; Oufir, Mouhssin

2018-01-01

In a screening of natural products for allosteric modulators of GABA A receptors (γ-aminobutyric acid type A receptor), piperine was identified as a compound targeting a benzodiazepine-independent binding site. Given that piperine is also an activator of TRPV1 (transient receptor potential vanilloid type 1) receptors involved in pain signaling and thermoregulation, a series of piperine analogs were prepared in several cycles of structural optimization, with the aim of separating GABA A and TRPV1 activating properties. We here investigated the metabolism of piperine and selected analogs in view of further cycles of lead optimization. Metabolic stability of the compounds was evaluated by incubation with pooled human liver microsomes, and metabolites were analyzed by UHPLC-Q-TOF-MS. CYP450 isoenzymes involved in metabolism of compounds were identified by reaction phenotyping with Silensomes™. Unbound fraction in whole blood was determined by rapid equilibrium dialysis. Piperine was the metabolically most stable compound. Aliphatic hydroxylation, and N- and O-dealkylation were the major routes of oxidative metabolism. Piperine was exclusively metabolized by CYP1A2, whereas CYP2C9 contributed significantly in the oxidative metabolism of all analogs. Extensive binding to blood constituents was observed for all compounds. Copyright © 2017 Elsevier B.V. All rights reserved.

Conformational Heterogeneity of Unbound Proteins Enhances Recognition in Protein-Protein Encounters.

PubMed

Pallara, Chiara; Rueda, Manuel; Abagyan, Ruben; Fernández-Recio, Juan

2016-07-12

To understand cellular processes at the molecular level we need to improve our knowledge of protein-protein interactions, from a structural, mechanistic, and energetic point of view. Current theoretical studies and computational docking simulations show that protein dynamics plays a key role in protein association and support the need for including protein flexibility in modeling protein interactions. Assuming the conformational selection binding mechanism, in which the unbound state can sample bound conformers, one possible strategy to include flexibility in docking predictions would be the use of conformational ensembles originated from unbound protein structures. Here we present an exhaustive computational study about the use of precomputed unbound ensembles in the context of protein docking, performed on a set of 124 cases of the Protein-Protein Docking Benchmark 3.0. Conformational ensembles were generated by conformational optimization and refinement with MODELLER and by short molecular dynamics trajectories with AMBER. We identified those conformers providing optimal binding and investigated the role of protein conformational heterogeneity in protein-protein recognition. Our results show that a restricted conformational refinement can generate conformers with better binding properties and improve docking encounters in medium-flexible cases. For more flexible cases, a more extended conformational sampling based on Normal Mode Analysis was proven helpful. We found that successful conformers provide better energetic complementarity to the docking partners, which is compatible with recent views of binding association. In addition to the mechanistic considerations, these findings could be exploited for practical docking predictions of improved efficiency.

Binding Properties of General Odorant Binding Proteins from the Oriental Fruit Moth, Grapholita molesta (Busck) (Lepidoptera: Tortricidae)

PubMed Central

Li, Guangwei; Chen, Xiulin; Li, Boliao; Zhang, Guohui; Li, Yiping; Wu, Junxiang

2016-01-01

Background The oriental fruit moth Grapholita molesta is a host-switching pest species. The adults highly depend on olfactory cues in locating optimal host plants and oviposition sites. Odorant binding proteins (OBPs) are thought to be responsible for recognizing and transporting hydrophobic odorants across the aqueous sensillum lymph to stimulate the odorant receptors (ORs) within the antennal sensilla and activate the olfactory signal transduction pathway. Exploring the physiological function of these OBPs could facilitate understanding insect chemical communications. Methodology/Principal Finding Two antennae-specific general OBPs (GOBPs) of G. molesta were expressed and purified in vitro. The binding affinities of G. molesta GOBP1 and 2 (GmolGOBP1 and 2) for sex pheromone components and host plant volatiles were measured by fluorescence ligand-binding assays. The distribution of GmolGOBP1 and 2 in the antennal sensillum were defined by whole mount fluorescence immunohistochemistry (WM-FIHC) experiments. The binding sites of GmolGOBP2 were predicted using homology modeling, molecular docking and site-directed mutagenesis. Both GmolGOBP1 and 2 are housing in sensilla basiconica and with no differences in male and female antennae. Recombinant GmolGOBP1 (rGmolGOBP1) exhibited broad binding properties towards host plant volatiles and sex pheromone components; rGmolGOBP2 could not effectively bind host plant volatiles but showed specific binding affinity with a minor sex pheromone component dodecanol. We chose GmolGOBP2 and dodecanol for further homology modeling, molecular docking, and site-directed mutagenesis. Binding affinities of mutants demonstrated that Thr9 was the key binding site and confirmed dodecanol bonding to protein involves a hydrogen bond. Combined with the pH effect on binding affinities of rGmolGOBP2, ligand binding and release of GmolGOBP2 were related to a pH-dependent conformational transition. Conclusion Two rGmolGOBPs exhibit different binding characteristics for tested ligands. rGmolGOBP1 has dual functions in recognition of host plant volatiles and sex pheromone components, while rGmolGOBP2 is mainly involved in minor sex pheromone component dodecanol perception. This study also provides empirical evidence for the predicted functions of key amino acids in recombinant protein ligand-binding characteristics. PMID:27152703

Synthesis, analgesic activity, and binding properties of some epibatidine analogs with a tropine skeleton.

PubMed

Rádl, S; Hafner, W; Budesínsky, M; Hejnová, L; Krejcí, I

2000-06-01

A series of epibatidine analogs and their positional isomers bearing an 8-azabicyclo[3.2.1]octane moiety is described. Also some of their simplified analogs bearing a 3-piperidine moiety are reported. Their receptor binding profiles (5-HT1A, 5-HT1B, M1, M2, neuronal nicotinic receptor) and analgesic activity (hot plate, acetic acid induced writhing) have been studied. Some of the compounds, especially those containing an 8-azabicyclo[3.2.1]oct-2-ene moiety possess high afinity for the nicotinic cholinergic receptor. The most analgesically active compounds are also highly toxic. Optimized structures (PM3-MOPAC, Alchemy 2000, Tripos Inc.) of compounds 1-9 were compared with that of epibatidine.

Pheromone discrimination by a pH-tuned polymorphism of the Bombyx mori pheromone-binding protein.

PubMed

Damberger, Fred F; Michel, Erich; Ishida, Yuko; Leal, Walter S; Wüthrich, Kurt

2013-11-12

The Bombyx mori pheromone-binding protein (BmorPBP) is known to adopt two different conformations. These are BmorPBP(A), where a regular helix formed by the C-terminal dodecapeptide segment, α7, occupies the ligand-binding cavity, and BmorPBP(B), where the binding site is free to accept ligands. NMR spectra of delipidated BmorPBP solutions at the physiological pH of the bulk sensillum lymph near pH 6.5 show only BmorPBP(A), and in mixtures, the two species are in slow exchange on the chemical shift frequency scale. This equilibrium has been monitored at variable pH and ligand concentrations, demonstrating that it is an intrinsic property of BmorPBP that is strongly affected by pH variation and ligand binding. This polymorphism tunes BmorPBP for optimal selective pheromone transport: Competition between α7 and lipophilic ligands for its binding cavity enables selective uptake of bombykol at the pore endings in the sensillum wall, whereas compounds with lower binding affinity can only be bound in the bulk sensillum lymph. After transport across the bulk sensillum lymph into the lower pH area near the dendritic membrane surface, bombykol is ejected near the receptor, whereas compounds with lower binding affinity are ejected before reaching the olfactory receptor, rendering them susceptible to degradation by enzymes present in the sensillum lymph.

Novel interactions of CAPS (Ca2+-dependent activator protein for secretion) with the three neuronal SNARE proteins required for vesicle fusion.

PubMed

Daily, Neil J; Boswell, Kristin L; James, Declan J; Martin, Thomas F J

2010-11-12

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca(2+)-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis.

Novel Interactions of CAPS (Ca2+-dependent Activator Protein for Secretion) with the Three Neuronal SNARE Proteins Required for Vesicle Fusion*

PubMed Central

Daily, Neil J.; Boswell, Kristin L.; James, Declan J.; Martin, Thomas F. J.

2010-01-01

CAPS (aka CADPS) is required for optimal vesicle exocytosis in neurons and endocrine cells where it functions to prime the exocytic machinery for Ca2+-triggered fusion. Fusion is mediated by trans complexes of the SNARE proteins VAMP-2, syntaxin-1, and SNAP-25 that bridge vesicle and plasma membrane. CAPS promotes SNARE complex formation on liposomes, but the SNARE binding properties of CAPS are unknown. The current work revealed that CAPS exhibits high affinity binding to syntaxin-1 and SNAP-25 and moderate affinity binding to VAMP-2. CAPS binding is specific for a subset of exocytic SNARE protein isoforms and requires membrane integration of the SNARE proteins. SNARE protein binding by CAPS is novel and mediated by interactions with the SNARE motifs in the three proteins. The C-terminal site for CAPS binding on syntaxin-1 does not overlap the Munc18-1 binding site and both proteins can co-reside on membrane-integrated syntaxin-1. As expected for a C-terminal binding site on syntaxin-1, CAPS stimulates SNARE-dependent liposome fusion with N-terminal truncated syntaxin-1 but exhibits impaired activity with C-terminal syntaxin-1 mutants. Overall the results suggest that SNARE complex formation promoted by CAPS may be mediated by direct interactions of CAPS with each of the three SNARE proteins required for vesicle exocytosis. PMID:20826818

Optimization of binding electrostatics: Charge complementarity in the barnase-barstar protein complex

PubMed Central

lee, Lee-Peng; Tidor, Bruce

2001-01-01

Theoretical and experimental studies have shown that the large desolvation penalty required for polar and charged groups frequently precludes their involvement in electrostatic interactions that contribute strongly to net stability in the folding or binding of proteins in aqueous solution near room temperature. We have previously developed a theoretical framework for computing optimized electrostatic interactions and illustrated use of the algorithm with simplified geometries. Given a receptor and model assumptions, the method computes the ligand-charge distribution that provides the most favorable balance of desolvation and interaction effects on binding. In this paper the method has been extended to treat complexes using actual molecular shapes. The barnase-barstar protein complex was investigated with barnase treated as a target receptor. The atomic point charges of barstar were varied to optimize the electrostatic binding free energy. Barnase and natural barstar form a tight complex (Kd ∼ 10−14 M) with many charged and polar groups near the interface that make this a particularly relevant system for investigating the role of electrostatic effects on binding. The results show that sets of barstar charges (resulting from optimization with different constraints) can be found that give rise to relatively large predicted improvements in electrostatic binding free energy. Principles for enhancing the effect of electrostatic interactions in molecular binding in aqueous environments are discussed in light of the optima. Our findings suggest that, in general, the enhancements in electrostatic binding free energy resulting from modification of polar and charged groups can be substantial. Moreover, a recently proposed definition of electrostatic complementarity is shown to be a useful tool for examining binding interfaces. Finally, calculational results suggest that wild-type barstar is closer to being affinity optimized than is barnase for their mutual binding, consistent with the known roles of these proteins. PMID:11266622

The importance of hydration thermodynamics in fragment-to-lead optimization.

PubMed

Ichihara, Osamu; Shimada, Yuzo; Yoshidome, Daisuke

2014-12-01

Using a computational approach to assess changes in solvation thermodynamics upon ligand binding, we investigated the effects of water molecules on the binding energetics of over 20 fragment hits and their corresponding optimized lead compounds. Binding activity and X-ray crystallographic data of published fragment-to-lead optimization studies from various therapeutically relevant targets were studied. The analysis reveals a distinct difference between the thermodynamic profile of water molecules displaced by fragment hits and those displaced by the corresponding optimized lead compounds. Specifically, fragment hits tend to displace water molecules with notably unfavorable excess entropies-configurationally constrained water molecules-relative to those displaced by the newly added moieties of the lead compound during the course of fragment-to-lead optimization. Herein we describe the details of this analysis with the goal of providing practical guidelines for exploiting thermodynamic signatures of binding site water molecules in the context of fragment-to-lead optimization. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization.

PubMed

Ellis, J Michael; Altman, Michael D; Cash, Brandon; Haidle, Andrew M; Kubiak, Rachel L; Maddess, Matthew L; Yan, Youwei; Northrup, Alan B

2016-12-08

Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain.

Carboxamide Spleen Tyrosine Kinase (Syk) Inhibitors: Leveraging Ground State Interactions To Accelerate Optimization

PubMed Central

2016-01-01

Optimization of a series of highly potent and kinome selective carbon-linked carboxamide spleen tyrosine kinase (Syk) inhibitors with favorable drug-like properties is described. A pervasive Ames liability in an analogous nitrogen-linked carboxamide series was obviated by replacement with a carbon-linked moiety. Initial efforts lacked on-target potency, likely due to strain induced between the hinge binding amide and solvent front heterocycle. Consideration of ground state and bound state energetics allowed rapid realization of improved solvent front substituents affording subnanomolar Syk potency and high kinome selectivity. These molecules were also devoid of mutagenicity risk as assessed via the Ames test using the TA97a Salmonella strain. PMID:27994755

Magnetic behavior in Cr{sub 2}@Ge{sub n} (1≤n≤12) clusters: A density functional investigation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dhaka, Kapil, E-mail: kapil.dhaka@pilani.bits-pilani.ac.in; Trivedi, Ravi, E-mail: kapil.dhaka@pilani.bits-pilani.ac.in; Bandyopadhyay, Debashis, E-mail: kapil.dhaka@pilani.bits-pilani.ac.in

2014-04-24

With a goal to produce magnetic moment in Cr{sub 2} Doped Ge{sub n} clusters which will be useful for practical applications, we have considered the structure and magnetic properties of Pure Germanium clusters and substitutionally doped it with Cr dimer to produce Cr{sub 2}@Ge{sub n} clusters. As the first step of calculation, geometrical optimizations of the nanoclusters have been done. These optimized geometries have been used in calculate the average binding energy per atom (BE), HOMO-LUMO gap and hence the relative stability of the clusters. These parameters have been demonstrated as structural and electronic properties of the clusters. Gap betweenmore » highest occupied molecular orbital and lowest unoccupied molecular orbital indicate cluster to be a potential motif for generating magnetic cluster assembled materials. Based on these values a comparative study on different sized clusters has been done in order to understand the origin of structures, electronic and magnetic properties of Cr{sub 2}@Ge{sub n} nanoclusters.« less

Structural study of biologically significant ligands with major birch pollen allergen Betv1 by docking and molecular dynamics simulation

PubMed Central

Kundu, Sangeeta; Roy, Debjani

2010-01-01

The major birch pollen allergen, Betv1 of Betula verrucosa is the main causative agent of birch pollen allergy in humans. Betv1 is capable of binding several physiological ligands including fatty acids, flavones, cytokinins and sterols. Until now, no structural information from crystallography or NMR is available regarding binding mode of any of these ligands into the binding pocket of Betv1. In the present study thirteen ligands have been successfully docked into the hydrophobic cavity of Betv1 and binding free energies of the complexes have been calculated using AutoDock 3.0.5. A linear relationship with correlation coefficient (R2) of 0.6 is obtained between ΔGbs values plotted against their corresponding IC50 values. The complex formed between Betv1 and the best docking pose for each ligand has been optimized by molecular dynamics simulation. Here, we describe the ligand binding of Betv1, which provides insight into the biological function of this protein. This knowledge is required for structural alteration or inhibition of some of these ligands in order to modify the allergenic properties of this protein. PMID:20978606

Optimized Standard Operating Procedures for the Analysis of Cerebrospinal Fluid Aβ42 and the Ratios of Aβ Isoforms Using Low Protein Binding Tubes.

PubMed

Vanderstichele, Hugo Marcel Johan; Janelidze, Shorena; Demeyer, Leentje; Coart, Els; Stoops, Erik; Herbst, Victor; Mauroo, Kimberley; Brix, Britta; Hansson, Oskar

2016-05-31

Reduced cerebrospinal fluid (CSF) concentration of amyloid-β1-42 (Aβ1-42) reflects the presence of amyloidopathy in brains of subjects with Alzheimer's disease (AD). To qualify the use of Aβ1-42/Aβ1-40 for improvement of standard operating procedures (SOP) for measurement of CSF Aβ with a focus on CSF collection, storage, and analysis. Euroimmun ELISAs for CSF Aβ isoforms were used to set up a SOP with respect to recipient properties (low binding, polypropylene), volume of tubes, freeze/thaw cycles, addition of detergents (Triton X-100, Tween-20) in collection or storage tubes or during CSF analysis. Data were analyzed with linear repeated measures and mixed effects models. Optimization of CSF analysis included a pre-wash of recipients (e.g., tubes, 96-well plates) before sample analysis. Using the Aβ1-42/Aβ1-40 ratio, in contrast to Aβ1-42, eliminated effects of tube type, additional freeze/thaw cycles, or effect of CSF volumes for polypropylene storage tubes. 'Low binding' tubes reduced the loss of Aβ when aliquoting CSF or in function of additional freeze/thaw cycles. Addition of detergent in CSF collection tubes resulted in an almost complete absence of variation in function of collection procedures, but affected the concentration of Aβ isoforms in the immunoassay. The ratio of Aβ1-42/Aβ1-40 is a more robust biomarker than Aβ1-42 toward (pre-) analytical interfering factors. Further, 'low binding' recipients and addition of detergent in collection tubes are able to remove effects of SOP-related confounding factors. Integration of the Aβ1-42/Aβ1-40 ratio and 'low-binding tubes' into guidance criteria may speed up worldwide standardization of CSF biomarker analysis.

A peek into tropomyosin binding and unfolding on the actin filament.

PubMed

Singh, Abhishek; Hitchcock-Degregori, Sarah E

2009-07-24

Tropomyosin is a prototypical coiled coil along its length with subtle variations in structure that allow interactions with actin and other proteins. Actin binding globally stabilizes tropomyosin. Tropomyosin-actin interaction occurs periodically along the length of tropomyosin. However, it is not well understood how tropomyosin binds actin. Tropomyosin's periodic binding sites make differential contributions to two components of actin binding, cooperativity and affinity, and can be classified as primary or secondary sites. We show through mutagenesis and analysis of recombinant striated muscle alpha-tropomyosins that primary actin binding sites have a destabilizing coiled-coil interface, typically alanine-rich, embedded within a non-interface recognition sequence. Introduction of an Ala cluster in place of the native, more stable interface in period 2 and/or period 3 sites (of seven) increased the affinity or cooperativity of actin binding, analysed by cosedimentation and differential scanning calorimetry. Replacement of period 3 with period 5 sequence, an unstable region of known importance for cooperative actin binding, increased the cooperativity of binding. Introduction of the fluorescent probe, pyrene, near the mutation sites in periods 2 and 3 reported local instability, stabilization by actin binding, and local unfolding before or coincident with dissociation from actin (measured using light scattering), and chain dissociation (analyzed using circular dichroism). This, and previous work, suggests that regions of tropomyosin involved in binding actin have non-interface residues specific for interaction with actin and an unstable interface that is locally stabilized upon binding. The destabilized interface allows residues on the coiled-coil surface to obtain an optimal conformation for interaction with actin by increasing the number of local substates that the side chains can sample. We suggest that local disorder is a property typical of coiled coil binding sites and proteins that have multiple binding partners, of which tropomyosin is one type.

Efficient engineering of chromosomal ribosome binding site libraries in mismatch repair proficient Escherichia coli.

PubMed

Oesterle, Sabine; Gerngross, Daniel; Schmitt, Steven; Roberts, Tania Michelle; Panke, Sven

2017-09-26

Multiplexed gene expression optimization via modulation of gene translation efficiency through ribosome binding site (RBS) engineering is a valuable approach for optimizing artificial properties in bacteria, ranging from genetic circuits to production pathways. Established algorithms design smart RBS-libraries based on a single partially-degenerate sequence that efficiently samples the entire space of translation initiation rates. However, the sequence space that is accessible when integrating the library by CRISPR/Cas9-based genome editing is severely restricted by DNA mismatch repair (MMR) systems. MMR efficiency depends on the type and length of the mismatch and thus effectively removes potential library members from the pool. Rather than working in MMR-deficient strains, which accumulate off-target mutations, or depending on temporary MMR inactivation, which requires additional steps, we eliminate this limitation by developing a pre-selection rule of genome-library-optimized-sequences (GLOS) that enables introducing large functional diversity into MMR-proficient strains with sequences that are no longer subject to MMR-processing. We implement several GLOS-libraries in Escherichia coli and show that GLOS-libraries indeed retain diversity during genome editing and that such libraries can be used in complex genome editing operations such as concomitant deletions. We argue that this approach allows for stable and efficient fine tuning of chromosomal functions with minimal effort.

Targeting a KH-domain protein with RNA decoys.

PubMed

Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

2002-09-01

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins.

Targeting a KH-domain protein with RNA decoys.

PubMed Central

Makeyev, Aleksandr V; Eastmond, Dawn L; Liebhaber, Stephen A

2002-01-01

RNA-binding proteins are involved in the regulation of many aspects of eukaryotic gene expression. Targeted interference with RNA-protein interactions could offer novel approaches to modulation of expression profiles, alteration of developmental pathways, and reversal of certain disease processes. Here we investigate a decoy strategy for the study of the alphaCP subgroup of KH-domain RNA-binding proteins. These poly(C)-binding proteins have been implicated in a wide spectrum of posttranscriptional controls. Three categories of RNA decoys to alphaCPs were studied: poly(C) homopolymers, native mRNA-binding sites, and a high-affinity structure selected from a combinatorial library. Native chemistry was found to be essential for alphaCP decoy action. Because alphaCP proteins are found in both the nucleus and cytoplasm, decoy cassettes were incorporated within both nuclear (U1 snRNA) and cytoplasmic (VA1 RNA) RNA frameworks. Several sequences demonstrated optimal decoy properties when assayed for protein-binding and decoy bioactivity in vitro. A subset of these transcripts was shown to mediate targeted inhibition of alphaCP-dependent translation when expressed in either the nucleus or cytoplasm of transfected cells. Significantly, these studies establish the feasibility of developing RNA decoys that can selectively target biologic functions of abundant and widely expressed RNA binding proteins. PMID:12358435

Maximizing in vivo target clearance by design of pH-dependent target binding antibodies with altered affinity to FcRn.

PubMed

Yang, Danlin; Giragossian, Craig; Castellano, Steven; Lasaro, Marcio; Xiao, Haiguang; Saraf, Himanshu; Hess Kenny, Cynthia; Rybina, Irina; Huang, Zhong-Fu; Ahlberg, Jennifer; Bigwarfe, Tammy; Myzithras, Maria; Waltz, Erica; Roberts, Simon; Kroe-Barrett, Rachel; Singh, Sanjaya

2017-10-01

Antibodies with pH-dependent binding to both target antigens and neonatal Fc receptor (FcRn) provide an alternative tool to conventional neutralizing antibodies, particularly for therapies where reduction in antigen level is challenging due to high target burden. However, the requirements for optimal binding kinetic framework and extent of pH dependence for these antibodies to maximize target clearance from circulation are not well understood. We have identified a series of naturally-occurring high affinity antibodies with pH-dependent target binding properties. By in vivo studies in cynomolgus monkeys, we show that pH-dependent binding to the target alone is not sufficient for effective target removal from circulation, but requires Fc mutations that increase antibody binding to FcRn. Affinity-enhanced pH-dependent FcRn binding that is double-digit nM at pH 7.4 and single-digit nM at pH 6 achieved maximal target reduction when combined with similar target binding affinities in reverse pH directions. Sustained target clearance below the baseline level was achieved 3 weeks after single-dose administration at 1.5 mg/kg. Using the experimentally derived mechanistic model, we demonstrate the essential kinetic interplay between target turnover and antibody pH-dependent binding during the FcRn recycling, and identify the key components for achieving maximal target clearance. These results bridge the demand for improved patient dosing convenience with the "know-how" of therapeutic modality by design.

Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides.

PubMed

Tran, Tuan; Childs-Disney, Jessica L; Liu, Biao; Guan, Lirui; Rzuczek, Suzanne; Disney, Matthew D

2014-04-18

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

PubMed Central

2015-01-01

We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide. PMID:24506227

Self-Consistent Charge Density Functional Tight-Binding Study of Poly(3,4-ethylenedioxythiophene): Poly(styrenesulfonate) Ammonia Gas Sensor.

PubMed

Marutaphan, Ampaiwan; Seekaew, Yotsarayuth; Wongchoosuk, Chatchawal

2017-12-01

Geometric and electronic properties of 3,4-ethylenedioxythiophene (EDOT), styrene sulfonate (SS), and EDOT: SS oligomers up to 10 repeating units were studied by the self-consistent charge density functional tight-binding (SCC-DFTB) method. An application of PEDOT:PSS for ammonia (NH 3 ) detection was highlighted and investigated both experimentally and theoretically. The results showed an important role of H-bonds in EDOT:SS oligomers complex conformation. Electrical conductivity of EDOT increased with increasing oligomers and doping SS due to enhancement of π conjugation. Printed PEDOT:PSS gas sensor exhibited relatively high response and selectivity to NH 3 . The SCC-DFTB calculation suggested domination of direct charge transfer process in changing of PEDOT:PSS conductivity upon NH 3 exposure at room temperature. The NH 3 molecules preferred to bind with PEDOT:PSS via physisorption. The most favorable adsorption site for PEDOT:PSS-NH 3 interaction was found to be at the nitrogen atom of NH 3 and hydrogen atoms of SS with an average optimal binding distance of 2.00 Å.

Multiobjective optimization of combinatorial libraries.

PubMed

Agrafiotis, D K

2002-01-01

Combinatorial chemistry and high-throughput screening have caused a fundamental shift in the way chemists contemplate experiments. Designing a combinatorial library is a controversial art that involves a heterogeneous mix of chemistry, mathematics, economics, experience, and intuition. Although there seems to be little agreement as to what constitutes an ideal library, one thing is certain: only one property or measure seldom defines the quality of the design. In most real-world applications, a good experiment requires the simultaneous optimization of several, often conflicting, design objectives, some of which may be vague and uncertain. In this paper, we discuss a class of algorithms for subset selection rooted in the principles of multiobjective optimization. Our approach is to employ an objective function that encodes all of the desired selection criteria, and then use a simulated annealing or evolutionary approach to identify the optimal (or a nearly optimal) subset from among the vast number of possibilities. Many design criteria can be accommodated, including diversity, similarity to known actives, predicted activity and/or selectivity determined by quantitative structure-activity relationship (QSAR) models or receptor binding models, enforcement of certain property distributions, reagent cost and availability, and many others. The method is robust, convergent, and extensible, offers the user full control over the relative significance of the various objectives in the final design, and permits the simultaneous selection of compounds from multiple libraries in full- or sparse-array format.

Identification of sequence-structure RNA binding motifs for SELEX-derived aptamers.

PubMed

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E; Przytycka, Teresa M

2012-06-15

Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. To close this gap we developed, Aptamotif, a computational method for the identification of sequence-structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process.

Reshaping the Energy Landscape Transforms the Mechanism and Binding Kinetics of DNA Threading Intercalation.

PubMed

Clark, Andrew G; Naufer, M Nabuan; Westerlund, Fredrik; Lincoln, Per; Rouzina, Ioulia; Paramanathan, Thayaparan; Williams, Mark C

2018-02-06

Molecules that bind DNA via threading intercalation show high binding affinity as well as slow dissociation kinetics, properties ideal for the development of anticancer drugs. To this end, it is critical to identify the specific molecular characteristics of threading intercalators that result in optimal DNA interactions. Using single-molecule techniques, we quantify the binding of a small metal-organic ruthenium threading intercalator (Δ,Δ-B) and compare its binding characteristics to a similar molecule with significantly larger threading moieties (Δ,Δ-P). The binding affinities of the two molecules are the same, while comparison of the binding kinetics reveals significantly faster kinetics for Δ,Δ-B. However, the kinetics is still much slower than that observed for conventional intercalators. Comparison of the two threading intercalators shows that the binding affinity is modulated independently by the intercalating section and the binding kinetics is modulated by the threading moiety. In order to thread DNA, Δ,Δ-P requires a "lock mechanism", in which a large length increase of the DNA duplex is required for both association and dissociation. In contrast, measurements of the force-dependent binding kinetics show that Δ,Δ-B requires a large DNA length increase for association but no length increase for dissociation from DNA. This contrasts strongly with conventional intercalators, for which almost no DNA length change is required for association but a large DNA length change must occur for dissociation. This result illustrates the fundamentally different mechanism of threading intercalation compared with conventional intercalation and will pave the way for the rational design of therapeutic drugs based on DNA threading intercalation.

Bio-inspired network optimization in soft materials — Insights from the plant cell wall

NASA Astrophysics Data System (ADS)

Vincent, R. R.; Cucheval, A.; Hemar, Y.; Williams, M. A. K.

2009-01-01

The dynamic-mechanical responses of ionotropic gels made from the biopolymer pectin have recently been investigated by microrheological experiments and found to exhibit behaviour indicative of semi-flexible polymer networks. In this work we investigate the gelling behaviour of pectin systems in which an enzyme (pectinmethylesterase, PME) is used to liberate ion-binding sites on initially inert polymers, while in the presence of ions. This is in contrast to the previous work, where it was the release of ions (rather than ion-binding groups) that was controlled and the polymers had pre-existing cross-linkable moieties. In stark contrast to the semi-flexible network paradigm of biological gels and the previous work on pectin, the gels studied herein exhibit the properties of chemically cross-linked networks of flexible polymers.

Designing molecular complexes using free-energy derivatives from liquid-state integral equation theory

NASA Astrophysics Data System (ADS)

Mrugalla, Florian; Kast, Stefan M.

2016-09-01

Complex formation between molecules in solution is the key process by which molecular interactions are translated into functional systems. These processes are governed by the binding or free energy of association which depends on both direct molecular interactions and the solvation contribution. A design goal frequently addressed in pharmaceutical sciences is the optimization of chemical properties of the complex partners in the sense of minimizing their binding free energy with respect to a change in chemical structure. Here, we demonstrate that liquid-state theory in the form of the solute-solute equation of the reference interaction site model provides all necessary information for such a task with high efficiency. In particular, computing derivatives of the potential of mean force (PMF), which defines the free-energy surface of complex formation, with respect to potential parameters can be viewed as a means to define a direction in chemical space toward better binders. We illustrate the methodology in the benchmark case of alkali ion binding to the crown ether 18-crown-6 in aqueous solution. In order to examine the validity of the underlying solute-solute theory, we first compare PMFs computed by different approaches, including explicit free-energy molecular dynamics simulations as a reference. Predictions of an optimally binding ion radius based on free-energy derivatives are then shown to yield consistent results for different ion parameter sets and to compare well with earlier, orders-of-magnitude more costly explicit simulation results. This proof-of-principle study, therefore, demonstrates the potential of liquid-state theory for molecular design problems.

Designing molecular complexes using free-energy derivatives from liquid-state integral equation theory.

PubMed

Mrugalla, Florian; Kast, Stefan M

2016-09-01

Complex formation between molecules in solution is the key process by which molecular interactions are translated into functional systems. These processes are governed by the binding or free energy of association which depends on both direct molecular interactions and the solvation contribution. A design goal frequently addressed in pharmaceutical sciences is the optimization of chemical properties of the complex partners in the sense of minimizing their binding free energy with respect to a change in chemical structure. Here, we demonstrate that liquid-state theory in the form of the solute-solute equation of the reference interaction site model provides all necessary information for such a task with high efficiency. In particular, computing derivatives of the potential of mean force (PMF), which defines the free-energy surface of complex formation, with respect to potential parameters can be viewed as a means to define a direction in chemical space toward better binders. We illustrate the methodology in the benchmark case of alkali ion binding to the crown ether 18-crown-6 in aqueous solution. In order to examine the validity of the underlying solute-solute theory, we first compare PMFs computed by different approaches, including explicit free-energy molecular dynamics simulations as a reference. Predictions of an optimally binding ion radius based on free-energy derivatives are then shown to yield consistent results for different ion parameter sets and to compare well with earlier, orders-of-magnitude more costly explicit simulation results. This proof-of-principle study, therefore, demonstrates the potential of liquid-state theory for molecular design problems.

Dicyanovinylnaphthalenes for neuroimaging of amyloids and relationships of electronic structures and geometries to binding affinities

PubMed Central

Petrič, Andrej; Johnson, Scott A.; Pham, Hung V.; Li, Ying; Čeh, Simon; Golobič, Amalija; Agdeppa, Eric D.; Timbol, Gerald; Liu, Jie; Keum, Gyochang; Satyamurthy, Nagichettiar; Kepe, Vladimir; Houk, Kendall N.; Barrio, Jorge R.

2012-01-01

The positron-emission tomography (PET) probe 2-(1-[6-[(2-fluoroethyl)(methyl)amino]-2-naphthyl]ethylidene) (FDDNP) is used for the noninvasive brain imaging of amyloid-β (Aβ) and other amyloid aggregates present in Alzheimer’s disease and other neurodegenerative diseases. A series of FDDNP analogs has been synthesized and characterized using spectroscopic and computational methods. The binding affinities of these molecules have been measured experimentally and explained through the use of a computational model. The analogs were created by systematically modifying the donor and the acceptor sides of FDDNP to learn the structural requirements for optimal binding to Aβ aggregates. FDDNP and its analogs are neutral, environmentally sensitive, fluorescent molecules with high dipole moments, as evidenced by their spectroscopic properties and dipole moment calculations. The preferred solution-state conformation of these compounds is directly related to the binding affinities. The extreme cases were a nonplanar analog t-butyl-FDDNP, which shows low binding affinity for Aβ aggregates (520 nM Ki) in vitro and a nearly planar tricyclic analog cDDNP, which displayed the highest binding affinity (10 pM Ki). Using a previously published X-ray crystallographic model of 1,1-dicyano-2-[6-(dimethylamino)naphthalen-2-yl]propene (DDNP) bound to an amyloidogenic Aβ peptide model, we show that the binding affinity is inversely related to the distortion energy necessary to avoid steric clashes along the internal surface of the binding channel. PMID:23012452

Modern bioanalysis of proteins by electrophoretic techniques.

PubMed

Krizkova, Sona; Ryvolova, Marketa; Masarik, Michal; Zitka, Ondrej; Adam, Vojtech; Hubalek, Jaromir; Eckschlager, Tomas; Kizek, Rene

2014-01-01

In 1957, protein rich in cysteine able to bind cadmium was isolated from horse kidney and named as metallothionein according to its structural properties. Further, this protein and metallothionein-like proteins have been found in tissues of other animal species, yeasts, fungi and plants. MT is as a potential cancer marker in the focus of interest, and its properties, functions, and behavior under various conditions are intensively studied. Our protocol describes separation of two major mammalian isoforms of MT (MT-1 and MT-2) using capillary electrophoresis (CE) coupled with UV detector. This protocol enables separation of MT isoforms and studying of their basic behavior as well as their quantification with detection limit in units of ng per μL. Sodium borate buffer (20 mM, pH 9.5) was optimized as a background electrolyte, and the separation was carried out in fused silica capillary with internal diameter of 75 μm and electric field intensity of 350 V/cm. Optimal detection wavelength was 254 nm.

Design of activated serine-containing catalytic triads with atomic level accuracy

PubMed Central

Rajagopalan, Sridharan; Wang, Chu; Yu, Kai; Kuzin, Alexandre P.; Richter, Florian; Lew, Scott; Miklos, Aleksandr E.; Matthews, Megan L.; Seetharaman, Jayaraman; Su, Min; Hunt, John. F.; Cravatt, Benjamin F.; Baker, David

2014-01-01

A challenge in the computational design of enzymes is that multiple properties must be simultaneously optimized -- substrate-binding, transition state stabilization, and product release -- and this has limited the absolute activity of successful designs. Here, we focus on a single critical property of many enzymes: the nucleophilicity of an active site residue that initiates catalysis. We design proteins with idealized serine-containing catalytic triads, and assess their nucleophilicity directly in native biological systems using activity-based organophosphate probes. Crystal structures of the most successful designs show unprecedented agreement with computational models, including extensive hydrogen bonding networks between the catalytic triad (or quartet) residues, and mutagenesis experiments demonstrate that these networks are critical for serine activation and organophosphate-reactivity. Following optimization by yeast-display, the designs react with organophosphate probes at rates comparable to natural serine hydrolases. Co-crystal structures with diisopropyl fluorophosphate bound to the serine nucleophile suggest the designs could provide the basis for a new class of organophosphate captures agents. PMID:24705591

Interactions of cisplatin analogues with lysozyme: a comparative analysis.

PubMed

Ferraro, Giarita; De Benedictis, Ilaria; Malfitano, Annamaria; Morelli, Giancarlo; Novellino, Ettore; Marasco, Daniela

2017-10-01

The biophysical characterization of drug binding to proteins plays a key role in structural biology and in the discovery and optimization of drug discovery processes. The search for optimal combinations of biophysical techniques that can correctly and efficiently identify and quantify binding of metal-based drugs to their final target is challenging, due to the physicochemical properties of these agents. Different cisplatin derivatives have shown different citotoxicities in most common cancer lines, suggesting that they exert their biological activity via different mechanisms of action. Here we carried out a comparative analysis, by studying the behaviours of three Pt-compounds under the same experimental conditions and binding assays to properly deepen the determinants of the different MAOs. Indeed we compared the results obtained using surface plasmon resonance, isothermal titration calorimetry, fluorescence spectroscopy and thermal shift assays based on circular dichroism experiments in the characterization of the formation of adducts obtained upon reaction of cisplatin, carboplatin and iodinated analogue of cisplatin, cis-Pt (NH 3 ) 2 I 2 , with the model protein hen egg white lysozyme, both at neutral and acid pHs. Further we reasoned on the applicability of employed techniques for the study the thermodynamics and kinetics of the reaction of a metallodrug with a protein and to reveal which information can be obtained using a combination of these analyses. Data were discussed on the light of the existing structural data collected on the platinated protein.

Macroscopic modeling and simulations of supercoiled DNA with bound proteins

NASA Astrophysics Data System (ADS)

Huang, Jing; Schlick, Tamar

2002-11-01

General methods are presented for modeling and simulating DNA molecules with bound proteins on the macromolecular level. These new approaches are motivated by the need for accurate and affordable methods to simulate slow processes (on the millisecond time scale) in DNA/protein systems, such as the large-scale motions involved in the Hin-mediated inversion process. Our approaches, based on the wormlike chain model of long DNA molecules, introduce inhomogeneous potentials for DNA/protein complexes based on available atomic-level structures. Electrostatically, treat those DNA/protein complexes as sets of effective charges, optimized by our discrete surface charge optimization package, in which the charges are distributed on an excluded-volume surface that represents the macromolecular complex. We also introduce directional bending potentials as well as non-identical bead hydrodynamics algorithm to further mimic the inhomogeneous effects caused by protein binding. These models thus account for basic elements of protein binding effects on DNA local structure but remain computational tractable. To validate these models and methods, we reproduce various properties measured by both Monte Carlo methods and experiments. We then apply the developed models to study the Hin-mediated inversion system in long DNA. By simulating supercoiled, circular DNA with or without bound proteins, we observe significant effects of protein binding on global conformations and long-time dynamics of the DNA on the kilo basepair length.

Structure-based design of Aurora A & B inhibitors

NASA Astrophysics Data System (ADS)

Poulsen, Anders; William, Anthony; Lee, Angeline; Blanchard, Stéphanie; Teo, Eeling; Deng, Weiping; Tu, Noah; Tan, Evelyn; Sun, Eric; Goh, Kay Lin; Ong, Wai Chung; Ng, Chee Pang; Goh, Kee Chuan; Bonday, Zahid

2008-12-01

The Aurora family of serine/threonine kinases are mitotic regulators involved in centrosome duplication, formation of the bipolar mitotic spindle and the alignment of the chromosomes along the spindle. These proteins are frequently overexpressed in tumor cells as compared to normal cells and are therefore potential therapeutic oncology targets. An Aurora A high throughput screen revealed a promising sub-micromolar indazole-benzimidazole lead. Modification of the benzimidazole portion of the lead to a C2 linker with a phenyl ring was proposed to achieve novelty. Docking revealed that a conjugated linker was optimal and the resulting compounds were equipotent with the lead. Further structure-guided optimization of substituents on the 5 & 6 position of the indazole led to single digit nanomolar potency. The homology between the Aurora A & Aurora B kinase domains is 71% but their binding sites only differ at residues 212 & 217 (Aurora A numbering). However interactions with only the latter residue may be used for obtaining selectivity. An analysis of published Aurora A and Aurora B X-ray structures reveals subtle differences in the shape of the binding sites. This was exploited by introduction of appropriately sized substituents in the 4 & 6 position of the indazole leading to Aurora B selective inhibitors. Finally we calculate the conformational energy penalty of the putative bioactive conformation of our inhibitors and show that this property correlates well with the Aurora A binding affinity.

Single-Molecule Imaging of an in Vitro-Evolved RNA Aptamer Reveals Homogeneous Ligand Binding Kinetics

PubMed Central

2009-01-01

Many studies of RNA folding and catalysis have revealed conformational heterogeneity, metastable folding intermediates, and long-lived states with distinct catalytic activities. We have developed a single-molecule imaging approach for investigating the functional heterogeneity of in vitro-evolved RNA aptamers. Monitoring the association of fluorescently labeled ligands with individual RNA aptamer molecules has allowed us to record binding events over the course of multiple days, thus providing sufficient statistics to quantitatively define the kinetic properties at the single-molecule level. The ligand binding kinetics of the highly optimized RNA aptamer studied here displays a remarkable degree of uniformity and lack of memory. Such homogeneous behavior is quite different from the heterogeneity seen in previous single-molecule studies of naturally derived RNA and protein enzymes. The single-molecule methods we describe may be of use in analyzing the distribution of functional molecules in heterogeneous evolving populations or even in unselected samples of random sequences. PMID:19572753

Effect of Stapling Architecture on Physiochemical Properties and Cell Permeability of Stapled α-Helical Peptides: A Comparative Study.

PubMed

Tian, Yuan; Jiang, Yanhong; Li, Jingxu; Wang, Dongyuan; Zhao, Hui; Li, Zigang

2017-11-02

Stapled peptides have emerged as a new class of targeting molecules with high binding affinity and specificity for intracellular undruggable targets. Their ability to penetrate cell membranes is exceptionally intriguing but remains elusively and controversially discussed. To understand the effect of stapling architectures on their physiochemical properties and to aid in promoting their cell permeability, we report herein a comparative study on the physiochemical properties and cell permeability of stapled α-helical peptides with different types of crosslinks. We highlight the decisive impact of the intrinsic properties of the crosslinks on cell permeability rather than the helical contents of the peptides in model amphipathic sequences targeting estrogen receptor-coactivator interaction. We envision this finding to shed further light on the chemical optimization of stapled α-helical peptides or macrocyclic cell-penetrating peptides for enhanced cell penetration. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Computational Methods Used in Hit-to-Lead and Lead Optimization Stages of Structure-Based Drug Discovery.

PubMed

Heifetz, Alexander; Southey, Michelle; Morao, Inaki; Townsend-Nicholson, Andrea; Bodkin, Mike J

2018-01-01

GPCR modeling approaches are widely used in the hit-to-lead (H2L) and lead optimization (LO) stages of drug discovery. The aims of these modeling approaches are to predict the 3D structures of the receptor-ligand complexes, to explore the key interactions between the receptor and the ligand and to utilize these insights in the design of new molecules with improved binding, selectivity or other pharmacological properties. In this book chapter, we present a brief survey of key computational approaches integrated with hierarchical GPCR modeling protocol (HGMP) used in hit-to-lead (H2L) and in lead optimization (LO) stages of structure-based drug discovery (SBDD). We outline the differences in modeling strategies used in H2L and LO of SBDD and illustrate how these tools have been applied in three drug discovery projects.

Characterization of ligand binding to melanocortin 4 receptors using fluorescent peptides with improved kinetic properties.

PubMed

Link, Reet; Veiksina, Santa; Rinken, Ago; Kopanchuk, Sergei

2017-03-15

Melanocortin 4 (MC 4 ) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC 4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC 4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ 1/2 =160min) and UTBC102 (τ 1/2 =7min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ 1/2 =224min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC 4 receptor specific ligands and could complement each other in these studies. Copyright © 2017 Elsevier B.V. All rights reserved.

Dynamically achieved active site precision in enzyme catalysis.

PubMed

Klinman, Judith P

2015-02-17

CONSPECTUS: The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes' enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme-substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C-H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed.

Modelling realistic TiO2 nanospheres: A benchmark study of SCC-DFTB against hybrid DFT

NASA Astrophysics Data System (ADS)

Selli, Daniele; Fazio, Gianluca; Di Valentin, Cristiana

2017-10-01

TiO2 nanoparticles (NPs) are nowadays considered fundamental building blocks for many technological applications. Morphology is found to play a key role with spherical NPs presenting higher binding properties and chemical activity. From the experimental point of view, the characterization of these nano-objects is extremely complex, opening a large room for computational investigations. In this work, TiO2 spherical NPs of different sizes (from 300 to 4000 atoms) have been studied with a two-scale computational approach. Global optimization to obtain stable and equilibrated nanospheres was performed with a self-consistent charge density functional tight-binding (SCC-DFTB) simulated annealing process, causing a considerable atomic rearrangement within the nanospheres. Those SCC-DFTB relaxed structures have been then optimized at the DFT(B3LYP) level of theory. We present a systematic and comparative SCC-DFTB vs DFT(B3LYP) study of the structural properties, with particular emphasis on the surface-to-bulk sites ratio, coordination distribution of surface sites, and surface energy. From the electronic point of view, we compare HOMO-LUMO and Kohn-Sham gaps, total and projected density of states. Overall, the comparisons between DFTB and hybrid density functional theory show that DFTB provides a rather accurate geometrical and electronic description of these nanospheres of realistic size (up to a diameter of 4.4 nm) at an extremely reduced computational cost. This opens for new challenges in simulations of very large systems and more extended molecular dynamics.

A general approach for developing system-specific functions to score protein-ligand docked complexes using support vector inductive logic programming.

PubMed

Amini, Ata; Shrimpton, Paul J; Muggleton, Stephen H; Sternberg, Michael J E

2007-12-01

Despite the increased recent use of protein-ligand and protein-protein docking in the drug discovery process due to the increases in computational power, the difficulty of accurately ranking the binding affinities of a series of ligands or a series of proteins docked to a protein receptor remains largely unsolved. This problem is of major concern in lead optimization procedures and has lead to the development of scoring functions tailored to rank the binding affinities of a series of ligands to a specific system. However, such methods can take a long time to develop and their transferability to other systems remains open to question. Here we demonstrate that given a suitable amount of background information a new approach using support vector inductive logic programming (SVILP) can be used to produce system-specific scoring functions. Inductive logic programming (ILP) learns logic-based rules for a given dataset that can be used to describe properties of each member of the set in a qualitative manner. By combining ILP with support vector machine regression, a quantitative set of rules can be obtained. SVILP has previously been used in a biological context to examine datasets containing a series of singular molecular structures and properties. Here we describe the use of SVILP to produce binding affinity predictions of a series of ligands to a particular protein. We also for the first time examine the applicability of SVILP techniques to datasets consisting of protein-ligand complexes. Our results show that SVILP performs comparably with other state-of-the-art methods on five protein-ligand systems as judged by similar cross-validated squares of their correlation coefficients. A McNemar test comparing SVILP to CoMFA and CoMSIA across the five systems indicates our method to be significantly better on one occasion. The ability to graphically display and understand the SVILP-produced rules is demonstrated and this feature of ILP can be used to derive hypothesis for future ligand design in lead optimization procedures. The approach can readily be extended to evaluate the binding affinities of a series of protein-protein complexes. (c) 2007 Wiley-Liss, Inc.

Effect of metal ions on photoluminescence, charge transport, magnetic and catalytic properties of all-inorganic colloidal nanocrystals and nanocrystal solids.

PubMed

Nag, Angshuman; Chung, Dae Sung; Dolzhnikov, Dmitriy S; Dimitrijevic, Nada M; Chattopadhyay, Soma; Shibata, Tomohiro; Talapin, Dmitri V

2012-08-22

Colloidal semiconductor nanocrystals (NCs) provide convenient "building blocks" for solution-processed solar cells, light-emitting devices, photocatalytic systems, etc. The use of inorganic ligands for colloidal NCs dramatically improved inter-NC charge transport, enabling fast progress in NC-based devices. Typical inorganic ligands (e.g., Sn(2)S(6)(4-), S(2-)) are represented by negatively charged ions that bind covalently to electrophilic metal surface sites. The binding of inorganic charged species to the NC surface provides electrostatic stabilization of NC colloids in polar solvents without introducing insulating barriers between NCs. In this work we show that cationic species needed for electrostatic balance of NC surface charges can also be employed for engineering almost every property of all-inorganic NCs and NC solids, including photoluminescence efficiency, electron mobility, doping, magnetic susceptibility, and electrocatalytic performance. We used a suite of experimental techniques to elucidate the impact of various metal ions on the characteristics of all-inorganic NCs and developed strategies for engineering and optimizing NC-based materials.

Tracing binding modes in hit-to-lead optimization: chameleon-like poses of aspartic protease inhibitors.

PubMed

Kuhnert, Maren; Köster, Helene; Bartholomäus, Ruben; Park, Ah Young; Shahim, Amir; Heine, Andreas; Steuber, Holger; Klebe, Gerhard; Diederich, Wibke E

2015-02-23

Successful lead optimization in structure-based drug discovery depends on the correct deduction and interpretation of the underlying structure-activity relationships (SAR) to facilitate efficient decision-making on the next candidates to be synthesized. Consequently, the question arises, how frequently a binding mode (re)-validation is required, to ensure not to be misled by invalid assumptions on the binding geometry. We present an example in which minor chemical modifications within one inhibitor series lead to surprisingly different binding modes. X-ray structure determination of eight inhibitors derived from one core scaffold resulted in four different binding modes in the aspartic protease endothiapepsin, a well-established surrogate for e.g. renin and β-secretase. In addition, we suggest an empirical metrics that might serve as an indicator during lead optimization to qualify compounds as candidates for structural revalidation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A Comparison Study for DNA Motif Modeling on Protein Binding Microarray.

PubMed

Wong, Ka-Chun; Li, Yue; Peng, Chengbin; Wong, Hau-San

2016-01-01

Transcription factor binding sites (TFBSs) are relatively short (5-15 bp) and degenerate. Identifying them is a computationally challenging task. In particular, protein binding microarray (PBM) is a high-throughput platform that can measure the DNA binding preference of a protein in a comprehensive and unbiased manner; for instance, a typical PBM experiment can measure binding signal intensities of a protein to all possible DNA k-mers (k = 8∼10). Since proteins can often bind to DNA with different binding intensities, one of the major challenges is to build TFBS (also known as DNA motif) models which can fully capture the quantitative binding affinity data. To learn DNA motif models from the non-convex objective function landscape, several optimization methods are compared and applied to the PBM motif model building problem. In particular, representative methods from different optimization paradigms have been chosen for modeling performance comparison on hundreds of PBM datasets. The results suggest that the multimodal optimization methods are very effective for capturing the binding preference information from PBM data. In particular, we observe a general performance improvement if choosing di-nucleotide modeling over mono-nucleotide modeling. In addition, the models learned by the best-performing method are applied to two independent applications: PBM probe rotation testing and ChIP-Seq peak sequence prediction, demonstrating its biological applicability.

Nonlinear optical and G-Quadruplex DNA stabilization properties of novel mixed ligand copper(II) complexes and coordination polymers: Synthesis, structural characterization and computational studies

NASA Astrophysics Data System (ADS)

Rajasekhar, Bathula; Bodavarapu, Navya; Sridevi, M.; Thamizhselvi, G.; RizhaNazar, K.; Padmanaban, R.; Swu, Toka

2018-03-01

The present study reports the synthesis and evaluation of nonlinear optical property and G-Quadruplex DNA Stabilization of five novel copper(II) mixed ligand complexes. They were synthesized from copper(II) salt, 2,5- and 2,3- pyridinedicarboxylic acid, diethylenetriamine and amide based ligand (AL). The crystal structure of these complexes were determined through X-ray diffraction and supported by ESI-MAS, NMR, UV-Vis and FT-IR spectroscopic methods. Their nonlinear optical property was studied using Gaussian09 computer program. For structural optimization and nonlinear optical property, density functional theory (DFT) based B3LYP method was used with LANL2DZ basis set for metal ion and 6-31G∗ for C,H,N,O and Cl atoms. The present work reveals that pre-polarized Complex-2 showed higher β value (29.59 × 10-30e.s.u) as compared to that of neutral complex-1 (β = 0.276 × 10-30e.s.u.) which may be due to greater advantage of polarizability. Complex-2 is expected to be a potential material for optoelectronic and photonic technologies. Docking studies using AutodockVina revealed that complex-2 has higher binding energy for both G-Quadruplex DNA (-8.7 kcal/mol) and duplex DNA (-10.1 kcal/mol). It was also observed that structure plays an important role in binding efficiency.

ProMateus—an open research approach to protein-binding sites analysis

PubMed Central

Neuvirth, Hani; Heinemann, Uri; Birnbaum, David; Tishby, Naftali; Schreiber, Gideon

2007-01-01

The development of bioinformatic tools by individual labs results in the abundance of parallel programs for the same task. For example, identification of binding site regions between interacting proteins is done using: ProMate, WHISCY, PPI-Pred, PINUP and others. All servers first identify unique properties of binding sites and then incorporate them into a predictor. Obviously, the resulting prediction would improve if the most suitable parameters from each of those predictors would be incorporated into one server. However, because of the variation in methods and databases, this is currently not feasible. Here, the protein-binding site prediction server is extended into a general protein-binding sites research tool, ProMateus. This web tool, based on ProMate's infrastructure enables the easy exploration and incorporation of new features and databases by the user, providing an evaluation of the benefit of individual features and their combination within a set framework. This transforms the individual research into a community exercise, bringing out the best from all users for optimized predictions. The analysis is demonstrated on a database of protein protein and protein-DNA interactions. This approach is basically different from that used in generating meta-servers. The implications of the open-research approach are discussed. ProMateus is available at http://bip.weizmann.ac.il/promate. PMID:17488838

Affinity-purified tetanus neurotoxin interaction with synaptic membranes: properties of a protease-sensitive receptor component

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lazarovici, P.; Yavin, E.

1986-11-04

The pharmacokinetic interaction of an affinity-purified /sup 125/I-labeled tetanotoxin fraction with guinea pig brain synaptosomal preparations was investigated. Binding of tetanotoxin was time- and temperature-dependent, was proportional to protein concentration, and was saturable at about 8 x 10/sup -9/ M as estimated by a solid-surface binding assay. Binding was optimal at pH 6.5 under low ionic strength buffer and was almost entirely blocked by gangliosides or antitoxin. In analogy to intact nerve cells, binding of toxin to membranes resulted in a tight association operationally defined as sequestration. Binding and sequestration were abolished after membrane pretreatment with sialidase. The enzyme couldmore » not dissociate the membrane-bound toxin formed at 4 or 37/sup 0/C under low ionic strength conditions, which is in part compatible with internalization as defined in nerve cell cultures. In the latter system the toxin could be removed at 4/sup 0/C but not at 37/sup 0/C. Binding was significantly reduced upon pretreatment of guinea pig brain membranes by a variety of hydrolytic enzymes. It is proposed that, in addition to a ganglioside, interaction of tetanotoxin with synaptic membranes is facilitated by a protein and may also require an appropriate lipid environment. These latter membrane constituents may play a pivotal role in the sequestration of the toxin.« less

Preparation of poly-L-lysine functionalized magnetic nanoparticles and their influence on viability of cancer cells

NASA Astrophysics Data System (ADS)

Khmara, I.; Koneracka, M.; Kubovcikova, M.; Zavisova, V.; Antal, I.; Csach, K.; Kopcansky, P.; Vidlickova, I.; Csaderova, L.; Pastorekova, S.; Zatovicova, M.

2017-04-01

This study was aimed at development of biocompatible amino-functionalized magnetic nanoparticles as carriers of specific antibodies able to detect and/or target cancer cells. Poly-L-lysine (PLL)-modified magnetic nanoparticle samples with different PLL/Fe3O4 content were prepared and tested to define the optimal PLL/Fe3O4 weight ratio. The samples were characterized for particle size and morphology (SEM, TEM and DLS), and surface properties (zeta potential measurements). The optimal PLL/Fe3O4 weight ratio of 1.0 based on both zeta potential and DLS measurements was in agreement with the UV/VIS measurements. Magnetic nanoparticles with the optimal PLL content were conjugated with antibody specific for the cancer biomarker carbonic anhydrase IX (CA IX), which is induced by hypoxia, a physiologic stress present in solid tumors and linked with aggressive tumor behavior. CA IX is localized on the cell surface with the antibody-binding epitope facing the extracellular space and is therefore suitable for antibody-based targeting of tumor cells. Here we showed that PLL/Fe3O4 magnetic nanoparticles exhibit cytotoxic activities in a cell type-dependent manner and bind to cells expressing CA IX when conjugated with the CA IX-specific antibody. These data support further investigations of the CA IX antibody-conjugated, magnetic field-guided/activated nanoparticles as tools in anticancer strategies.

[11C]Harmine Binding to Brain Monoamine Oxidase A: Test-Retest Properties and Noninvasive Quantification.

PubMed

Zanderigo, Francesca; D'Agostino, Alexandra E; Joshi, Nandita; Schain, Martin; Kumar, Dileep; Parsey, Ramin V; DeLorenzo, Christine; Mann, J John

2018-02-08

Inhibition of the isoform A of monoamine oxidase (MAO-A), a mitochondrial enzyme catalyzing deamination of monoamine neurotransmitters, is useful in treatment of depression and anxiety disorders. [ 11 C]harmine, a MAO-A PET radioligand, has been used to study mood disorders and antidepressant treatment. However, [ 11 C]harmine binding test-retest characteristics have to date only been partially investigated. Furthermore, since MAO-A is ubiquitously expressed, no reference region is available, thus requiring arterial blood sampling during PET scanning. Here, we investigate [ 11 C]harmine binding measurements test-retest properties; assess effects of using a minimally invasive input function estimation on binding quantification and repeatability; and explore binding potentials estimation using a reference region-free approach. Quantification of [ 11 C]harmine distribution volume (V T ) via kinetic models and graphical analyses was compared based on absolute test-retest percent difference (TRPD), intraclass correlation coefficient (ICC), and identifiability. The optimal procedure was also used with a simultaneously estimated input function in place of the measured curve. Lastly, an approach for binding potentials quantification in absence of a reference region was evaluated. [ 11 C]harmine V T estimates quantified using arterial blood and kinetic modeling showed average absolute TRPD values of 7.7 to 15.6 %, and ICC values between 0.56 and 0.86, across brain regions. Using simultaneous estimation (SIME) of input function resulted in V T estimates close to those obtained using arterial input function (r = 0.951, slope = 1.073, intercept = - 1.037), with numerically but not statistically higher test-retest difference (range 16.6 to 22.0 %), but with overall poor ICC values, between 0.30 and 0.57. Prospective studies using [ 11 C]harmine are possible given its test-retest repeatability when binding is quantified using arterial blood. Results with SIME of input function show potential for simplifying data acquisition by replacing arterial catheterization with one arterial blood sample at 20 min post-injection. Estimation of [ 11 C]harmine binding potentials remains a challenge that warrants further investigation.

A ruthenium dimer complex with a flexible linker slowly threads between DNA bases in two distinct steps.

PubMed

Bahira, Meriem; McCauley, Micah J; Almaqwashi, Ali A; Lincoln, Per; Westerlund, Fredrik; Rouzina, Ioulia; Williams, Mark C

2015-10-15

Several multi-component DNA intercalating small molecules have been designed around ruthenium-based intercalating monomers to optimize DNA binding properties for therapeutic use. Here we probe the DNA binding ligand [μ-C4(cpdppz)2(phen)4Ru2](4+), which consists of two Ru(phen)2dppz(2+) moieties joined by a flexible linker. To quantify ligand binding, double-stranded DNA is stretched with optical tweezers and exposed to ligand under constant applied force. In contrast to other bis-intercalators, we find that ligand association is described by a two-step process, which consists of fast bimolecular intercalation of the first dppz moiety followed by ∼10-fold slower intercalation of the second dppz moiety. The second step is rate-limited by the requirement for a DNA-ligand conformational change that allows the flexible linker to pass through the DNA duplex. Based on our measured force-dependent binding rates and ligand-induced DNA elongation measurements, we are able to map out the energy landscape and structural dynamics for both ligand binding steps. In addition, we find that at zero force the overall binding process involves fast association (∼10 s), slow dissociation (∼300 s), and very high affinity (Kd ∼10 nM). The methodology developed in this work will be useful for studying the mechanism of DNA binding by other multi-step intercalating ligands and proteins. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mechanics of composite actin networks: in vitro and cellular perspectives

NASA Astrophysics Data System (ADS)

Upadhyaya, Arpita

2014-03-01

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not well understood. The ABP, palladin, is essential for the integrity of cell morphology and movement during development. Palladin coexists with alpha-actinin in stress fibers and focal adhesions and binds to both actin and alpha-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we have characterized the micro-structure and mechanics of actin networks crosslinked with palladin and alpha-actinin. Our studies on composite networks of alpha-actinin/palladin/actin show that palladin and alpha-actinin synergistically determine network viscoelasticity. We have further examined the role of palladin in cellular force generation and mechanosensing. Traction force microscopy revealed that TAFs are sensitive to substrate stiffness as they generate larger forces on substrates of increased stiffness. Contrary to expectations, knocking down palladin increased the forces generated by cells, and also inhibited the ability to sense substrate stiffness for very stiff gels. This was accompanied by significant differences in the actin organization and adhesion dynamics of palladin knock down cells. Perturbation experiments also suggest altered myosin activity in palladin KD cells. Our results suggest that the actin crosslinkers such as palladin and myosin motors coordinate for optimal cell function and to prevent aberrant behavior as in cancer metastasis.

The Influence of Adnectin Binding on the Extracellular Domain of Epidermal Growth Factor Receptor

NASA Astrophysics Data System (ADS)

Iacob, Roxana E.; Chen, Guodong; Ahn, Joomi; Houel, Stephane; Wei, Hui; Mo, Jingjie; Tao, Li; Cohen, Daniel; Xie, Dianlin; Lin, Zheng; Morin, Paul E.; Doyle, Michael L.; Tymiak, Adrienne A.; Engen, John R.

2014-12-01

The precise and unambiguous elucidation and characterization of interactions between a high affinity recognition entity and its cognate protein provides important insights for the design and development of drugs with optimized properties and efficacy. In oncology, one important target protein has been shown to be the epidermal growth factor receptor (EGFR) through the development of therapeutic anticancer antibodies that are selective inhibitors of EGFR activity. More recently, smaller protein derived from the 10th type III domain of human fibronectin termed an adnectin has also been shown to inhibit EGFR in clinical studies. The mechanism of EGFR inhibition by either an adnectin or an antibody results from specific binding of the high affinity protein to the extracellular portion of EGFR (exEGFR) in a manner that prevents phosphorylation of the intracellular kinase domain of the receptor and thereby blocks intracellular signaling. Here, the structural changes induced upon binding were studied by probing the solution conformations of full length exEGFR alone and bound to a cognate adnectin through hydrogen/deuterium exchange mass spectrometry (HDX MS). The effects of binding in solution were identified and compared with the structure of a bound complex determined by X-ray crystallography.

Functionalization of ( n, 0) CNTs ( n = 3-16) by uracil: DFT studies

NASA Astrophysics Data System (ADS)

Mirzaei, Mahmoud; Harismah, Kun; Jafari, Elham; Gülseren, Oğuz; Rad, Ali Shokuhi

2018-01-01

Density functional theory (DFT) calculations were performed to investigate stabilities and properties for uracil (U)-functionalized carbon nanotubes (CNTs). To this aim, the optimized molecular properties were evaluated for ( n, 0) models of CNTs ( n = 3-16) in the original and U-functionalized forms. The results indicated that the dipole moments and energy gaps were independent of tubular diameters whereas the binding energies showed that the U-functionalization could be better achieved for n = 8-11 curvatures of ( n, 0) CNTs. Further studies based on the evaluated atomic-scale properties, including quadrupole coupling constants ( C Q ), indicated that the electronic properties of atoms could detect the effects of diameters variations of ( n, 0) CNTs, in which the effects were very much significant for the atoms around the U-functionalization regions. Finally, the achieved results of singular U, original CNTs, and CNT-U hybrids were compared to each other to demonstrate the stabilities and properties for the U-functionalized ( n, 0) CNTs.

Dynamics of Receptor-Mediated Nanoparticle Internalization into Endothelial Cells

PubMed Central

Gonzalez-Rodriguez, David; Barakat, Abdul I.

2015-01-01

Nanoparticles offer a promising medical tool for targeted drug delivery, for example to treat inflamed endothelial cells during the development of atherosclerosis. To inform the design of such therapeutic strategies, we develop a computational model of nanoparticle internalization into endothelial cells, where internalization is driven by receptor-ligand binding and limited by the deformation of the cell membrane and cytoplasm. We specifically consider the case of nanoparticles targeted against ICAM-1 receptors, of relevance for treating atherosclerosis. The model computes the kinetics of the internalization process, the dynamics of binding, and the distribution of stresses exerted between the nanoparticle and the cell membrane. The model predicts the existence of an optimal nanoparticle size for fastest internalization, consistent with experimental observations, as well as the role of bond characteristics, local cell mechanical properties, and external forces in the nanoparticle internalization process. PMID:25901833

Examination of a Method to Determine the Reference Region for Calculating the Specific Binding Ratio in Dopamine Transporter Imaging.

PubMed

Watanabe, Ayumi; Inoue, Yusuke; Asano, Yuji; Kikuchi, Kei; Miyatake, Hiroki; Tokushige, Takanobu

2017-01-01

The specific binding ratio (SBR) was first reported by Tossici-Bolt et al. for quantitative indicators for dopamine transporter (DAT) imaging. It is defined as the ratio of the specific binding concentration of the striatum to the non-specific binding concentration of the whole brain other than the striatum. The non-specific binding concentration is calculated based on the region of interest (ROI), which is set 20 mm inside the outer contour, defined by a threshold technique. Tossici-Bolt et al. used a 50% threshold, but sometimes we couldn't define the ROI of non-specific binding concentration (reference region) and calculate SBR appropriately with a 50% threshold. Therefore, we sought a new method for determining the reference region when calculating SBR. We used data from 20 patients who had undergone DAT imaging in our hospital, to calculate the non-specific binding concentration by the following methods, the threshold to define a reference region was fixed at some specific values (the fixing method) and reference region was visually optimized by an examiner at every examination (the visual optimization method). First, we assessed the reference region of each method visually, and afterward, we quantitatively compared SBR calculated based on each method. In the visual assessment, the scores of the fixing method at 30% and visual optimization method were higher than the scores of the fixing method at other values, with or without scatter correction. In the quantitative assessment, the SBR obtained by visual optimization of the reference region, based on consensus of three radiological technologists, was used as a baseline (the standard method). The values of SBR showed good agreement between the standard method and both the fixing method at 30% and the visual optimization method, with or without scatter correction. Therefore, the fixing method at 30% and the visual optimization method were equally suitable for determining the reference region.

Structure-based lead optimization to improve antiviral potency and ADMET properties of phenyl-1H-pyrrole-carboxamide entry inhibitors targeted to HIV-1 gp120.

PubMed

Curreli, Francesca; Belov, Dmitry S; Kwon, Young Do; Ramesh, Ranjith; Furimsky, Anna M; O'Loughlin, Kathleen; Byrge, Patricia C; Iyer, Lalitha V; Mirsalis, Jon C; Kurkin, Alexander V; Altieri, Andrea; Debnath, Asim K

2018-05-12

We are continuing our concerted effort to optimize our first lead entry antagonist, NBD-11021, which targets the Phe43 cavity of the HIV-1 envelope glycoprotein gp120, to improve antiviral potency and ADMET properties. In this report, we present a structure-based approach that helped us to generate working hypotheses to modify further a recently reported advanced lead entry antagonist, NBD-14107, which showed significant improvement in antiviral potency when tested in a single-cycle assay against a large panel of Env-pseudotyped viruses. We report here the synthesis of twenty-nine new compounds and evaluation of their antiviral activity in a single-cycle and multi-cycle assay to derive a comprehensive structure-activity relationship (SAR). We have selected three inhibitors with the high selectivity index for testing against a large panel of 55 Env-pseudotyped viruses representing a diverse set of clinical isolates of different subtypes. The antiviral activity of one of these potent inhibitors, 55 (NBD-14189), against some clinical isolates was as low as 63 nM. We determined the sensitivity of CD4-binding site mutated-pseudoviruses to these inhibitors to confirm that they target HIV-1 gp120. Furthermore, we assessed their ADMET properties and compared them to the clinical candidate attachment inhibitor, BMS-626529. The ADMET data indicate that some of these new inhibitors have comparable ADMET properties to BMS-626529 and can be optimized further to potential clinical candidates. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Discovery and Cocrystal Structure of Benzodiazepinedione HDM2 Antagonists that Activate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grasberger,B.; Lu, T.; Schubert, C.

2005-01-01

HDM2 binds to an {alpha}-helical transactivation domain of p53, inhibiting its tumor suppressive functions. A miniaturized thermal denaturation assay was used to screen chemical libraries, resulting in the discovery of a novel series of benzodiazepinedione antagonists of the HDM2-p53 interaction. The X-ray crystal structure of improved antagonists bound to HDM2 reveals their {alpha}-helix mimetic properties. These optimized molecules increase the transcription of p53 target genes and decrease proliferation of tumor cells expressing wild-type p53.

More than a simple lipophilic contact: a detailed thermodynamic analysis of nonbasic residues in the s1 pocket of thrombin.

PubMed

Baum, Bernhard; Mohamed, Menshawy; Zayed, Mohamed; Gerlach, Christof; Heine, Andreas; Hangauer, David; Klebe, Gerhard

2009-07-03

The field of medicinal chemistry aims to design and optimize small molecule leads into drug candidates that may positively interfere with pathological disease situations in humans or combat the growth of infective pathogens. From the plethora of crystal structures of protein-inhibitor complexes we have learned how molecules recognize each other geometrically, but we still have rather superficial understanding of why they bind to each other. This contribution surveys a series of 26 thrombin inhibitors with small systematic structural differences to elucidate the rationale for their widely deviating binding affinity from 185 microM to 4 nM as recorded by enzyme kinetic measurements. Five well-resolved (resolution 2.30 - 1.47 A) crystal structures of thrombin-inhibitor complexes and an apo-structure of the uncomplexed enzyme (1.50 A) are correlated with thermodynamic data recorded by isothermal titration calorimetry with 12 selected inhibitors from the series. Taking solubility data into account, the variation in physicochemical properties allows conclusions to be reached about the relative importance of the enthalpic binding features as well as to estimate the importance of the parameters more difficult to capture, such as residual ligand entropy and desolvation properties. The collected data reveal a comprehensive picture of the thermodynamic signature that explains the so far poorly understood attractive force experienced by m-chloro-benzylamides to thrombin.

Identification of sequence–structure RNA binding motifs for SELEX-derived aptamers

PubMed Central

Hoinka, Jan; Zotenko, Elena; Friedman, Adam; Sauna, Zuben E.; Przytycka, Teresa M.

2012-01-01

Motivation: Systematic Evolution of Ligands by EXponential Enrichment (SELEX) represents a state-of-the-art technology to isolate single-stranded (ribo)nucleic acid fragments, named aptamers, which bind to a molecule (or molecules) of interest via specific structural regions induced by their sequence-dependent fold. This powerful method has applications in designing protein inhibitors, molecular detection systems, therapeutic drugs and antibody replacement among others. However, full understanding and consequently optimal utilization of the process has lagged behind its wide application due to the lack of dedicated computational approaches. At the same time, the combination of SELEX with novel sequencing technologies is beginning to provide the data that will allow the examination of a variety of properties of the selection process. Results: To close this gap we developed, Aptamotif, a computational method for the identification of sequence–structure motifs in SELEX-derived aptamers. To increase the chances of identifying functional motifs, Aptamotif uses an ensemble-based approach. We validated the method using two published aptamer datasets containing experimentally determined motifs of increasing complexity. We were able to recreate the author's findings to a high degree, thus proving the capability of our approach to identify binding motifs in SELEX data. Additionally, using our new experimental dataset, we illustrate the application of Aptamotif to elucidate several properties of the selection process. Contact: przytyck@ncbi.nlm.nih.gov, Zuben.Sauna@fda.hhs.gov PMID:22689764

Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity.

PubMed

Vermersch, P S; Lemon, D D; Tesmer, J J; Quiocho, F A

1991-07-16

In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions. Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography. ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc. Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal). The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar. We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara. The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes. We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Self-Consistent Optimization of Excited States within Density-Functional Tight-Binding.

PubMed

Kowalczyk, Tim; Le, Khoa; Irle, Stephan

2016-01-12

We present an implementation of energies and gradients for the ΔDFTB method, an analogue of Δ-self-consistent-field density functional theory (ΔSCF) within density-functional tight-binding, for the lowest singlet excited state of closed-shell molecules. Benchmarks of ΔDFTB excitation energies, optimized geometries, Stokes shifts, and vibrational frequencies reveal that ΔDFTB provides a qualitatively correct description of changes in molecular geometries and vibrational frequencies due to excited-state relaxation. The accuracy of ΔDFTB Stokes shifts is comparable to that of ΔSCF-DFT, and ΔDFTB performs similarly to ΔSCF with the PBE functional for vertical excitation energies of larger chromophores where the need for efficient excited-state methods is most urgent. We provide some justification for the use of an excited-state reference density in the DFTB expansion of the electronic energy and demonstrate that ΔDFTB preserves many of the properties of its parent ΔSCF approach. This implementation fills an important gap in the extended framework of DFTB, where access to excited states has been limited to the time-dependent linear-response approach, and affords access to rapid exploration of a valuable class of excited-state potential energy surfaces.

Improvement of LysM-Mediated Surface Display of Designed Ankyrin Repeat Proteins (DARPins) in Recombinant and Nonrecombinant Strains of Lactococcus lactis and Lactobacillus Species

PubMed Central

Zadravec, Petra; Štrukelj, Borut

2015-01-01

Safety and probiotic properties make lactic acid bacteria (LAB) attractive hosts for surface display of heterologous proteins. Protein display on nonrecombinant microorganisms is preferred for therapeutic and food applications due to regulatory requirements. We displayed two designed ankyrin repeat proteins (DARPins), each possessing affinity for the Fc region of human IgG, on the surface of Lactococcus lactis by fusing them to the Usp45 secretion signal and to the peptidoglycan-binding C terminus of AcmA, containing lysine motif (LysM) repeats. Growth medium containing a secreted fusion protein was used to test its heterologous binding to 10 strains of species of the genus Lactobacillus, using flow cytometry, whole-cell enzyme-linked immunosorbent assay (ELISA), and fluorescence microscopy. The fusion proteins bound to the surfaces of all lactobacilli; however, binding to the majority of bacteria was only 2- to 5-fold stronger than that of the control. Lactobacillus salivarius ATCC 11741 demonstrated exceptionally strong binding (32- to 55-fold higher than that of the control) and may therefore be an attractive host for nonrecombinant surface display. Genomic comparison of the species indicated the exopolysaccharides of Lb. salivarius as a possible reason for the difference. Additionally, a 15-fold concentration-dependent increase in nonrecombinant surface display on L. lactis was demonstrated by growing bacteria with sublethal concentrations of the antibiotics chloramphenicol and erythromycin. Nonrecombinant surface display on LAB, based on LysM repeats, was optimized by selecting Lactobacillus salivarius ATCC 11741 as the optimal host and by introducing antibiotics as additives for increasing surface display on L. lactis. Additionally, effective display of DARPins on the surfaces of nonrecombinant LAB has opened up several new therapeutic possibilities. PMID:25576617

Structure-based affinity maturation of a chimeric anti-ricin antibody C4C13.

PubMed

Luo, Longlong; Luo, Qun; Guo, Leiming; Lv, Ming; Lin, Zhou; Geng, Jing; Li, Xinying; Li, Yan; Shen, Beifen; Qiao, Chunxia; Feng, Jiannan

2014-01-01

Ricin is a highly lethal toxin. Anti-ricin chimeric monoclonal antibody (mAb) C4C13 was prepared in our lab; however, its binding affinity was much weaker than that of the parent antibody 4C13. In this study, based on the computer-guided homology modeling and conformational optimization methods, the 3-D structure of C4C13 variable regions Fv was constructed and optimized. Using molecular docking and dynamics simulation methods, the 3-D complex structure of ricin and C4C13 Fv was obtained. Considering the orientation property, surface electrostatic distribution, residues chemical and physical character and intermolecular hydrogen bond, the binding mode and key residues were predicted. According to C4C13 Fv fragment and ricin complementary binding surface, electrostatic attraction periphery and van der Waals interaction interface, three mutants (i.e., M1 (N(H102)F, W(H103)Y); M2 (W(H103)Y) and M3 (R(L90)G)) were designed, in which M1 and M2 were predicted to possess higher antigen-binding activity than C4C13, while M3 was weaker. The relative affinity assays by ELISA showed that M1 and M2 mutations had higher affinity (9.6 and 18.3 nmol/L) than C4C13 (130 nmol/L) and M3 had weaker affinity (234.5 nmol/L) than C4C13. The results showed that the modeling complex structure of the antigen (ricin) and antibody (C4C13) is reasonable. Our work offered affinity maturated antibodies by site mutations, which were beneficial for valuable anti-ricin antibody design and preparation in future.

Extending rule-based methods to model molecular geometry and 3D model resolution.

PubMed

Hoard, Brittany; Jacobson, Bruna; Manavi, Kasra; Tapia, Lydia

2016-08-01

Computational modeling is an important tool for the study of complex biochemical processes associated with cell signaling networks. However, it is challenging to simulate processes that involve hundreds of large molecules due to the high computational cost of such simulations. Rule-based modeling is a method that can be used to simulate these processes with reasonably low computational cost, but traditional rule-based modeling approaches do not include details of molecular geometry. The incorporation of geometry into biochemical models can more accurately capture details of these processes, and may lead to insights into how geometry affects the products that form. Furthermore, geometric rule-based modeling can be used to complement other computational methods that explicitly represent molecular geometry in order to quantify binding site accessibility and steric effects. We propose a novel implementation of rule-based modeling that encodes details of molecular geometry into the rules and binding rates. We demonstrate how rules are constructed according to the molecular curvature. We then perform a study of antigen-antibody aggregation using our proposed method. We simulate the binding of antibody complexes to binding regions of the shrimp allergen Pen a 1 using a previously developed 3D rigid-body Monte Carlo simulation, and we analyze the aggregate sizes. Then, using our novel approach, we optimize a rule-based model according to the geometry of the Pen a 1 molecule and the data from the Monte Carlo simulation. We use the distances between the binding regions of Pen a 1 to optimize the rules and binding rates. We perform this procedure for multiple conformations of Pen a 1 and analyze the impact of conformation and resolution on the optimal rule-based model. We find that the optimized rule-based models provide information about the average steric hindrance between binding regions and the probability that antibodies will bind to these regions. These optimized models quantify the variation in aggregate size that results from differences in molecular geometry and from model resolution.

Calculating an optimal box size for ligand docking and virtual screening against experimental and predicted binding pockets.

PubMed

Feinstein, Wei P; Brylinski, Michal

2015-01-01

Computational approaches have emerged as an instrumental methodology in modern research. For example, virtual screening by molecular docking is routinely used in computer-aided drug discovery. One of the critical parameters for ligand docking is the size of a search space used to identify low-energy binding poses of drug candidates. Currently available docking packages often come with a default protocol for calculating the box size, however, many of these procedures have not been systematically evaluated. In this study, we investigate how the docking accuracy of AutoDock Vina is affected by the selection of a search space. We propose a new procedure for calculating the optimal docking box size that maximizes the accuracy of binding pose prediction against a non-redundant and representative dataset of 3,659 protein-ligand complexes selected from the Protein Data Bank. Subsequently, we use the Directory of Useful Decoys, Enhanced to demonstrate that the optimized docking box size also yields an improved ranking in virtual screening. Binding pockets in both datasets are derived from the experimental complex structures and, additionally, predicted by eFindSite. A systematic analysis of ligand binding poses generated by AutoDock Vina shows that the highest accuracy is achieved when the dimensions of the search space are 2.9 times larger than the radius of gyration of a docking compound. Subsequent virtual screening benchmarks demonstrate that this optimized docking box size also improves compound ranking. For instance, using predicted ligand binding sites, the average enrichment factor calculated for the top 1 % (10 %) of the screening library is 8.20 (3.28) for the optimized protocol, compared to 7.67 (3.19) for the default procedure. Depending on the evaluation metric, the optimal docking box size gives better ranking in virtual screening for about two-thirds of target proteins. This fully automated procedure can be used to optimize docking protocols in order to improve the ranking accuracy in production virtual screening simulations. Importantly, the optimized search space systematically yields better results than the default method not only for experimental pockets, but also for those predicted from protein structures. A script for calculating the optimal docking box size is freely available at www.brylinski.org/content/docking-box-size. Graphical AbstractWe developed a procedure to optimize the box size in molecular docking calculations. Left panel shows the predicted binding pose of NADP (green sticks) compared to the experimental complex structure of human aldose reductase (blue sticks) using a default protocol. Right panel shows the docking accuracy using an optimized box size.

Solubility and binding properties of PEGylated lysozyme derivatives with increasing molecular weight on hydrophobic-interaction chromatographic resins.

PubMed

Müller, Egbert; Josic, Djuro; Schröder, Tim; Moosmann, Anna

2010-07-09

Dynamic binding capacities and resolution of PEGylated lysozyme derivatives with varying molecular weights of poly (ethylene) glycol (PEG) with 5 kDa, 10 kDa and 30 kDa for HIC resins and columns are presented. To find the optimal range for the operating conditions, solubility studies were performed by high-throughput analyses in a 96-well plate format, and optimal salt concentrations and pH values were determined. The solubility of PEG-proteins was strongly influenced by the length of the PEG moiety. Large differences in the solubilities of PEGylated lysozymes in two different salts, ammonium sulfate and sodium chloride were found. Solubility of PEGylated lysozyme derivatives in ammonium sulfate decreases with increased length of attached PEG chains. In sodium chloride all PEGylated lysozyme derivatives are fully soluble in a concentration range between 0.1 mg protein/ml and 10 mg protein/ml. The binding capacities for PEGylated lysozyme to HIC resins are dependent on the salt type and molecular weight of the PEG polymer. In both salt solutions, ammonium sulfate and sodium chloride, the highest binding capacity of the resin was found for 5 kDa PEGylated lysozyme. For both native lysozyme and 30 kDa mono-PEGylated lysozyme the binding capacities were lower. In separation experiments on a TSKgel Butyl-NPR hydrophobic-interaction column with ammonium sulfate as mobile phase, the elution order was: native lysozyme, 5 kDa mono-PEGylated lysozyme and oligo-PEGylated lysozyme. This elution order was found to be reversed when sodium chloride was used. Furthermore, the resolution of the three mono-PEGylated forms was not possible with this column and ammonium sulfate as mobile phase. In 4 M sodium chloride a resolution of all PEGylated lysozyme forms was achieved. A tentative explanation for these phenomena can be the increased solvation of the PEG polymers in sodium chloride which changes the usual attractive hydrophobic forces in ammonium sulfate to more repulsive hydration forces in this hydrotrophic salt.

Deletion of uncharacterized domain from α-1,3-glucanase of Bacillus circulans KA-304 enhances heterologous enzyme production in Escherichia coli.

PubMed

Yano, Shigekazu; Suyotha, Wasana; Zanma, Sumika; Konno, Hiroyuki; Cherdvorapong, Vipavee; Wakayama, Mamoru

2018-05-08

α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. k cat /K m values of AgΔUCD and Agl-KA were 11.4 and 11.1 s -1 mg -1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

Controlling n-type doping in MoO 3

DOE PAGES

Peelaers, H.; Chabinyc, M. L.; Van de Walle, C. G.

2017-02-27

Here, we study the electronic properties of native defects and intentional dopant impurities in MoO 3, a widely used transparent conductor. Using first-principles hybrid functional calculations, we show that electron polarons can be self-trapped, but they can also bind to defects; thus, they play an important role in understanding the properties of doped MoO 3. Our calculations show that oxygen vacancies can cause unintentional n-type doping in MoO 3. Mo vacancies are unlikely to form. Tc and Re impurities on the Mo site and halogens (F, Cl, and Br) on the O site all act as shallow donors but trapmore » electron polarons. Fe, Ru, and Os impurities are amphoteric and will compensate n-type MoO 3. Mn dopants are also amphoteric, and they show interesting magnetic properties. These results support the design of doping approaches that optimally exploit functionality.« less

Dynamically Achieved Active Site Precision in Enzyme Catalysis

PubMed Central

2015-01-01

Conspectus The grand challenge in enzymology is to define and understand all of the parameters that contribute to enzymes’ enormous rate accelerations. The property of hydrogen tunneling in enzyme reactions has moved the focus of research away from an exclusive focus on transition state stabilization toward the importance of the motions of the heavy atoms of the protein, a role for reduced barrier width in catalysis, and the sampling of a protein conformational landscape to achieve a family of protein substates that optimize enzyme–substrate interactions and beyond. This Account focuses on a thermophilic alcohol dehydrogenase for which the chemical step of hydride transfer is rate determining across a wide range of experimental conditions. The properties of the chemical coordinate have been probed using kinetic isotope effects, indicating a transition in behavior below 30 °C that distinguishes nonoptimal from optimal C–H activation. Further, the introduction of single site mutants has the impact of either enhancing or eliminating the temperature dependent transition in catalysis. Biophysical probes, which include time dependent hydrogen/deuterium exchange and fluorescent lifetimes and Stokes shifts, have also been pursued. These studies allow the correlation of spatially resolved transitions in protein motions with catalysis. It is now possible to define a long-range network of protein motions in ht-ADH that extends from a dimer interface to the substrate binding domain across to the cofactor binding domain, over a distance of ca. 30 Å. The ongoing challenge to obtaining spatial and temporal resolution of catalysis-linked protein motions is discussed. PMID:25539048

Phage display of engineered binding proteins.

PubMed

Levisson, Mark; Spruijt, Ruud B; Winkel, Ingrid Nolla; Kengen, Servé W M; van der Oost, John

2014-01-01

In current purification processes optimization of the capture step generally has a large impact on cost reduction. At present, valuable biomolecules are often produced in relatively low concentrations and, consequently, the eventual selective separation from complex mixtures can be rather inefficient. A separation technology based on a very selective high-affinity binding may overcome these problems. Proteins in their natural environment manifest functionality by interacting specifically and often with relatively high affinity with other molecules, such as substrates, inhibitors, activators, or other proteins. At present, antibodies are the most commonly used binding proteins in numerous applications. However, antibodies do have limitations, such as high production costs, low stability, and a complex patent landscape. A novel approach is therefore to use non-immunoglobulin engineered binding proteins in affinity purification. In order to obtain engineered binders with a desired specificity, a large mutant library of the new to-be-developed binding protein has to be created and screened for potential binders. A powerful technique to screen and select for proteins with desired properties from a large pool of variants is phage display. Here, we indicate several criteria for potential binding protein scaffolds and explain the principle of M13 phage display. In addition, we describe experimental protocols for the initial steps in setting up a M13 phage display system based on the pComb3X vector, including construction of the phagemid vector, production of phages displaying the protein of interest, and confirmation of display on the M13 phage.

Ligand-protein docking using a quantum stochastic tunneling optimization method.

PubMed

Mancera, Ricardo L; Källblad, Per; Todorov, Nikolay P

2004-04-30

A novel hybrid optimization method called quantum stochastic tunneling has been recently introduced. Here, we report its implementation within a new docking program called EasyDock and a validation with the CCDC/Astex data set of ligand-protein complexes using the PLP score to represent the ligand-protein potential energy surface and ScreenScore to score the ligand-protein binding energies. When taking the top energy-ranked ligand binding mode pose, we were able to predict the correct crystallographic ligand binding mode in up to 75% of the cases. By using this novel optimization method run times for typical docking simulations are significantly shortened. Copyright 2004 Wiley Periodicals, Inc. J Comput Chem 25: 858-864, 2004

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus

PubMed Central

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying

2016-01-01

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. PMID:26801571

Molecular Structural Basis for the Cold Adaptedness of the Psychrophilic β-Glucosidase BglU in Micrococcus antarcticus.

PubMed

Miao, Li-Li; Hou, Yan-Jie; Fan, Hong-Xia; Qu, Jie; Qi, Chao; Liu, Ying; Li, De-Feng; Liu, Zhi-Pei

2016-01-22

Psychrophilic enzymes play crucial roles in cold adaptation of microbes and provide useful models for studies of protein evolution, folding, and dynamic properties. We examined the crystal structure (2.2-Å resolution) of the psychrophilic β-glucosidase BglU, a member of the glycosyl hydrolase 1 (GH1) enzyme family found in the cold-adapted bacterium Micrococcus antarcticus. Structural comparison and sequence alignment between BglU and its mesophilic and thermophilic counterpart enzymes (BglB and GlyTn, respectively) revealed two notable features distinct to BglU: (i) a unique long-loop L3 (35 versus 7 amino acids in others) involved in substrate binding and (ii) a unique amino acid, His299 (Tyr in others), involved in the stabilization of an ordered water molecule chain. Shortening of loop L3 to 25 amino acids reduced low-temperature catalytic activity, substrate-binding ability, the optimal temperature, and the melting temperature (Tm). Mutation of His299 to Tyr increased the optimal temperature, the Tm, and the catalytic activity. Conversely, mutation of Tyr301 to His in BglB caused a reduction in catalytic activity, thermostability, and the optimal temperature (45 to 35°C). Loop L3 shortening and H299Y substitution jointly restored enzyme activity to the level of BglU, but at moderate temperatures. Our findings indicate that loop L3 controls the level of catalytic activity at low temperatures, residue His299 is responsible for thermolability (particularly heat lability of the active center), and long-loop L3 and His299 are jointly responsible for the psychrophilic properties. The described structural basis for the cold adaptedness of BglU will be helpful for structure-based engineering of new cold-adapted enzymes and for the production of mutants useful in a variety of industrial processes at different temperatures. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Vibrational, structural and electronic properties investigation by DFT calculations and molecular docking studies with DNA topoisomerase II of strychnobrasiline type alkaloids: A theoretical approach for potentially bioactive molecules

NASA Astrophysics Data System (ADS)

Costa, Renyer A.; Oliveira, Kelson M. T.; Costa, Emmanoel Vilaça; Pinheiro, Maria L. B.

2017-10-01

A combined experimental and theoretical DFT study of the structural, vibrational and electronic properties of strychnobrasiline and 12-hydroxy-10,11-dimethoxystrychnobrasiline is presented using the Becke three-parameter Lee-Yang-Parr function (B3LYP) and 6-311G(2d,p) basis set. The theoretical geometry optimization data were compared with the X-ray data for a similar structure in the associated literature, showing close values. The calculated HOMO-LUMO gap values showed that the presence of substituents in the benzene ring influences the quantum properties which are directly related to the reactive properties. Theoretical UV spectra agreed well with the measured experimental data, with bands assigned. In addition, Natural Bond Orbitals (NBOs), Mapped molecular electrostatic potential surface (MEPS) and NLO calculations were also performed at the same theory level. The theoretical vibrational analysis revealed several characteristic vibrations that may be used as a diagnostic tool for other strychnobrasiline type alkaloids, simplifying their identification and structural characterization. Molecular docking calculations with DNA Topoisomerase II-DNA complex showed binding free energies values of -8.0 and -9.5 kcal/mol for strychnobrasiline and 12-hydroxy-10,11-dimethoxystrychnobrasiline respectively, while for amsacrine, used for the treatment of leukemia, the binding free energy ΔG presented a value of -10.0 kcal/mol, suggesting that strychnobrasiline derivative alkaloids might exhibit an antineoplastic activity.

Aptamer-based liposomes improve specific drug loading and release.

PubMed

Plourde, Kevin; Derbali, Rabeb Mouna; Desrosiers, Arnaud; Dubath, Céline; Vallée-Bélisle, Alexis; Leblond, Jeanne

2017-04-10

Aptamer technology has shown much promise in cancer therapeutics for its targeting abilities. However, its potential to improve drug loading and release from nanocarriers has not been thoroughly explored. In this study, we employed drug-binding aptamers to actively load drugs into liposomes. We designed a series of DNA aptamer sequences specific to doxorubicin, displaying multiple binding sites and various binding affinities. The binding ability of aptamers was preserved when incorporated into cationic liposomes, binding up to 15equivalents of doxorubicin per aptamer, therefore drawing the drug into liposomes. Optimization of the charge and drug/aptamer ratios resulted in ≥80% encapsulation efficiency of doxorubicin, ten times higher than classical passively-encapsulating liposomal formulations and similar to a pH-gradient active loading strategy. In addition, kinetic release profiles and cytotoxicity assay on HeLa cells demonstrated that the release and therapeutic efficacy of liposomal doxorubicin could be controlled by the aptamer's structure. Our results suggest that the aptamer exhibiting a specific intermediate affinity is the best suited to achieve high drug loading while maintaining efficient drug release and therapeutic activity. This strategy was successfully applied to tobramycin, a hydrophilic drug suffering from low encapsulation into liposomes, where its loading was improved six-fold using aptamers. Overall, we demonstrate that aptamers could act, in addition to their targeting properties, as multifunctional excipients for liposomal formulations. Copyright © 2017 Elsevier B.V. All rights reserved.

Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

PubMed Central

Almaqwashi, Ali A.; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C.

2016-01-01

There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

SU-F-SPS-08: Measuring the Interaction Of DDR Cell Receptors and Extracellular Matrix Collagen in Prostate Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dong, J; Sarkar, A; Hoffmann, P

Purpose: Discoidin domain receptors (DDR) have recently been recognized as important players in cancer progression. DDRs are cell receptors that interact with collagen, an extracellular matrix (ECM) protein. However the detailed mechanism of their interaction is unclear. Here we attempted to examine their interaction in terms of structural (surface topography), mechanical (rupture force), and kinetic (binding probability) information on the single molecular scale with the use of atomic force microscopy (AFM). Methods: The Quantitative Nano-mechanical property Mapping (QNM) mode of AFM allowed to assess the cells in liquid growth media at their optimal physiological while being viable. Human benign prostatemore » hyperplasia (BPH-1) cell line was genetically regulated to suppress DDR expression (DDR- cells) and was compared with naturally DDR expressing cells (DDR+). Results: Binding force measurements (n = 1000) were obtained before and after the two groups were treated with fibronectin (FN), an integrin-inhibiting antibody to block the binding of integrin. The quantification indicates that cells containing DDR bind with collagen at a most probable force of 80.3–83.0 ±7.6 pN. The probability of them binding is 0.167 when other interactions (mainly due to integrin-collagen binding) are minimized. Conclusion: Together with further force measurements at different pulling speeds will determine dissociation rate, binding distance and activation barrier. These parameters in benign cells provides some groundwork in understanding DDR’s behavior in various cell microenvironments such as in malignant tumor cells. Funding supported by Richard Barber Interdisciplinary Research Program of Wayne State University.« less

Application of enhanced electronegative multimodal chromatography as the primary capture step for immunoglobulin G purification.

PubMed

Wang, Yanli; Chen, Quan; Xian, Mo; Nian, Rui; Xu, Fei

2018-06-01

In recent studies, electronegative multimodal chromatography with Eshmuno HCX was demonstrated to be a highly promising recovery step for direct immunoglobulin G (IgG) capture from undiluted cell culture fluid. In this study, the binding properties of HCX to IgG at different pH/salt combinations were systematically studied, and its purification performance was significantly enhanced by lowering the washing pH and conductivity after high capacity binding of IgG under its optimal conditions. A single polishing step gave an end-product with non-histone host cell protein (nh-HCP) below 1 ppm, DNA less than 1 ppb, which aggregates less than 0.5% and an overall IgG recovery of 86.2%. The whole non-affinity chromatography based two-column-step process supports direct feed loading without buffer adjustment, thus extraordinarily boosting the overall productivity and cost-savings.

Three-dimensional metal-intercalated covalent organic frameworks for near-ambient energy storage

PubMed Central

Gao, Fei; Ding, Zijing; Meng, Sheng

2013-01-01

A new form of nanoporous material, metal intercalated covalent organic framework (MCOF) is proposed and its energy storage property revealed. Employing density functional and thermodynamical analysis, we find that stable, chemically active, porous materials could form by stacking covalent organic framework (COF) layers with metals as a gluing agent. Metal acts as active sites, while its aggregation is suppressed by a binding energy significantly larger than the corresponding cohesive energy of bulk metals. Two important parameters, metal binding and metal-metal separation, are tuned by selecting suitable building blocks and linkers when constructing COF layers. Systematic searches among a variety of elements and organic molecules identify Ca-intercalated COF with diphenylethyne units as optimal material for H2 storage, reaching a striking gravimetric density ~ 5 wt% at near-ambient conditions (300 K, 20 bar), in comparison to < 0.1 wt% for bare COF-1 under the same condition. PMID:23698018

Modular assembly of proteins on nanoparticles.

PubMed

Ma, Wenwei; Saccardo, Angela; Roccatano, Danilo; Aboagye-Mensah, Dorothy; Alkaseem, Mohammad; Jewkes, Matthew; Di Nezza, Francesca; Baron, Mark; Soloviev, Mikhail; Ferrari, Enrico

2018-04-16

Generally, the high diversity of protein properties necessitates the development of unique nanoparticle bio-conjugation methods, optimized for each different protein. Here we describe a universal bio-conjugation approach which makes use of a new recombinant fusion protein combining two distinct domains. The N-terminal part is Glutathione S-Transferase (GST) from Schistosoma japonicum, for which we identify and characterize the remarkable ability to bind gold nanoparticles (GNPs) by forming gold-sulfur bonds (Au-S). The C-terminal part of this multi-domain construct is the SpyCatcher from Streptococcus pyogenes, which provides the ability to capture recombinant proteins encoding a SpyTag. Here we show that SpyCatcher can be immobilized covalently on GNPs through GST without the loss of its full functionality. We then show that GST-SpyCatcher activated particles are able to covalently bind a SpyTag modified protein by simple mixing, through the spontaneous formation of an unusual isopeptide bond.

Preparation and characterization of tannase immobilized onto carboxyl-functionalized superparamagnetic ferroferric oxide nanoparticles.

PubMed

Wu, Changzheng; Xu, Caiyun; Ni, Hui; Yang, Qiuming; Cai, Huinong; Xiao, Anfeng

2016-04-01

Tannase from Aspergillus tubingensis was immobilized onto carboxyl-functionalized Fe3O4 nanoparticles (CMNPs), and conditions affecting tannase immobilization were investigated. Successful binding between CMNPs and tannase was confirmed by Fourier transform infrared spectroscopy and thermogravimetric analysis. Vibrating sample magnetometry and X-ray diffraction showed that the CMNPs and immobilized tannase exhibit distinct magnetic responses and superparamagnetic properties. Free and immobilized tannase exhibited identical optimal temperatures of 50°C and differing pH optima at 6 and 7, respectively. The thermal, pH, and storage stabilities of the immobilized tannase were superior to those of free tannase. After six cycles of catalytic hydrolysis of propyl gallate, the immobilized tannase maintained over 60% of its initial activity. The Michaelis constant (Km) of the immobilized enzyme indicated its higher affinity for substrate binding than the free enzyme. Copyright © 2016 Elsevier Ltd. All rights reserved.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries.

PubMed

Tullila, Antti; Nevanen, Tarja K

2017-05-31

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library.

Utilization of Multi-Immunization and Multiple Selection Strategies for Isolation of Hapten-Specific Antibodies from Recombinant Antibody Phage Display Libraries

PubMed Central

Tullila, Antti; Nevanen, Tarja K.

2017-01-01

Phage display technology provides a powerful tool for the development of novel recombinant antibodies. In this work, we optimized and streamlined the recombinant antibody discovery process for haptens as an example. A multi-immunization approach was used in order to avoid the need for construction of multiple antibody libraries. Selection methods were developed to utilize the full potential of the recombinant antibody library by applying four different elution conditions simultaneously. High-throughput immunoassays were used to analyse the binding properties of the individual antibody clones. Different carrier proteins were used in the immunization, selection, and screening phases to avoid enrichment of the antibodies for the carrier protein epitopes. Novel recombinant antibodies against mycophenolic acid and ochratoxin A, with affinities up to 39 nM and 34 nM, respectively, were isolated from a multi-immunized fragment antigen-binding (Fab) library. PMID:28561803

Point defects in Cd(Zn)Te and TlBr: Theory

NASA Astrophysics Data System (ADS)

Lordi, Vincenzo

2013-09-01

The effects of various crystal defects on the performances of CdTe, CdZnxTe (CZT), and TlBr for room-temperature high-energy radiation detection are examined using first-principles theoretical methods. The predictive, parameter-free, atomistic approaches used provide fundamental understanding of defect properties that are difficult to measure and also allow rapid screening of possibilities for material engineering, such as optimal doping and annealing conditions. Several recent examples from the author's work are reviewed, including: (i) accurate calculations of the thermodynamic and electronic properties of native point defects and point defect complexes in CdTe and CZT; (ii) the effects of Zn alloying on the native point defect properties in CZT; (iii) point defect diffusion and binding leading to Te clustering in Cd(Zn)Te; (iv) the profound effect of native point defects—principally vacancies—on the intrinsic material properties of TlBr, particularly its electronic and ionic conductivity; and (v) a study on doping TlBr to independently control the electronic and ionic conductivity.

Chemotactic properties and absence of the formyl peptide receptor in ferret (Mustela putorius furo) neutrophils.

PubMed

Nakata, Makoto; Otsubo, Kouji; Kikuchi, Tomoko; Itou, Takuya; Sakai, Takeo

2010-02-01

This study describes a chemotaxis assay of ferret polymorphonuclear cells (PMNs). The optimal conditions for this chemotaxis assay were investigated for three chemoattractants: zymosan activated serum (ZAS), recombinant human interleukin-8 (rhIL-8) and N-formyl-Met-Leu- Phe (fMLF). In this study, ferret polymorphonuclear cells (PMNs) reacted to ZAS and rhIL-8, but not fMLF. The optimal concentration of ZAS and rhIL-8 were 5% and 100 ng/ml, respectively. The optimal incubation time of each reagent was 60 min. Due to the lack of response shown from fMLF, the existence of formyl peptide receptors (FPR) on ferret PMNs was investigated by evaluating FPR binding using flow cytometry. The receptor was not detected, implying that ferret neutrophils may lack FPR. This study confirms the fundamental experimental conditions for ferret PMNs chemotaxis and elucidates new findings concerning FPR in ferret neutrophils. Copyright 2009 Elsevier Ltd. All rights reserved.

Systematic optimization model and algorithm for binding sequence selection in computational enzyme design

PubMed Central

Huang, Xiaoqiang; Han, Kehang; Zhu, Yushan

2013-01-01

A systematic optimization model for binding sequence selection in computational enzyme design was developed based on the transition state theory of enzyme catalysis and graph-theoretical modeling. The saddle point on the free energy surface of the reaction system was represented by catalytic geometrical constraints, and the binding energy between the active site and transition state was minimized to reduce the activation energy barrier. The resulting hyperscale combinatorial optimization problem was tackled using a novel heuristic global optimization algorithm, which was inspired and tested by the protein core sequence selection problem. The sequence recapitulation tests on native active sites for two enzyme catalyzed hydrolytic reactions were applied to evaluate the predictive power of the design methodology. The results of the calculation show that most of the native binding sites can be successfully identified if the catalytic geometrical constraints and the structural motifs of the substrate are taken into account. Reliably predicting active site sequences may have significant implications for the creation of novel enzymes that are capable of catalyzing targeted chemical reactions. PMID:23649589

Peptide-Directed PdAu Nanoscale Surface Segregation: Toward Controlled Bimetallic Architecture for Catalytic Materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bedford, Nicholas M.; Showalter, Allison R.; Woehl, Taylor J.

Bimetallic nanoparticles are of immense scientific and technological interest given the synergistic properties observed when mixing two different metallic species at the nanoscale. This is particularly prevalent in catalysis, where bimetallic nanoparticles often exhibit improved catalytic activity and durability over their monometallic counterparts. Yet despite intense research efforts, little is understood regarding how to optimize bimetallic surface composition and structure synthetically using rational design principles. Recently, it has been demonstrated that peptide-enabled routes for nanoparticle synthesis result in materials with sequence-dependent catalytic properties, providing an opportunity for rational design through sequence manipulation. In this study, bimetallic PdAu nanoparticles are synthesizedmore » with a small set of peptides containing known Pd and Au binding motifs. The resulting nanoparticles were extensively characterized using high-resolution scanning transmission electron microscopy, X-ray absorption spectroscopy and high-energy X-ray diffraction coupled to atomic pair distribution function analysis. Structural information obtained from synchrotron radiation methods were then used to generate model nanoparticle configurations using reverse Monte Carlo simulations, which illustrate sequence-dependence in both surface structure and surface composition. Replica exchange solute tempering molecular dynamic simulations were also used to predict the modes of peptide binding on monometallic surfaces, indicating that different sequences bind to the metal interfaces via different mechanisms. As a testbed reaction, electrocatalytic methanol oxidation experiments were performed, wherein differences in catalytic activity are clearly observed in materials with identical bimetallic composition. Finally, taken together, this study indicates that peptides could be used to arrive at bimetallic surfaces with enhanced catalytic properties, which could be leveraged for rational bimetallic nanoparticle design using peptide-enabled approaches.« less

Peptide-Directed PdAu Nanoscale Surface Segregation: Toward Controlled Bimetallic Architecture for Catalytic Materials

DOE PAGES

Bedford, Nicholas M.; Showalter, Allison R.; Woehl, Taylor J.; ...

2016-09-01

Bimetallic nanoparticles are of immense scientific and technological interest given the synergistic properties observed when mixing two different metallic species at the nanoscale. This is particularly prevalent in catalysis, where bimetallic nanoparticles often exhibit improved catalytic activity and durability over their monometallic counterparts. Yet despite intense research efforts, little is understood regarding how to optimize bimetallic surface composition and structure synthetically using rational design principles. Recently, it has been demonstrated that peptide-enabled routes for nanoparticle synthesis result in materials with sequence-dependent catalytic properties, providing an opportunity for rational design through sequence manipulation. In this study, bimetallic PdAu nanoparticles are synthesizedmore » with a small set of peptides containing known Pd and Au binding motifs. The resulting nanoparticles were extensively characterized using high-resolution scanning transmission electron microscopy, X-ray absorption spectroscopy and high-energy X-ray diffraction coupled to atomic pair distribution function analysis. Structural information obtained from synchrotron radiation methods were then used to generate model nanoparticle configurations using reverse Monte Carlo simulations, which illustrate sequence-dependence in both surface structure and surface composition. Replica exchange solute tempering molecular dynamic simulations were also used to predict the modes of peptide binding on monometallic surfaces, indicating that different sequences bind to the metal interfaces via different mechanisms. As a testbed reaction, electrocatalytic methanol oxidation experiments were performed, wherein differences in catalytic activity are clearly observed in materials with identical bimetallic composition. Finally, taken together, this study indicates that peptides could be used to arrive at bimetallic surfaces with enhanced catalytic properties, which could be leveraged for rational bimetallic nanoparticle design using peptide-enabled approaches.« less

Parameterizing the binding properties of dissolved organic matter with default values skews the prediction of copper solution speciation and ecotoxicity in soil.

PubMed

Djae, Tanalou; Bravin, Matthieu N; Garnier, Cédric; Doelsch, Emmanuel

2017-04-01

Parameterizing speciation models by setting the percentage of dissolved organic matter (DOM) that is reactive (% r-DOM) toward metal cations at a single 65% default value is very common in predictive ecotoxicology. The authors tested this practice by comparing the free copper activity (pCu 2+  = -log 10 [Cu 2+ ]) measured in 55 soil sample solutions with pCu 2+ predicted with the Windermere humic aqueous model (WHAM) parameterized by default. Predictions of Cu toxicity to soil organisms based on measured or predicted pCu 2+ were also compared. Default WHAM parameterization substantially skewed the prediction of measured pCu 2+ by up to 2.7 pCu 2+ units (root mean square residual = 0.75-1.3) and subsequently the prediction of Cu toxicity for microbial functions, invertebrates, and plants by up to 36%, 45%, and 59% (root mean square residuals ≤9 %, 11%, and 17%), respectively. Reparametrizing WHAM by optimizing the 2 DOM binding properties (i.e., % r-DOM and the Cu complexation constant) within a physically realistic value range much improved the prediction of measured pCu 2+ (root mean square residual = 0.14-0.25). Accordingly, this WHAM parameterization successfully predicted Cu toxicity for microbial functions, invertebrates, and plants (root mean square residual ≤3.4%, 4.4%, and 5.8%, respectively). Thus, it is essential to account for the real heterogeneity in DOM binding properties for relatively accurate prediction of Cu speciation in soil solution and Cu toxic effects on soil organisms. Environ Toxicol Chem 2017;36:898-905. © 2016 SETAC. © 2016 SETAC.

Engineering and characterization of fluorogenic glycine riboswitches

PubMed Central

Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

2016-01-01

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (kon), and dissociation (koff) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. kon and koff were in the order of 10−3s−1 and 10−2s−1, respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. PMID:27220466

Synthesis, characterization, crystal structure and theoretical study of a compound with benzodiazole ring: antimicrobial activity and DNA binding.

PubMed

Latha, P; Kodisundaram, P; Sundararajan, M L; Jeyakumar, T

2014-08-14

2-(Thiophen-2-yl)-1-((thiophen-2-yl)methyl)-1H-1,3-benzodiazole (HL) is synthesized and characterized by elemental analysis, UV-Vis, FT-IR, (1)H, (13)C NMR, mass spectra, scanning electron microscope (SEM) and single crystal X-ray diffraction. The crystal structure is stabilized by intermolecular CH⋯N and CH⋯π interactions. The molecular structure is also optimized at the B3LYP/6-31G level using density functional theory (DFT). The structural parameters from the theory are nearer to those of crystal, the calculated total energy of coordination is -1522.814a.u. The energy of HOMO-LUMO and the energy gap are -0.20718, -0.04314, 0.16404a.u, respectively. All data obtained from the spectral studies support the structural properties of the compound HL. The benzimidazole ring is essentially planar. The in vitro biological screening effects of the synthesized compound is tested against four bacterial and four fungal strains by well diffusion method. Antioxidant property and DNA binding behaviour of the compound has been investigated using spectrophotometric method. Copyright © 2014 Elsevier B.V. All rights reserved.

Multiparameter structural optimization of single-walled carbon nanotube composites: toward record strength, stiffness, and toughness.

PubMed

Shim, Bong Sup; Zhu, Jian; Jan, Edward; Critchley, Kevin; Ho, Szushen; Podsiadlo, Paul; Sun, Kai; Kotov, Nicholas A

2009-07-28

Efficient coupling of mechanical properties of SWNTs with the matrix leading to the transfer of unique mechanical properties of SWNTs to the macroscopic composites is a tremendous challenge of today's materials science. The typical mechanical properties of known SWNT composites, such as strength, stiffness, and toughness, are assessed in an introductory survey where we focused on concrete numerical parameters characterizing mechanical properties. Obtaining ideal stress transfer will require fine optimization of nanotube-polymer interface. SWNT nanocomposites were made here by layer-by-layer (LBL) assembly with poly(vinyl alcohol) (PVA), and the first example of optimization in respect to key parameters determining the connectivity at the graphene-polymer interface, namely, degree of SWNT oxidation and cross-linking chemistry, was demonstrated. The resulting SWNT-PVA composites demonstrated tensile strength (σ(ult)) = 504.5 ± 67.3 MPa, stiffness (E) = 15.6 ± 3.8 GPa, and toughness (K) = 121.2 ± 19.2 J/g with maximum values recorded at σ(ult) = 600.1 MPa, E = 20.6 GPa, and K = 152.1 J/g. This represents the strongest and stiffest nonfibrous SWNT composites made to date outperforming other bulk composites by 2-10 times. Its high performance is attributed to both high nanotube content and efficient stress transfer. The resulting LBL composite is also one of the toughest in this category of materials and exceeding the toughness of Kevlar by 3-fold. Our observation suggests that the strengthening and toughening mechanism originates from the synergistic combination of high degree of SWNT exfoliation, efficient SWNT-PVA binding, crack surface roughening, and fairly efficient distribution of local stress over the SWNT network. The need for a multiscale approach in designing SWNT composites is advocated.

Nickel binding and [NiFe]-hydrogenase maturation by the metallochaperone SlyD with a single metal-binding site in Escherichia coli.

PubMed

Kaluarachchi, Harini; Altenstein, Matthias; Sugumar, Sonia R; Balbach, Jochen; Zamble, Deborah B; Haupt, Caroline

2012-03-16

SlyD (sensitive to lysis D) is a nickel metallochaperone involved in the maturation of [NiFe]-hydrogenases in Escherichia coli (E. coli) and specifically contributes to the nickel delivery step during enzyme biosynthesis. This protein contains a C-terminal metal-binding domain that is rich in potential metal-binding residues that enable SlyD to bind multiple nickel ions with high affinity. The SlyD homolog from Thermus thermophilus does not contain the extended cysteine- and histidine-rich C-terminal tail of the E. coli protein, yet it binds a single Ni(II) ion tightly. To investigate whether a single metal-binding motif can functionally replace the full-length domain, we generated a truncation of E. coli SlyD, SlyD155. Ni(II) binding to SlyD155 was investigated by using isothermal titration calorimetry, NMR and electrospray ionization mass spectrometry measurements. This in vitro characterization revealed that SlyD155 contains a single metal-binding motif with high affinity for nickel. Structural characterization by X-ray absorption spectroscopy and NMR indicated that nickel was coordinated in an octahedral geometry with at least two histidines as ligands. Heterodimerization between SlyD and another hydrogenase accessory protein, HypB, is essential for optimal hydrogenase maturation and was confirmed for SlyD155 via cross-linking experiments and NMR titrations, as were conserved chaperone and peptidyl-prolyl isomerase activities. Although these properties of SlyD are preserved in the truncated version, it does not modulate nickel binding to HypB in vitro or contribute to the maturation of [NiFe]-hydrogenases in vivo, unlike the full-length protein. This study highlights the importance of the unusual metal-binding domain of E. coli SlyD in hydrogenase biogenesis. Copyright © 2012 Elsevier Ltd. All rights reserved.

THE MECHANISM OF ACTION OF COLCHICINE

PubMed Central

Wilson, Leslie; Meza, Isaura

1973-01-01

The thermal depolymerization procedure of Stephens (1970. J. Mol. Biol. 47:353) has been employed for solubilization of Strongylocentrotus purpuratus sperm tail outer doublet microtubules with the use of a buffer during solubilization which is of optimal pH and ionic strength for the preservation of colchicine binding activity of chick embryo brain tubulin. Colchicine binding values were corrected for first-order decay during heat solubilization at 50°C (t½ = 5.4 min) and incubation with colchicine at 37°C in the presence of vinblastine sulfate (t½ = 485 min). The colchicine binding properties of heat-solubilized outer doublet tubulin were qualitatively identical with those of other soluble forms of tubulin. The solubilized tubulin (mol wt, 115,000) bound 0.9 ± 0.2 mol of colchicine per mol of tubulin, with a binding constant of 6.3 x 105 liters/mol at 37°C. The colchicine binding reaction was both time and temperature dependent, and the binding of colchicine was prevented in a competitive manner by podophyllotoxin (Ki = 1.3 x 10-6 M). The first-order decay of colchicine binding activity was substantially decreased by the addition of the vinca alkaloids, vinblastine sulfate or vincristine sulfate, thus demonstrating the presence of a vinca alkaloid binding site(s) on the outer doublet tubulin. Tubulin contained within the assembled microtubules did not decay. Intact outer doublet microtubules bound less than 0.001 mol of colchicine per mol of tubulin contained in the microtubules, under conditions where soluble tubulin would have bound 1 mol of colchicine per mol of tubulin (saturating concentration of colchicine, no decay of colchicine binding activity). The presence of colchicine had no effect on the rate of solubilization of outer doublet microtubules during incubation at 37°C. Therefore, the colchicine binding site on tubulin is blocked (not available to bind colchicine) when the tubulin is in the assembled outer doublet microtubules. PMID:4747924

Optimal charges in lead progression: a structure-based neuraminidase case study.

PubMed

Armstrong, Kathryn A; Tidor, Bruce; Cheng, Alan C

2006-04-20

Collective experience in structure-based lead progression has found electrostatic interactions to be more difficult to optimize than shape-based ones. A major reason for this is that the net electrostatic contribution observed includes a significant nonintuitive desolvation component in addition to the more intuitive intermolecular interaction component. To investigate whether knowledge of the ligand optimal charge distribution can facilitate more intuitive design of electrostatic interactions, we took a series of small-molecule influenza neuraminidase inhibitors with known protein cocrystal structures and calculated the difference between the optimal and actual charge distributions. This difference from the electrostatic optimum correlates with the calculated electrostatic contribution to binding (r(2) = 0.94) despite small changes in binding modes caused by chemical substitutions, suggesting that the optimal charge distribution is a useful design goal. Furthermore, detailed suggestions for chemical modification generated by this approach are in many cases consistent with observed improvements in binding affinity, and the method appears to be useful despite discrete chemical constraints. Taken together, these results suggest that charge optimization is useful in facilitating generation of compound ideas in lead optimization. Our results also provide insight into design of neuraminidase inhibitors.

Inhibition of human Na(v)1.5 sodium channels by strychnine and its analogs.

PubMed

Yuan, Chunhua; Sun, Lirong; Zhang, Meng; Li, Shuji; Wang, Xuemin; Gao, Tianming; Zhu, Xinhong

2011-08-15

Strychnine and brucine from the seeds of the plant Strychnos nux vomica have been shown to have interesting pharmacological effects on several neurotransmitter receptors. In this study, we have characterized the pharmacological properties of strychnine and its analogs on human Na(v)1.5 channels to assess their potential therapeutic advantage in certain arrhythmias. Among the eight alkaloids, only strychnine and icajine exhibited inhibition potency on the Na(v)1.5 channel with the half-maximum inhibition (IC(50)) values of 83.1μM and 104.6μM, respectively. Structure-function analysis indicated that the increased bulky methoxy groups on the phenyl ring or the negatively charged oxygen atom may account for this lack of inhibition on the Na(v)1.5 channel. Strychnine and icajine may bind to the channel by cation-π interactions. The substitution with a large side chain on the phenyl ring or the increased molecular volume may alter the optimized position for the compound close to the binding sites of the channel. Strychnine and icajine bind to the Na(v)1.5 channel with a new mechanism that is different from TTX and local anesthetics. They bind to the outer vestibule of the channel pore with fast association and dissociation rates at resting state. Strychnine and icajine had little effect on steady-state fast inactivation but markedly shifted the slow inactivation of Na(v)1.5 currents toward more hyperpolarized potentials. The property of icajine influencing slow-inactivated state of Na(v)1.5 channel would be potential therapeutic advantages in certain arrhythmias. Copyright © 2011 Elsevier Inc. All rights reserved.

Effect of temperature acclimation on red blood cell oxygen affinity in Pacific bluefin tuna (Thunnus orientalis) and yellowfin tuna (Thunnus albacares).

PubMed

Lilly, Laura E; Bonaventura, Joseph; Lipnick, Michael S; Block, Barbara A

2015-03-01

Hemoglobin-oxygen (Hb-O2) binding properties are central to aerobic physiology, and must be optimized for an animal's aerobic requirements and environmental conditions, both of which can vary widely with seasonal changes or acutely with diving. In the case of tunas, the matter is further complicated by large regional temperature differences between tissues within the same animal. This study investigates the effects of thermal acclimation on red blood cell Hb-O2 binding in Pacific bluefin tuna (T. orientalis) and yellowfin tuna (T. albacares) maintained in captive tanks at acclimation temperatures of 17°, 20° and 24 °C. Oxygen binding properties of acclimated tuna isolated red blood cells were examined under varying experimental temperatures (15°-35 °C) and CO2 levels (0%, 0.5% and 1.5%). Results for Pacific bluefin tuna produced temperature-independence at 17 °C- and 20 °C-acclimation temperatures and significant reverse temperature-dependence at 24 °C-acclimation in the absence of CO2, with instances of reverse temperature-dependence in 17 °C- and 24 °C-acclimations at 0.5% and 1.5% CO2. In contrast, yellowfin tuna produced normal temperature-dependence at each acclimation temperature at 0% CO2, temperature-independence at 0.5% and 1.5% CO2, and significant reverse temperature-dependence at 17 °C-acclimation and 0.5% CO2. Thermal acclimation of Pacific bluefin tuna increased O2 binding affinity of the 17 °C-acclimation group, and produced a significantly steeper oxygen equilibrium curve slope (nH) at 24 °C-acclimation compared to the other acclimation temperatures. We discuss the potential implications of these findings below. Copyright © 2014 Elsevier Inc. All rights reserved.

Tight binding simulation study on zigzag single-walled carbon nanotubes

NASA Astrophysics Data System (ADS)

Sharma, Deepa; Jaggi, Neena; Gupta, Vishu

2018-01-01

Tight binding simulation studies using the density functional tight binding (DFTB) model have been performed on various zigzag single-walled carbon-nanotubes (SWCNTs) to investigate their electronic properties using DFTB module of the Material Studio Software version 7.0. Various combinations of different eigen-solvers and charge mixing schemes available in the DFTB Module have been tried to chalk out the electronic structure. The analytically deduced values of the bandgap of (9, 0) SWCNT were compared with the experimentally determined value reported in the literature. On comparison, it was found that the tight binding approximations tend to drastically underestimate the bandgap values. However, the combination of Anderson charge mixing method with standard eigensolver when implemented using the smart algorithm was found to produce fairly close results. These optimized model parameters were then used to determine the band structures of various zigzag SWCNTs. (9, 0) Single-walled Nanotube which is extensively being used for sensing NH3, CH4 and NO2 has been picked up as a reference material since its experimental bandgap value has been reported in the literature. It has been found to exhibit a finite energy bandgap in contrast to its expected metallic nature. The study is of utmost significance as it not only probes and validates the simulation route for predicting suitable properties of nanomaterials but also throws light on the comparative efficacy of the different approximation and rationalization quantum mechanical techniques used in simulation studies. Such simulation studies if used intelligently prove to be immensely useful to the material scientists as they not only save time and effort but also pave the way to new experiments by making valuable predictions.

Ultrafast Excited-state Deactivation of Flavins Bound to Dodecin*

PubMed Central

Staudt, Heike; Oesterhelt, Dieter; Grininger, Martin; Wachtveitl, Josef

2012-01-01

Dodecins, a group of flavin-binding proteins with a dodecameric quaternary structure, are able to incorporate two flavins within each of their six identical binding pockets building an aromatic tetrade with two tryptophan residues. Dodecin from the archaeal Halobacterium salinarum is a riboflavin storage device. We demonstrate that unwanted side reactions induced by reactive riboflavin species and degradation of riboflavin are avoided by ultrafast depopulation of the reactive excited state of riboflavin. Intriguingly, in this process, the staggered riboflavin dimers do not interact in ground and photoexcited states. Rather, within the tetrade assembly, each riboflavin is kept under the control of the respective adjacent tryptophan, which suggests that the stacked arrangement is a matter of optimizing the flavin load. We further identify an electron transfer in combination with a proton transfer as a central element of the effective excited state depopulation mechanism. Structural and functional comparisons of the archaeal dodecin with bacterial homologs reveal diverging evolution. Bacterial dodecins bind the flavin FMN instead of riboflavin and exhibit a clearly different binding pocket design with inverse incorporations of flavin dimers. The different adoption of flavin changes photochemical properties, making bacterial dodecin a comparably less efficient quencher of flavins. This supports a functional role different for bacterial and archaeal dodecins. PMID:22451648

Structural and electronic properties of carbon nanotube-reinforced epoxy resins.

PubMed

Suggs, Kelvin; Wang, Xiao-Qian

2010-03-01

Nanocomposites of cured epoxy resin reinforced by single-walled carbon nanotubes exhibit a plethora of interesting behaviors at the molecular level. We have employed a combination of force-field-based molecular mechanics and first-principles calculations to study the corresponding binding and charge-transfer behavior. The simulation study of various nanotube species and curing agent configurations provides insight into the optimal structures in lieu of interfacial stability. An analysis of charge distributions of the epoxy functionalized semiconducting and metallic tubes reveals distinct level hybridizations. The implications of these results for understanding dispersion mechanism and future nano reinforced composite developments are discussed.

Modified pavement cement concrete

NASA Astrophysics Data System (ADS)

Botsman, L. N.; Ageeva, M. S.; Botsman, A. N.; Shapovalov, S. M.

2018-03-01

The paper suggests design principles of pavement cement concrete, which covers optimization of compositions and structures at the stage of mixture components selection due to the use of plasticizing agents and air-retaining substances that increase the viability of a concrete mixture. It also demonstrates advisability of using plasticizing agents together with air-retaining substances when developing pavement concrete compositions, which provides for the improvement of physical and mechanical properties of concrete and the reduction of cement binding agent consumption thus preserving strength indicators. The paper shows dependences of the main physical-mechanical parameters of concrete on cement consumption, a type and amount of additives.

Identification of potent, selective, CNS-targeted inverse agonists of the ghrelin receptor.

PubMed

McClure, Kim F; Jackson, Margaret; Cameron, Kimberly O; Kung, Daniel W; Perry, David A; Orr, Suvi T M; Zhang, Yingxin; Kohrt, Jeffrey; Tu, Meihua; Gao, Hua; Fernando, Dilinie; Jones, Ryan; Erasga, Noe; Wang, Guoqiang; Polivkova, Jana; Jiao, Wenhua; Swartz, Roger; Ueno, Hirokazu; Bhattacharya, Samit K; Stock, Ingrid A; Varma, Sam; Bagdasarian, Victoria; Perez, Sylvie; Kelly-Sullivan, Dawn; Wang, Ruduan; Kong, Jimmy; Cornelius, Peter; Michael, Laura; Lee, Eunsun; Janssen, Ann; Steyn, Stefanus J; Lapham, Kimberly; Goosen, Theunis

2013-10-01

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine-piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties. Copyright © 2013 Elsevier Ltd. All rights reserved.

Rapid comparison of protein binding site surfaces with Property Encoded Shape Distributions (PESD)

PubMed Central

Das, Sourav; Kokardekar, Arshad

2009-01-01

Patterns in shape and property distributions on the surface of binding sites are often conserved across functional proteins without significant conservation of the underlying amino-acid residues. To explore similarities of these sites from the viewpoint of a ligand, a sequence and fold-independent method was created to rapidly and accurately compare binding sites of proteins represented by property-mapped triangulated Gauss-Connolly surfaces. Within this paradigm, signatures for each binding site surface are produced by calculating their property-encoded shape distributions (PESD), a measure of the probability that a particular property will be at a specific distance to another on the molecular surface. Similarity between the signatures can then be treated as a measure of similarity between binding sites. As postulated, the PESD method rapidly detected high levels of similarity in binding site surface characteristics even in cases where there was very low similarity at the sequence level. In a screening experiment involving each member of the PDBBind 2005 dataset as a query against the rest of the set, PESD was able to retrieve a binding site with identical E.C. (Enzyme Commission) numbers as the top match in 79.5% of cases. The ability of the method in detecting similarity in binding sites with low sequence conservations were compared with state-of-the-art binding site comparison methods. PMID:19919089

A "Reverse-Schur" Approach to Optimization With Linear PDE Constraints: Application to Biomolecule Analysis and Design.

PubMed

Bardhan, Jaydeep P; Altman, Michael D; Tidor, B; White, Jacob K

2009-01-01

We present a partial-differential-equation (PDE)-constrained approach for optimizing a molecule's electrostatic interactions with a target molecule. The approach, which we call reverse-Schur co-optimization, can be more than two orders of magnitude faster than the traditional approach to electrostatic optimization. The efficiency of the co-optimization approach may enhance the value of electrostatic optimization for ligand-design efforts-in such projects, it is often desirable to screen many candidate ligands for their viability, and the optimization of electrostatic interactions can improve ligand binding affinity and specificity. The theoretical basis for electrostatic optimization derives from linear-response theory, most commonly continuum models, and simple assumptions about molecular binding processes. Although the theory has been used successfully to study a wide variety of molecular binding events, its implications have not yet been fully explored, in part due to the computational expense associated with the optimization. The co-optimization algorithm achieves improved performance by solving the optimization and electrostatic simulation problems simultaneously, and is applicable to both unconstrained and constrained optimization problems. Reverse-Schur co-optimization resembles other well-known techniques for solving optimization problems with PDE constraints. Model problems as well as realistic examples validate the reverse-Schur method, and demonstrate that our technique and alternative PDE-constrained methods scale very favorably compared to the standard approach. Regularization, which ordinarily requires an explicit representation of the objective function, can be included using an approximate Hessian calculated using the new BIBEE/P (boundary-integral-based electrostatics estimation by preconditioning) method.

A “Reverse-Schur” Approach to Optimization With Linear PDE Constraints: Application to Biomolecule Analysis and Design

PubMed Central

Bardhan, Jaydeep P.; Altman, Michael D.

2009-01-01

We present a partial-differential-equation (PDE)-constrained approach for optimizing a molecule’s electrostatic interactions with a target molecule. The approach, which we call reverse-Schur co-optimization, can be more than two orders of magnitude faster than the traditional approach to electrostatic optimization. The efficiency of the co-optimization approach may enhance the value of electrostatic optimization for ligand-design efforts–in such projects, it is often desirable to screen many candidate ligands for their viability, and the optimization of electrostatic interactions can improve ligand binding affinity and specificity. The theoretical basis for electrostatic optimization derives from linear-response theory, most commonly continuum models, and simple assumptions about molecular binding processes. Although the theory has been used successfully to study a wide variety of molecular binding events, its implications have not yet been fully explored, in part due to the computational expense associated with the optimization. The co-optimization algorithm achieves improved performance by solving the optimization and electrostatic simulation problems simultaneously, and is applicable to both unconstrained and constrained optimization problems. Reverse-Schur co-optimization resembles other well-known techniques for solving optimization problems with PDE constraints. Model problems as well as realistic examples validate the reverse-Schur method, and demonstrate that our technique and alternative PDE-constrained methods scale very favorably compared to the standard approach. Regularization, which ordinarily requires an explicit representation of the objective function, can be included using an approximate Hessian calculated using the new BIBEE/P (boundary-integral-based electrostatics estimation by preconditioning) method. PMID:23055839

Characterization of the swine adipocyte A1 adenosine receptor using an optimized assay system.

PubMed

Dong, Q; Schuchman, J; Carey, G B

1994-07-01

The radioligand binding assay of A1 adenosine receptors in adipocyte crude plasma membrane from Yucatan miniature swine was optimized by evaluating 17 factors involved in the assay. Significant effects of CHAPS, adenosine deaminase, EDTA, pre-rinsing glass fiber filters and pH were found for the binding measurements. Using the optimized procedure, [3H]8-cyclopentyl-1,3-dipropylxanthine, ([3H]-DPCPX) binding to A1 adenosine receptors in swine subcutaneous adipocyte crude plasma membrane was measured; Bmax and Kd values were 479 +/- 77 fmol/mg protein and 0.87 +/- 0.10 nM, respectively. Values for mesenteric adipose tissue from sedentary swine and subcutaneous adipose tissue from exercise-trained swine were also measured.

Computational design of a pH-sensitive IgG binding protein.

PubMed

Strauch, Eva-Maria; Fleishman, Sarel J; Baker, David

2014-01-14

Computational design provides the opportunity to program protein-protein interactions for desired applications. We used de novo protein interface design to generate a pH-dependent Fc domain binding protein that buries immunoglobulin G (IgG) His-433. Using next-generation sequencing of naïve and selected pools of a library of design variants, we generated a molecular footprint of the designed binding surface, confirming the binding mode and guiding further optimization of the balance between affinity and pH sensitivity. In biolayer interferometry experiments, the optimized design binds IgG with a Kd of ∼ 4 nM at pH 8.2, and approximately 500-fold more weakly at pH 5.5. The protein is extremely stable, heat-resistant and highly expressed in bacteria, and allows pH-based control of binding for IgG affinity purification and diagnostic devices.

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi.

PubMed

Parker, Greg S; Maity, Tuhin Subhra; Bass, Brenda L

2008-12-26

Double-stranded RNA (dsRNA)-binding proteins facilitate Dicer functions in RNA interference. Caenorhabditis elegans RDE-4 facilitates cleavage of long dsRNA to small interfering RNA (siRNA), while human trans-activation response RNA-binding protein (TRBP) functions downstream to pass siRNA to the RNA-induced silencing complex. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based on the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds noncooperatively. Our studies offer a paradigm for how dsRNA-binding proteins, which are not sequence specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRNA-binding motif 2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation.

Comparative Analysis of Binding Kinetics and Thermodynamics of Dipeptidyl Peptidase-4 Inhibitors and Their Relationship to Structure.

PubMed

Schnapp, Gisela; Klein, Thomas; Hoevels, Yvette; Bakker, Remko A; Nar, Herbert

2016-08-25

The binding kinetics and thermodynamics of dipeptidyl peptidase (DPP)-4 inhibitors (gliptins) were investigated using surface plasmon resonance and isothermal titration calorimetry. Binding of gliptins to DPP-4 is a rapid electrostatically driven process. Off-rates were generally slow partly because of reversible covalent bond formation by some gliptins, and partly because of strong and extensive interactions. Binding of all gliptins is enthalpy-dominated due to strong ionic interactions and strong solvent-shielded hydrogen bonds. Using a congeneric series of molecules which represented the intermediates in the lead optimization program of linagliptin, the onset of slow binding kinetics and development of the thermodynamic repertoire were analyzed in the context of incremental changes of the chemical structures. All compounds rapidly associated, and therefore the optimization of affinity and residence time is highly correlated. The major contributor to the increasing free energy of binding was a strong increase of binding enthalpy, whereas entropic contributions remained low and constant despite significant addition of lipophilicity.

Microfluidic system for facilitated quantification of nanoparticle accumulation to cells under laminar flow

PubMed Central

Kusunose, Jiro; Zhang, Hua; Gagnon, M. Karen J.; Pan, Tingrui; Simon, Scott I.; Ferrara, Katherine W.

2012-01-01

The identification of novel, synthetic targeting ligands to endothelial receptors has led to the rapid development of targeted nanoparticles for drug, gene and imaging probe delivery. Central to development and optimization are effective models for assessing particle binding in vitro. Here, we developed a simple and cost effective method to quantitatively assess nanoparticle accumulation under physiologically-relevant laminar flow. We designed reversibly vacuum–sealed PDMS microfluidic chambers compatible with 35 mm petri dishes, which deliver uniform or gradient shear stress. These chambers have sufficient surface area for facile cell collection for particle accumulation quantitation through FACS. We tested this model by synthesizing and flowing liposomes coated with APN (KD ~ 300 µM) and VCAM-1-targeting (KD ~ 30 µM) peptides over HUVEC. Particle binding significantly increased with ligand concentration (up to 6 mol%) and decreased with excess PEG. While the accumulation of particles with the lower affinity ligand decreased with shear, accumulation of those with the higher affinity ligand was highest in a low shear environment (2.4 dyne/cm2), as compared with greater shear or the absence of shear. We describe here a robust flow chamber model that is applied to optimize the properties of 100 nm liposomes targeted to inflamed endothelium. PMID:22855121

Accurate Binding Free Energy Predictions in Fragment Optimization.

PubMed

Steinbrecher, Thomas B; Dahlgren, Markus; Cappel, Daniel; Lin, Teng; Wang, Lingle; Krilov, Goran; Abel, Robert; Friesner, Richard; Sherman, Woody

2015-11-23

Predicting protein-ligand binding free energies is a central aim of computational structure-based drug design (SBDD)--improved accuracy in binding free energy predictions could significantly reduce costs and accelerate project timelines in lead discovery and optimization. The recent development and validation of advanced free energy calculation methods represents a major step toward this goal. Accurately predicting the relative binding free energy changes of modifications to ligands is especially valuable in the field of fragment-based drug design, since fragment screens tend to deliver initial hits of low binding affinity that require multiple rounds of synthesis to gain the requisite potency for a project. In this study, we show that a free energy perturbation protocol, FEP+, which was previously validated on drug-like lead compounds, is suitable for the calculation of relative binding strengths of fragment-sized compounds as well. We study several pharmaceutically relevant targets with a total of more than 90 fragments and find that the FEP+ methodology, which uses explicit solvent molecular dynamics and physics-based scoring with no parameters adjusted, can accurately predict relative fragment binding affinities. The calculations afford R(2)-values on average greater than 0.5 compared to experimental data and RMS errors of ca. 1.1 kcal/mol overall, demonstrating significant improvements over the docking and MM-GBSA methods tested in this work and indicating that FEP+ has the requisite predictive power to impact fragment-based affinity optimization projects.

Data quality in drug discovery: the role of analytical performance in ligand binding assays

NASA Astrophysics Data System (ADS)

Wätzig, Hermann; Oltmann-Norden, Imke; Steinicke, Franziska; Alhazmi, Hassan A.; Nachbar, Markus; El-Hady, Deia Abd; Albishri, Hassan M.; Baumann, Knut; Exner, Thomas; Böckler, Frank M.; El Deeb, Sami

2015-09-01

Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

Computational design of nanoparticle drug delivery systems for selective targeting

NASA Astrophysics Data System (ADS)

Duncan, Gregg A.; Bevan, Michael A.

2015-09-01

Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues.Ligand-functionalized nanoparticles capable of selectively binding to diseased versus healthy cell populations are attractive for improved efficacy of nanoparticle-based drug and gene therapies. However, nanoparticles functionalized with high affinity targeting ligands may lead to undesired off-target binding to healthy cells. In this work, Monte Carlo simulations were used to quantitatively determine net surface interactions, binding valency, and selectivity between targeted nanoparticles and cell surfaces. Dissociation constant, KD, and target membrane protein density, ρR, are explored over a range representative of healthy and cancerous cell surfaces. Our findings show highly selective binding to diseased cell surfaces can be achieved with multiple, weaker affinity targeting ligands that can be further optimized by varying the targeting ligand density, ρL. Using the approach developed in this work, nanomedicines can be optimally designed for exclusively targeting diseased cells and tissues. Electronic supplementary information (ESI) available: Movie showing simulation renderings of targeted (ρL = 1820/μm2, KD = 120 μM) nanoparticle selective binding to cancer (ρR = 256/μm2) vs. healthy (ρR = 64/μm2) cell surfaces. Target membrane proteins have linear color scale depending on binding energy ranging from white when unbound (URL = 0) to red when tightly bound (URL = UM). See DOI: 10.1039/c5nr03691g

Characterization of complexes between phenethylamine enantiomers and β-cyclodextrin derivatives by capillary electrophoresis-Determination of binding constants and complex mobilities.

PubMed

Wahl, Joachim; Furuishi, Takayuki; Yonemochi, Etsuo; Meinel, Lorenz; Holzgrabe, Ulrike

2017-04-01

To optimize chiral separation conditions and to improve the knowledge of enantioseparation, it is important to know the binding constants K between analytes and cyclodextrins and the electrophoretic mobilities of the temporarily formed analyte-cyclodextrin-complexes. K values for complexes between eight phenethylamine enantiomers, namely ephedrine, pseudoephedrine, methylephedrine and norephedrine, and four different β-cyclodextrin derivatives were determined by affinity capillary electrophoresis. The binding constants were calculated from the electrophoretic mobility values of the phenethylamine enantiomers at increasing concentrations of cyclodextrins in running buffer. Three different linear plotting methods (x-reciprocal, y-reciprocal, double reciprocal) and nonlinear regression were used for the determination of binding constants with β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin, methyl-β-cyclodextrin and 6-O-α-maltosyl-β-cyclodextrin. The cyclodextrin concentration in a 50 mM phosphate buffer pH 3.0 was varied from 0 to 12 mM. To investigate the influence of the binding constant values on the enantioseparation the observed electrophoretic selectivities were compared with the obtained K values and the calculated enantiomer-cyclodextrin-complex mobilities. The different electrophoretic mobilities of the temporarily formed complexes were crucial factors for the migration order and enantioseparation of ephedrine derivatives. To verify the apparent binding constants determined by capillary electrophoresis, a titration process using ephedrine enantiomers and β-cyclodextrin was carried out. Furthermore, the isothermal titration calorimetry measurements gave information about the thermal properties of the complexes. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A method for predicting individual residue contributions to enzyme specificity and binding-site energies, and its application to MTH1.

PubMed

Stewart, James J P

2016-11-01

A new method for predicting the energy contributions to substrate binding and to specificity has been developed. Conventional global optimization methods do not permit the subtle effects responsible for these properties to be modeled with sufficient precision to allow confidence to be placed in the results, but by making simple alterations to the model, the precisions of the various energies involved can be improved from about ±2 kcal mol -1 to ±0.1 kcal mol -1 . This technique was applied to the oxidized nucleotide pyrophosphohydrolase enzyme MTH1. MTH1 is unusual in that the binding and reaction sites are well separated-an advantage from a computational chemistry perspective, as it allows the energetics involved in docking to be modeled without the need to consider any issues relating to reaction mechanisms. In this study, two types of energy terms were investigated: the noncovalent interactions between the binding site and the substrate, and those responsible for discriminating between the oxidized nucleotide 8-oxo-dGTP and the normal dGTP. Both of these were investigated using the semiempirical method PM7 in the program MOPAC. The contributions of the individual residues to both the binding energy and the specificity of MTH1 were calculated by simulating the effect of mutations. Where comparisons were possible, all calculated results were in agreement with experimental observations. This technique provides fresh insight into the binding mechanism that enzymes use for discriminating between possible substrates.

Targeted Radiotherapy of Estrogen Receptor Positive Tumors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raghavan Rajagopalan

The overall objectives of the proposal were to develop estrogen receptor (ER) binding small molecule radiopharmaceuticals for targeted radiotherapy of ER positive (ER+) tumors. In particular, this proposal focused on embedding a {sup 186,188}Re or a {sup 32}P radionuclide into an estrogen steroidal framework by isosteric substitution such that the resulting structure is topologically similar to the estrogen (estrogen mimic). The estrogen mimic molecules expected to bind to the ER and exhibit biodistribution akin to that of native estrogen due to structural mimicry. It is anticipated that the {sup 186,188}Re- or a {sup 32}P-containing estrogen mimics will be useful formore » targeted molecular radiotherapy of ER+ tumors. It is well established that the in vivo target tissue uptake of estrogen like steroidal molecules is related to the binding of the steroids to sex hormone binding globulin (SHBG). SHBG is important in the uptake of estrogens and testosterone in target tissues by SHBG receptors on the cell surface. However, hitherto the design of estrogen like small molecule radiopharmaceuticals was focused on optimizing ER binding characteristics without emphasis on SHBG binding properties. Consequently, even the molecules with good ER affinity in vitro, performed poorly in biodistribution studies. Based on molecular modeling studies the proposal focused on developing estrogen mimics 1-3 which were topologically similar to native estrogens, and form hydrogen bonds in ER and SHBG in the same manner as those of native estrogens. To this end the technical objectives of the proposal focused on synthesizing the rhenium-estrone and estradiol mimics 1 and 2 respectively, and phosphorous estradiol mimic 3 and to assess their stability and in vitro binding characteristics to ER and SHBG.« less

Metal cofactor modulated folding and target recognition of HIV-1 NCp7.

PubMed

Ren, Weitong; Ji, Dongqing; Xu, Xiulian

2018-01-01

The HIV-1 nucleocapsid 7 (NCp7) plays crucial roles in multiple stages of HIV-1 life cycle, and its biological functions rely on the binding of zinc ions. Understanding the molecular mechanism of how the zinc ions modulate the conformational dynamics and functions of the NCp7 is essential for the drug development and HIV-1 treatment. In this work, using a structure-based coarse-grained model, we studied the effects of zinc cofactors on the folding and target RNA(SL3) recognition of the NCp7 by molecular dynamics simulations. After reproducing some key properties of the zinc binding and folding of the NCp7 observed in previous experiments, our simulations revealed several interesting features in the metal ion modulated folding and target recognition. Firstly, we showed that the zinc binding makes the folding transition states of the two zinc fingers less structured, which is in line with the Hammond effect observed typically in mutation, temperature or denaturant induced perturbations to protein structure and stability. Secondly, We showed that there exists mutual interplay between the zinc ion binding and NCp7-target recognition. Binding of zinc ions enhances the affinity between the NCp7 and the target RNA, whereas the formation of the NCp7-RNA complex reshapes the intrinsic energy landscape of the NCp7 and increases the stability and zinc affinity of the two zinc fingers. Thirdly, by characterizing the effects of salt concentrations on the target RNA recognition, we showed that the NCp7 achieves optimal balance between the affinity and binding kinetics near the physiologically relevant salt concentrations. In addition, the effects of zinc binding on the inter-domain conformational flexibility and folding cooperativity of the NCp7 were also discussed.

Semirational Approach for Ultrahigh Poly(3-hydroxybutyrate) Accumulation in Escherichia coli by Combining One-Step Library Construction and High-Throughput Screening.

PubMed

Li, Teng; Ye, Jianwen; Shen, Rui; Zong, Yeqing; Zhao, Xuejin; Lou, Chunbo; Chen, Guo-Qiang

2016-11-18

As a product of a multistep enzymatic reaction, accumulation of poly(3-hydroxybutyrate) (PHB) in Escherichia coli (E. coli) can be achieved by overexpression of the PHB synthesis pathway from a native producer involving three genes phbC, phbA, and phbB. Pathway optimization by adjusting expression levels of the three genes can influence properties of the final product. Here, we reported a semirational approach for highly efficient PHB pathway optimization in E. coli based on a phbCAB operon cloned from the native producer Ralstonia entropha (R. entropha). Rationally designed ribosomal binding site (RBS) libraries with defined strengths for each of the three genes were constructed based on high or low copy number plasmids in a one-pot reaction by an oligo-linker mediated assembly (OLMA) method. Strains with desired properties were evaluated and selected by three different methodologies, including visual selection, high-throughput screening, and detailed in-depth analysis. Applying this approach, strains accumulating 0%-92% PHB contents in cell dry weight (CDW) were achieved. PHB with various weight-average molecular weights (M w ) of 2.7-6.8 × 10 6 were also efficiently produced in relatively high contents. These results suggest that the semirational approach combining library design, construction, and proper screening is an efficient way to optimize PHB and other multienzyme pathways.

Probing the General Time Scale Question of Boronic Acid Binding with Sugars in Aqueous Solution at Physiological pH

PubMed Central

Ni, Nanting; Laughlin, Sarah; Wang, Yingji; Feng, You; Zheng, Yujun

2012-01-01

The boronic acid group is widely used in chemosensor design due to its ability to reversibly bind diol-containing compounds. The thermodynamic properties of the boronic acid-diol binding process have been investigated extensively. However, there are few studies of the kinetic properties of such binding processes. In this report, stopped-flow method was used for the first time to study the kinetic properties of the binding between three model arylboronic acids, 4-, 5-, and 8-isoquinolinylboronic acids, and various sugars. With all the boronic acid-diol pair sexamined, reactions were complete within seconds. The kon values with various sugars follow the order of D-fructose >D-tagatose>D-mannose >D-glucose. This trend tracks the thermodynamic binding affinities for these sugars and demonstrates that the “on” rate is the key factor determining the binding constant. PMID:22464680

A first-principles study on adsorption behaviors of pristine and Li-decorated graphene sheets toward hydrazine molecules

NASA Astrophysics Data System (ADS)

Zeng, Huadong; Cheng, Xinlu; Wang, Wei

2018-03-01

The adsorption behaviors and properties of hydrazine (N2H4) molecules on pristine and Li-decorated graphene sheets were investigated by means of first-principles based on density functional theory. We systematically analyzed the optimal geometry, average binding energy, charge transfer, charge density difference and density of states of N2H4 molecules adsorbed on pristine and Li-decorated graphene sheets. It is found that the interaction between single N2H4 molecule and pristine graphene is weak physisorption with the low binding energy of -0.026 eV, suggesting that the pristine graphene sheet is insensitive to the presence of N2H4 molecule. However, it is markedly enhanced after lithium decoration with the high binding energy of -1.004 eV, verifying that the Li-decorated graphene sheet is significantly sensitive to detect N2H4 molecule. Meanwhile, the effects of the concentrations of N2H4 molecules on two different substrates were studied detailedly. For pristine graphene substrate, the average binding energy augments apparently with increasing the number of N2H4 molecules, which is mainly attributed to the van der Waals interactions and hydrogen bonds among N2H4 clusters. Li-decorated graphene sheet has still a strong affinity to N2H4 molecules despite the corresponding average binding energy emerges a contrary tendency. Overall, Li-decorated graphene sheet could be considered as a potential gas sensor in field of hydrazine molecules.

Superbinder SH2 domains act as antagonists of cell signaling.

PubMed

Kaneko, Tomonori; Huang, Haiming; Cao, Xuan; Li, Xing; Li, Chengjun; Voss, Courtney; Sidhu, Sachdev S; Li, Shawn S C

2012-09-25

Protein-ligand interactions mediated by modular domains, which often play important roles in regulating cellular functions, are generally of moderate affinities. We examined the Src homology 2 (SH2) domain, a modular domain that recognizes phosphorylated tyrosine (pTyr) residues, to investigate how the binding affinity of a modular domain for its ligand influences the structure and cellular function of the protein. We used the phage display method to perform directed evolution of the pTyr-binding residues in the SH2 domain of the tyrosine kinase Fyn and identified three amino acid substitutions that critically affected binding. We generated three SH2 domain triple-point mutants that were "superbinders" with much higher affinities for pTyr-containing peptides than the natural domain. Crystallographic analysis of one of these superbinders revealed that the superbinder SH2 domain recognized the pTyr moiety in a bipartite binding mode: A hydrophobic surface encompassed the phenyl ring, and a positively charged site engaged the phosphate. When expressed in mammalian cells, the superbinder SH2 domains blocked epidermal growth factor receptor signaling and inhibited anchorage-independent cell proliferation, suggesting that pTyr superbinders might be explored for therapeutic applications and useful as biological research tools. Although the SH2 domain fold can support much higher affinity for its ligand than is observed in nature, our results suggest that natural SH2 domains are not optimized for ligand binding but for specificity and flexibility, which are likely properties important for their function in signaling and regulatory processes.

The vibrational spectroscopic studies and molecular property analysis of Estradiol, Tamoxifen and their interaction by density functional theory

NASA Astrophysics Data System (ADS)

Borah, Mukunda Madhab; Gomti Devi, Th.

2018-07-01

In the present work Tamoxifen, Estradiol and their interaction are studied using the experimental and theoretical methodologies. The spectral characterization was made by using Raman, FTIR, DFT and VEDA calculation. The optimization of the molecules have been studied using basis set B3LYP/6-31 G(d,p). Complete vibrational assignment of Tamoxifen, Estradiol and Estradiol + Tamoxifen have been attempted and the potential energy distribution and normal mode analysis had also been carried out to determine the contributions of bond oscillators in each normal mode. We have optimized several binding modes of Estradiol and Tamoxifen and taken the lowest energy conformer in our interest. The molecular geometry, HOMO-LUMO energy gap, molecular hardness (η), ionization energy (IE), electron affinity (EA), total energy and dipole moment were analyzed. The observed experimental and the scaled theoretical results were found in good agreement.

Identification and in Vivo Evaluation of Liver X Receptor β-Selective Agonists for the Potential Treatment of Alzheimer’s Disease

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stachel, Shawn J.; Zerbinatti, Celina; Rudd, Michael T.

2016-04-14

Herein, we describe the development of a functionally selective liver X receptor β (LXRβ) agonist series optimized for Emax selectivity, solubility, and physical properties to allow efficacy and safety studies in vivo. Compound 9 showed central pharmacodynamic effects in rodent models, evidenced by statistically significant increases in apolipoprotein E (apoE) and ATP-binding cassette transporter levels in the brain, along with a greatly improved peripheral lipid safety profile when compared to those of full dual agonists. These findings were replicated by subchronic dosing studies in non-human primates, where cerebrospinal fluid levels of apoE and amyloid-β peptides were increased concomitantly with anmore » improved peripheral lipid profile relative to that of nonselective compounds. These results suggest that optimization of LXR agonists for Emax selectivity may have the potential to circumvent the adverse lipid-related effects of hepatic LXR activity.« less

Optimization of a binding fragment targeting the "enlarged methionine pocket" leads to potent Trypanosoma brucei methionyl-tRNA synthetase inhibitors.

PubMed

Huang, Wenlin; Zhang, Zhongsheng; Ranade, Ranae M; Gillespie, J Robert; Barros-Álvarez, Ximena; Creason, Sharon A; Shibata, Sayaka; Verlinde, Christophe L M J; Hol, Wim G J; Buckner, Frederick S; Fan, Erkang

2017-06-15

Potent inhibitors of Trypanosoma brucei methionyl-tRNA synthetase were previously designed using a structure-guided approach. Compounds 1 and 2 were the most active compounds in the cyclic and linear linker series, respectively. To further improve cellular potency, SAR investigation of a binding fragment targeting the "enlarged methionine pocket" (EMP) was performed. The optimization led to the identification of a 6,8-dichloro-tetrahydroquinoline ring as a favorable fragment to bind the EMP. Replacement of 3,5-dichloro-benzyl group (the EMP binding fragment) of inhibitor 2 using this tetrahydroquinoline fragment resulted in compound 13, that exhibited an EC 50 of 4nM. Copyright © 2017 Elsevier Ltd. All rights reserved.

Efficient computation of optimal oligo-RNA binding.

PubMed

Hodas, Nathan O; Aalberts, Daniel P

2004-01-01

We present an algorithm that calculates the optimal binding conformation and free energy of two RNA molecules, one or both oligomeric. This algorithm has applications to modeling DNA microarrays, RNA splice-site recognitions and other antisense problems. Although other recent algorithms perform the same calculation in time proportional to the sum of the lengths cubed, O((N1 + N2)3), our oligomer binding algorithm, called bindigo, scales as the product of the sequence lengths, O(N1*N2). The algorithm performs well in practice with the aid of a heuristic for large asymmetric loops. To demonstrate its speed and utility, we use bindigo to investigate the binding proclivities of U1 snRNA to mRNA donor splice sites.

dsRNA binding properties of RDE-4 and TRBP reflect their distinct roles in RNAi

PubMed Central

Parker, Greg S.; Maity, Tuhin Subhra; Bass, Brenda L.

2008-01-01

SUMMARY dsRNA binding proteins (dsRBPs) facilitate Dicer functions in RNAi. C. elegans RDE-4 facilitates cleavage of long dsRNA to siRNA, while human TRBP functions downstream to pass siRNA to RISC. We show that these distinct in vivo roles are reflected in in vitro binding properties. RDE-4 preferentially binds long dsRNA, while TRBP binds siRNA with an affinity that is independent of dsRNA length. These properties are mechanistically based in the fact that RDE-4 binds cooperatively, via contributions from multiple domains, while TRBP binds non-cooperatively. Our studies offer a paradigm for how dsRBPs, which are not sequence-specific, discern dsRNA length. Additionally, analyses of the ability of RDE-4 deletion constructs and RDE-4/TRBP chimeras to reconstitute Dicer activity suggest RDE-4 promotes activity using its dsRBM2 to bind dsRNA, its linker region to interact with Dicer, and its C-terminus for Dicer activation. PMID:18948111

Postprocessing of docked protein-ligand complexes using implicit solvation models.

PubMed

Lindström, Anton; Edvinsson, Lotta; Johansson, Andreas; Andersson, C David; Andersson, Ida E; Raubacher, Florian; Linusson, Anna

2011-02-28

Molecular docking plays an important role in drug discovery as a tool for the structure-based design of small organic ligands for macromolecules. Possible applications of docking are identification of the bioactive conformation of a protein-ligand complex and the ranking of different ligands with respect to their strength of binding to a particular target. We have investigated the effect of implicit water on the postprocessing of binding poses generated by molecular docking using MM-PB/GB-SA (molecular mechanics Poisson-Boltzmann and generalized Born surface area) methodology. The investigation was divided into three parts: geometry optimization, pose selection, and estimation of the relative binding energies of docked protein-ligand complexes. Appropriate geometry optimization afforded more accurate binding poses for 20% of the complexes investigated. The time required for this step was greatly reduced by minimizing the energy of the binding site using GB solvation models rather than minimizing the entire complex using the PB model. By optimizing the geometries of docking poses using the GB(HCT+SA) model then calculating their free energies of binding using the PB implicit solvent model, binding poses similar to those observed in crystal structures were obtained. Rescoring of these poses according to their calculated binding energies resulted in improved correlations with experimental binding data. These correlations could be further improved by applying the postprocessing to several of the most highly ranked poses rather than focusing exclusively on the top-scored pose. The postprocessing protocol was successfully applied to the analysis of a set of Factor Xa inhibitors and a set of glycopeptide ligands for the class II major histocompatibility complex (MHC) A(q) protein. These results indicate that the protocol for the postprocessing of docked protein-ligand complexes developed in this paper may be generally useful for structure-based design in drug discovery.

A universal label-free fluorescent aptasensor based on Ru complex and quantum dots for adenosine, dopamine and 17β-estradiol detection.

PubMed

Huang, Hailiang; Shi, Shuo; Gao, Xing; Gao, Ruru; Zhu, Ying; Wu, Xuewen; Zang, Ruimin; Yao, Tianming

2016-05-15

Based on specific aptamer binding properties, a strategy for adenosine, dopamine and 17β-estradiol detection was realised by employing Ru complex and quantum dots (QDs) as fluorescence probes. Ru complex, which could quench the fluorescence of QDs, preferred to bind with aptamer DNA and resulted in the fluorescence rise of QDs. When the aptamer DNA was incubated with the target first, it could not bind with Ru complex and the fluorescence of QDs was quenched. Under the optimal condition, the fluorescence intensity was linearly proportional to the concentration of adenosine, dopamine and 17β-estradiol with a limit of detection (LOD) of 101 nM, 19 nM and 37 nM, respectively. The experiments in fetal bovine serum were also carried out with good results. This universal method was rapid, label-free, low-cost, easy-operating and highly repeatable for the detection of adenosine, dopamine and 17β-estradiol. Qualitative detection by naked eyes was also available without complex instruments. It could also be extended to detect various analytes, such as metal ions, proteins and small molecules by using appropriate aptamers. Copyright © 2015 Elsevier B.V. All rights reserved.

A new synthesis route for Os-complex modified redox polymers for potential biofuel cell applications.

PubMed

Pöller, Sascha; Beyl, Yvonne; Vivekananthan, Jeevanthi; Guschin, Dmitrii A; Schuhmann, Wolfgang

2012-10-01

A new synthesis route for Os-complex modified redox polymers was developed. Instead of ligand exchange reactions for coordinative binding of suitable precursor Os-complexes at the polymer, Os-complexes already exhibiting the final ligand shell containing a suitable functional group were bound to the polymer via an epoxide opening reaction. By separation of the polymer synthesis from the ligand exchange reaction at the Os-complex, the modification of the same polymer backbone with different Os-complexes or the binding of the same Os-complex to a number of different polymer backbones becomes feasible. In addition, the Os-complex can be purified and characterized prior to its binding to the polymer. In order to further understand and optimize suitable enzyme/redox polymer systems concerning their potential application in biosensors or biofuel cells, a series of redox polymers was synthesized and used as immobilization matrix for Trametes hirsuta laccase. The properties of the obtained biofuel cell cathodes were compared with similar biocatalytic interfaces derived from redox polymers obtained via ligand exchange reaction of the parent Os-complex with a ligand integrated into the polymer backbone during the polymer synthesis. Copyright © 2011 Elsevier B.V. All rights reserved.

A sequence-specific transcription activator motif and powerful synthetic variants that bind Mediator using a fuzzy protein interface.

PubMed

Warfield, Linda; Tuttle, Lisa M; Pacheco, Derek; Klevit, Rachel E; Hahn, Steven

2014-08-26

Although many transcription activators contact the same set of coactivator complexes, the mechanism and specificity of these interactions have been unclear. For example, do intrinsically disordered transcription activation domains (ADs) use sequence-specific motifs, or do ADs of seemingly different sequence have common properties that encode activation function? We find that the central activation domain (cAD) of the yeast activator Gcn4 functions through a short, conserved sequence-specific motif. Optimizing the residues surrounding this short motif by inserting additional hydrophobic residues creates very powerful ADs that bind the Mediator subunit Gal11/Med15 with high affinity via a "fuzzy" protein interface. In contrast to Gcn4, the activity of these synthetic ADs is not strongly dependent on any one residue of the AD, and this redundancy is similar to that of some natural ADs in which few if any sequence-specific residues have been identified. The additional hydrophobic residues in the synthetic ADs likely allow multiple faces of the AD helix to interact with the Gal11 activator-binding domain, effectively forming a fuzzier interface than that of the wild-type cAD.

dsRNA binding characterization of full length recombinant wild type and mutants Zaire ebolavirus VP35.

PubMed

Zinzula, Luca; Esposito, Francesca; Pala, Daniela; Tramontano, Enzo

2012-03-01

The Ebola viruses (EBOVs) VP35 protein is a multifunctional major virulence factor involved in EBOVs replication and evasion of the host immune system. EBOV VP35 is an essential component of the viral RNA polymerase, it is a key participant of the nucleocapsid assembly and it inhibits the innate immune response by antagonizing RIG-I like receptors through its dsRNA binding function and, hence, by suppressing the host type I interferon (IFN) production. Insights into the VP35 dsRNA recognition have been recently revealed by structural and functional analysis performed on its C-terminus protein. We report the biochemical characterization of the Zaire ebolavirus (ZEBOV) full-length recombinant VP35 (rVP35)-dsRNA binding function. We established a novel in vitro magnetic dsRNA binding pull down assay, determined the rVP35 optimal dsRNA binding parameters, measured the rVP35 equilibrium dissociation constant for heterologous in vitro transcribed dsRNA of different length and short synthetic dsRNA of 8bp, and validated the assay for compound screening by assessing the inhibitory ability of auryntricarboxylic acid (IC(50) value of 50μg/mL). Furthermore, we compared the dsRNA binding properties of full length wt rVP35 with those of R305A, K309A and R312A rVP35 mutants, which were previously reported to be defective in dsRNA binding-mediated IFN inhibition, showing that the latter have measurably increased K(d) values for dsRNA binding and modified migration patterns in mobility shift assays with respect to wt rVP35. Overall, these results provide the first characterization of the full-length wt and mutants VP35-dsRNA binding functions. Copyright © 2012 Elsevier B.V. All rights reserved.

QuickVina: accelerating AutoDock Vina using gradient-based heuristics for global optimization.

PubMed

Handoko, Stephanus Daniel; Ouyang, Xuchang; Su, Chinh Tran To; Kwoh, Chee Keong; Ong, Yew Soon

2012-01-01

Predicting binding between macromolecule and small molecule is a crucial phase in the field of rational drug design. AutoDock Vina, one of the most widely used docking software released in 2009, uses an empirical scoring function to evaluate the binding affinity between the molecules and employs the iterated local search global optimizer for global optimization, achieving a significantly improved speed and better accuracy of the binding mode prediction compared its predecessor, AutoDock 4. In this paper, we propose further improvement in the local search algorithm of Vina by heuristically preventing some intermediate points from undergoing local search. Our improved version of Vina-dubbed QVina-achieved a maximum acceleration of about 25 times with the average speed-up of 8.34 times compared to the original Vina when tested on a set of 231 protein-ligand complexes while maintaining the optimal scores mostly identical. Using our heuristics, larger number of different ligands can be quickly screened against a given receptor within the same time frame.

E-novo: an automated workflow for efficient structure-based lead optimization.

PubMed

Pearce, Bradley C; Langley, David R; Kang, Jia; Huang, Hongwei; Kulkarni, Amit

2009-07-01

An automated E-Novo protocol designed as a structure-based lead optimization tool was prepared through Pipeline Pilot with existing CHARMm components in Discovery Studio. A scaffold core having 3D binding coordinates of interest is generated from a ligand-bound protein structural model. Ligands of interest are generated from the scaffold using an R-group fragmentation/enumeration tool within E-Novo, with their cores aligned. The ligand side chains are conformationally sampled and are subjected to core-constrained protein docking, using a modified CHARMm-based CDOCKER method to generate top poses along with CDOCKER energies. In the final stage of E-Novo, a physics-based binding energy scoring function ranks the top ligand CDOCKER poses using a more accurate Molecular Mechanics-Generalized Born with Surface Area method. Correlation of the calculated ligand binding energies with experimental binding affinities were used to validate protocol performance. Inhibitors of Src tyrosine kinase, CDK2 kinase, beta-secretase, factor Xa, HIV protease, and thrombin were used to test the protocol using published ligand crystal structure data within reasonably defined binding sites. In-house Respiratory Syncytial Virus inhibitor data were used as a more challenging test set using a hand-built binding model. Least squares fits for all data sets suggested reasonable validation of the protocol within the context of observed ligand binding poses. The E-Novo protocol provides a convenient all-in-one structure-based design process for rapid assessment and scoring of lead optimization libraries.

Structural computational modeling of RNA aptamers.

PubMed

Xu, Xiaojun; Dickey, David D; Chen, Shi-Jie; Giangrande, Paloma H

2016-07-01

RNA aptamers represent an emerging class of biologics that can be easily adapted for personalized and precision medicine. Several therapeutic aptamers with desirable binding and functional properties have been developed and evaluated in preclinical studies over the past 25years. However, for the majority of these aptamers, their clinical potential has yet to be realized. A significant hurdle to the clinical adoption of this novel class of biologicals is the limited information on their secondary and tertiary structure. Knowledge of the RNA's structure would greatly facilitate and expedite the post-selection optimization steps required for translation, including truncation (to reduce costs of manufacturing), chemical modification (to enhance stability and improve safety) and chemical conjugation (to improve drug properties for combinatorial therapy). Here we describe a structural computational modeling methodology that when coupled to a standard functional assay, can be used to determine key sequence and structural motifs of an RNA aptamer. We applied this methodology to enable the truncation of an aptamer to prostate specific membrane antigen (PSMA) with great potential for targeted therapy that had failed previous truncation attempts. This methodology can be easily applied to optimize other aptamers with therapeutic potential. Copyright © 2016. Published by Elsevier Inc.

DHA supplementation: current implications in pregnancy and childhood.

PubMed

Rogers, Lynette K; Valentine, Christina J; Keim, Sarah A

2013-04-01

Dietary supplementation with ω-3 long chain fatty acids including docosahexaenoic acid (DHA) has increased in popularity in recent years and adequate DHA supplementation during pregnancy and early childhood is of clinical importance. Some evidence has been built for the neuro-cognitive benefits of supplementation with long chain polyunsaturated fatty acids (LCPUFA) such as DHA during pregnancy; however, recent data indicate that the anti-inflammatory properties may be of at least equal significance. Adequate DHA availability in the fetus/infant optimizes brain and retinal maturation in part by influencing neurotransmitter pathways. The anti-inflammatory properties of LCPUFA are largely mediated through modulation of signaling either directly through binding to receptors or through changes in lipid raft formation and receptor presentation. Our goal is to review the current findings on DHA supplementation, specifically in pregnancy and infant neurodevelopment, as a pharmacologic agent with both preventative and therapeutic value. Given the overall benefits of DHA, maternal and infant supplementation may improve neurological outcomes especially in vulernable populations. However, optimal composition of the supplement and dosing and treatment strategies still need to be determined to lend support for routine supplementation. Copyright © 2012 Elsevier Ltd. All rights reserved.

On the role of electrostatics in protein-protein interactions

NASA Astrophysics Data System (ADS)

Zhang, Zhe; Witham, Shawn; Alexov, Emil

2011-06-01

The role of electrostatics in protein-protein interactions and binding is reviewed in this paper. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and the basic electrostatic effects occurring upon the formation of the complex are discussed. The effect of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated which indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartments. The similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity.

On the role of electrostatics on protein-protein interactions

PubMed Central

Zhang, Zhe; Witham, Shawn; Alexov, Emil

2011-01-01

The role of electrostatics on protein-protein interactions and binding is reviewed in this article. A brief outline of the computational modeling, in the framework of continuum electrostatics, is presented and basic electrostatic effects occurring upon the formation of the complex are discussed. The role of the salt concentration and pH of the water phase on protein-protein binding free energy is demonstrated and indicates that the increase of the salt concentration tends to weaken the binding, an observation that is attributed to the optimization of the charge-charge interactions across the interface. It is pointed out that the pH-optimum (pH of optimal binding affinity) varies among the protein-protein complexes, and perhaps is a result of their adaptation to particular subcellular compartment. At the end, the similarities and differences between hetero- and homo-complexes are outlined and discussed with respect to the binding mode and charge complementarity. PMID:21572182

Capture and separation of l-histidine through optimized zinc-decorated magnetic silica spheres.

PubMed

Cardoso, Vanessa F; Sebastián, Víctor; Silva, Carlos J R; Botelho, Gabriela; Lanceros-Méndez, Senentxu

2017-09-01

Zinc-decorated magnetic silica spheres were developed, optimized and tested for the capture and separation of l-histidine. The magnetic silica spheres were prepared using a simple sol-gel method and show excellent magnetic characteristics, adsorption capacity toward metal ions, and stability in aqueous solution in a wide pH range. The binding capacity of zinc-decorated magnetic silica spheres to histidine proved to be strongly influenced by the morphology, composition and concentration of metal at the surface of the magnetic silica spheres and therefore these parameters should be carefully controlled in order to maximize the performance for protein purification purposes. Optimized zinc-decorated magnetic silica spheres demonstrate a binding capacity to l-histidine of approximately 44mgg -1 at the optimum binding pH buffer. Copyright © 2017 Elsevier B.V. All rights reserved.

Protein crystallography and site-direct mutagenesis analysis of the poly(ethylene terephthalate) hydrolase PETase from Ideonella sakaiensis.

PubMed

Liu, Bing; He, Lihui; Wang, Liping; Li, Tao; Li, Changcheng; Liu, Huayi; Luo, Yunzi; Bao, Rui

2018-03-30

Compared with traditional recycle strategies, biodegradation provides a sustainable solution for poly (ethylene terephthalate) (PET) wastes disposal. PETase, a newly identified enzyme from Ideonella sakaiensis, has high efficiency and specificity towards PET, which provides a prominent prospect on PET degradation. Based on the biochemical analysis, we propose that the wide substrate-binding pocket is critical for its excellent property on crystallized PET hydrolysis. Structure-guided site-directed mutagenesis exhibited improvement in PETase catalytic efficiency, providing valuable insight on how the molecular engineering of PETase can optimize its application in biocatalysis. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Using Atomic Force Microscopy to Characterize the Conformational Properties of Proteins and Protein-DNA Complexes That Carry Out DNA Repair.

PubMed

LeBlanc, Sharonda; Wilkins, Hunter; Li, Zimeng; Kaur, Parminder; Wang, Hong; Erie, Dorothy A

2017-01-01

Atomic force microscopy (AFM) is a scanning probe technique that allows visualization of single biomolecules and complexes deposited on a surface with nanometer resolution. AFM is a powerful tool for characterizing protein-protein and protein-DNA interactions. It can be used to capture snapshots of protein-DNA solution dynamics, which in turn, enables the characterization of the conformational properties of transient protein-protein and protein-DNA interactions. With AFM, it is possible to determine the stoichiometries and binding affinities of protein-protein and protein-DNA associations, the specificity of proteins binding to specific sites on DNA, and the conformations of the complexes. We describe methods to prepare and deposit samples, including surface treatments for optimal depositions, and how to quantitatively analyze images. We also discuss a new electrostatic force imaging technique called DREEM, which allows the visualization of the path of DNA within proteins in protein-DNA complexes. Collectively, these methods facilitate the development of comprehensive models of DNA repair and provide a broader understanding of all protein-protein and protein-nucleic acid interactions. The structural details gleaned from analysis of AFM images coupled with biochemistry provide vital information toward establishing the structure-function relationships that govern DNA repair processes. © 2017 Elsevier Inc. All rights reserved.

Study of iridium silicide monolayers using density functional theory

NASA Astrophysics Data System (ADS)

Popis, Minh D.; Popis, Sylvester V.; Oncel, Nuri; Hoffmann, Mark R.; ćakır, Deniz

2018-02-01

In this study, we investigated physical and electronic properties of possible two-dimensional structures formed by Si (silicon) and Ir (iridium). To this end, different plausible structures were modeled by using density functional theory and the cohesive energies calculated for the geometry of optimized structures, with the lowest equilibrium lattice constants. Among several candidate structures, we identified three mechanically (via elastic constants and Young's modulus), dynamically (via phonon calculations), and thermodynamically stable iridium silicide monolayer structures. The lowest energy structure has a chemical formula of Ir2Si4 (called r-IrSi2), with a rectangular lattice (Pmmn space group). Its cohesive energy was calculated to be -0.248 eV (per IrSi2 unit) with respect to bulk Ir and bulk Si. The band structure indicates that the Ir2Si4 monolayer exhibits metallic properties. Other stable structures have hexagonal (P-3m1) and tetragonal (P4/nmm) cell structures with 0.12 and 0.20 eV/f.u. higher cohesive energies, respectively. Our calculations showed that Ir-Si monolayers are reactive. Although O2 molecules exothermically dissociate on the surface of the free-standing iridium silicide monolayers with large binding energies, H2O molecules bind to the monolayers with a rather weak interaction.

Engineering and characterization of fluorogenic glycine riboswitches.

PubMed

Ketterer, Simon; Gladis, Lukas; Kozica, Adnan; Meier, Matthias

2016-07-08

A set of 12 fluorogenic glycine riboswitches with different thermodynamic and kinetic response properties was engineered. For the design of functional riboswitches, a three-part RNA approach was applied based on the idea of linking a RNA sensor, transmitter and actuator part together. For the RNA sensor and actuator part, we used the tandem glycine aptamer structure from Bacillus subtillis, and fluorogenic aptamer Spinach, respectively. To achieve optimal signal transduction from the sensor to the actuator, a riboswitch library with variable transmitter was screened with a microfluidic large-scale integration chip. This allowed us to establish the complete thermodynamic binding profiles of the riboswitch library. Glycine dissociation constants of the 12 strong fluorescence response riboswitches varied between 99.7 and 570 μM. Furthermore, the kinetic glycine binding (k(on)), and dissociation (k(off)) rates, and corresponding energy barriers of the 10 strongest fluorescence response riboswitches were determined with the same chip platform. k(on) and k(off) were in the order of 10(-3)s(-1) and 10(-2)s(-1), respectively. Conclusively, we demonstrate that systematic screening of synthetic and natural linked RNA parts with microfluidic chip technology is an effective approach to rapidly generate fluorogenic metabolite riboswitches with a broad range of biophysical response properties. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Nanofiber adsorbents for high productivity downstream processing.

PubMed

Hardick, Oliver; Dods, Stewart; Stevens, Bob; Bracewell, Daniel G

2013-04-01

Electrospun polymeric nanofiber adsorbents offer an alternative ligand support surface for bioseparations. Their non-woven fiber structure with diameters in the sub-micron range creates a remarkably high surface area. To improve the purification productivity of biological molecules by chromatography, cellulose nanofiber adsorbents were fabricated and assembled into a cartridge and filter holder format with a volume of 0.15 mL, a bed height of 0.3 mm and diameter of 25 mm. The present study investigated the performance of diethylaminoethyl (DEAE) derivatized regenerated cellulose nanofiber adsorbents based on criteria including mass transfer and flow properties, binding capacity, and fouling effects. Our results show that nanofibers offer higher flow and mass transfer properties. The non-optimized DEAE-nanofiber adsorbents indicate a binding capacity of 10% that of packed bed systems with BSA as a single component system. However, they operate reproducibly at flowrates of a hundred times that of packed beds, resulting in a potential productivity increase of 10-fold. Lifetime studies showed that this novel adsorbent material operated reproducibly with complex feed material (centrifuged and 0.45 µm filtered yeast homogenate) and harsh cleaning-in-place conditions over multiple cycles. DEAE nanofibers showed superior operating performance in permeability and fouling over conventional adsorbents indicating their potential for bioseparation applications. Copyright © 2012 Wiley Periodicals, Inc.

High-Throughput Method for Ranking the Affinity of Peptide Ligands Selected from Phage Display Libraries

PubMed Central

González-Techera, A.; Umpiérrez-Failache, M.; Cardozo, S.; Obal, G.; Pritsch, O.; Last, J. A.; Gee, S. J.; Hammock, B. D.; González-Sapienza, G.

2010-01-01

The use of phage display peptide libraries allows rapid isolation of peptide ligands for any target selector molecule. However, due to differences in peptide expression and the heterogeneity of the phage preparations, there is no easy way to compare the binding properties of the selected clones, which operates as a major “bottleneck” of the technology. Here, we present the development of a new type of library that allows rapid comparison of the relative affinity of the selected peptides in a high-throughput screening format. As a model system, a phage display peptide library constructed on a phagemid vector that contains the bacterial alkaline phosphatase gene (BAP) was selected with an antiherbicide antibody. Due to the intrinsic switching capacity of the library, the selected peptides were transferred “en masse” from the phage coat protein to BAP. This was coupled to an optimized affinity ELISA where normalized amounts of the peptide–BAP fusion allow direct comparison of the binding properties of hundreds of peptide ligands. The system was validated by plasmon surface resonance experiments using synthetic peptides, showing that the method discriminates among the affinities of the peptides within 3 orders of magnitude. In addition, the peptide–BAP protein can find direct application as a tracer reagent. PMID:18393454

Photophysical and photochemical properties of Bauhinia megalandra (Caesalpinaceae) extracts as new PDT photosensitizer

NASA Astrophysics Data System (ADS)

Vargas Tovar, Franklin R.; Rivas, C.; Estrada, O.; Marcano O., Aristides A.; Echevarria, Lorenzo; Diaz, Yrene; Alexander, I.; Rodriguez, L.; Padron, L.; Rivera, I. R.

2004-10-01

Recently new photosensitizers, chlorophyll "a and b" derivatives, for photodynamic therapy (PDT) have been presented. It already passed complete pre-clinical investigations. This prompted us to carry out an extensive study of photophysical properties of chlorine derivatives, important both for optimization of their clinic applications and for study of mechanisms of chlorine PDT&. The fresh leaves of Bauhinia megalandra (Caesalpinaceae) were extracted with methanol by percolation, and re-extract with a mixture of methanol-water (1:1), the insoluble fraction was then separated by column chromatography [RP18/hexane-ethylacetate (9:1)] to obtain four fractions named 1 to 4. These compounds were identified by NMR data. We found that 3 and 4 efficiently generates singlet oxygen when irradiated with visible light. Detection of the singlet oxygen was fulfilled by its reaction with histidine and detected by bleaching p-nitrosodimethylaniline under 440 nm irradiation. The quantum yields of singlet oxygen determined by us were 0.088 (1), 0.151 (2), 0.219 (3) and 0.301 (4). We measured absorption and fluorescence spectra of compounds 1 to 4 (Mg-chlorophyll-a, Pheophytin, Mg-chlorophyll-b and chlorophyll-b respectively) in different media and in aqueous solutions of human serum albumin. The association constant of the compounds 1, 2, 3 and 4 in the presence of HSA were estimated. The binding and quenching studies suggest that only 1 and 3 may serve as a useful fluorescence probe for structure/function studies of different chlorophyll binding proteins. No photoinduced binding was observed after irradiation by all the studied compounds in presence of human serum albumin.

Spectral and catalytic properties of aryl-alcohol oxidase, a fungal flavoenzyme acting on polyunsaturated alcohols

PubMed Central

2005-01-01

Spectral and catalytic properties of the flavoenzyme AAO (aryl-alcohol oxidase) from Pleurotus eryngii were investigated using recombinant enzyme. Unlike most flavoprotein oxidases, AAO does not thermodynamically stabilize a flavin semiquinone radical and forms no sulphite adduct. AAO catalyses the oxidative dehydrogenation of a wide range of unsaturated primary alcohols with hydrogen peroxide production. This differentiates the enzyme from VAO (vanillyl-alcohol oxidase), which is specific for phenolic compounds. Moreover, AAO is optimally active in the pH range of 5–6, whereas VAO has an optimum at pH 10. Kinetic studies showed that AAO is most active with p-anisyl alcohol and 2,4-hexadien-1-ol. AAO converts m- and p-chlorinated benzyl alcohols at a similar rate as it does benzyl alcohol, but introduction of a p-methoxy substituent in benzyl alcohol increases the reaction rate approx. 5-fold. AAO also exhibits low activity on aromatic aldehydes. 19F NMR analysis showed that fluorinated benzaldehydes are converted into the corresponding benzoic acids. Inhibition studies revealed that the AAO active site can bind a wide range of aromatic ligands, chavicol (4-allylphenol) and p-anisic (4-methoxybenzoic) acid being the best competitive inhibitors. Uncompetitive inhibition was observed with 4-methoxybenzylamine. The properties described above render AAO a unique oxidase. The possible mechanism of AAO binding and oxidation of substrates is discussed in the light of the results of the inhibition and kinetic studies. PMID:15813702

A Rhizavidin Monomer with Nearly Multimeric Avidin-Like Binding Stability Against Biotin Conjugates.

PubMed

Lee, Jeong Min; Kim, Jung A; Yen, Tzu-Chi; Lee, In Hwan; Ahn, Byungjun; Lee, Younghoon; Hsieh, Chia-Lung; Kim, Ho Min; Jung, Yongwon

2016-03-01

Developing a monomeric form of an avidin-like protein with highly stable biotin binding properties has been a major challenge in biotin-avidin linking technology. Here we report a monomeric avidin-like protein-enhanced monoavidin-with off-rates almost comparable to those of multimeric avidin proteins against various biotin conjugates. Enhanced monoavidin (eMA) was developed from naturally dimeric rhizavidin by optimally maintaining protein rigidity during monomerization and additionally shielding the bound biotin by diverse engineering of the surface residues. eMA allowed the monovalent and nonperturbing labeling of head-group-biotinylated lipids in bilayer membranes. In addition, we fabricated an unprecedented 24-meric avidin probe by fusing eMA to a multimeric cage protein. The 24-meric avidin and eMA were utilized to demonstrate how artificial clustering of cell-surface proteins greatly enhances the internalization rates of assembled proteins on live cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Biological Evaluation and X-ray Co-crystal Structures of Cyclohexylpyrrolidine Ligands for Trypanothione Reductase, an Enzyme from the Redox Metabolism of Trypanosoma.

PubMed

De Gasparo, Raoul; Brodbeck-Persch, Elke; Bryson, Steve; Hentzen, Nina B; Kaiser, Marcel; Pai, Emil F; Krauth-Siegel, R Luise; Diederich, François

2018-05-08

The tropical diseases human African trypanosomiasis, Chagas disease, and the various forms of leishmaniasis are caused by parasites of the family of trypanosomatids. These protozoa possess a unique redox metabolism based on trypanothione and trypanothione reductase (TR), making TR a promising drug target. We report the optimization of properties and potency of cyclohexylpyrrolidine inhibitors of TR by structure-based design. The best inhibitors were freely soluble and showed competitive inhibition constants (K i ) against Trypanosoma (T.) brucei TR and T. cruzi TR and in vitro activities (half-maximal inhibitory concentration, IC 50 ) against these parasites in the low micromolar range, with high selectivity against human glutathione reductase. X-ray co-crystal structures confirmed the binding of the ligands to the hydrophobic wall of the "mepacrine binding site" with the new, solubility-providing vectors oriented toward the surface of the large active site. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expression levels of MHC class I molecules are inversely correlated with promiscuity of peptide binding

PubMed Central

Chappell, Paul E; Meziane, El Kahina; Harrison, Michael; Magiera, Łukasz; Hermann, Clemens; Mears, Laura; Wrobel, Antoni G; Durant, Charlotte; Nielsen, Lise Lotte; Buus, Søren; Ternette, Nicola; Mwangi, William; Butter, Colin; Nair, Venugopal; Ahyee, Trudy; Duggleby, Richard; Madrigal, Alejandro; Roversi, Pietro; Lea, Susan M; Kaufman, Jim

2015-01-01

Highly polymorphic major histocompatibility complex (MHC) molecules are at the heart of adaptive immune responses, playing crucial roles in many kinds of disease and in vaccination. We report that breadth of peptide presentation and level of cell surface expression of class I molecules are inversely correlated in both chickens and humans. This relationship correlates with protective responses against infectious pathogens including Marek's disease virus leading to lethal tumours in chickens and human immunodeficiency virus infection progressing to AIDS in humans. We propose that differences in peptide binding repertoire define two groups of MHC class I molecules strategically evolved as generalists and specialists for different modes of pathogen resistance. We suggest that differences in cell surface expression level ensure the development of optimal peripheral T cell responses. The inverse relationship of peptide repertoire and expression is evidently a fundamental property of MHC molecules, with ramifications extending beyond immunology and medicine to evolutionary biology and conservation. DOI: http://dx.doi.org/10.7554/eLife.05345.001 PMID:25860507

Inhibition and Regulation of the Ergothioneine Biosynthetic Methyltransferase EgtD.

PubMed

Misson, Laëtitia; Burn, Reto; Vit, Allegra; Hildesheim, Julia; Beliaeva, Mariia A; Blankenfeldt, Wulf; Seebeck, Florian P

2018-05-18

Ergothioneine is an emerging factor in cellular redox homeostasis in bacteria, fungi, plants, and animals. Reports that ergothioneine biosynthesis may be important for the pathogenicity of bacteria and fungi raise the question as to how this pathway is regulated and whether the corresponding enzymes may be therapeutic targets. The first step in ergothioneine biosynthesis is catalyzed by the methyltransferase EgtD that converts histidine into N-α-trimethylhistidine. This report examines the kinetic, thermodynamic and structural basis for substrate, product, and inhibitor binding by EgtD from Mycobacterium smegmatis. This study reveals an unprecedented substrate binding mechanism and a fine-tuned affinity landscape as determinants for product specificity and product inhibition. Both properties are evolved features that optimize the function of EgtD in the context of cellular ergothioneine production. On the basis of these findings, we developed a series of simple histidine derivatives that inhibit methyltransferase activity at low micromolar concentrations. Crystal structures of inhibited complexes validate this structure- and mechanism-based design strategy.

Improving Nonspecific Binding and Solubility: Bicycloalkyl Groups and Cubanes as para-Phenyl Bioisosteres.

PubMed

Auberson, Yves P; Brocklehurst, Cara; Furegati, Markus; Fessard, Thomas C; Koch, Guido; Decker, Andrea; La Vecchia, Luigi; Briard, Emmanuelle

2017-04-20

Bicycloalkyl groups have been previously described as phenyl group bioisosteres. This article describes the synthesis of new building blocks allowing their introduction into complex molecules, and explores their use as a means to modify the physicochemical properties of drug candidates and improve the quality of imaging agents. In particular, the replacement of an aromatic ring with a bicyclo[1.1.1]pentane-1,3-diyl (BCP) group improves aqueous solubility by at least 50-fold, and markedly decreases nonspecific binding (NSB) as measured by CHI(IAM), the chromatographic hydrophobicity index on immobilized artificial membranes. Structural variations with the bicyclo[2.2.2]octane-1,4-diyl group led to more lipophilic molecules and did not show the same benefits regarding NSB or solubility, whereas substitutions with cubane-1,4-diyl showed improvements for both parameters. These results confirm the potential advantages of both BCP and cubane motifs as bioisosteric replacements for optimizing para-phenyl-substituted molecules. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Evaluation of [(201)Tl](III) Vancomycin in normal rats.

PubMed

Jalilian, Amir Reza; Hosseini, Mohammad Amin; Majdabadi, Abbas; Saddadi, Fariba

2008-01-01

Tl-201 has potential in the preparation of radiolabelled compounds similar to its homologues, like In-111 and radiogallium. In this paper, recently prepared [(201)Tl](III) vancomycin complex ([(201)Tl](III)VAN) has been evaluated for its biological properties. [(201)Tl](III)VAN was prepared according to the optimized conditions followed by biodistribution studies in normal rats for up to 52 h. The Staphylococcus aurous specific binding was checked in vitro. The complex was finally injected to normal rats. Tracer SPECT images were obtained in normal animals and compared to those of (67)Ga-citrate. Freshly-prepared [(201)Tl](III)VAN batches (radiochemical yield > 99%, radiochemical purity > 98%, specific activity approximately 1.2 Ci/mmol) showed a similar biodistribution to that of unlabeled vancomycin. The microorganism binding ratios were 3 and 9 for tracer (201)Tl(3+) and tracer (201)Tl(III)DTPA, respectively, suggesting the preservation of the tracer bioactivity. As a nonspecific cell penetrating tracer, [(201)Tl](III)DTPA was used.

The amyloid fold of Gad m 1 epitopes governs IgE binding

PubMed Central

Sánchez, Rosa; Martínez, Javier; Castro, Ana; Pedrosa, María; Quirce, Santiago; Rodríguez-Pérez, Rosa; Gasset, María

2016-01-01

Amyloids are polymeric structural states formed from locally or totally unfolded protein chains that permit surface reorganizations, stability enhancements and interaction properties that are absent in the precursor monomers. β-Parvalbumin, the major allergen in fish allergy, forms amyloids that are recognized by IgE in the patient sera, suggesting a yet unknown pathological role for these assemblies. We used Gad m 1 as the fish β-parvalbumin model and a combination of approaches, including peptide arrays, recombinant wt and mutant chains, biophysical characterizations, protease digestions, mass spectrometry, dot-blot and ELISA assays to gain insights into the role of amyloids in the IgE interaction. We found that Gad m 1 immunoreactive regions behave as sequence-dependent conformational epitopes that provide a 1000-fold increase in affinity and the structural repetitiveness required for optimal IgE binding and cross-linking upon folding into amyloids. These findings support the amyloid state as a key entity in type I food allergy. PMID:27597317

Exploring high-affinity binding properties of octamer peptides by principal component analysis of tetramer peptides.

PubMed

Kume, Akiko; Kawai, Shun; Kato, Ryuji; Iwata, Shinmei; Shimizu, Kazunori; Honda, Hiroyuki

2017-02-01

To investigate the binding properties of a peptide sequence, we conducted principal component analysis (PCA) of the physicochemical features of a tetramer peptide library comprised of 512 peptides, and the variables were reduced to two principal components. We selected IL-2 and IgG as model proteins and the binding affinity to these proteins was assayed using the 512 peptides mentioned above. PCA of binding affinity data showed that 16 and 18 variables were suitable for localizing IL-2 and IgG high-affinity binding peptides, respectively, into a restricted region of the PCA plot. We then investigated whether the binding affinity of octamer peptide libraries could be predicted using the identified region in the tetramer PCA. The results show that octamer high-affinity binding peptides were also concentrated in the tetramer high-affinity binding region of both IL-2 and IgG. The average fluorescence intensity of high-affinity binding peptides was 3.3- and 2.1-fold higher than that of low-affinity binding peptides for IL-2 and IgG, respectively. We conclude that PCA may be used to identify octamer peptides with high- or low-affinity binding properties from data from a tetramer peptide library. Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Representation of Ion–Protein Interactions Using the Drude Polarizable Force-Field

PubMed Central

2016-01-01

Small metal ions play critical roles in numerous biological processes. Of particular interest is how metalloenzymes are allosterically regulated by the binding of specific ions. Understanding how ion binding affects these biological processes requires atomic models that accurately treat the microscopic interactions with the protein ligands. Theoretical approaches at different levels of sophistication can contribute to a deeper understanding of these systems, although computational models must strike a balance between accuracy and efficiency in order to enable long molecular dynamics simulations. In this study, we present a systematic effort to optimize the parameters of a polarizable force field based on classical Drude oscillators to accurately represent the interactions between ions (K+, Na+, Ca2+, and Cl–) and coordinating amino-acid residues for a set of 30 biologically important proteins. By combining ab initio calculations and experimental thermodynamic data, we derive a polarizable force field that is consistent with a wide range of properties, including the geometries and interaction energies of gas-phase ion/protein-like model compound clusters, and the experimental solvation free-energies of the cations in liquids. The resulting models display significant improvements relative to the fixed-atomic-charge additive CHARMM C36 force field, particularly in their ability to reproduce the many-body electrostatic nonadditivity effects estimated from ab initio calculations. The analysis clarifies the fundamental limitations of the pairwise additivity assumption inherent in classical fixed-charge force fields, and shows its dramatic failures in the case of Ca2+ binding sites. These optimized polarizable models, amenable to computationally efficient large-scale MD simulations, set a firm foundation and offer a powerful avenue to study the roles of the ions in soluble and membrane transport proteins. PMID:25578354

Rational Design of Highly Potent and Slow-Binding Cytochrome bc1 Inhibitor as Fungicide by Computational Substitution Optimization

PubMed Central

Hao, Ge-Fei; Yang, Sheng-Gang; Huang, Wei; Wang, Le; Shen, Yan-Qing; Tu, Wen-Long; Li, Hui; Huang, Li-Shar; Wu, Jia-Wei; Berry, Edward A.; Yang, Guang-Fu

2015-01-01

Hit to lead (H2L) optimization is a key step for drug and agrochemical discovery. A critical challenge for H2L optimization is the low efficiency due to the lack of predictive method with high accuracy. We described a new computational method called Computational Substitution Optimization (CSO) that has allowed us to rapidly identify compounds with cytochrome bc1 complex inhibitory activity in the nanomolar and subnanomolar range. The comprehensively optimized candidate has proved to be a slow binding inhibitor of bc1 complex, ~73-fold more potent (Ki = 4.1 nM) than the best commercial fungicide azoxystrobin (AZ; Ki = 297.6 nM) and shows excellent in vivo fungicidal activity against downy mildew and powdery mildew disease. The excellent correlation between experimental and calculated binding free-energy shifts together with further crystallographic analysis confirmed the prediction accuracy of CSO method. To the best of our knowledge, CSO is a new computational approach to substitution-scanning mutagenesis of ligand and could be used as a general strategy of H2L optimisation in drug and agrochemical design.

[Strategy of molecular design of drugs: the unification of macro-properties and micro-structures of a molecule].

PubMed

Guo, Zong-Ru

2008-03-01

The interaction of a drug with the organism involves both the disposition of a drug by the organism and the action of a drug on the organism. The disposition of various exogenous substances, including drugs, complies with general rules. The underlying physical and chemical changes to different drugs in view of time and space, i. e. pharmacokinetics, share common characteristics, that is the tout ensemble of a molecule and its macroscopic properties convey direct effect on the pharmacokinetic behavior as the tendency and consequence of biological evolution. The action of a drug on the organism, on the other hand, implicates the physico-chemical binding of a drug molecule to the target protein, which induces pharmacological and toxicological effects. The biological reactions, no matter beneficial or adverse, are all specific and individual manifestation of the drug molecule and determined by the interactive binding between definitive atoms or groups of the drug molecule and the macromolecular target in three-dimension. Such critical atoms, groups, or fragments responsible for the interaction reflect the microscopic structures of drug molecules and are called pharmacophore. In this context, a drug molecule is presumed as an assembly of macroscopic property and microscopic structure, with the macroscopic properties determining the absorption, distribution, metabolism and elimination of drugs and the microscopic structure coining pharmacological action. The knowledge of the internal relationship between macroscopy/microscopy and PK/PD conduces to comprehension of drug action and guides molecular drug design, because this conception facilitates the identification of structural features necessary for biological response, and the determination of factors modulating the physico-chemical and pharmacokinetic properties. The factors determining macro-properties include molecular weight, solubility, charge, lipophilicity (partition), and polar surface area, etc., which are destined by molecular scaffolds and/or side chain(s) apart from pharmacophore. The features of micro-structures contributing to specific activity contain hydrogen bonding donor and acceptor, positive and negative charge centers, hydrophobic centers and centers of aromatic rings. Different combinations and spacial arrangements of these features determine the distinct activity presented. The macro-property and micro-structure are integrated into a single molecule, and are inseparable. The macro-property reflects overall contribution of atoms and groups in the micro-structure. On the other hand, structural changes aimed to adjust macroscopic property usually alter the relative position of the microscopic structure. The goal of molecular drug design is to integrate the macroscopic and microscopic factors in optimized manner. In the early stage of molecular design, both macroscopic property and microscopic structure should be considered to make pharmacodynamics, pharmacokinetics, and physico-chemical properties in optimal match. Therefore, it required the existence of structural overlapping among acceptable pharmacokinetics, visible developing potential and specific pharmacodynamics. The larger the scope of overlapping, the higher the possibility to be a drug.

Human lactoferrin binding to Porphyromonas gingivalis, Prevotella intermedia and Prevotella melaninogenica.

PubMed

Kalfas, S; Andersson, M; Edwardsson, S; Forsgren, A; Naidu, A S

1991-12-01

Human isolates of Porphyromonas gingivalis (n = 16), Prevotella intermedia n = 82) and Prevotella melaninogenica (n = 18) from diseased periodontal pockets were examined for interaction with human lactoferrin (HLf) in a standardized 125I-labeled protein binding assay. The highest HLf binding was found in P. intermedia strains, followed by P. gingivalis and P. melaninogenica. Further characterization of the interaction was performed with 1 representative strain from each species. HLf binding to P. gingivalis reached a saturation instantly and was optimal at pH 5.0-6.5. The corresponding values for P. melaninogenica were 90 min and pH 3.0-5.5. The HLf binding to the 2 strains seem to be nonspecific. In contrast, P. intermedia demonstrated specific binding, and a time-saturability within 60 min with an optimal uptake at pH 6.0-7.5. Scatchard analysis implied 45,000 receptors per cell with an affinity constant of 5.5 x 10(-7) M on P. intermedia strain 4H. The binding capacity in all 3 strains was affected by the culture medium. HLf binding components in these strains were susceptible to heat or proteases. Binding was eliminated in P. gingivalis and was enhanced in P. intermedia and P. melaninogenica by periodate treatment. Unlabeled HLf or bovine lactoferrin effectively displaced labeled HLf binding. Various proteins and carbohydrates did not inhibit HLf binding. Our data suggest that HLf binds to these periodontitis-associated species and that this mechanism is distinct from the previously known ligand interactions in oral bacteria.

Nicotinic acetylcholine receptor probed with a photoactivatable agonist: improved labeling specificity by addition of CeIV/glutathione. Extension to laser flash photolabeling.

PubMed

Grutter, T; Goeldner, M; Kotzyba-Hibert, F

1999-06-08

The molecular structure of Torpedo marmorata acetylcholine binding sites has been investigated previously by photoaffinity labeling. However, besides the nicotine molecule [Middleton et al. (1991) Biochemistry 30, 6987-6997], all other photosensitive probes used for this purpose interacted only with closed receptor states. In the perspective of mapping the functional activated state, we synthesized and developed a new photoactivatable agonist of nAChR capable of alkylation of the acetylcholine (ACh) binding sites, as reported previously [Kotzyba-Hibert et al. (1997) Bioconjugate Chem. 8, 472-480]. Here, we describe the setup of experimental conditions that were made in order to optimize the photolabeling reaction and in particular its specificity. We found that subsequent addition of the oxidant ceric ion (CeIV) and reduced glutathione before the photolabeling step lowered considerably nonspecific labeling (over 90% protection with d-tubocurarine) without affecting the binding properties of the ACh binding sites. As a consequence, irradiation at 360 nm for 20 min in these new conditions gave satisfactory coupling yields (7.5%). A general mechanism was proposed to explain the successive reactions occurring and their drastic effect on the specificity of the labeling reaction. Last, these incubation conditions can be extended to nanosecond pulsed laser photolysis leading to the same specific photoincorporation as for usual irradiations (8.5% coupling yield of ACh binding sites, 77% protection with carbamylcholine). Laser flash photocoupling of a diazocyclohexadienoyl probe on nAChR was achieved for the first time. Taken together, these data indicate that future investigation of the molecular dynamics of allosteric transitions occurring at the activated ACh binding sites should be possible.

Combined glyco- and protein-Fc engineering simultaneously enhance cytotoxicity and half-life of a therapeutic antibody.

PubMed

Monnet, Céline; Jorieux, Sylvie; Souyris, Nathalie; Zaki, Ouafa; Jacquet, Alexandra; Fournier, Nathalie; Crozet, Fabien; de Romeuf, Christophe; Bouayadi, Khalil; Urbain, Rémi; Behrens, Christian K; Mondon, Philippe; Fontayne, Alexandre

2014-01-01

While glyco-engineered monoclonal antibodies (mAbs) with improved antibody-dependent cell-mediated cytotoxicity (ADCC) are reaching the market, extensive efforts have also been made to improve their pharmacokinetic properties to generate biologically superior molecules. Most therapeutic mAbs are human or humanized IgG molecules whose half-life is dependent on the neonatal Fc receptor FcRn. FcRn reduces IgG catabolism by binding to the Fc domain of endocytosed IgG in acidic lysosomal compartments, allowing them to be recycled into the blood. Fc-engineered mAbs with increased FcRn affinity resulted in longer in vivo half-life in animal models, but also in healthy humans. These Fc-engineered mAbs were obtained by alanine scanning, directed mutagenesis or in silico approach of the FcRn binding site. In our approach, we applied a random mutagenesis technology (MutaGen™) to generate mutations evenly distributed over the whole Fc sequence of human IgG1. IgG variants with improved FcRn-binding were then isolated from these Fc-libraries using a pH-dependent phage display selection process. Two successive rounds of mutagenesis and selection were performed to identify several mutations that dramatically improve FcRn binding. Notably, many of these mutations were unpredictable by rational design as they were located distantly from the FcRn binding site, validating our random molecular approach. When produced on the EMABling(®) platform allowing effector function increase, our IgG variants retained both higher ADCC and higher FcRn binding. Moreover, these IgG variants exhibited longer half-life in human FcRn transgenic mice. These results clearly demonstrate that glyco-engineering to improve cytotoxicity and protein-engineering to increase half-life can be combined to further optimize therapeutic mAbs.

Characterization of the DNA binding properties of polyomavirus capsid protein

NASA Technical Reports Server (NTRS)

Chang, D.; Cai, X.; Consigli, R. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

The DNA binding properties of the polyomavirus structural proteins VP1, VP2, and VP3 were studied by Southwestern analysis. The major viral structural protein VP1 and host-contributed histone proteins of polyomavirus virions were shown to exhibit DNA binding activity, but the minor capsid proteins VP2 and VP3 failed to bind DNA. The N-terminal first five amino acids (Ala-1 to Lys-5) were identified as the VP1 DNA binding domain by genetic and biochemical approaches. Wild-type VP1 expressed in Escherichia coli (RK1448) exhibited DNA binding activity, but the N-terminal truncated VP1 mutants (lacking Ala-1 to Lys-5 and Ala-1 to Cys-11) failed to bind DNA. The synthetic peptide (Ala-1 to Cys-11) was also shown to have an affinity for DNA binding. Site-directed mutagenesis of the VP1 gene showed that the point mutations at Pro-2, Lys-3, and Arg-4 on the VP1 molecule did not affect DNA binding properties but that the point mutation at Lys-5 drastically reduced DNA binding affinity. The N-terminal (Ala-1 to Lys-5) region of VP1 was found to be essential and specific for DNA binding, while the DNA appears to be non-sequence specific. The DNA binding domain and the nuclear localization signal are located in the same N-terminal region.

Experimental design, modeling and optimization of polyplex formation between DNA oligonucleotides and branched polyethylenimine.

PubMed

Clima, Lilia; Ursu, Elena L; Cojocaru, Corneliu; Rotaru, Alexandru; Barboiu, Mihail; Pinteala, Mariana

2015-09-28

The complexes formed by DNA and polycations have received great attention owing to their potential application in gene therapy. In this study, the binding efficiency between double-stranded oligonucleotides (dsDNA) and branched polyethylenimine (B-PEI) has been quantified by processing of the images captured from the gel electrophoresis assays. The central composite experimental design has been employed to investigate the effects of controllable factors on the binding efficiency. On the basis of experimental data and the response surface methodology, a multivariate regression model has been constructed and statistically validated. The model has enabled us to predict the binding efficiency depending on experimental factors, such as concentrations of dsDNA and B-PEI as well as the initial pH of solution. The optimization of the binding process has been performed using simplex and gradient methods. The optimal conditions determined for polyplex formation have yielded a maximal binding efficiency close to 100%. In order to reveal the mechanism of complex formation at the atomic-scale, a molecular dynamic simulation has been carried out. According to the computation results, B-PEI amine hydrogen atoms have interacted with oxygen atoms from dsDNA phosphate groups. These interactions have led to the formation of hydrogen bonds between macromolecules, stabilizing the polyplex structure.

Density functional theory analysis and molecular docking evaluation of 1-(2, 5-dichloro-4-sulfophenyl)-3-methyl-5-pyrazolone as COX2 inhibitor against inflammatory diseases

NASA Astrophysics Data System (ADS)

Kavitha, T.; Velraj, G.

2017-08-01

The molecular structure of 1-(2, 5-Dichloro-4-Sulfophenyl)-3-Methyl-5-Pyrazolone (DSMP) was optimized using DFT/B3LYP/6-31++G(d,p) level and its corresponding experimental as well as theoretical FT-IR, FT-Raman vibrational frequencies and UV-Vis spectral analysis were carried out. The vibrational assignments and total energy distributions of each vibration were presented with the aid of Veda 4xx software. The molecular electrostatic potential, HOMO-LUMO energies, global and local reactivity descriptors and natural bond orbitals were analyzed in order to find the most possible reactive sites of the molecule and it was found that DSMP molecule possess enhanced nucleophilic activity. One of the common known COX2 inhibitor, celecoxib (CXB) was also found to exhibit similar reactivity properties and hence DSMP was also expected to inhibit COX enzymes. In order to detect the COX inhibition nature of DSMP, molecular docking analysis was carried out with the help of Autodock software. For that, the optimized structure was in turn used for docking DSMP with COX enzymes. The binding energy scores and inhibitory constant values reveal that the DSMP molecule possess good binding affinity and low inhibition constant towards COX2 enzyme and hence it can be used as an anti-inflammatory drug after carrying out necessary biological tests.

Development of solid-phase microextraction coupled with liquid chromatography for analysis of tramadol in brain tissue using its molecularly imprinted polymer.

PubMed

Habibi-Khorasani, Monireh; Mohammadpour, Amir Hooshang; Mohajeri, Seyed Ahmad

2017-02-01

In this work, performance of a molecularly imprinted polymer (MIP) as a selective solid-phase microextraction sorbent for the extraction and enrichment of tramadol in aqueous solution and rabbit brain tissue, is described. Binding properties of MIPs were studied in comparison with their nonimprinted polymer (NIP). Ten milligrams of the optimized MIP was then evaluated as a sorbent, for preconcentration, in molecularly imprinted solid-phase microextraction (MISPME) of tramadol from aqueous solution and rabbit brain tissue. The analytical method was calibrated in the range of 0.004 ppm (4 ng mL -1 ) and 10 ppm (10 μg mL -1 ) in aqueous media and in the ranges of 0.01 and 10 ppm in rabbit brain tissue, respectively. The results indicated significantly higher binding affinity of MIPs to tramadol, in comparison with NIP. The MISPME procedure was developed and optimized with a recovery of 81.12-107.54% in aqueous solution and 76.16-91.20% in rabbit brain tissue. The inter- and intra-day variation values were <8.24 and 5.06%, respectively. Finally the calibrated method was applied for determination of tramadol in real rabbit brain tissue samples after administration of a lethal dose. Our data demonstrated the potential of MISPME for rapid, sensitive and cost-effective sample analysis. Copyright © 2016 John Wiley & Sons, Ltd.

An ultra-tunable platform for molecular engineering of high-performance crystalline porous materials

DOE PAGES

Zhai, Quan -Guo; Bu, Xianhui; Mao, Chengyu; ...

2016-12-07

Metal-organic frameworks are a class of crystalline porous materials with potential applications in catalysis, gas separation and storage, and so on. Of great importance is the development of innovative synthetic strategies to optimize porosity, composition and functionality to target specific applications. Here we show a platform for the development of metal-organic materials and control of their gas sorption properties. This platform can accommodate a large variety of organic ligands and homo- or hetero-metallic clusters, which allows for extraordinary tunability in gas sorption properties. Even without any strong binding sites, most members of this platform exhibit high gas uptake capacity. Asmore » a result, the high capacity is accomplished with an isosteric heat of adsorption as low as 20 kJ mol –1 for carbon dioxide, which could bring a distinct economic advantage because of the significantly reduced energy consumption for activation and regeneration of adsorbents.« less

Analysis of the Structures and Properties of (GaSb)n (n = 4-9) Clusters through Density Functional Theory.

PubMed

Lu, Qi Liang; Luo, Qi Quan; Huang, Shou Guo; Li, Yi De; Wan, Jian Guo

2016-07-07

An optimization strategy combining global semiempirical quantum mechanical search with all-electron density functional theory was adopted to determine the lowest energy structure of (GaSb)n clusters up to n = 9. The growth pattern of the clusters differed from those of previously reported group III-V binary clusters. A cagelike configuration was found for cluster sizes n ≤ 7. The structure of (GaSb)6 deviated from that of other III-V clusters. Competition existed between core-shell and hollow cage structures of (GaSb)7. Novel noncagelike structures were energetically preferred over the cages for the (GaSb)8 and (GaSb)9 clusters. Electronic properties, such as vertical ionization potential, adiabatic electron affinities, HOMO-LUMO gaps, and average on-site charges on Ga or Sb atoms, as well as binding energies, were computed.

Unified modelling of the thermoelectric properties in SrTiO3

NASA Astrophysics Data System (ADS)

Bouzerar, G.; Thébaud, S.; Adessi, Ch.; Debord, R.; Apreutesei, M.; Bachelet, R.; Pailhès, S.

2017-06-01

Thermoelectric materials are opening a promising pathway to address energy conversion issues governed by a competition between thermal and electronic transport. Improving the efficiency is a difficult task, a challenge that requires new strategies to unearth optimized compounds. We present a theory of thermoelectric transport in electron-doped SrTiO3, based on a realistic tight-binding model that includes relevant scattering processes. We compare our calculations against a wide panel of experimental data, both bulk and thin films. We find a qualitative and quantitative agreement over both a wide range of temperatures and carrier concentrations, from light to heavily doped. Moreover, the results appear insensitive to the nature of the dopant La, B, Gd and Nb. Thus, the quantitative success found in the case of SrTiO3, reveals an efficient procedure to explore new routes to improve the thermoelectric properties in oxides.

RNA-binding properties and mapping of the RNA-binding domain from the movement protein of Prunus necrotic ringspot virus.

PubMed

Herranz, M Carmen; Pallás, Vicente

2004-03-01

The movement protein (MP) of Prunus necrotic ringspot virus (PNRSV) is involved in intercellular virus transport. In this study, putative RNA-binding properties of the PNRSV MP were studied. The PNRSV MP was produced in Escherichia coli using an expression vector. Electrophoretic mobility shift assays (EMSAs) using DIG-labelled riboprobes demonstrated that PNRSV MP bound ssRNA cooperatively without sequence specificity. Two different ribonucleoprotein complexes were found to be formed depending on the molar MP : PNRSV RNA ratio. The different responses of the complexes to urea treatment strongly suggested that they have different structural properties. Deletion mutagenesis followed by Northwestern analysis allowed location of a nucleic acid binding domain to aa 56-88. This 33 aa RNA-binding motif is the smallest region delineated among members of the family Bromoviridae for which RNA-binding properties have been demonstrated. This domain is highly conserved within all phylogenetic subgroups previously described for PNRSV isolates. Interestingly, the RNA-binding domain described here and the one described for Alfamovirus are located at the N terminus of their corresponding MPs, whereas similar domains previously characterized in members of the genera Bromovirus and Cucumovirus are present at the C terminus, strongly reflecting their corresponding phylogenetic relationships. The evolutionary implications of this observation are discussed.

Introducing folding stability into the score function for computational design of RNA-binding peptides boosts the probability of success.

PubMed

Xiao, Xingqing; Agris, Paul F; Hall, Carol K

2016-05-01

A computational strategy that integrates our peptide search algorithm with atomistic molecular dynamics simulation was used to design rational peptide drugs that recognize and bind to the anticodon stem and loop domain (ASL(Lys3)) of human tRNAUUULys3 for the purpose of interrupting HIV replication. The score function of the search algorithm was improved by adding a peptide stability term weighted by an adjustable factor λ to the peptide binding free energy. The five best peptide sequences associated with five different values of λ were determined using the search algorithm and then input in atomistic simulations to examine the stability of the peptides' folded conformations and their ability to bind to ASL(Lys3). Simulation results demonstrated that setting an intermediate value of λ achieves a good balance between optimizing the peptide's binding ability and stabilizing its folded conformation during the sequence evolution process, and hence leads to optimal binding to the target ASL(Lys3). Thus, addition of a peptide stability term significantly improves the success rate for our peptide design search. © 2016 Wiley Periodicals, Inc.

Ligand Residence Time at G-protein-Coupled Receptors-Why We Should Take Our Time To Study It.

PubMed

Hoffmann, C; Castro, M; Rinken, A; Leurs, R; Hill, S J; Vischer, H F

2015-09-01

Over the past decade the kinetics of ligand binding to a receptor have received increasing interest. The concept of drug-target residence time is becoming an invaluable parameter for drug optimization. It holds great promise for drug development, and its optimization is thought to reduce off-target effects. The success of long-acting drugs like tiotropium support this hypothesis. Nonetheless, we know surprisingly little about the dynamics and the molecular detail of the drug binding process. Because protein dynamics and adaptation during the binding event will change the conformation of the protein, ligand binding will not be the static process that is often described. This can cause problems because simple mathematical models often fail to adequately describe the dynamics of the binding process. In this minireview we will discuss the current situation with an emphasis on G-protein-coupled receptors. These are important membrane protein drug targets that undergo conformational changes upon agonist binding to communicate signaling information across the plasma membrane of cells. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

How Much Binding Affinity Can be Gained by Filling a Cavity?

PubMed Central

Kawasaki, Yuko; Chufan, Eduardo E.; Lafont, Virginie; Hidaka, Koushi; Kiso, Yoshiaki; Amzel, L. Mario; Freire, Ernesto

2011-01-01

Binding affinity optimization is critical during drug development. Here we evaluate the thermodynamic consequences of filling a binding cavity with functionalities of increasing van der Waals radii (-H, -F, -Cl and CH3) that improve the geometric fit without participating in hydrogen bonding or other specific interactions. We observe a binding affinity increase of two orders of magnitude. There appears to be three phases in the process. The first phase is associated with the formation of stable van der Waals interactions. This phase is characterized by a gain in binding enthalpy and a loss in binding entropy, attributed to a loss of conformational degrees of freedom. For the specific case presented in this paper, the enthalpy gain amounts to −1.5 kcal/mol while the entropic losses amount to +0.9 kcal/mol resulting in a net 3.5-fold affinity gain. The second phase is characterized by simultaneous enthalpic and entropic gains. This phase improves the binding affinity 25-fold. The third phase represents the collapse of the trend and is triggered by the introduction of chemical functionalities larger than the binding cavity itself (CH(CH3)2). It is characterized by large enthalpy and affinity losses. The thermodynamic signatures associated with each phase provide guidelines for lead optimization. PMID:20028396

How Does Mg2+ Modulate the RNA Folding Mechanism: A Case Study of the G:C W:W Trans Basepair.

PubMed

Halder, Antarip; Roy, Rohit; Bhattacharyya, Dhananjay; Mitra, Abhijit

2017-07-25

Reverse Watson-Crick G:C basepairs (G:C W:W Trans) occur frequently in different functional RNAs. This is one of the few basepairs whose gas-phase-optimized isolated geometry is inconsistent with the corresponding experimental geometry. Several earlier studies indicate that through post-transcriptional modification, direct protonation, or coordination with Mg 2+ , accumulation of positive charge near N7 of guanine can stabilize the experimental geometry. Interestingly, recent studies reveal significant variation in the position of putatively bound Mg 2+ . This, in conjunction with recently raised doubts regarding some of the Mg 2+ assignments near the imino nitrogen of guanine, is suggestive of the existence of multiple Mg 2+ binding modes for this basepair. Our detailed investigation of Mg 2+ -bound G:C W:W Trans pairs occurring in high-resolution RNA crystal structures shows that they are found in 14 different contexts, eight of which display Mg 2+ binding at the Hoogsteen edge of guanine. Further examination of occurrences in these eight contexts led to the characterization of three different Mg 2+ binding modes: 1) direct binding via N7 coordination, 2) direct binding via O6 coordination, and 3) binding via hydrogen-bonding interaction with the first-shell water molecules. In the crystal structures, the latter two modes are associated with a buckled and propeller-twisted geometry of the basepair. Interestingly, respective optimized geometries of these different Mg 2+ binding modes (optimized using six different DFT functionals) are consistent with their corresponding experimental geometries. Subsequent interaction energy calculations at the MP2 level, and decomposition of its components, suggest that for G:C W:W Trans , Mg 2+ binding can fine tune the basepair geometries without compromising with their stability. Our results, therefore, underline the importance of the mode of binding of Mg 2+ ions in shaping RNA structure, folding and function. Copyright © 2017. Published by Elsevier Inc.

Binding Free Energy Calculations for Lead Optimization: Assessment of Their Accuracy in an Industrial Drug Design Context.

PubMed

Homeyer, Nadine; Stoll, Friederike; Hillisch, Alexander; Gohlke, Holger

2014-08-12

Correctly ranking compounds according to their computed relative binding affinities will be of great value for decision making in the lead optimization phase of industrial drug discovery. However, the performance of existing computationally demanding binding free energy calculation methods in this context is largely unknown. We analyzed the performance of the molecular mechanics continuum solvent, the linear interaction energy (LIE), and the thermodynamic integration (TI) approach for three sets of compounds from industrial lead optimization projects. The data sets pose challenges typical for this early stage of drug discovery. None of the methods was sufficiently predictive when applied out of the box without considering these challenges. Detailed investigations of failures revealed critical points that are essential for good binding free energy predictions. When data set-specific features were considered accordingly, predictions valuable for lead optimization could be obtained for all approaches but LIE. Our findings lead to clear recommendations for when to use which of the above approaches. Our findings also stress the important role of expert knowledge in this process, not least for estimating the accuracy of prediction results by TI, using indicators such as the size and chemical structure of exchanged groups and the statistical error in the predictions. Such knowledge will be invaluable when it comes to the question which of the TI results can be trusted for decision making.

Point Defect Properties of Cd(Zn)Te and TlBr for Room-Temperature Gamma Radiation Detectors

NASA Astrophysics Data System (ADS)

Lordi, Vincenzo

2013-03-01

The effects of various crystal defects in CdTe, Cd1-xZnxTe (CZT), and TlBr are critical for their performance as room-temperature gamma radiation detectors. We use predictive first principles theoretical methods to provide fundamental, atomic scale understanding of the defect properties of these materials to enable design of optimal growth and processing conditions, such as doping, annealing, and stoichiometry. Several recent cases will be reviewed, including (i) accurate calculations of the thermodynamic and electronic properties of native point defects and point defect complexes in CdTe and CZT; (ii) the effects of Zn alloying on the native point defect properties of CZT; (iii) point defect diffusion and binding related to Te clustering in Cd(Zn)Te; (iv) the profound effect of native point defects--principally vacancies--on the intrinsic material properties of TlBr, particularly electronic and ionic conductivity; (v) tailored doping of TlBr to independently control the electronic and ionic conductivity; and (vi) the effects of metal impurities on the electronic properties and device performance of TlBr detectors. Prepared by LLNL under Contract DE-AC52-07NA27344 with support from the National Nuclear Security Administration Office of Nonproliferation and Verification Research and Development NA-22.

Utilization of a modified special-cubic design and an electronic tongue for bitterness masking formulation optimization.

PubMed

Li, Lianli; Naini, Venkatesh; Ahmed, Salah U

2007-10-01

A unique modification of simplex design was applied to an electronic tongue (E-Tongue) analysis in bitterness masking formulation optimization. Three formulation variables were evaluated in the simplex design, i.e. concentrations of two taste masking polymers, Amberlite and Carbopol, and pH of the granulating fluid. Response of the design was a bitterness distance measured using an E-Tongue by applying a principle component analysis, which represents taste masking efficiency of the formulation. The smaller the distance, the better the bitterness masking effect. Contour plots and polynomial equations of the bitterness distance response were generated as a function of formulation composition and pH. It was found that interactions between polymer and pH reduced the bitterness of the formulation, attributed to pH-dependent ionization and complexation properties of the ionic polymers, thus keeping the drug out of solution and unavailable to bitterness perception. At pH 4.9 and an Amberlite/Carbopol ratio of 1.4:1 (w/w), the optimal taste masking formulation was achieved and in agreement with human gustatory sensation study results. Therefore, adopting a modified simplex experimental design on response measured using an E-Tongue provided an efficient approach to taste masking formulation optimization using ionic binding polymers. (c) 2007 Wiley-Liss, Inc.

Major histocompatibility class I molecules present Urtica dioica agglutinin, a superantigen of vegetal origin, to T lymphocytes.

PubMed

Rovira, P; Buckle, M; Abastado, J P; Peumans, W J; Truffa-Bachi, P

1999-05-01

The Urtica dioica agglutinin (UDA) shares with the superantigens the property of activating T cell subsets bearing particular Vbeta segments of the TCR. However, UDA is a lectin capable of binding to many glycoproteins on cell membranes. The implication of MHC versus other glycoproteins in UDA presentation was presently studied. Using mutant mice lacking MHC class I (MHC-I), MHC class II (MHC-II) or both MHC antigens, we provided evidence that MHC-I and MHC-II molecules serve as UDA receptors. Presentation by either one of these molecules ensured similar T cell responses and co-stimulatory signals were mandatory for optimal T cell activation and proliferation both in MHC-I and MHC-II contexts. Remarkably, in the absence of MHC molecules, UDA could not be efficiently presented to T cells by other glycosylated proteins. Surface plasmon resonance studies were used to confirm the binding of UDA to MHC-I molecules using a fusion protein consisting of MHC-I domains and beta2-microglobulin. The results indicated that the interaction between UDA and MHC-I molecules implicated lectin-binding site(s) of UDA. Taken together, our data demonstrate that, in addition to MHC-II antigens, MHC-I molecules serve as an alternative ligand for UDA.

Acid-base and copper-binding properties of three organic matter fractions isolated from a forest floor soil solution

NASA Astrophysics Data System (ADS)

van Schaik, Joris W. J.; Kleja, Dan B.; Gustafsson, Jon Petter

2010-02-01

Vast amounts of knowledge about the proton- and metal-binding properties of dissolved organic matter (DOM) in natural waters have been obtained in studies on isolated humic and fulvic (hydrophobic) acids. Although macromolecular hydrophilic acids normally make up about one-third of DOM, their proton- and metal-binding properties are poorly known. Here, we investigated the acid-base and Cu-binding properties of the hydrophobic (fulvic) acid fraction and two hydrophilic fractions isolated from a soil solution. Proton titrations revealed a higher total charge for the hydrophilic acid fractions than for the hydrophobic acid fraction. The most hydrophilic fraction appeared to be dominated by weak acid sites, as evidenced by increased slope of the curve of surface charge versus pH at pH values above 6. The titration curves were poorly predicted by both Stockholm Humic Model (SHM) and NICA-Donnan model calculations using generic parameter values, but could be modelled accurately after optimisation of the proton-binding parameters (pH ⩽ 9). Cu-binding isotherms for the three fractions were determined at pH values of 4, 6 and 9. With the optimised proton-binding parameters, the SHM model predictions for Cu binding improved, whereas the NICA-Donnan predictions deteriorated. After optimisation of Cu-binding parameters, both models described the experimental data satisfactorily. Iron(III) and aluminium competed strongly with Cu for binding sites at both pH 4 and pH 6. The SHM model predicted this competition reasonably well, but the NICA-Donnan model underestimated the effects significantly at pH 6. Overall, the Cu-binding behaviour of the two hydrophilic acid fractions was very similar to that of the hydrophobic acid fraction, despite the differences observed in proton-binding characteristics. These results show that for modelling purposes, it is essential to include the hydrophilic acid fraction in the pool of 'active' humic substances.

Stabilization of marly soils with portland cement

NASA Astrophysics Data System (ADS)

Piskunov, Maksim; Karzin, Evgeny; Lukina, Valentina; Lukinov, Vitaly; Kholkin, Anatolii

2017-10-01

Stabilization of marlous soils with Portland cement will increase the service life of motor roads in areas where marl is used as a local road construction material. The result of the conducted research is the conclusion about the principal possibility of stabilization of marlous soils with Portland cement, and about the optimal percentage of the mineral part and the binding agent. When planning the experiment, a simplex-lattice plan was implemented, which makes it possible to obtain a mathematical model for changing the properties of a material in the form of polynomials of incomplete third order. Brands were determined for compressive strength according to GOST 23558-94 and variants of stabilized soils were proposed for road construction.

Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(ʟ-Lysine): Experimental Study and Modeling Approach.

PubMed

Vasiliu, Tudor; Cojocaru, Corneliu; Rotaru, Alexandru; Pricope, Gabriela; Pinteala, Mariana; Clima, Lilia

2017-06-17

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(ʟ-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4.

Optimization of Polyplex Formation between DNA Oligonucleotide and Poly(l-Lysine): Experimental Study and Modeling Approach

PubMed Central

Vasiliu, Tudor; Cojocaru, Corneliu; Rotaru, Alexandru; Pricope, Gabriela; Pinteala, Mariana; Clima, Lilia

2017-01-01

The polyplexes formed by nucleic acids and polycations have received a great attention owing to their potential application in gene therapy. In our study, we report experimental results and modeling outcomes regarding the optimization of polyplex formation between the double-stranded DNA (dsDNA) and poly(l-Lysine) (PLL). The quantification of the binding efficiency during polyplex formation was performed by processing of the images captured from the gel electrophoresis assays. The design of experiments (DoE) and response surface methodology (RSM) were employed to investigate the coupling effect of key factors (pH and N/P ratio) affecting the binding efficiency. According to the experimental observations and response surface analysis, the N/P ratio showed a major influence on binding efficiency compared to pH. Model-based optimization calculations along with the experimental confirmation runs unveiled the maximal binding efficiency (99.4%) achieved at pH 5.4 and N/P ratio 125. To support the experimental data and reveal insights of molecular mechanism responsible for the polyplex formation between dsDNA and PLL, molecular dynamics simulations were performed at pH 5.4 and 7.4. PMID:28629130

Understanding and predicting binding between human leukocyte antigens (HLAs) and peptides by network analysis.

PubMed

Luo, Heng; Ye, Hao; Ng, Hui; Shi, Leming; Tong, Weida; Mattes, William; Mendrick, Donna; Hong, Huixiao

2015-01-01

As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Network analysis is a useful approach for analyzing large and sparse datasets such as the HLA-peptide binding dataset. The modules identified from the network analysis clustered peptides and HLAs with similar sequences and properties of amino acids. Nebula performed well in the predictions of HLA-peptide binding. We demonstrated that network analysis coupled with Nebula is an efficient approach to understand and predict HLA-peptide binding interactions and thus, could further our understanding of ADRs.

Understanding and predicting binding between human leukocyte antigens (HLAs) and peptides by network analysis

PubMed Central

2015-01-01

Background As the major histocompatibility complex (MHC), human leukocyte antigens (HLAs) are one of the most polymorphic genes in humans. Patients carrying certain HLA alleles may develop adverse drug reactions (ADRs) after taking specific drugs. Peptides play an important role in HLA related ADRs as they are the necessary co-binders of HLAs with drugs. Many experimental data have been generated for understanding HLA-peptide binding. However, efficiently utilizing the data for understanding and accurately predicting HLA-peptide binding is challenging. Therefore, we developed a network analysis based method to understand and predict HLA-peptide binding. Methods Qualitative Class I HLA-peptide binding data were harvested and prepared from four major databases. An HLA-peptide binding network was constructed from this dataset and modules were identified by the fast greedy modularity optimization algorithm. To examine the significance of signals in the yielded models, the modularity was compared with the modularity values generated from 1,000 random networks. The peptides and HLAs in the modules were characterized by similarity analysis. The neighbor-edges based and unbiased leverage algorithm (Nebula) was developed for predicting HLA-peptide binding. Leave-one-out (LOO) validations and two-fold cross-validations were conducted to evaluate the performance of Nebula using the constructed HLA-peptide binding network. Results Nine modules were identified from analyzing the HLA-peptide binding network with a highest modularity compared to all the random networks. Peptide length and functional side chains of amino acids at certain positions of the peptides were different among the modules. HLA sequences were module dependent to some extent. Nebula archived an overall prediction accuracy of 0.816 in the LOO validations and average accuracy of 0.795 in the two-fold cross-validations and outperformed the method reported in the literature. Conclusions Network analysis is a useful approach for analyzing large and sparse datasets such as the HLA-peptide binding dataset. The modules identified from the network analysis clustered peptides and HLAs with similar sequences and properties of amino acids. Nebula performed well in the predictions of HLA-peptide binding. We demonstrated that network analysis coupled with Nebula is an efficient approach to understand and predict HLA-peptide binding interactions and thus, could further our understanding of ADRs. PMID:26424483

Effect of Dispersion on Surface Interactions of Cobalt(II) Octaethylporphyrin Monolayer on Au(111) and HOPG(0001) Substrates: a Comparative First Principles Study

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chilukuri, Bhaskar; Mazur, Ursula; Hipps, Kerry W.

A density functional theory study of a cobalt(II) octaethylporphyrin (CoOEP) monolayer on Au(111) and HOPG(0001) surfaces was performed under periodic boundary conditions. Calculations with and without dispersion corrections are performed and the effect of van der Waals forces on the interface properties is analyzed. Calculations have determined that the CoOEP molecule tends to bind at the 3-fold and the 6-fold center sites on Au(111) and HOPG(0001), respectively. Geometric optimizations at the center binding sites have indicated that the porphyrin molecules (in the monolayer) lie flat on both substrates. Calculations also reveal that the CoOEP monolayer binds slightly more strongly tomore » Au(111) than to HOPG(0001). Charge density difference plots disclose that charge is redistributed mostly around the porphyrin plane and the first layer of the substrates. Dispersion interactions cause a larger substrate to molecule charge pushback on Au(111) than on HOPG. CoOEP adsorption tends to lower the work functions of either substrate, qualitatively agreeing with the experimental photoelectron spectroscopic data. Comparison of the density of states (DOS) of the isolated CoOEP molecule with that on gold and HOPG substrates showed significant band shifts around the Fermi energy due to intermolecular orbital hybridization. Simulated STM images were plotted with the Tersoff–Hamann approach using the local density of states, which also agree with the experimental results. This study elucidates the role of dispersion for better describing porphyrin–substrate interactions. A DFT based overview of geometric, adsorption and electronic properties of a porphyrin monolayer on conductive surfaces is presented.« less

A novel DNA nanosensor based on CdSe/ZnS quantum dots and synthesized Fe3O4 magnetic nanoparticles.

PubMed

Hushiarian, Roozbeh; Yusof, Nor Azah; Abdullah, Abdul Halim; Ahmad, Shahrul Ainliah Alang; Dutse, Sabo Wada

2014-04-09

Although nanoparticle-enhanced biosensors have been extensively researched, few studies have systematically characterized the roles of nanoparticles in enhancing biosensor functionality. This paper describes a successful new method in which DNA binds directly to iron oxide nanoparticles for use in an optical biosensor. A wide variety of nanoparticles with different properties have found broad application in biosensors because their small physical size presents unique chemical, physical, and electronic properties that are different from those of bulk materials. Of all nanoparticles, magnetic nanoparticles are proving to be a versatile tool, an excellent case in point being in DNA bioassays, where magnetic nanoparticles are often used for optimization of the hybridization and separation of target DNA. A critical step in the successful construction of a DNA biosensor is the efficient attachment of biomolecules to the surface of magnetic nanoparticles. To date, most methods of synthesizing these nanoparticles have led to the formation of hydrophobic particles that require additional surface modifications. As a result, the surface to volume ratio decreases and nonspecific bindings may occur so that the sensitivity and efficiency of the device deteriorates. A new method of large-scale synthesis of iron oxide (Fe3O4) nanoparticles which results in the magnetite particles being in aqueous phase, was employed in this study. Small modifications were applied to design an optical DNA nanosensor based on sandwich hybridization. Characterization of the synthesized particles was carried out using a variety of techniques and CdSe/ZnS core-shell quantum dots were used as the reporter markers in a spectrofluorophotometer. We showed conclusively that DNA binds to the surface of ironoxide nanoparticles without further surface modifications and that these magnetic nanoparticles can be efficiently utilized as biomolecule carriers in biosensing devices.

Tuning reactivity of diphenylpropynone derivatives with metal-associated amyloid-β species via structural modifications.

PubMed

Liu, Yuzhong; Kochi, Akiko; Pithadia, Amit S; Lee, Sanghyun; Nam, Younwoo; Beck, Michael W; He, Xiaoming; Lee, Dongkuk; Lim, Mi Hee

2013-07-15

A diphenylpropynone derivative, DPP2, has been recently demonstrated to target metal-associated amyloid-β (metal-Aβ) species implicated in Alzheimer's disease (AD). DPP2 was shown to interact with metal-Aβ species and subsequently control Aβ aggregation (reactivity) in vitro; however, its cytotoxicity has limited further biological applications. In order to improve reactivity toward Aβ species and lower cytotoxicity, along with gaining an understanding of a structure-reactivity-cytotoxicity relationship, we designed, prepared, and characterized a series of small molecules (C1/C2, P1/P2, and PA1/PA2) as structurally modified DPP2 analogues. A similar metal binding site to that of DPP2 was contained in these compounds while their structures were varied to afford different interactions and reactivities with metal ions, Aβ species, and metal-Aβ species. Distinct reactivities of our chemical family toward in vitro Aβ aggregation in the absence and presence of metal ions were observed. Among our chemical series, the compound (C2) with a relatively rigid backbone and a dimethylamino group was observed to noticeably regulate both metal-free and metal-mediated Aβ aggregation to different extents. Using our compounds, cell viability was significantly improved, compared to that with DPP2. Lastly, modifications on the DPP framework maintained the structural properties for potential blood-brain barrier (BBB) permeability. Overall, our studies demonstrated that structural variations adjacent to the metal binding site of DPP2 could govern different metal binding properties, interactions with Aβ and metal-Aβ species, reactivity toward metal-free and metal-induced Aβ aggregation, and cytotoxicity of the compounds, establishing a structure-reactivity-cytotoxicity relationship. This information could help gain insight into structural optimization for developing nontoxic chemical reagents toward targeting metal-Aβ species and modulating their reactivity in biological systems.

Effect of dispersion on surface interactions of cobalt(II) octaethylporphyrin monolayer on Au(111) and HOPG(0001) substrates: a comparative first principles study.

PubMed

Chilukuri, Bhaskar; Mazur, Ursula; Hipps, K W

2014-07-21

A density functional theory study of a cobalt(II) octaethylporphyrin (CoOEP) monolayer on Au(111) and HOPG(0001) surfaces was performed under periodic boundary conditions. Calculations with and without dispersion corrections are performed and the effect of van der Waals forces on the interface properties is analyzed. Calculations have determined that the CoOEP molecule tends to bind at the 3-fold and the 6-fold center sites on Au(111) and HOPG(0001), respectively. Geometric optimizations at the center binding sites have indicated that the porphyrin molecules (in the monolayer) lie flat on both substrates. Calculations also reveal that the CoOEP monolayer binds slightly more strongly to Au(111) than to HOPG(0001). Charge density difference plots disclose that charge is redistributed mostly around the porphyrin plane and the first layer of the substrates. Dispersion interactions cause a larger substrate to molecule charge pushback on Au(111) than on HOPG. CoOEP adsorption tends to lower the work functions of either substrate, qualitatively agreeing with the experimental photoelectron spectroscopic data. Comparison of the density of states (DOS) of the isolated CoOEP molecule with that on gold and HOPG substrates showed significant band shifts around the Fermi energy due to intermolecular orbital hybridization. Simulated STM images were plotted with the Tersoff-Hamann approach using the local density of states, which also agree with the experimental results. This study elucidates the role of dispersion for better describing porphyrin-substrate interactions. A DFT based overview of geometric, adsorption and electronic properties of a porphyrin monolayer on conductive surfaces is presented.

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors.

PubMed

Nongonierma, Alice B; Mooney, Catherine; Shields, Denis C; FitzGerald, Richard J

2014-07-01

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC50 216μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC50 values of 3.5 and 44.2μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure-activity relationship modeling, rather than on docking, in computationally selecting peptides for screening. Copyright © 2014 Elsevier Inc. All rights reserved.

Cyclometalated gold(III) trioxadiborrin complexes: studies of the bonding and excited states.

PubMed

Ayoub, Nicholas A; Browne, Amberle R; Anderson, Bryce L; Gray, Thomas G

2016-03-07

Trioxadiborrins are chelating ligands that assemble in dehydration reactions of boronic acids. They are structurally related to β-diketonate ligands, but have a 2-charge. Little is known of the bonding properties of trioxadiborrin ligands. Presented here are density-functional theory (DFT) studies of cyclometalated gold(III) trioxadiborrins. Substituent effects are evaluated, and comparison is made to the cyclometalating 2-(4-tolyl)pyridine (tpy) ligand on gold. The tpy ligand binds more strongly than any trioxadiborrin ligand considered here, and the two ligands bind competitively to gold. The 1,3-diphenyl trioxadiborrin ligand of 1 has a larger absolute binding enthalpy to gold than its β-diketonate analogue. Conjugation between boron and aryl substituents delocalizes charge and attenuates the trioxadiborrin's binding capacity. Steric effects that disrupt conjugation between boron and aryl substituents cause the trioxadiborrin to chelate more tightly. Fragment bond orders are divided into in-plane and out-of-plane contributions for square planar 1. In-plane bonding accounts for 88% of bond order between (tpy)Au2+ and the trioxadiborrin ligand. Cyclometalated gold(III) trioxadiborrin complexes were previously shown to be phosphorescent. Spin-unrestricted triplet-state geometry optimizations find that the ten largest excited-state distortions all occur on the tpy ligand. A plot of spin density in triplet 1 shows spin to reside predominantly on tpy. The 77 K luminescence spectrum of 1 is reported here. Time-dependent DFT and configuration interaction singles calculations (corrected for doubles excitations) overestimate the emission energy by ∼ 0.12 eV.

A Novel Selective Inverse Agonist of the CB2 Receptor as a Radiolabeled Tool Compound for Kinetic Binding Studies.

PubMed

Martella, Andrea; Sijben, Huub; Rufer, Arne C; Grether, Uwe; Fingerle, Juergen; Ullmer, Christoph; Hartung, Thomas; IJzerman, Adriaan P; van der Stelt, Mario; Heitman, Laura H

2017-10-01

The endocannabinoid system, and in particular the cannabinoid type 2 receptor (CB2R), raised the interest of many medicinal chemistry programs for its therapeutic relevance in several (patho)physiologic processes. However, the physico-chemical properties of tool compounds for CB2R (e.g., the radioligand [ 3 H]CP55,940) are not optimal, despite the research efforts in developing effective drugs to target this system. At the same time, the importance of drug-target binding kinetics is growing since the kinetic binding profile of a ligand may provide important insights for the resulting in vivo efficacy. In this context we synthesized and characterized [ 3 H]RO6957022, a highly selective CB2R inverse agonist, as a radiolabeled tool compound. In equilibrium and kinetic binding experiments [ 3 H]RO6957022 showed high affinity for human CB2R with fast association ( k on ) and moderate dissociation ( k off ) kinetics. To demonstrate the robustness of [ 3 H]RO6957022 binding, affinity studies were carried out for a wide range of CB2R reference ligands, spanning the range of full, partial, and inverse agonists. Finally, we used [ 3 H]RO6957022 to study the kinetic binding profiles (i.e., k on and k off values) of selected synthetic and endogenous (i.e., 2-arachidonoylglycerol, anandamide, and noladin ether) CB2R ligands by competition association experiments. All tested ligands, and in particular the endocannabinoids, displayed distinct kinetic profiles, shedding more light on their mechanism of action and the importance of association rates in the determination of CB2R affinity. Altogether, this study shows that the use of a novel tool compound, i.e., [ 3 H]RO6957022, can support the development of novel ligands with a repertoire of kinetic binding profiles for CB2R. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

Quantum mechanical, spectroscopic study (FT-IR and FT - Raman), NBO analysis, HOMO-LUMO, first order hyperpolarizability and docking studies of a non-steroidal anti-inflammatory compound

NASA Astrophysics Data System (ADS)

Sakthivel, S.; Alagesan, T.; Muthu, S.; Abraham, Christina Susan; Geetha, E.

2018-03-01

Experimental and theoretical studies on the optimized geometrical structure, electronic and vibrational characteristics of (+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoic acid are presented employing B3LYP/6-311++G (d,p) basis set. Simulated FT-IR and FT-Raman spectra were in concurrence with the observed spectra attained in a spectral range of FT-IR (4000 - 400 cm-1) and FT-Raman (4000 - 100 cm-1). Quantum chemical calculations and the comprehensive vibrational assignments of wavenumbers of the optimized geometry using Potential Energy Distribution (PED) were calculated with scaled quantum mechanics. The infrared intensities and Raman intensities of (+)-(S)-2-(6-methoxynaphthalen-2-yl) propanoic acid were reported. Frontier molecular orbital analysis and reactivity parameters were calculated. Molecular Electrostatic Potential (MEP), Natural Bond Orbital (NBO) analysis, Non Linear Optical (NLO) behavior and thermodynamic properties were studied. In addition, the Mulliken charge distribution and Fukui function were analyzed. Molecular docking was used to dock in the title molecule into the active site of the protein 5L9B which belongs to the class of proteins exhibiting the property as a HIF1A (Hypoxia-inducible factor 1-alpha) expression inhibitor and the minimum binding energy was detected to be -6.2 kcal/mol.

Graphene oxide as an optimal candidate material for methane storage.

PubMed

Chouhan, Rajiv K; Ulman, Kanchan; Narasimhan, Shobhana

2015-07-28

Methane, the primary constituent of natural gas, binds too weakly to nanostructured carbons to meet the targets set for on-board vehicular storage to be viable. We show, using density functional theory calculations, that replacing graphene by graphene oxide increases the adsorption energy of methane by 50%. This enhancement is sufficient to achieve the optimal binding strength. In order to gain insight into the sources of this increased binding, that could also be used to formulate design principles for novel storage materials, we consider a sequence of model systems that progressively take us from graphene to graphene oxide. A careful analysis of the various contributions to the weak binding between the methane molecule and the graphene oxide shows that the enhancement has important contributions from London dispersion interactions as well as electrostatic interactions such as Debye interactions, aided by geometric curvature induced primarily by the presence of epoxy groups.

Role of Molecular Dynamics and Related Methods in Drug Discovery.

PubMed

De Vivo, Marco; Masetti, Matteo; Bottegoni, Giovanni; Cavalli, Andrea

2016-05-12

Molecular dynamics (MD) and related methods are close to becoming routine computational tools for drug discovery. Their main advantage is in explicitly treating structural flexibility and entropic effects. This allows a more accurate estimate of the thermodynamics and kinetics associated with drug-target recognition and binding, as better algorithms and hardware architectures increase their use. Here, we review the theoretical background of MD and enhanced sampling methods, focusing on free-energy perturbation, metadynamics, steered MD, and other methods most consistently used to study drug-target binding. We discuss unbiased MD simulations that nowadays allow the observation of unsupervised ligand-target binding, assessing how these approaches help optimizing target affinity and drug residence time toward improved drug efficacy. Further issues discussed include allosteric modulation and the role of water molecules in ligand binding and optimization. We conclude by calling for more prospective studies to attest to these methods' utility in discovering novel drug candidates.

Analysis of calcium-induced conformational changes in calcium-binding allergens and quantitative determination of their IgE binding properties.

PubMed

Parody, Nuria; Fuertes, Miguel Angel; Alonso, Carlos; Pico de Coaña, Yago

2013-01-01

The polcalcin family is one of the most epidemiologically relevant families of calcium-binding allergens. Polcalcins are potent plant allergens that contain one or several EF-hand motifs and their allergenicity is primarily associated with the Ca(2+)-bound form of the protein. Conformation, stability, as well as IgE recognition of calcium-binding allergens greatly depend on the presence of protein-bound calcium ions. We describe a protocol that uses three techniques (SDS-PAGE, circular dichroism spectroscopy, and ELISA) to describe the effects that calcium has on the structural changes in an allergen and its IgE binding properties.

Atomistic Tight-Binding Theory Applied to Structural and Optical Properties of Silicon Nanodisks

NASA Astrophysics Data System (ADS)

Sukkabot, Worasak

2018-05-01

The use of ultrathin crystalline silicon (c-Si) wafers in solar cells necessitates a highly effective light absorber to compensate for poor light absorption. One route to overcoming this problem is to use a periodic array of Si nanodisks on ultrathin c-Si. In the present manuscript, we numerically investigate the effects of the geometrical parameters of the Si nanodisks, including disk diameter (D) and length (L), on the structural and optical properties, using atomistic tight-binding theory. These computations confirm that the electronic structure and optical properties are sensitive to the structural parameters. As the disk diameter and length increase, the single-electron energies decrease, and the single-hole energies increase. These calculations also reveal that, because of the quantum confinement effect, the optical band gaps gradually decrease independently of the increasing disk diameter and length. The optical spectra can be tuned across the visible region by varying the disk diameter and length, which is a useful feature for optimizing light absorption in solar cell applications. As the disk diameter and length increased, the optical intensities also increased; however, the atomistic electron-hole interactions and ground electron-hole wave function overlap progressively decreased. The ground electron-hole wave function overlap, Stokes shift, and fine structure splitting decreased as the disk diameter and length were increased. Thus, Si nanodisks with a large diameter and length might be a suitable candidate source of entangled photons. The Si nanodisks in this study also show promise for applications to solar cells based on ultrathin c-Si wafers.

Theoretical insights into effects of molar ratios on stabilities, mechanical properties and detonation performance of CL-20/RDX cocrystal explosives by molecular dynamics simulation

NASA Astrophysics Data System (ADS)

Hang, Gui-yun; Yu, Wen-li; Wang, Tao; Wang, Jin-tao; Li, Zhen

2017-08-01

The CL-20/RDX cocrystal models with different molar ratios were established by substitution method and molecular dynamics (MD) simulation method was applied to investigate the influences of molar ratios on mechanical properties, stabilities and detonation performance of cocrystal explosives. The crystal parameters, structures, binding energies, mechanical properties and some detonation parameters of different cocrystal explosives were got and compared. The results illustrate that the molar ratio has a direct influence on properties of cocrystal explosive and each of the cocrystal model holds different mechanical properties, binding energies and detonation parameters. The mechanical properties of CL-20/RDX cocrystal explosive can be effectively improved and the cocrystal model with molar ratio in 1:1 has the best mechanical properties. Besides, it has the highest binding energy, so the stability and compatibility is the best. The detonation parameters show that the cocrystal explosive has better detonation performance than RDX. In a word, the cocrystal explosive with molar ratio in 1:1 has the best mechanical properties, highest binding energy and excellent energy density and detonation performance, it is quite promising and can satisfy the requirements of high energy density compounds (HEDC). This paper could offer some theoretical instructions and novel insights for the CL-20 cocrystal explosive designing.

Quantitative theory of hydrophobic effect as a driving force of protein structure

PubMed Central

Perunov, Nikolay; England, Jeremy L

2014-01-01

Various studies suggest that the hydrophobic effect plays a major role in driving the folding of proteins. In the past, however, it has been challenging to translate this understanding into a predictive, quantitative theory of how the full pattern of sequence hydrophobicity in a protein shapes functionally important features of its tertiary structure. Here, we extend and apply such a phenomenological theory of the sequence-structure relationship in globular protein domains, which had previously been applied to the study of allosteric motion. In an effort to optimize parameters for the model, we first analyze the patterns of backbone burial found in single-domain crystal structures, and discover that classic hydrophobicity scales derived from bulk physicochemical properties of amino acids are already nearly optimal for prediction of burial using the model. Subsequently, we apply the model to studying structural fluctuations in proteins and establish a means of identifying ligand-binding and protein–protein interaction sites using this approach. PMID:24408023

Modulation of the fibrillogenesis inhibition properties of two transthyretin ligands by halogenation.

PubMed

Cotrina, Ellen Y; Pinto, Marta; Bosch, Lluís; Vilà, Marta; Blasi, Daniel; Quintana, Jordi; Centeno, Nuria B; Arsequell, Gemma; Planas, Antoni; Valencia, Gregorio

2013-11-27

The amyloidogenic protein transthyretin (TTR) is thought to aggregate into amyloid fibrils by tetramer dissociation which can be inhibited by a number of small molecule compounds. Our analysis of a series of crystallographic protein-inhibitor complexes has shown no clear correlation between the observed molecular interactions and the in vitro activity of the inhibitors. From this analysis, it emerged that halogen bonding (XB) could be mediating some key interactions. Analysis of the halogenated derivatives of two well-known TTR inhibitors has shown that while flufenamic acid affinity for TTR was unchanged by halogenation, diflunisal gradually improves binding up to 1 order of magnitude after iodination through interactions that can be interpreted as a suboptimal XB (carbonyl Thr106: I...O distance 3.96-4.05 Å; C-I...O angle 152-156°) or as rather optimized van der Waals contacts or as a mixture of both. These results illustrate the potential of halogenation strategies in designing and optimizing TTR fibrillogenesis inhibitors.

Enhancing the cellular anti-proliferation activity of pyridazinones as c-met inhibitors using docking analysis.

PubMed

Xing, Weiqiang; Ai, Jing; Jin, Shiyu; Shi, Zhangxing; Peng, Xia; Wang, Lang; Ji, Yinchun; Lu, Dong; Liu, Yang; Geng, Meiyu; Hu, Youhong

2015-05-05

A series of 2, 6-disubstituted pyridazinone derivatives were evaluated and optimized for their c-Met inhibitory activity in enzyme and cellular assay. An analysis of the SAR results arising from computer modeling analysis of members of the library led to the proposal that in order to obtain optimal inhibitory activity in cellular systems the lipophilic/hydrophilic properties of individual structural fragments in the inhibitors need to match those of corresponding binding pockets in the enzyme. Guided by this proposal, the quinoline-pyridazinone 8a, containing hydrophobic 6-indolyl pyridazinone and quinoline moieties along with a hydrophilic morpholine terminal group, was designed and synthesized. The results of studies with this substance showed that it is a selective c-Met inhibitor with both a high enzyme inhibition IC50 value of 4.2 nM and a high EBC-1 cell proliferation inhibition IC50 value of 17 nM. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Optimized ratiometric calcium sensors for functional in vivo imaging of neurons and T lymphocytes.

PubMed

Thestrup, Thomas; Litzlbauer, Julia; Bartholomäus, Ingo; Mues, Marsilius; Russo, Luigi; Dana, Hod; Kovalchuk, Yuri; Liang, Yajie; Kalamakis, Georgios; Laukat, Yvonne; Becker, Stefan; Witte, Gregor; Geiger, Anselm; Allen, Taylor; Rome, Lawrence C; Chen, Tsai-Wen; Kim, Douglas S; Garaschuk, Olga; Griesinger, Christian; Griesbeck, Oliver

2014-02-01

The quality of genetically encoded calcium indicators (GECIs) has improved dramatically in recent years, but high-performing ratiometric indicators are still rare. Here we describe a series of fluorescence resonance energy transfer (FRET)-based calcium biosensors with a reduced number of calcium binding sites per sensor. These 'Twitch' sensors are based on the C-terminal domain of Opsanus troponin C. Their FRET responses were optimized by a large-scale functional screen in bacterial colonies, refined by a secondary screen in rat hippocampal neuron cultures. We tested the in vivo performance of the most sensitive variants in the brain and lymph nodes of mice. The sensitivity of the Twitch sensors matched that of synthetic calcium dyes and allowed visualization of tonic action potential firing in neurons and high resolution functional tracking of T lymphocytes. Given their ratiometric readout, their brightness, large dynamic range and linear response properties, Twitch sensors represent versatile tools for neuroscience and immunology.

A sensitive capacitive immunosensor for direct detection of human heart fatty acid-binding protein (h-FABP).

PubMed

Mihailescu, Carmen-Marinela; Stan, Dana; Iosub, Rodica; Moldovan, Carmen; Savin, Mihaela

2015-01-01

The fabrication of a capacitive interdigitated immunosensor (CID) based on a mixed self-assembled monolayer (mSAM) film for the direct detection of heart fatty-acid binding protein (h-FABP) without any labeling is described. The capacitance changes of mSAMs vs. homogenous ordered self-assembled monolayers (hSAMs) on gold work electrodes/covalently bonded antibodies/buffered medium are utilized for monitoring the specific antibody-antigen interaction. Capacitance measurements in the absence and presence of Faradaic currents were performed. The electrochemical properties of mixed monolayers were compared with those of a pure monolayer of 11-mercaptoundecanoic acid (MUA) self-assembled on gold surfaces. Taking into account the stability of the studied monolayers during the electrochemical experiments with the Faradaic process, the best SAM functionalization method was used for developing a sensitive capacitive immunosensor with a non-Faradaic process for direct immune detection of human h-FABP. Under the optimized conditions, the proposed mixed self-assembled monolayer (mSAM1) on gold electrode exhibited good insulating properties such as a capacitive behavior when detecting h-FABP from human serum in the range of 98 pg ml(-1)-100 ng ml(-1), with a detection limit of 0.836 ng ml(-1) comparative with a homogenous self-assembled monolayer (hSAM). Copyright © 2014 Elsevier B.V. All rights reserved.

Binding properties and colour of Bologna sausage made with varying fat levels, protein levels and cooking temperatures.

PubMed

Carballo, J; Mota, N; Barreto, G; Colmenero, F J

1995-01-01

A little-studied procedure for adjusting the properties of low-fat products is to use the influence that both composition and certain processing factors exert on these properties. The object of the present work was to assess the effects of protein level (P, ranging from 10% to 16%), fat level (F, ranging from 10.1% to 22%) and cooking temperature (HT, ranging from 77 °C to 105 °C) on the binding properties and colour of meat emulsions. Protein content was the variable that most influenced total expressible fluid (TEF) and purge loss. Heating rate had scarcely any effect on the binding properties of Bologna sausages. Analysis of variance indicated that the regression models for parameters L, a and b were not significant.

Hydrogen-Deuterium Exchange Mass Spectrometry Reveals Calcium Binding Properties and Allosteric Regulation of Downstream Regulatory Element Antagonist Modulator (DREAM).

PubMed

Zhang, Jun; Li, Jing; Craig, Theodore A; Kumar, Rajiv; Gross, Michael L

2017-07-18

Downstream regulatory element antagonist modulator (DREAM) is an EF-hand Ca 2+ -binding protein that also binds to a specific DNA sequence, downstream regulatory elements (DRE), and thereby regulates transcription in a calcium-dependent fashion. DREAM binds to DRE in the absence of Ca 2+ but detaches from DRE under Ca 2+ stimulation, allowing gene expression. The Ca 2+ binding properties of DREAM and the consequences of the binding on protein structure are key to understanding the function of DREAM. Here we describe the application of hydrogen-deuterium exchange mass spectrometry (HDX-MS) and site-directed mutagenesis to investigate the Ca 2+ binding properties and the subsequent conformational changes of full-length DREAM. We demonstrate that all EF-hands undergo large conformation changes upon calcium binding even though the EF-1 hand is not capable of binding to Ca 2+ . Moreover, EF-2 is a lower-affinity site compared to EF-3 and -4 hands. Comparison of HDX profiles between wild-type DREAM and two EF-1 mutated constructs illustrates that the conformational changes in the EF-1 hand are induced by long-range structural interactions. HDX analyses also reveal a conformational change in an N-terminal leucine-charged residue-rich domain (LCD) remote from Ca 2+ -binding EF-hands. This LCD domain is responsible for the direct interaction between DREAM and cAMP response element-binding protein (CREB) and regulates the recruitment of the co-activator, CREB-binding protein. These long-range interactions strongly suggest how conformational changes transmit the Ca 2+ signal to CREB-mediated gene transcription.

Tuning transport properties of graphene three-terminal structures by mechanical deformation

NASA Astrophysics Data System (ADS)

Torres, V.; Faria, D.; Latgé, A.

2018-04-01

Straintronic devices made of carbon-based materials have been pushed up due to the graphene high mechanical flexibility and the possibility of interesting changes in transport properties. Properly designed strained systems have been proposed to allow optimized transport responses that can be explored in experimental realizations. In multiterminal systems, comparisons between schemes with different geometries are important to characterize the modifications introduced by mechanical deformations, especially if the deformations are localized at a central part of the system or extended in a large region. Then, in the present analysis, we study the strain effects on the transport properties of triangular and hexagonal graphene flakes, with zigzag and armchair edges, connected to three electronic terminals, formed by semi-infinite graphene nanoribbons. Using the Green's function formalism with circular renormalization schemes, and a single band tight-binding approximation, we find that resonant tunneling transport becomes relevant and is more affected by localized deformations in the hexagonal graphene flakes. Moreover, triangular systems with deformation extended to the leads, like longitudinal three-folded type, are shown as an interesting scenario for building nanoscale waveguides for electronic current.

Formulation, physicochemical characterization and stability study of lithium-loaded microemulsion system.

PubMed

Mouri, Abdelkader; Legrand, Philippe; El Ghzaoui, Abdeslam; Dorandeu, Christophe; Maurel, Jean Claude; Devoisselle, Jean-Marie

2016-04-11

Lithium biocompatible microemulsion based on Peceol(®), lecithin, ethanol and water was studied in attempt to identify the optimal compositions in term of drug content, physicochemical properties and stability. Lithium solubilization in microemulsion was found to be compatible with a drug-surfactant binding model. Lithium ions were predominantly solubilized within lecithin head group altering significantly the interfacial properties of the system. Pseudo-ternary phase diagrams of drug free and drug loaded microemulsions were built at constant ethanol/lecithin weight ratio (40/60). Lithium loaded microemulsion has totally disappeared in the Peceol(®) rich part of phase diagram; critical fractions of lecithin and ethanol were required for the formation of stable microemulsion. The effect of lithium concentration on the properties and physical stability of microemulsions were studied using microscopy, Karl Fischer titrations, rheology analyses, conductivity measurements and centrifugation tests. The investigated microemulsions were found to be stable under accelerated storage conditions. The systems exhibited low viscosity and behaved as Newtonian fluid and no structural transition was shown. Copyright © 2016 Elsevier B.V. All rights reserved.

Gadolinium-Based Contrast Agents for MR Cancer Imaging

PubMed Central

Zhou, Zhuxian; Lu, Zheng-Rong

2013-01-01

Magnetic resonance imaging (MRI) is a clinical imaging modality effective for anatomical and functional imaging of diseased soft tissues, including solid tumors. MRI contrast agents have been routinely used for detecting tumor at an early stage. Gadolinium based contrast agents are the most commonly used contrast agents in clinical MRI. There have been significant efforts to design and develop novel Gd(III) contrast agents with high relaxivity, low toxicity and specific tumor binding. The relaxivity of the Gd(III) contrast agents can be increased by proper chemical modification. The toxicity of Gd(III) contrast agents can be reduced by increasing the agents’ thermodynamic and kinetic stability, as well as optimizing their pharmacokinetic properties. The increasing knowledge in the field of cancer genomics and biology provides an opportunity for designing tumor-specific contrast agents. Various new Gd(III) chelates have been designed and evaluated in animal models for more effective cancer MRI. This review outlines the design and development, physicochemical properties, and in vivo properties of several classes of Gd(III)-based MR contrast agents for tumor imaging. PMID:23047730

New Parameters for Higher Accuracy in the Computation of Binding Free Energy Differences upon Alanine Scanning Mutagenesis on Protein-Protein Interfaces.

PubMed

Simões, Inês C M; Costa, Inês P D; Coimbra, João T S; Ramos, Maria J; Fernandes, Pedro A

2017-01-23

Knowing how proteins make stable complexes enables the development of inhibitors to preclude protein-protein (P:P) binding. The identification of the specific interfacial residues that mostly contribute to protein binding, denominated as hot spots, is thus critical. Here, we refine an in silico alanine scanning mutagenesis protocol, based on a residue-dependent dielectric constant version of the Molecular Mechanics/Poisson-Boltzmann Surface Area method. We have used a large data set of structurally diverse P:P complexes to redefine the residue-dependent dielectric constants used in the determination of binding free energies. The accuracy of the method was validated through comparison with experimental data, considering the per-residue P:P binding free energy (ΔΔG binding ) differences upon alanine mutation. Different protocols were tested, i.e., a geometry optimization protocol and three molecular dynamics (MD) protocols: (1) one using explicit water molecules, (2) another with an implicit solvation model, and (3) a third where we have carried out an accelerated MD with explicit water molecules. Using a set of protein dielectric constants (within the range from 1 to 20) we showed that the dielectric constants of 7 for nonpolar and polar residues and 11 for charged residues (and histidine) provide optimal ΔΔG binding predictions. An overall mean unsigned error (MUE) of 1.4 kcal mol -1 relative to the experiment was achieved in 210 mutations only with geometry optimization, which was further reduced with MD simulations (MUE of 1.1 kcal mol -1 for the MD employing explicit solvent). This recalibrated method allows for a better computational identification of hot spots, avoiding expensive and time-consuming experiments or thermodynamic integration/ free energy perturbation/ uBAR calculations, and will hopefully help new drug discovery campaigns in their quest of searching spots of interest for binding small drug-like molecules at P:P interfaces.

Pyrene maleimide as a probe of microenvironmental and dynamics properties of protein binding sites

NASA Astrophysics Data System (ADS)

Benci, S.; Vaccari, S.; Schianchi, G.; Locatelli, Donata; Vaghi, P.; Bottiroli, Giovanni F.

1995-01-01

N-(1-Pyrene)maleimide is highly fluorescent upon covalent binding with sulfhydryl and amino groups of the proteins. Multiexponential fluorescence decays were observed for the dye bound to different proteins even when a single binding site is involved. The lack of information about the fluorescence decay of free dye does not allow to define the variations of fluorescence parameter following the conjugation and their correlation with the binding properties of the fluorophore. In this work, a study of the fluorescence of the probe, free in solution, bound to different antibodies and to the antigen-antibody complex both in solution and in cell, has been performed. The experimental results showed that chemico-physical properties of the medium influence the fluorescence decay of the probe in both the free and bound forms, although to a different extent. The variations of fluorescence decay and anisotropy of the bound probe are related to the electronic characteristics of microenvironment and show an increased stabilization of the probe binding site with the increasing complexity of the substrate. The sensitivity of the fluorescence properties of the probe to the binding site environment opens interesting perspectives concerning the application of Py- maleimide fluorochromization to assess the degree of specificity of immunocytochemical labelling.

Computational studies of metal-metal and metal-ligand interactions

NASA Technical Reports Server (NTRS)

Barnes, Leslie A.

1992-01-01

The geometric structure of Cr(CO)6 is optimized at the modified coupled-pair functional (MCPF), single and double excitation coupled-cluster (CCSD) and CCSD(T) levels of theory (including a perturbational estimate for connected triple excitations), and the force constants for the totally symmetric representation are determined. The geometry of Cr(CO)5 is partially optimized at the MCPF, CCSD and CCSD(T) levels of theory. Comparison with experimental data shows that the CCSD(T) method gives the best results for the structures and force constants, and that remaining errors are probably due to deficiencies in the one-particle basis sets used for CO. A detailed comparison of the properties of free CO is therefore given, at both the MCPF and CCSD/CCSD(T) levels of treatment, using a variety of basis sets. With very large one-particle basis sets, the SSCD(T) method gives excellent results for the bond distance, dipole moment and harmonic frequency of free CO. The total binding energies of Cr(CO)6 and Cr(CO)5 are also determined at the MCPF, CCSD and CCSD(T) levels of theory. The CCSD(T) method gives a much larger total binding energy than either the MCPF or CCSD methods. An analysis of the basis set superposition error (BSSE) at the MCPF level of treatment points out limitations in the one-particle basis used here and in a previous study. Calculations using larger basis sets reduced the BSSE, but the total binding energy of Cr(CO)6 is still significantly smaller than the experimental value, although the first CO bond dissociation energy of Cr(CO)6 is well described. An investigation of 3s3p correlation reveals only a small effect. The remaining discrepancy between the experimental and theoretical total binding energy of Cr(CO)6 is probably due to limitations in the one-particle basis, rather than limitations in the correlation treatment. In particular an additional d function and an f function on each C and O are needed to obtain quantitative results. This is underscored by the fact that even using a very large primitive se (1042 primitive functions contracted to 300 basis functions), the superposition error for the total binding energy of Cr(CO)6 is 22 kcal/mol at the MCPF level of treatment.

Molecular Docking Study on Galantamine Derivatives as Cholinesterase Inhibitors.

PubMed

Atanasova, Mariyana; Yordanov, Nikola; Dimitrov, Ivan; Berkov, Strahil; Doytchinova, Irini

2015-06-01

A training set of 22 synthetic galantamine derivatives binding to acetylcholinesterase was docked by GOLD and the protocol was optimized in terms of scoring function, rigidity/flexibility of the binding site, presence/absence of a water molecule inside and radius of the binding site. A moderate correlation was found between the affinities of compounds expressed as pIC50 values and their docking scores. The optimized docking protocol was validated by an external test set of 11 natural galantamine derivatives and the correlation coefficient between the docking scores and the pIC50 values was 0.800. The derived relationship was used to analyze the interactions between galantamine derivatives and AChE. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle.

DTIC Science & Technology

1996-08-01

J-4030 TITLE: The In Vivo DNA Binding Properties of Wild-Type and Mutant p53 Proteins in Mammary Cell Lines During the Course of Cell Cycle PRINCIPAL...The In Vivo DNA Binding Properties of 5. FUNDING NUMBERS Wild-Type and Mutant p53 Proteins in Mammary Cell Lines DAMD17-94-J-4030 During the Course of...ABSTRACT (Maximum 200 Using a pair of murine cell lines, one lacking p53 and a derivative cell line containing temperature sensitive p53 val 135

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

PubMed Central

Webb, C F; Cadman, H F; Wallis, M

1986-01-01

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes. PMID:3790086

Attractors in Sequence Space: Agent-Based Exploration of MHC I Binding Peptides.

PubMed

Jäger, Natalie; Wisniewska, Joanna M; Hiss, Jan A; Freier, Anja; Losch, Florian O; Walden, Peter; Wrede, Paul; Schneider, Gisbert

2010-01-12

Ant Colony Optimization (ACO) is a meta-heuristic that utilizes a computational analogue of ant trail pheromones to solve combinatorial optimization problems. The size of the ant colony and the representation of the ants' pheromone trails is unique referring to the given optimization problem. In the present study, we employed ACO to generate novel peptides that stabilize MHC I protein on the plasma membrane of a murine lymphoma cell line. A jury of feedforward neural network classifiers served as fitness function for peptide design by ACO. Bioactive murine MHC I H-2K(b) stabilizing as well as nonstabilizing octapeptides were designed, synthesized and tested. These peptides reveal residue motifs that are relevant for MHC I receptor binding. We demonstrate how the performance of the implemented ACO algorithm depends on the colony size and the size of the search space. The actual peptide design process by ACO constitutes a search path in sequence space that can be visualized as trajectories on a self-organizing map (SOM). By projecting the sequence space on a SOM we visualize the convergence of the different solutions that emerge during the optimization process in sequence space. The SOM representation reveals attractors in sequence space for MHC I binding peptides. The combination of ACO and SOM enables systematic peptide optimization. This technique allows for the rational design of various types of bioactive peptides with minimal experimental effort. Here, we demonstrate its successful application to the design of MHC-I binding and nonbinding peptides which exhibit substantial bioactivity in a cell-based assay. Copyright © 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Effect of PEG-PDMAEMA Block Copolymer Architecture on Polyelectrolyte Complex Formation with Heparin.

PubMed

Välimäki, Salla; Khakalo, Alexey; Ora, Ari; Johansson, Leena-Sisko; Rojas, Orlando J; Kostiainen, Mauri A

2016-09-12

Heparin is a naturally occurring polyelectrolyte consisting of a sulfated polysaccharide backbone. It is widely used as an anticoagulant during major surgical operations. However, the associated bleeding risks require rapid neutralization after the operation. The only clinically approved antidote for heparin is protamine sulfate, which is, however, ineffective against low molecular weight heparin and can cause severe adverse reactions in patients. In this study, the facile synthesis of cationic-neutral diblock copolymers and their effective heparin binding is presented. Poly(ethylene glycol)-poly(2-(dimethylamino)ethyl methacrylate) (PEG-PDMAEMA) block copolymers were synthesized in two steps via atom-transfer radical polymerization (ATRP) using PEG as a macroinitiator. Solution state binding between heparin and a range of PEG-PDMAEMA block copolymers and one homopolymer was studied with dynamic light scattering and methylene blue displacement assay. Also in vitro binding in plasma was studied by utilizing a chromogenic heparin anti-Xa assay. Additionally, quartz crystal microbalance and multiparametric surface plasmon resonance were used to study the surface adsorption kinetics of the polymers on a heparin layer. It was shown that the block copolymers and heparin form electrostatically bound complexes with varying colloidal properties, where the block lengths play a key role in controlling the heparin binding affinity, polyelectrolyte complex size and surface charge. With the optimized polymers (PEG114PDMAEMA52 and PEG114PDMAEMA100), heparin could be neutralized in a dose-dependent manner, and bound efficiently into small neutral complexes, with a hydrodynamic radius less than 100 nm. These complexes had only a limited effect on cell viability. Based on these studies, our approach paves the way for the development of new polymeric heparin binding agents.

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13.

PubMed

Cruz, Jorddy N; Costa, José F S; Khayat, André S; Kuca, Kamil; Barros, Carlos A L; Neto, A M J C

2018-05-04

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG bind ) obtained for the Tam16 lead molecule was of -51.43 kcal/mol. When comparing this result with the ΔG bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC 50 for Tam16 is the smallest necessary to inhibit the Pks13 (IC 50  = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.

Reactions of guanine with methyl chloride and methyl bromide: O6-methylation versus charge transfer complex formation

NASA Astrophysics Data System (ADS)

Shukla, P. K.; Mishra, P. C.; Suhai, S.

Density functional theory (DFT) at the B3LYP/6-31+G* and B3LYP/AUG-cc-pVDZ levels was employed to study O6-methylation of guanine due to its reactions with methyl chloride and methyl bromide and to obtain explanation as to why the methyl halides cause genotoxicity and possess mutagenic and carcinogenic properties. Geometries of the various isolated species involved in the reactions, reactant complexes (RCs), and product complexes (PCs) were optimized in gas phase. Transition states connecting the reactant complexes with the product complexes were also optimized in gas phase at the same levels of theory. The reactant complexes, product complexes, and transition states were solvated in aqueous media using the polarizable continuum model (PCM) of the self-consistent reaction field theory. Zero-point energy (ZPE) correction to total energy and the corresponding thermal energy correction to enthalpy were made in each case. The reactant complexes of the keto form of guanine with methyl chloride and methyl bromide in water are appreciably more stable than the corresponding complexes involving the enol form of guanine. The nature of binding in the product complexes was found to be of the charge transfer type (O6mG+ · X-, X dbond Cl, Br). Binding of HCl, HBr, and H2O molecules to the PCs obtained with the keto form of guanine did not alter the positions of the halide anions in the PCs, and the charge transfer character of the PCs was also not modified due to this binding. Further, the complexes obtained due to the binding of HCl, HBr, and H2O molecules to the PCs had greater stability than the isolated PCs. The reaction barriers involved in the formation of PCs were found to be quite high (?50 kcal/mol). Mechanisms of genotoxicity, mutagenesis and carcinogenesis caused by the methyl halides appear to involve charge transfer-type complex formation. Thus the mechanisms of these processes involving the methyl halides appear to be quite different from those that involve the other strongly carcinogenic methylating agents.

Nutraceutical, Anti-Inflammatory, and Immune Modulatory Effects of β-Glucan Isolated from Yeast

PubMed Central

Bacha, Umar; Iqbal, Sanaullah; Anjum, Aftab Ahmad

2017-01-01

β-Glucan is a dietary fibre, found in many natural sources, and controls chronic metabolic diseases effectively. However, β-glucan from the yeast has rarely been investigated. Objectively, conditions were optimized to isolate β-glucan from the yeast (max. 66% yield); those optimized conditions included 1.0 M NaOH, pH 7.0, and 90°C. The purity and identity of the isolated β-glucan were characterized through FT-IR, SEM, DSC, and physicofunctional properties. The obtained results from DSC revealed highly stable β-glucan (m.p., 125°C) with antioxidant activity (TAC value 0.240 ± 0.0021 µg/mg, H2O2 scavenging 38%), which has promising bile acid binding 40.463% and glucose control (in vitro). In line with these results, we evaluated the in vivo anti-inflammatory potential, that is, myeloperoxidase activity and reduction in MDA and NO; protective effect on proteins and keeping viscosity within normal range exhibited improvement. Also, the in vivo cholesterol binding and reduction in the skin thickness by β-glucan were highly encouraging. Finally, our results confirmed that yeast β-glucan is effective against some of the inflammatory and oxidative stress markers studied in this investigation. In general, the effect of 4%  β-glucan was more noticeable versus 2%  β-glucan. Therefore, our results support the utilization of β-glucan as a novel, economically cheap, and functional food ingredient. PMID:28913359

Ground-state properties of 4He and 16O extrapolated from lattice QCD with pionless EFT

DOE Office of Scientific and Technical Information (OSTI.GOV)

Contessi, L.; Lovato, A.; Pederiva, F.

Here, we extend the prediction range of Pionless Effective Field Theory with an analysis of the ground state of 16O in leading order. To renormalize the theory, we use as input both experimental data and lattice QCD predictions of nuclear observables, which probe the sensitivity of nuclei to increased quark masses. The nuclear many-body Schrödinger equation is solved with the Auxiliary Field Diffusion Monte Carlo method. For the first time in a nuclear quantum Monte Carlo calculation, a linear optimization procedure, which allows us to devise an accurate trial wave function with a large number of variational parameters, is adopted.more » The method yields a binding energy of 4He which is in good agreement with experiment at physical pion mass and with lattice calculations at larger pion masses. At leading order we do not find any evidence of a 16O state which is stable against breakup into four 4He, although higher-order terms could bind 16O.« less

Ground-state properties of 4He and 16O extrapolated from lattice QCD with pionless EFT

DOE PAGES

Contessi, L.; Lovato, A.; Pederiva, F.; ...

2017-07-26

Here, we extend the prediction range of Pionless Effective Field Theory with an analysis of the ground state of 16O in leading order. To renormalize the theory, we use as input both experimental data and lattice QCD predictions of nuclear observables, which probe the sensitivity of nuclei to increased quark masses. The nuclear many-body Schrödinger equation is solved with the Auxiliary Field Diffusion Monte Carlo method. For the first time in a nuclear quantum Monte Carlo calculation, a linear optimization procedure, which allows us to devise an accurate trial wave function with a large number of variational parameters, is adopted.more » The method yields a binding energy of 4He which is in good agreement with experiment at physical pion mass and with lattice calculations at larger pion masses. At leading order we do not find any evidence of a 16O state which is stable against breakup into four 4He, although higher-order terms could bind 16O.« less

Synthesis of galactosyl compounds for targeted gene delivery.

PubMed

Ren, T; Zhang, G; Liu, D

2001-11-01

Cell-specific DNA delivery offers a great potential for targeted gene therapy. Toward this end, we have synthesized a series of compounds carrying galactose residues as a targeting ligand for asialoglycoprotein receptors of hepatocytes and primary amine groups as a functional domain for DNA binding. Biological activity of these galactosyl compounds in DNA delivery was evaluated in HepG2 and BL-6 cells and compared with respect to the number of galactose residues as well as primary amine groups in each molecule. Transfection experiments using a firefly luciferase gene as a reporter revealed that compounds with multivalent binding properties were more active in DNA delivery. An optimal transfection activity in HepG2 cells requires seven primary amine groups and a minimum of two galactose residues in each molecule. The transfection activity of compounds carrying multi-galactose residues can be inhibited by asialofetuin, a natural substrate for asialoglycoprotein receptors of hepatocytes, suggesting that gene transfer by these galactosyl compounds is asialoglycoprotein receptor-mediated. These results provide direct evidence in support of our new strategy for the use of small and synthetic compounds for cell specific and targeted gene delivery.

Adaptation of avian influenza A (H6N1) virus from avian to human receptor-binding preference

PubMed Central

Wang, Fei; Qi, Jianxun; Bi, Yuhai; Zhang, Wei; Wang, Min; Zhang, Baorong; Wang, Ming; Liu, Jinhua; Yan, Jinghua; Shi, Yi; Gao, George F

2015-01-01

The receptor-binding specificity of influenza A viruses is a major determinant for the host tropism of the virus, which enables interspecies transmission. In 2013, the first human case of infection with avian influenza A (H6N1) virus was reported in Taiwan. To gather evidence concerning the epidemic potential of H6 subtype viruses, we performed comprehensive analysis of receptor-binding properties of Taiwan-isolated H6 HAs from 1972 to 2013. We propose that the receptor-binding properties of Taiwan-isolated H6 HAs have undergone three major stages: initially avian receptor-binding preference, secondarily obtaining human receptor-binding capacity, and recently human receptor-binding preference, which has been confirmed by receptor-binding assessment of three representative virus isolates. Mutagenesis work revealed that E190V and G228S substitutions are important to acquire the human receptor-binding capacity, and the P186L substitution could reduce the binding to avian receptor. Further structural analysis revealed how the P186L substitution in the receptor-binding site of HA determines the receptor-binding preference change. We conclude that the human-infecting H6N1 evolved into a human receptor preference. PMID:25940072

Computational Design of Ligand Binding Proteins with High Affinity and Selectivity

PubMed Central

Dou, Jiayi; Doyle, Lindsey; Nelson, Jorgen W.; Schena, Alberto; Jankowski, Wojciech; Kalodimos, Charalampos G.; Johnsson, Kai; Stoddard, Barry L.; Baker, David

2014-01-01

The ability to design proteins with high affinity and selectivity for any given small molecule would have numerous applications in biosensing, diagnostics, and therapeutics, and is a rigorous test of our understanding of the physiochemical principles that govern molecular recognition phenomena. Attempts to design ligand binding proteins have met with little success, however, and the computational design of precise molecular recognition between proteins and small molecules remains an “unsolved problem”1. We describe a general method for the computational design of small molecule binding sites with pre-organized hydrogen bonding and hydrophobic interfaces and high overall shape complementary to the ligand, and use it to design protein binding sites for the steroid digoxigenin (DIG). Of 17 designs that were experimentally characterized, two bind DIG; the highest affinity design has the lowest predicted interaction energy and the most pre-organized binding site in the set. A comprehensive binding-fitness landscape of this design generated by library selection and deep sequencing was used to guide optimization of binding affinity to a picomolar level, and two X-ray co-crystal structures of optimized complexes show atomic level agreement with the design models. The designed binder has a high selectivity for DIG over the related steroids digitoxigenin, progesterone, and β-estradiol, which can be reprogrammed through the designed hydrogen-bonding interactions. Taken together, the binding fitness landscape, co-crystal structures, and thermodynamic binding parameters illustrate how increases in binding affinity can result from distal sequence changes that limit the protein ensemble to conformers making the most energetically favorable interactions with the ligand. The computational design method presented here should enable the development of a new generation of biosensors, therapeutics, and diagnostics. PMID:24005320

Comparative study of the molecularly imprinted polymers prepared by reversible addition-fragmentation chain transfer "bulk" polymerization and traditional radical "bulk" polymerization.

PubMed

Ma, Yue; Pan, Guoqing; Zhang, Ying; Guo, Xianzhi; Zhang, Huiqi

2013-05-01

Bisphenol A (BPA) and propranolol-imprinted polymers have been prepared via both reversible addition-fragmentation chain transfer "bulk" polymerization (RAFTBP) and traditional radical "bulk" polymerization (TRBP) under similar reaction conditions, and their equilibrium binding properties were compared in detail for the first time. The chemical compositions, specific surface areas, equilibrium bindings, and selectivity of the obtained molecularly imprinted polymers (MIPs) were systematically characterized. The experimental results showed that the MIPs with molecular imprinting effects and quite fast binding kinetics could be readily prepared via RAFTBP, but they did not show improved template binding properties in comparison with those prepared via TRBP, which is in sharp contrast to many previous reports. This could be attributed to the heavily interrupted equilibrium between the dormant species and active radicals in the RAFT mechanism because of the occurrence of fast gelation during RAFTBP. The findings presented here strongly demonstrates that the application of controlled radical polymerizations (CRPs) in molecular imprinting does not always benefit the binding properties of the resultant MIPs, which is of significant importance for the rational use of CRPs in generating MIPs with improved properties. Copyright © 2013 John Wiley & Sons, Ltd.

Ion-binding properties of the ClC chloride selectivity filter

PubMed Central

Lobet, Séverine; Dutzler, Raimund

2006-01-01

The ClC channels are members of a large protein family of chloride (Cl−) channels and secondary active Cl− transporters. Despite their diverse functions, the transmembrane architecture within the family is conserved. Here we present a crystallographic study on the ion-binding properties of the ClC selectivity filter in the close homolog from Escherichia coli (EcClC). The ClC selectivity filter contains three ion-binding sites that bridge the extra- and intracellular solutions. The sites bind Cl− ions with mM affinity. Despite their close proximity within the filter, the three sites can be occupied simultaneously. The ion-binding properties are found conserved from the bacterial transporter EcClC to the human Cl− channel ClC-1, suggesting a close functional link between ion permeation in the channels and active transport in the transporters. In resemblance to K+ channels, ions permeate the ClC channel in a single file, with mutual repulsion between the ions fostering rapid conduction. PMID:16341087

Comparative study of classification algorithms for immunosignaturing data

PubMed Central

2012-01-01

Background High-throughput technologies such as DNA, RNA, protein, antibody and peptide microarrays are often used to examine differences across drug treatments, diseases, transgenic animals, and others. Typically one trains a classification system by gathering large amounts of probe-level data, selecting informative features, and classifies test samples using a small number of features. As new microarrays are invented, classification systems that worked well for other array types may not be ideal. Expression microarrays, arguably one of the most prevalent array types, have been used for years to help develop classification algorithms. Many biological assumptions are built into classifiers that were designed for these types of data. One of the more problematic is the assumption of independence, both at the probe level and again at the biological level. Probes for RNA transcripts are designed to bind single transcripts. At the biological level, many genes have dependencies across transcriptional pathways where co-regulation of transcriptional units may make many genes appear as being completely dependent. Thus, algorithms that perform well for gene expression data may not be suitable when other technologies with different binding characteristics exist. The immunosignaturing microarray is based on complex mixtures of antibodies binding to arrays of random sequence peptides. It relies on many-to-many binding of antibodies to the random sequence peptides. Each peptide can bind multiple antibodies and each antibody can bind multiple peptides. This technology has been shown to be highly reproducible and appears promising for diagnosing a variety of disease states. However, it is not clear what is the optimal classification algorithm for analyzing this new type of data. Results We characterized several classification algorithms to analyze immunosignaturing data. We selected several datasets that range from easy to difficult to classify, from simple monoclonal binding to complex binding patterns in asthma patients. We then classified the biological samples using 17 different classification algorithms. Using a wide variety of assessment criteria, we found ‘Naïve Bayes’ far more useful than other widely used methods due to its simplicity, robustness, speed and accuracy. Conclusions ‘Naïve Bayes’ algorithm appears to accommodate the complex patterns hidden within multilayered immunosignaturing microarray data due to its fundamental mathematical properties. PMID:22720696

Effect of enzymatic deamidation of soy protein by protein-glutaminase on the flavor-binding properties of the protein under aqueous conditions.

PubMed

Suppavorasatit, Inthawoot; Cadwallader, Keith R

2012-08-15

The effect of the enzymatic deamidation by protein-glutaminase (PG) on flavor-binding properties of soy protein isolate (SPI) under aqueous conditions was evaluated by a modified equilibrium dialysis (ultrafiltration) technique. Binding parameters, such as number of binding sites (n) and binding constants (K), were derived from Klotz plots. The partial deamidation of SPI by PG (43.7% degree of deamidation) decreased overall flavor-binding affinity (nK) at 25 °C for both vanillin and maltol by approximately 9- and 4-fold, respectively. The thermodynamic parameters of binding indicated that the flavor-protein interactions were spontaneous (negative ΔG°) and that the driving force of the interactions shifted from entropy to enthalpy driven as a result of deamidation. Deamidation of soy protein caused a change in the mechanism of binding from hydrophobic interactions or covalent bonding (Schiff base formation) to weaker van der Waals forces or hydrogen bonding.

ProBiS-CHARMMing: Web Interface for Prediction and Optimization of Ligands in Protein Binding Sites.

PubMed

Konc, Janez; Miller, Benjamin T; Štular, Tanja; Lešnik, Samo; Woodcock, H Lee; Brooks, Bernard R; Janežič, Dušanka

2015-11-23

Proteins often exist only as apo structures (unligated) in the Protein Data Bank, with their corresponding holo structures (with ligands) unavailable. However, apoproteins may not represent the amino-acid residue arrangement upon ligand binding well, which is especially problematic for molecular docking. We developed the ProBiS-CHARMMing web interface by connecting the ProBiS ( http://probis.cmm.ki.si ) and CHARMMing ( http://www.charmming.org ) web servers into one functional unit that enables prediction of protein-ligand complexes and allows for their geometry optimization and interaction energy calculation. The ProBiS web server predicts ligands (small compounds, proteins, nucleic acids, and single-atom ligands) that may bind to a query protein. This is achieved by comparing its surface structure against a nonredundant database of protein structures and finding those that have binding sites similar to that of the query protein. Existing ligands found in the similar binding sites are then transposed to the query according to predictions from ProBiS. The CHARMMing web server enables, among other things, minimization and potential energy calculation for a wide variety of biomolecular systems, and it is used here to optimize the geometry of the predicted protein-ligand complex structures using the CHARMM force field and to calculate their interaction energies with the corresponding query proteins. We show how ProBiS-CHARMMing can be used to predict ligands and their poses for a particular binding site, and minimize the predicted protein-ligand complexes to obtain representations of holoproteins. The ProBiS-CHARMMing web interface is freely available for academic users at http://probis.nih.gov.

Design, synthesis and biological evaluation of (S)-valine thiazole-derived cyclic and non-cyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1)

PubMed Central

Singh, Satyakam; Prasad, Nagarajan Rajendra; Kapoor, Khyati; Chufan, Eduardo E.; Patel, Bhargav A.; Ambudkar, Suresh V.; Talele, Tanaji T.

2014-01-01

Multidrug resistance (MDR) caused by ATP-binding cassette (ABC) transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure to cancer chemotherapy. Previously, selenazole containing cyclic peptides were reported as P-gp inhibitors and these were also used for co-crystallization with mouse P-gp, which has 87% homology to human P-gp. It has been reported that human P-gp, can simultaneously accommodate 2-3 moderate size molecules at the drug binding pocket. Our in-silico analysis based on the homology model of human P-gp spurred our efforts to investigate the optimal size of (S)-valine-derived thiazole units that can be accommodated at drug-binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)-valine-derived thiazole units to investigate the optimal size, lipophilicity and the structural form (linear and cyclic) of valine-derived thiazole peptides that can accommodate well in the P-gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear- (13) and cyclic-trimer (17) derivatives of QZ59S-SSS were found to be the most and equally potent inhibitors of human P-gp (IC50 = 1.5 μM). Cyclic trimer and linear trimer being equipotent, future studies can be focused on non-cyclic counterparts of cyclic peptides maintaining linear trimer length. Binding model of the linear trimer (13) within the drug-binding site on the homology model of human P-gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the non-cyclic form. PMID:24288265

Design, synthesis, and biological evaluation of (S)-valine thiazole-derived cyclic and noncyclic peptidomimetic oligomers as modulators of human P-glycoprotein (ABCB1).

PubMed

Singh, Satyakam; Prasad, Nagarajan Rajendra; Kapoor, Khyati; Chufan, Eduardo E; Patel, Bhargav A; Ambudkar, Suresh V; Talele, Tanaji T

2014-01-03

Multidrug resistance caused by ATP binding cassette transporter P-glycoprotein (P-gp) through extrusion of anticancer drugs from the cells is a major cause of failure in cancer chemotherapy. Previously, selenazole-containing cyclic peptides were reported as P-gp inhibitors and were also used for co-crystallization with mouse P-gp, which has 87 % homology to human P-gp. It has been reported that human P-gp can simultaneously accommodate two to three moderately sized molecules at the drug binding pocket. Our in silico analysis, based on the homology model of human P-gp, spurred our efforts to investigate the optimal size of (S)-valine-derived thiazole units that can be accommodated at the drug binding pocket. Towards this goal, we synthesized varying lengths of linear and cyclic derivatives of (S)-valine-derived thiazole units to investigate the optimal size, lipophilicity, and structural form (linear or cyclic) of valine-derived thiazole peptides that can be accommodated in the P-gp binding pocket and affects its activity, previously an unexplored concept. Among these oligomers, lipophilic linear (13) and cyclic trimer (17) derivatives of QZ59S-SSS were found to be the most and equally potent inhibitors of human P-gp (IC50 =1.5 μM). As the cyclic trimer and linear trimer compounds are equipotent, future studies should focus on noncyclic counterparts of cyclic peptides maintaining linear trimer length. A binding model of the linear trimer 13 within the drug binding site on the homology model of human P-gp represents an opportunity for future optimization, specifically replacing valine and thiazole groups in the noncyclic form. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A practical approach to automate randomized design of experiments for ligand-binding assays.

PubMed

Tsoi, Jennifer; Patel, Vimal; Shih, Judy

2014-03-01

Design of experiments (DOE) is utilized in optimizing ligand-binding assay by modeling factor effects. To reduce the analyst's workload and error inherent with DOE, we propose the integration of automated liquid handlers to perform the randomized designs. A randomized design created from statistical software was imported into custom macro converting the design into a liquid-handler worklist to automate reagent delivery. An optimized assay was transferred to a contract research organization resulting in a successful validation. We developed a practical solution for assay optimization by integrating DOE and automation to increase assay robustness and enable successful method transfer. The flexibility of this process allows it to be applied to a variety of assay designs.

Facile characterization of aptamer kinetic and equilibrium binding properties using surface plasmon resonance

PubMed Central

Chang, Andrew L.; McKeague, Maureen; Smolke, Christina D.

2015-01-01

Nucleic acid aptamers find widespread use as targeting and sensing agents in nature and biotechnology. Their ability to bind an extensive range of molecular targets, including small molecules, proteins, and ions, with high affinity and specificity enables their use in diverse diagnostic, therapeutic, imaging, and gene-regulatory applications. Here, we describe methods for characterizing aptamer kinetic and equilibrium binding properties using a surface plasmon resonance-based platform. This aptamer characterization platform is broadly useful for studying aptamer–ligand interactions, comparing aptamer properties, screening functional aptamers during in vitro selection processes, and prototyping aptamers for integration into nucleic acid devices. PMID:25432760

Investigating DNA Binding and Conformational Variation in Temperature Sensitive p53 Cancer Mutants Using QM-MM Simulations

PubMed Central

Koulgi, Shruti; Achalere, Archana; Sonavane, Uddhavesh; Joshi, Rajendra

2015-01-01

The tp53 gene is found to be mutated in 50% of all the cancers. The p53 protein, a product of tp53 gene, is a multi-domain protein. It consists of a core DNA binding domain (DBD) which is responsible for its binding and transcription of downstream target genes. The mutations in p53 protein are responsible for creating cancerous conditions and are found to be occurring at a high frequency in the DBD region of p53. Some of these mutations are also known to be temperature sensitive (ts) in nature. They are known to exhibit partial or strong binding with DNA in the temperature range (298–306 K). Whereas, at 310 K and above they show complete loss in binding. We have analyzed the changes in binding and conformational behavior at 300 K and 310 K for three of the ts-mutants viz., V143A, R249S and R175H. QM-MM simulations have been performed on the wild type and the above mentioned ts-mutants for 30 ns each. The optimal estimate of free energy of binding for a particular number of interface hydrogen bonds was calculated using the maximum likelihood method as described by Chodera et. al (2007). This parameter has been observed to be able to mimic the binding affinity of the p53 ts-mutants at 300 K and 310 K. Thus the correlation between MM-GBSA free energy of binding and hydrogen bonds formed by the interface residues between p53 and DNA has revealed the temperature dependent nature of these mutants. The role of main chain dihedrals was obtained by performing dihedral principal component analysis (PCA). This analysis, suggests that the conformational variations in the main chain dihedrals (ϕ and ψ) of the p53 ts-mutants may have caused reduction in the overall stability of the protein. The solvent exposure of the side chains of the interface residues were found to hamper the binding of the p53 to the DNA. Solvent Accessible Surface Area (SASA) also proved to be a crucial property in distinguishing the conformers obtained at 300 K and 310 K for the three ts-mutants from the wild type at 300 K. PMID:26579714

Quantitation of the calcium and membrane binding properties of the C2 domains of dysferlin.

PubMed

Abdullah, Nazish; Padmanarayana, Murugesh; Marty, Naomi J; Johnson, Colin P

2014-01-21

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca(2+) sensitive, the Ca(2+) binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca(2+) and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca(2+) with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with Kd values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar Kd value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca(2+) enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca(2+) albeit with varying affinity and stoichiometry. Copyright © 2014 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Global optimization of small bimetallic Pd-Co binary nanoalloy clusters: a genetic algorithm approach at the DFT level.

PubMed

Aslan, Mikail; Davis, Jack B A; Johnston, Roy L

2016-03-07

The global optimisation of small bimetallic PdCo binary nanoalloys are systematically investigated using the Birmingham Cluster Genetic Algorithm (BCGA). The effect of size and composition on the structures, stability, magnetic and electronic properties including the binding energies, second finite difference energies and mixing energies of Pd-Co binary nanoalloys are discussed. A detailed analysis of Pd-Co structural motifs and segregation effects is also presented. The maximal mixing energy corresponds to Pd atom compositions for which the number of mixed Pd-Co bonds is maximised. Global minimum clusters are distinguished from transition states by vibrational frequency analysis. HOMO-LUMO gap, electric dipole moment and vibrational frequency analyses are made to enable correlation with future experiments.

Targeted polymeric therapeutic nanoparticles: design, development and clinical translation†

PubMed Central

Kamaly, Nazila; Xiao, Zeyu; Valencia, Pedro M.; Radovic-Moreno, Aleksandar F.; Farokhzad, Omid C.

2013-01-01

Polymeric materials have been used in a range of pharmaceutical and biotechnology products for more than 40 years. These materials have evolved from their earlier use as biodegradable products such as resorbable sutures, orthopaedic implants, macroscale and microscale drug delivery systems such as microparticles and wafers used as controlled drug release depots, to multifunctional nanoparticles (NPs) capable of targeting, and controlled release of therapeutic and diagnostic agents. These newer generations of targeted and controlled release polymeric NPs are now engineered to navigate the complex in vivo environment, and incorporate functionalities for achieving target specificity, control of drug concentration and exposure kinetics at the tissue, cell, and subcellular levels. Indeed this optimization of drug pharmacology as aided by careful design of multifunctional NPs can lead to improved drug safety and efficacy, and may be complimentary to drug enhancements that are traditionally achieved by medicinal chemistry. In this regard, polymeric NPs have the potential to result in a highly differentiated new class of therapeutics, distinct from the original active drugs used in their composition, and distinct from first generation NPs that largely facilitated drug formulation. A greater flexibility in the design of drug molecules themselves may also be facilitated following their incorporation into NPs, as drug properties (solubility, metabolism, plasma binding, biodistribution, target tissue accumulation) will no longer be constrained to the same extent by drug chemical composition, but also become in-part the function of the physicochemical properties of the NP. The combination of optimally designed drugs with optimally engineered polymeric NPs opens up the possibility of improved clinical outcomes that may not be achievable with the administration of drugs in their conventional form. In this critical review, we aim to provide insights into the design and development of targeted polymeric NPs and to highlight the challenges associated with the engineering of this novel class of therapeutics, including considerations of NP design optimization, development and biophysicochemical properties. Additionally, we highlight some recent examples from the literature, which demonstrate current trends and novel concepts in both the design and utility of targeted polymeric NPs (444 references). PMID:22388185

Binding free energy analysis of protein-protein docking model structures by evERdock.

PubMed

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-14

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Binding free energy analysis of protein-protein docking model structures by evERdock

NASA Astrophysics Data System (ADS)

Takemura, Kazuhiro; Matubayasi, Nobuyuki; Kitao, Akio

2018-03-01

To aid the evaluation of protein-protein complex model structures generated by protein docking prediction (decoys), we previously developed a method to calculate the binding free energies for complexes. The method combines a short (2 ns) all-atom molecular dynamics simulation with explicit solvent and solution theory in the energy representation (ER). We showed that this method successfully selected structures similar to the native complex structure (near-native decoys) as the lowest binding free energy structures. In our current work, we applied this method (evERdock) to 100 or 300 model structures of four protein-protein complexes. The crystal structures and the near-native decoys showed the lowest binding free energy of all the examined structures, indicating that evERdock can successfully evaluate decoys. Several decoys that show low interface root-mean-square distance but relatively high binding free energy were also identified. Analysis of the fraction of native contacts, hydrogen bonds, and salt bridges at the protein-protein interface indicated that these decoys were insufficiently optimized at the interface. After optimizing the interactions around the interface by including interfacial water molecules, the binding free energies of these decoys were improved. We also investigated the effect of solute entropy on binding free energy and found that consideration of the entropy term does not necessarily improve the evaluations of decoys using the normal model analysis for entropy calculation.

Extended Lagrangian formulation of charge-constrained tight-binding molecular dynamics.

PubMed

Cawkwell, M J; Coe, J D; Yadav, S K; Liu, X-Y; Niklasson, A M N

2015-06-09

The extended Lagrangian Born-Oppenheimer molecular dynamics formalism [Niklasson, Phys. Rev. Lett., 2008, 100, 123004] has been applied to a tight-binding model under the constraint of local charge neutrality to yield microcanonical trajectories with both precise, long-term energy conservation and a reduced number of self-consistent field optimizations at each time step. The extended Lagrangian molecular dynamics formalism restores time reversal symmetry in the propagation of the electronic degrees of freedom, and it enables the efficient and accurate self-consistent optimization of the chemical potential and atomwise potential energy shifts in the on-site elements of the tight-binding Hamiltonian that are required when enforcing local charge neutrality. These capabilities are illustrated with microcanonical molecular dynamics simulations of a small metallic cluster using an sd-valent tight-binding model for titanium. The effects of weak dissipation on the propagation of the auxiliary degrees of freedom for the chemical potential and on-site Hamiltonian matrix elements that is used to counteract the accumulation of numerical noise during trajectories was also investigated.

Chlorozotocin

PubMed Central

Panasci, Lawrence C.; Green, Dianna; Schein, Philip S.

1979-01-01

Chlorozotocin is a chloroethyl nitrosourea with a glucose carrier that has curative activity for the murine L1210 leukemia, but is nonmyelosuppressive in mice. To determine the mechanism for this unique property of reduced bone marrow toxicity, comparative studies were conducted with chlorozotocin and CCNU, a myelotoxic chloroethyl nitrosourea. Suspensions of L1210 leukemia and murine bone marrow cells were incubated for 2 h with 0.1 mM [14C]-chloroethyl chlorozotocin or CCNU. Chlorozotocin demonstrated a fourfold increased covalent binding of the chloroethyl group to L1210 nuclei when compared to equimolar CCNU. Chlorozotocin alkylation of L1210 cells resulted in the binding of 57 pmol of [14C]ethyl group/mg of DNA, which represented a 2.3-fold increased alkylation when compared to CCNU. In marked contrast, the binding of the chloroethyl group to bone marrow nuclei was equivalent for both drugs. In addition, chlorozotocin alkylation of murine bone marrow DNA, 45 pmol of [14C]ethyl group/mg of DNA, was equivalent to that of CCNU. The ratio of L1210:bone marrow DNA alkylation was 1.3 for chlorozotocin compared to 0.6 for CCNU. The intracellular carbamoylation of L1210 and bone marrow protein by CCNU was 400- to 600-fold greater than that produced by chlorozotocin. After a 2-h exposure to 0.1, 0.05, or 0.01 mM drug, both chlorozotocin and CCNU produced a reduction in the cloning efficiency of L1210 cells that was dose dependent. However, chlorozotocin was significantly more cytotoxic than CCNU at all three molar concentrations (P < 0.01). Chlorozotocin, 0.1 mM, reduced L1210 DNA synthesis to 1% of control by 48 h, in contrast to 16% with equimolar CCNU (P < 0.01). In mice bearing 105 L1210 cells, chlorozotocin produced its optimal antitumor activity (332% increased life span [ILS]) at doses of 48-64 μmol/kg, with >50% indefinite survivors. In contrast, CCNU at the same molar doses resulted in only a 191% ILS; a CCNU dose of 128 μmol/kg was required for comparable optimal L1210 antitumor activity, 413% ILS. On a molar basis, the dose of chlorozotocin that produced optimal in vivo L1210 antitumor activity was one-third to one-half that of CCNU. Chlorozotocin, unlike CCNU, produced no murine bone marrow toxicity at its optimal therapeutic dose. This unique combination of antitumor activity without myelosuppression can be correlated with the advantageous ratio of L1210:bone marrow in vitro DNA alkylation by chlorozotocin (1.3) as compared to equimolar CCNU (0.6). PMID:158033

Accurate ensemble molecular dynamics binding free energy ranking of multidrug-resistant HIV-1 proteases.

PubMed

Sadiq, S Kashif; Wright, David W; Kenway, Owain A; Coveney, Peter V

2010-05-24

Accurate calculation of important thermodynamic properties, such as macromolecular binding free energies, is one of the principal goals of molecular dynamics simulations. However, single long simulation frequently produces incorrectly converged quantitative results due to inadequate sampling of conformational space in a feasible wall-clock time. Multiple short (ensemble) simulations have been shown to explore conformational space more effectively than single long simulations, but the two methods have not yet been thermodynamically compared. Here we show that, for end-state binding free energy determination methods, ensemble simulations exhibit significantly enhanced thermodynamic sampling over single long simulations and result in accurate and converged relative binding free energies that are reproducible to within 0.5 kcal/mol. Completely correct ranking is obtained for six HIV-1 protease variants bound to lopinavir with a correlation coefficient of 0.89 and a mean relative deviation from experiment of 0.9 kcal/mol. Multidrug resistance to lopinavir is enthalpically driven and increases through a decrease in the protein-ligand van der Waals interaction, principally due to the V82A/I84V mutation, and an increase in net electrostatic repulsion due to water-mediated disruption of protein-ligand interactions in the catalytic region. Furthermore, we correctly rank, to within 1 kcal/mol of experiment, the substantially increased chemical potency of lopinavir binding to the wild-type protease compared to saquinavir and show that lopinavir takes advantage of a decreased net electrostatic repulsion to confer enhanced binding. Our approach is dependent on the combined use of petascale computing resources and on an automated simulation workflow to attain the required level of sampling and turn around time to obtain the results, which can be as little as three days. This level of performance promotes integration of such methodology with clinical decision support systems for the optimization of patient-specific therapy.

Pharmacokinetics and concentration-effect relationships of therapeutic monoclonal antibodies and fusion proteins.

PubMed

Ternant, David; Paintaud, Gilles

2005-09-01

Although monoclonal antibodies (mAbs) constitute a major advance in therapeutics, their pharmacokinetic (PK) and pharmacodynamic (PD) properties are not fully understood. Saturable mechanisms are thought to occur in distribution and elimination of mAbs, which are protected from degradation by the Brambell's receptor (FcRn). The binding of mAbs to their target antigen explains part of their nonlinear PK and PD properties. The interindividual variability in mAb PK can be explained by several factors, including immune response against the biodrug and differences in the number of antigenic sites. The concentration-effect relationships of mAbs are complex and dependent on their mechanism of action. Interindividual differences in mAb PD can be explained by factors such as genetics and clinical status. PK and concentration-effect studies are necessary to design optimal dosing regimens. Because of their above-mentioned characteristics, the interindividual variability in their dose-response relationships must be studied by PK-PD modelling.

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds.

PubMed

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-03-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

New Thiazolyl-triazole Schiff Bases: Synthesis and Evaluation of the Anti-Candida Potential.

PubMed

Stana, Anca; Enache, Alexandra; Vodnar, Dan Cristian; Nastasă, Cristina; Benedec, Daniela; Ionuț, Ioana; Login, Cezar; Marc, Gabriel; Oniga, Ovidiu; Tiperciuc, Brîndușa

2016-11-22

In the context of the dangerous phenomenon of fungal resistance to the available therapies, we present here the chemical synthesis of a new series of thiazolyl-triazole Schiff bases B1 - B15 , which were in vitro assessed for their anti- Candida potential. Compound B10 was found to be more potent against Candida spp. when compared with the reference drugs Fluconazole and Ketoconazole. A docking study of the newly synthesized Schiff bases was performed, and results showed good binding affinity in the active site of co-crystallized Itraconazole-lanosterol 14α-demethylase isolated from Saccharomyces cerevisiae . An in silico ADMET (absorption, distribution, metabolism, excretion, toxicity) study was done in order to predict some pharmacokinetic and pharmacotoxicological properties. The Schiff bases showed good drug-like properties. The results of in vitro anti- Candida activity, a docking study and ADMET prediction revealed that the newly synthesized compounds have potential anti- Candida activity and evidenced the most active derivative, B10 , which can be further optimized as a lead compound.

Two-dimensional materials from high-throughput computational exfoliation of experimentally known compounds

NASA Astrophysics Data System (ADS)

Mounet, Nicolas; Gibertini, Marco; Schwaller, Philippe; Campi, Davide; Merkys, Andrius; Marrazzo, Antimo; Sohier, Thibault; Castelli, Ivano Eligio; Cepellotti, Andrea; Pizzi, Giovanni; Marzari, Nicola

2018-02-01

Two-dimensional (2D) materials have emerged as promising candidates for next-generation electronic and optoelectronic applications. Yet, only a few dozen 2D materials have been successfully synthesized or exfoliated. Here, we search for 2D materials that can be easily exfoliated from their parent compounds. Starting from 108,423 unique, experimentally known 3D compounds, we identify a subset of 5,619 compounds that appear layered according to robust geometric and bonding criteria. High-throughput calculations using van der Waals density functional theory, validated against experimental structural data and calculated random phase approximation binding energies, further allowed the identification of 1,825 compounds that are either easily or potentially exfoliable. In particular, the subset of 1,036 easily exfoliable cases provides novel structural prototypes and simple ternary compounds as well as a large portfolio of materials to search from for optimal properties. For a subset of 258 compounds, we explore vibrational, electronic, magnetic and topological properties, identifying 56 ferromagnetic and antiferromagnetic systems, including half-metals and half-semiconductors.

Identification of glycosaminoglycans using high-performance liquid chromatography on a hydroxyapatite column.

PubMed

Narita, H; Takeda, Y; Takagaki, K; Nakamura, T; Harata, S; Endo, M

1995-11-20

Glycosaminoglycans (heparin, heparan sulfate, dermatan sulfate, chondroitin sulfate, and hyaluronic acid) were labeled with a fluorescent reagent, 2-aminopyridine. The fluoro-labeled glycosaminoglycans were subjected to high-performance liquid chromatography on a hydroxyapatite column. The binding property of each glycosaminoglycan to hydroxyapatite was different. The structural properties of glycosaminoglycans bound to hydroxyapatite were then investigated using chemical desulfated or enzymic depolymerized glycosaminoglycans. This revealed that the sulfate content and molecular weight of the glycosaminoglycans correlated with their binding properties to hydroxyapatite. Desulfated dermatan sulfate but not desulfated chondroitin 6-sulfate bound to the hydroxyapatite. These data indicate that iduronic acid residues of glycosaminoglycans are important for the binding property. The method described which uses hydroxyapatite columns facilitates rapid separation and microanalysis of the glycosaminoglycans, especially dermatan sulfate and chondroitin sulfate.

Contacts between the factor TUF and RPG sequences.

PubMed

Vignais, M L; Huet, J; Buhler, J M; Sentenac, A

1990-08-25

The yeast TUF factor binds specifically to RPG-like sequences involved in multiple functions at enhancers, silencers, and telomeres. We have characterized the interaction of TUF with its optimal binding sequence, rpg-1 (1-ACACCCATACATTT-14), using a gel DNA-binding assay in combination with methylation protection and mutagenesis experiments. As many as 10 base pairs appear to be engaged in factor binding. Analysis of a collection of 30 different RPG mutants demonstrated the importance of 8 base pairs at position 2, 3, 4, 5, 6, 7, 10, and 12 and the critical role of the central GC pair at position 5. Methylation protection data on four different natural sites confirmed a close contact at positions 4, 5, 6, and 10 and suggested additional contacts at base pairs 8, 12, and 13. The derived consensus sequence was RCAAYCCRYNCAYY. A quantitative band shift analysis was used to determine the equilibrium dissociation constant for the complex of TUF and its optimal binding site rpg-1. The specific dissociation constant (K8) was found to be 1.3 x 10(-11) M. The comparison of the K8 value with the dissociation constant obtained for nonspecific DNA sites (Kn8 = 8.7 x 10(-6) M) shows the high binding selectivity of TUF for its specific RPG target.

Computational screening of functional groups for capture of toxic industrial chemicals in porous materials.

PubMed

Kim, Ki Chul; Fairen-Jimenez, David; Snurr, Randall Q

2017-12-06

A thermodynamic analysis using quantum chemical methods was carried out to identify optimal functional group candidates that can be included in metal-organic frameworks and activated carbons for the selective capture of toxic industrial chemicals (TICs) in humid air. We calculated the binding energies of 14 critical TICs plus water with a series of 10 functional groups attached to a naphthalene ring model. Using vibrational calculations, the free energies of adsorption were calculated in addition to the binding energies. Our results show that, in these systems, the binding energies and free energies follow similar trends. We identified copper(i) carboxylate as the optimal functional group (among those studied) for the selective binding of the majority of the TICs in humid air, and this functional group exhibits especially strong binding for sulfuric acid. Further thermodynamic analysis shows that the presence of water weakens the binding strength of sulfuric acid with the copper carboxylate group. Our calculations predict that functionalization of aromatic rings would be detrimental to selective capture of COCl 2 , CO 2 , and Cl 2 under humid conditions. Finally, we found that forming an ionic complex, H 3 O + HSO 4 - , between H 2 SO 4 and H 2 O via proton transfer is not favorable on copper carboxylate.

Characterization and modification of phage T7 DNA polymerase for use in DNA sequencing; Progress report, June 1, 1990--May 31, 1993

DOE Office of Scientific and Technical Information (OSTI.GOV)

Richardson, C.C.

1993-12-31

This project focuses on the DNA polymerase (gene 5 protein) of phage T7 for use in DNA sequence analysis. Gene 5 protein interacts with accessory proteins to acquire properties essential for DNA replication. One goal is to understand these interactions in order to modify the proteins for use in DNA sequencing. E. coli thioredoxin, binds to gene 5 protein and clamps it to a primer-template. They have analyzed the binding of gene 5 protein-thioredoxin to primer-templates and have defined the optimal conditions to form an extremely stable complex with a dNTP in the polymerase catalytic site. The spatial proximity ofmore » these components has been determined using fluorescence emission anisotropy. The T7 DNA binding protein, the gene 2.5 protein, interacts with gene 5 protein and gene 4 protein to increase processivity and primer synthesis, respectively. Mutant gene 2.5 proteins have been isolated that do not interact with T7 DNA polymerase and can not support T7 growth. The nucleotide binding site of the T7 helicase has been identified and mutations affecting the site provide information on how the hydrolysis of NTPs fuel its unidirectional translocation. The sequence, GTC, has been shown to be necessary and sufficient for recognition by the T7 primase. The T7 gene 5.5 protein interacts with the E. coli nucleoid protein, H-NS, and also overcomes the phage {lambda} rex restriction system.« less

Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts

USDA-ARS?s Scientific Manuscript database

The surface properties of aged (stored for 10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labeled with fluorescein isothiocyanate while aged oocysts did not. In ...

Docking-based Screening of Ficus religiosa Phytochemicals as Inhibitors of Human Histamine H2 Receptor.

PubMed

Chaudhary, Amit; Yadav, Birendra Singh; Singh, Swati; Maurya, Pramod Kumar; Mishra, Alok; Srivastva, Shweta; Varadwaj, Pritish Kumar; Singh, Nand Kumar; Mani, Ashutosh

2017-10-01

Ficus religiosa L. is generally known as Peepal and belongs to family Moraceae . The tree is a source of many compounds having high medicinal value. In gastrointestinal tract, histamine H2 receptors have key role in histamine-stimulated gastric acid secretion. Their over stimulation causes its excessive production which is responsible for gastric ulcer. This study aims to screen the range of phytochemicals present in F. religiosa for binding with human histamine H2 and identify therapeutics for a gastric ulcer from the plant. In this work, a 3D-structure of human histamine H2 receptor was modeled by using homology modeling and the predicted model was validated using PROCHECK. Docking studies were also performed to assess binding affinities between modeled receptor and 34 compounds. Molecular dynamics simulations were done to identify most stable receptor-ligand complexes. Absorption, distribution, metabolism, excretion, and screening was done to evaluate pharmacokinetic properties of compounds. The results suggest that seven ligands, namely, germacrene, bergaptol, lanosterol, Ergost-5-en-3beta-ol, α-amyrin acetate, bergapten, and γ-cadinene showed better binding affinities. Among seven phytochemicals, lanosterol and α-amyrin acetate were found to have greater stability during simulation studies. These two compounds may be a suitable therapeutic agent against histamine H2 receptor. This study was performed to screen antiulcer compounds from F. religiosa . Molecular modeling, molecular docking and MD simulation studies were performed with selected phytochemicals from F. religiosa . The analysis suggests that Lanosterol and α-amyrin may be a suitable therapeutic agent against histamine H2 receptor. This study facilitates initiation of the herbal drug discovery process for the antiulcer activity. Abbreviations used: ADMET: Absorption, distribution, metabolism, excretion and toxicity, DOPE: Discrete Optimized Potential Energy, OPLS: Optimized potential for liquid simulations, RMSD: Root-mean-square deviation, HOA: Human oral absorption, MW: Molecular weight, SP: Standard-precision, XP: Extra-precision, GPCRs: G protein-coupled receptors, SASA: Solvent accessible surface area, Rg: Radius of gyration, NHB: Number of hydrogen bond.

Design, Synthesis, and Evaluation of Dihydrobenzo[cd]indole-6-sulfonamide as TNF-α Inhibitors.

PubMed

Deng, Xiaobing; Zhang, Xiaoling; Tang, Bo; Liu, Hongbo; Shen, Qi; Liu, Ying; Lai, Luhua

2018-01-01

Tumor necrosis factor-α (TNF-α) plays a pivotal role in inflammatory response. Dysregulation of TNF can lead to a variety of disastrous pathological effects, including auto-inflammatory diseases. Antibodies that directly targeting TNF-α have been proven effective in suppressing symptoms of these disorders. Compared to protein drugs, small molecule drugs are normally orally available and less expensive. Till now, peptide and small molecule TNF-α inhibitors are still in the early stage of development, and much more efforts should be made. In a previously study, we reported a TNF-α inhibitor, EJMC-1 with modest activity. Here, we optimized this compound by shape screen and rational design. In the first round, we screened commercial compound library for EJMC-1 analogs based on shape similarity. Out of the 68 compounds tested, 20 compounds showed better binding affinity than EJMC-1 in the SPR competitive binding assay. These 20 compounds were tested in cell assay and the most potent compound was 2-oxo-N-phenyl-1,2-dihydrobenzo[ cd ]indole-6-sulfonamide ( S10 ) with an IC 50 of 14 μM, which was 2.2-fold stronger than EJMC-1 . Based on the docking analysis of S10 and EJMC-1 binding with TNF-α, in the second round, we designed S10 analogs, purchased seven of them, and synthesized seven new compounds. The best compound, 4e showed an IC 50 -value of 3 μM in cell assay, which was 14-fold stronger than EJMC-1 . 4e was among the most potent TNF-α organic compound inhibitors reported so far. Our study demonstrated that 2-oxo-N-phenyl-1,2-dihydrobenzo[ cd ]indole-6-sulfonamide analogs could be developed as potent TNF-α inhibitors. 4e can be further optimized for its activity and properties. Our study provides insights into designing small molecule inhibitors directly targeting TNF-α and for protein-protein interaction inhibitor design.

Design, Synthesis, and Evaluation of Dihydrobenzo[cd]indole-6-sulfonamide as TNF-alpha Inhibitors

NASA Astrophysics Data System (ADS)

Deng, Xiaobing; Zhang, Xiaoling; Tang, Bo; Liu, Hongbo; Shen, Qi; Liu, Ying; Lai, Luhua

2018-04-01

Tumor necrosis factor-α (TNF-α) plays a pivotal role in inflammatory response. Dysregulation of TNF can lead to a variety of disastrous pathological effects, including auto-inflammatory diseases. Antibodies that directly targeting TNF-α have been proven effective in suppressing symptoms of these disorders. Compared to protein drugs, small molecule drugs are normally orally available and less expensive. Till now, peptide and small molecule TNF-α inhibitors are still in the early stage of development, and much more efforts should be made. In a previously study, we reported a TNF-α inhibitor, EJMC-1 with modest activity. Here, we optimized this compound by shape screen and rational design. In the first round, we screened commercial compound library for EJMC-1 analogs based on shape similarity. Out of the 68 compounds tested, 20 compounds showed better binding affinity than EJMC-1 in the SPR competitive binding assay. These 20 compounds were tested in cell assay and the most potent compound was 2-oxo-N-phenyl-1,2-dihydrobenzo[cd]indole-6-sulfonamide (S10) with an IC50 of 14 M, which was 2.2-fold stronger than EJMC-1. Based on the docking analysis of S10 and EJMC-1 binding with TNF-α, in the second round, we designed S10 analogues, purchased 7 of them and synthesized 7 new compounds. The best compound, 4e showed an IC50 value of 3 M in cell assay, which was 14-fold stronger than EJMC-1. 4e was among the most potent TNF-α organic compound inhibitors reported so far. Our study demonstrated that 2-oxo-N-phenyl-1,2-dihydrobenzo[cd]indole-6-sulfonamide analogues could be developed as potent TNF-α inhibitors. 4e can be further optimized for its activity and properties. Our study provides insights into designing small molecule inhibitors directly targeting TNF-α and for protein-protein interaction inhibitor design.

Antivirulence Isoquinolone Mannosides: Optimization of the Biaryl Aglycone for FimH Lectin Binding Affinity and Efficacy in the Treatment of Chronic UTI.

PubMed

Jarvis, Cassie; Han, Zhenfu; Kalas, Vasilios; Klein, Roger; Pinkner, Jerome S; Ford, Bradley; Binkley, Jana; Cusumano, Corinne K; Cusumano, Zachary; Mydock-McGrane, Laurel; Hultgren, Scott J; Janetka, James W

2016-02-17

Uropathogenic E. coli (UPEC) employ the mannose-binding adhesin FimH to colonize the bladder epithelium during urinary tract infection (UTI). Previously reported FimH antagonists exhibit good potency and efficacy, but low bioavailability and a short half-life in vivo. In a rational design strategy, we obtained an X-ray structure of lead mannosides and then designed mannosides with improved drug-like properties. We show that cyclizing the carboxamide onto the biphenyl B-ring aglycone of biphenyl mannosides into a fused heterocyclic ring, generates new biaryl mannosides such as isoquinolone 22 (2-methyl-4-(1-oxo-1,2-dihydroisoquinolin-7-yl)phenyl α-d-mannopyranoside) with enhanced potency and in vivo efficacy resulting from increased oral bioavailability. N-Substitution of the isoquinolone aglycone with various functionalities produced a new potent subseries of FimH antagonists. All analogues of the subseries have higher FimH binding affinity than unsubstituted lead 22, as determined by thermal shift differential scanning fluorimetry assay. Mannosides with pyridyl substitution on the isoquinolone group inhibit bacteria-mediated hemagglutination and prevent biofilm formation by UPEC with single-digit nanomolar potency, which is unprecedented for any FimH antagonists or any other antivirulence compounds reported to date. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Elucidating the interaction of clofazimine with bovine liver catalase; a comprehensive spectroscopic and molecular docking approach.

PubMed

Zaman, Masihuz; Nusrat, Saima; Zakariya, Syed Mohammad; Khan, Mohsin Vahid; Ajmal, Mohammad Rehan; Khan, Rizwan Hasan

2017-08-01

Nowadays, understanding of interface between protein and drugs has become an active research area of interest. These types of interactions provide structural guidelines in drug design with greater clinical efficacy. Thus, structural changes in catalase induced by clofazimine were monitored by various biophysical techniques including UV-visible spectrometer, fluorescence spectroscopy, circular dichroism, and dynamic light scattering techniques. Increase in absorption spectra (UV-visible spectrum) confers the complex formation between drug and protein. Fluorescence quenching with a binding constants of 2.47 × 10 4  M -1 revealed that clofazimine binds with protein. Using fluorescence resonance energy transfer, the distance (r) between the protein (donor) and drug (acceptor) was found to be 2.89 nm. Negative Gibbs free energy change (ΔG°) revealed that binding process is spontaneous. In addition, an increase in α-helicity was observed by far-UV circular dichroism spectra by adding clofazimine to protein. Dynamic light scattering results indicate that topology of bovine liver catalase was slightly altered in the presence of clofazimine. Hydrophobic interactions are the main forces between clofazimine and catalase interaction as depicted by molecular docking studies. Apart from hydrophobic interactions, some hydrogen bonding was also observed during docking method. The results obtained from the present study may establish abundant in optimizing the properties of ligand-protein mixtures relevant for numerous formulations. Copyright © 2017 John Wiley & Sons, Ltd.

Identification and characterization of a salivary-pellicle-binding peptide by phage display.

PubMed

Cukkemane, Nivedita; Bikker, Floris J; Nazmi, Kamran; Brand, Henk S; Veerman, Enno C I

2014-05-01

Dental biofilms are associated with oral diseases, making their control necessary. One way to control them is to prevent initial bacterial adherence to the salivary pellicle and thereby eventually decrease binding of late colonizing potential pathogens. The goal of this study was to generate a salivary-pellicle-binding peptide (SPBP) with antifouling activity towards primary colonizing bacteria. In order to achieve this goal we aimed to: (i) identify novel SPBPs by phage display; (ii) characterize the binding and antifouling properties of the selected SPBPs. A library of 2×10(9) phages displaying a random sequence of 12-mer peptides was used to identify peptides that bound selectively to the in vitro salivary pellicle. Three rounds of panning resulted in the selection of 10 pellicle-binding phages, each displaying a novel peptide sequence. The peptides were synthesized and their binding to the in vitro salivary pellicle was characterized in the presence and absence of calcium ions and Tween-20. The antifouling property of hydroxyapatite (HA) and saliva-coated HA discs treated with and without SPBPs were evaluated against Streptococcus gordonii. Ten unique SPBPs were identified using the phage display. One of these peptides, SPBP 10 (NSAAVRAYSPPS), exhibited significant binding to the in vitro salivary pellicle which was neither influenced by calcium ions, nor affected by up to 0.5% Tween-20. Its antifouling property against S. gordonii was significantly higher on the treated surfaces than on untreated surfaces. Use of the phage display library enabled us to find a specific SPBP with antifouling property towards S. gordonii. Copyright © 2014 Elsevier Ltd. All rights reserved.

Immunopharmacotherapeutic Manifolds and Modulation of Cocaine Overdose

PubMed Central

Treweek, Jennifer B.; Roberts, Amanda J.; Janda, Kim D.

2011-01-01

Cocaine achieves its psychostimulant, reinforcing properties through selectively blocking dopamine transporters, and this neurobiological mechanism impedes the use of classical receptor-antagonist pharmacotherapies to outcompete cocaine at CNS sites. Passive immunization with monoclonal antibodies (mAb) specific for cocaine circumvents this problem as drug is sequestered in the periphery prior to entry into the brain. To optimize an immunopharmacotherapeutic strategy for reversing severe cocaine toxicity, the therapeutic properties of mAb GNC92H2 IgG were compared to those of its engineered formats in a mouse overdose model. Whereas the extended half-life of an IgG justifies its application to the prophylactic treatment of addiction, the rapid, thorough biodistribution of mAb-based fragments, including F(ab')2, Fab and scFv, may correlate to accelerated scavenging of cocaine and reversal of toxicity. To test this hypothesis, mice were administered the anti-cocaine IgG (180 mg/kg, i.v.) or GNC92H2-based agent after receiving an LD50 cocaine dose (93 mg/kg, i.p.), and the timeline of overdose symptoms was recorded. All formats lowered the rate of lethality despite the >100-fold molar excess of drug to antibody binding capacity. However, only F(ab')2-92H2 and Fab-92H2 significantly attenuated the progression of premorbid behaviors, and Fab-92H2 prevented seizure generation in a percentage of mice. The calculation of serum half-life of each format demonstrated that the pharmacokinetic profile of Fab-92H2 (elimination half-life, t1/2 ∼ 100 minutes) best approximated that of cocaine. These results not only confirm the importance of highly specific and tight drug binding by the mAb, but also highlight the benefit of aligning the pharmacokinetic and pharmacodynamic properties of the immunopharmacotherapeutic with the targeted drug. PMID:21356233

Theoretical study of hydrogen bond interactions of fluvastatin with ι-carrageenan and λ-carrageenan.

PubMed

Papadopoulos, Anastasios G; Sigalas, Michael P

2011-07-01

The binding of the reductase inhibitor drug fluvastatin, hydroxy-3-methylglutaryl coenzyme A, with the hydrophilic ι- or λ-carrageenan polymers, serving as potential controllers of the drug's release rate, have been studied at the density functional level of theory with the B3LYP exchange correlation functional. Three low energy conformers of fluvastatin have been calculated. The vibrational spectroscopic properties calculated for the most stable conformer were in satisfactory agreement with the experimental data. A series of hydrogen bonded complexes of the most stable conformer of fluvastatin anion with low molecular weight models of the polymers have been fully optimized. In almost all, intermolecular H-bonds are formed between the sulfate groups of ι- or λ-carrageenan and fluvastatin's hydroxyls, resulting in a red shift of the fluvastatin's O - H stretching vibrations. Cooperative intramolecular H-bonds within fluvastatin or ι-, λ-carrageenan are also present. The BSSE and ZPE corrected interaction energies were estimated in the range 281-318 kJ mol⁻¹ for ι-carrageenan - fluvastatin and 145-200 kJ mol⁻¹ for λ-carrageenan - fluvastatin complexes. The electron density (ρ (bcp)) and Laplacian (∇²ρ (bcp)) properties at critical points of the intermolecular hydrogen bonds, estimated by AIM (atoms in molecules) calculations, have a low and positive character (∇²ρ(bcp) > 0), consistent with the electrostatic character of the hydrogen bonds. The structural and energetic data observed, as well as the extent of the red shift of the fluvastatin's O - H stretching vibrations upon complex formation and the properties of electron density show a stronger binding of fluvastatin to ι- than to λ-carrageenan.

Combination scaffolds of salmon fibrin, hyaluronic acid, and laminin for human neural stem cell and vascular tissue engineering

PubMed Central

Arulmoli, Janahan; Wright, Heather J.; Phan, Duc T.T.; Sheth, Urmi; Que, Richard A.; Botten, Giovanni A.; Keating, Mark; Botvinick, Elliot L.; Pathak, Medha M.; Zarembinski, Thomas I.; Yanni, Daniel S.; Razorenova, Olga V.; Hughes, Christopher C.W.; Flanagan, Lisa A.

2017-01-01

Human neural stem/progenitor cells (hNSPCs) are good candidates for treating central nervous system (CNS) trauma since they secrete beneficial trophic factors and differentiate into mature CNS cells; however, many cells die after transplantation. This cell death can be ameliorated by inclusion of a biomaterial scaffold, making identification of optimal scaffolds for hNSPCs a critical research focus. We investigated the properties of fibrin-based scaffolds and their effects on hNSPCs and found that fibrin generated from salmon fibrinogen and thrombin stimulates greater hNSPC proliferation than mammalian fibrin. Fibrin scaffolds degrade over the course of a few days in vivo, so we sought to develop a novel scaffold that would retain the beneficial properties of fibrin but degrade more slowly to provide longer support for hNSPCs. We found combination scaffolds of salmon fibrin with interpenetrating networks (IPNs) of hyaluronic acid (HA) with and without laminin polymerize more effectively than fibrin alone and generate compliant hydrogels matching the physical properties of brain tissue. Furthermore, combination scaffolds support hNSPC proliferation and differentiation while significantly attenuating the cell-mediated degradation seen with fibrin alone. HNSPCs express two fibrinogen-binding integrins, αVβ1 and α5β1, and several laminin binding integrins (α7β1, α6β1, α3β1) that can mediate interaction with the scaffold. Lastly, to test the ability of scaffolds to support vascularization, we analyzed human cord blood-derived endothelial cells alone and in co-culture with hNSPCs and found enhanced vessel formation and complexity in co-cultures within combination scaffolds. Overall, combination scaffolds of fibrin, HA, and laminin are excellent biomaterials for hNSPCs. PMID:27475528

Activation of spleen cells by ArtinM may account for its immunomodulatory properties.

PubMed

Silva, Thiago Aparecido da; Souza, Maria Aparecida de; Cecílio, Nerry Tatiana; Roque-Barreira, Maria Cristina

2014-09-01

ArtinM is a D-mannose-binding lectin extracted from Artocarpus heterophyllus that promotes interleukin-12 production by macrophages and dendritic cells. This property is considered responsible for T helper 1 immunity induced in vivo after ArtinM administration. In this study, we investigated the effect of native (jArtinM) and recombinant (rArtinM) forms of lectin on murine spleen cells and isolated T lymphocytes. We found that ArtinM binds to the surface of spleen cells. This interaction, which was blocked by D-mannose, induced cell activation, as manifested by increased mitochondrial activity, interleukin-2 production, and cell proliferation. We verified that a 30-times higher concentration of rArtinM was required to trigger optimal activation of spleen cells compared with that needed with jArtinM, although these proteins have identical sugar recognition properties and use the same signaling molecules to trigger cell activation. Because the distinction between native and recombinant is restricted to their tertiary structure (tetrameric and monomeric, respectively), we postulated that the multi-valence of jArtinM accounts for its superiority in promoting clustering of cell surface glycoreceptors and activation. The jArtinM and rArtinM activation effect exerted on spleen cells was reproduced on purified CD4(+) T cells. Our results suggest that ArtinM interaction with T cells leads to responses that may act in concert with the interleukin-12 produced by antigen-presenting cells to modulate immunity toward the T helper 1 axis. Further studies are necessary to dissect ArtinM/T-cell interactions to more fully understand the immunomodulation induced by carbohydrate recognition.

A Spectrophotometric Assay Optimizing Conditions for Pepsin Activity.

ERIC Educational Resources Information Center

Harding, Ethelynda E.; Kimsey, R. Scott

1998-01-01

Describes a laboratory protocol optimizing the conditions for the assay of pepsin activity using the Coomasie Blue dye binding assay of protein concentration. The dye bonds through strong, noncovalent interactions to basic and aromatic amino acid residues. (DDR)

Optimization of ionic conductivity in doped ceria

PubMed Central

Andersson, David A.; Simak, Sergei I.; Skorodumova, Natalia V.; Abrikosov, Igor A.; Johansson, Börje

2006-01-01

Oxides with the cubic fluorite structure, e.g., ceria (CeO2), are known to be good solid electrolytes when they are doped with cations of lower valence than the host cations. The high ionic conductivity of doped ceria makes it an attractive electrolyte for solid oxide fuel cells, whose prospects as an environmentally friendly power source are very promising. In these electrolytes, the current is carried by oxygen ions that are transported by oxygen vacancies, present to compensate for the lower charge of the dopant cations. Ionic conductivity in ceria is closely related to oxygen-vacancy formation and migration properties. A clear physical picture of the connection between the choice of a dopant and the improvement of ionic conductivity in ceria is still lacking. Here we present a quantum-mechanical first-principles study of the influence of different trivalent impurities on these properties. Our results reveal a remarkable correspondence between vacancy properties at the atomic level and the macroscopic ionic conductivity. The key parameters comprise migration barriers for bulk diffusion and vacancy–dopant interactions, represented by association (binding) energies of vacancy–dopant clusters. The interactions can be divided into repulsive elastic and attractive electronic parts. In the optimal electrolyte, these parts should balance. This finding offers a simple and clear way to narrow the search for superior dopants and combinations of dopants. The ideal dopant should have an effective atomic number between 61 (Pm) and 62 (Sm), and we elaborate that combinations of Nd/Sm and Pr/Gd show enhanced ionic conductivity, as compared with that for each element separately. PMID:16478802

Optimization of ionic conductivity in doped ceria.

PubMed

Andersson, David A; Simak, Sergei I; Skorodumova, Natalia V; Abrikosov, Igor A; Johansson, Börje

2006-03-07

Oxides with the cubic fluorite structure, e.g., ceria (CeO2), are known to be good solid electrolytes when they are doped with cations of lower valence than the host cations. The high ionic conductivity of doped ceria makes it an attractive electrolyte for solid oxide fuel cells, whose prospects as an environmentally friendly power source are very promising. In these electrolytes, the current is carried by oxygen ions that are transported by oxygen vacancies, present to compensate for the lower charge of the dopant cations. Ionic conductivity in ceria is closely related to oxygen-vacancy formation and migration properties. A clear physical picture of the connection between the choice of a dopant and the improvement of ionic conductivity in ceria is still lacking. Here we present a quantum-mechanical first-principles study of the influence of different trivalent impurities on these properties. Our results reveal a remarkable correspondence between vacancy properties at the atomic level and the macroscopic ionic conductivity. The key parameters comprise migration barriers for bulk diffusion and vacancy-dopant interactions, represented by association (binding) energies of vacancy-dopant clusters. The interactions can be divided into repulsive elastic and attractive electronic parts. In the optimal electrolyte, these parts should balance. This finding offers a simple and clear way to narrow the search for superior dopants and combinations of dopants. The ideal dopant should have an effective atomic number between 61 (Pm) and 62 (Sm), and we elaborate that combinations of Nd/Sm and Pr/Gd show enhanced ionic conductivity, as compared with that for each element separately.

Molecular dynamics study of binding energies, mechanical properties, and detonation performances of bicyclo-HMX-based PBXs.

PubMed

Qiu, Ling; Xiao, Heming

2009-05-15

To investigate the effect of polymer binders on the monoexplosive, molecular dynamics simulations were performed to study the binding energies, mechanical properties, and detonation performances of the bicyclo-HMX-based polymer-bonded explosives (PBXs). The results show that the binding energies on different crystalline surfaces of bicyclo-HMX decrease in the order of (010)>(100)>(001). On each crystalline surface, binding properties of different polymers with the same chain segment are different from each other, while those of the polymers in the same content decrease in the sequence of PVDF>F(2311)>F(2314) approximately PCTFE. The mechanical properties of a dozen of model systems (elastic coefficients, various moduli, Cauchy pressure, and Poisson's ratio) have been obtained. It is found that mechanical properties are effectively improved by adding small amounts of fluorine polymers, and the overall effect of fluorine polymers on three crystalline surfaces of bicyclo-HMX changes in the order of (010)>(001) approximately (100). In comparison with the base explosive, detonation performances of the PBXs decrease slightly, but they are still superior to TNT. These suggestions may be useful for the formulation design of bicyclo-HMX-based PBXs.

Exploring the potential of 3D Zernike descriptors and SVM for protein-protein interface prediction.

PubMed

Daberdaku, Sebastian; Ferrari, Carlo

2018-02-06

The correct determination of protein-protein interaction interfaces is important for understanding disease mechanisms and for rational drug design. To date, several computational methods for the prediction of protein interfaces have been developed, but the interface prediction problem is still not fully understood. Experimental evidence suggests that the location of binding sites is imprinted in the protein structure, but there are major differences among the interfaces of the various protein types: the characterising properties can vary a lot depending on the interaction type and function. The selection of an optimal set of features characterising the protein interface and the development of an effective method to represent and capture the complex protein recognition patterns are of paramount importance for this task. In this work we investigate the potential of a novel local surface descriptor based on 3D Zernike moments for the interface prediction task. Descriptors invariant to roto-translations are extracted from circular patches of the protein surface enriched with physico-chemical properties from the HQI8 amino acid index set, and are used as samples for a binary classification problem. Support Vector Machines are used as a classifier to distinguish interface local surface patches from non-interface ones. The proposed method was validated on 16 classes of proteins extracted from the Protein-Protein Docking Benchmark 5.0 and compared to other state-of-the-art protein interface predictors (SPPIDER, PrISE and NPS-HomPPI). The 3D Zernike descriptors are able to capture the similarity among patterns of physico-chemical and biochemical properties mapped on the protein surface arising from the various spatial arrangements of the underlying residues, and their usage can be easily extended to other sets of amino acid properties. The results suggest that the choice of a proper set of features characterising the protein interface is crucial for the interface prediction task, and that optimality strongly depends on the class of proteins whose interface we want to characterise. We postulate that different protein classes should be treated separately and that it is necessary to identify an optimal set of features for each protein class.

Carbohydrate recognition: A minimalistic approach to binding

NASA Astrophysics Data System (ADS)

Kubik, Stefan

2012-09-01

Synthetic receptors with properties resembling those of carbohydrate-binding proteins are known, but they are structurally rather complex. Elaborate structures are, however, not always required to bind carbohydrates in water -- much simpler compounds can be just as effective.

Nanoparticles engineered to bind cellular motors for efficient delivery.

PubMed

Dalmau-Mena, Inmaculada; Del Pino, Pablo; Pelaz, Beatriz; Cuesta-Geijo, Miguel Ángel; Galindo, Inmaculada; Moros, María; de la Fuente, Jesús M; Alonso, Covadonga

2018-03-30

Dynein is a cytoskeletal molecular motor protein that transports cellular cargoes along microtubules. Biomimetic synthetic peptides designed to bind dynein have been shown to acquire dynamic properties such as cell accumulation and active intra- and inter-cellular motion through cell-to-cell contacts and projections to distant cells. On the basis of these properties dynein-binding peptides could be used to functionalize nanoparticles for drug delivery applications. Here, we show that gold nanoparticles modified with dynein-binding delivery sequences become mobile, powered by molecular motor proteins. Modified nanoparticles showed dynamic properties, such as travelling the cytosol, crossing intracellular barriers and shuttling the nuclear membrane. Furthermore, nanoparticles were transported from one cell to another through cell-to-cell contacts and quickly spread to distant cells through cell projections. The capacity of these motor-bound nanoparticles to spread to many cells and increasing cellular retention, thus avoiding losses and allowing lower dosage, could make them candidate carriers for drug delivery.

Peculiarities of binding composition production in vortex jet mill

NASA Astrophysics Data System (ADS)

Zagorodnyuk, L. Kh; Lesovik, V. S.; Sumskoy, D. A.; Elistratkin, M. Yu; Makhortov, D. S.

2018-03-01

The article investigates the disintegration of perlite production waste in a vortex jet mill; the regularities of milling were established. Binding compositions were obtained at different ratios of cement vs. perlite sand production waste in the vortex jet mill in various milling regimes. The peculiarities of milling processes were studied, and technological and physicomechanical properties of the binding compositions were determined as well. The microstructure of the cement stones made of activated Portland cement and binding compositions in the vortex jet mill was elucidated by electron microscopy. The open pores of the cement-binding compositions prepared using perlite fillers were found to be filled by newgrowths at different stages of collective growth. The microstructure of the binding compositions is dense due to rationally proportioned composition, effective mineral filler— perlite waste — that creates additional substrates for internal composite microstructure formation, mechanochemical activation of raw mixture, which allows obtaining composites with required properties.

Emergent properties of nuclei from ab initio coupled-cluster calculations

NASA Astrophysics Data System (ADS)

Hagen, G.; Hjorth-Jensen, M.; Jansen, G. R.; Papenbrock, T.

2016-06-01

Emergent properties such as nuclear saturation and deformation, and the effects on shell structure due to the proximity of the scattering continuum and particle decay channels are fascinating phenomena in atomic nuclei. In recent years, ab initio approaches to nuclei have taken the first steps towards tackling the computational challenge of describing these phenomena from Hamiltonians with microscopic degrees of freedom. This endeavor is now possible due to ideas from effective field theories, novel optimization strategies for nuclear interactions, ab initio methods exhibiting a soft scaling with mass number, and ever-increasing computational power. This paper reviews some of the recent accomplishments. We also present new results. The recently optimized chiral interaction NNLO{}{{sat}} is shown to provide an accurate description of both charge radii and binding energies in selected light- and medium-mass nuclei up to 56Ni. We derive an efficient scheme for including continuum effects in coupled-cluster computations of nuclei based on chiral nucleon-nucleon and three-nucleon forces, and present new results for unbound states in the neutron-rich isotopes of oxygen and calcium. The coupling to the continuum impacts the energies of the {J}π =1/{2}-,3/{2}-,7/{2}-,3/{2}+ states in {}{17,23,25}O, and—contrary to naive shell-model expectations—the level ordering of the {J}π =3/{2}+,5/{2}+,9/{2}+ states in {}{53,55,61}Ca. ).

HdeB chaperone activity is coupled to its intrinsic dynamic properties

PubMed Central

Ding, Jienv; Yang, Chengfeng; Niu, Xiaogang; Hu, Yunfei; Jin, Changwen

2015-01-01

Enteric bacteria encounter extreme acidity when passing through hosts’ stomach. Since the bacterial periplasmic space quickly equilibrates with outer environment, an efficient acid resistance mechanism is essential in preventing irreversible protein denaturation/aggregation and maintaining bacteria viability. HdeB, along with its homolog HdeA, was identified as a periplasmic acid-resistant chaperone. Both proteins exist as homodimers and share similar monomeric structures under neutral pH, while showing different dimeric packing interfaces. Previous investigations show that HdeA functions through an acid-induced dimer-to-monomer transition and partial unfolding at low pH (pH 2–3), resulting in exposure of hydrophobic surfaces that bind substrate proteins. In contrast, HdeB appears to have a much higher optimal activation pH (pH 4–5), under which condition the protein maintains a well-folded dimer and the mechanism for its chaperone activity remains elusive. Herein, we present an NMR study of HdeB to investigate its dynamic properties. Our results reveal that HdeB undergoes significant micro- to milli-second timescale conformational exchanges at neutral to near-neutral pH, under the later condition it exhibits optimal activity. The current study indicates that HdeB activation is coupled to its intrinsic dynamics instead of structural changes, and therefore its functional mechanism is apparently different from HdeA. PMID:26593705

Comparative evaluation of several docking tools for docking small molecule ligands to DC-SIGN.

PubMed

Jug, Gregor; Anderluh, Marko; Tomašič, Tihomir

2015-06-01

Five docking tools, namely AutoDock, FRED, CDOCKER, FlexX and GOLD, have been critically examined, with the aim of selecting those most appropriate for use as docking tools for docking molecules to the lectin dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN). This lectin has been selected for its rather non-druggable binding site, which enables complex interactions that guide the binding of the core monosaccharide. Since optimal orientation is crucial for forming coordination bonds, it was important to assess whether the selected docking tools could reproduce the optimal binding conformation for several oligosaccharides that are known to bind DC-SIGN. Our results show that even widely used docking programs have certain limitations when faced with a rather shallow and featureless binding site, as is the case of DC-SIGN. The FRED docking software (OpenEye Scientific Software, Inc.) was found to score as the best tool for docking ligands to DC-SIGN. The performance of FRED was further assessed on another lectin, Langerin. We have demonstrated that this validated docking protocol could be used for docking to other lectins similar to DC-SIGN.

Synergistic Interactions between Chemokine Receptor Elements in Recognition of Interleukin-8 by Soluble Receptor Mimics†

PubMed Central

Barter, Emily F.; Stone, Martin J.

2012-01-01

Interleukin-8 (IL-8 or CXCL8), the archetypal member of the CXC chemokine subfamily, stimulates neutrophil chemotaxis by activation of the receptors CXCR1/IL8RA and CXCR2/IL8RB. Previous mutational studies have implicated both the N-terminal and third extracellular loop (E3) regions of these receptors in binding to IL-8. To investigate the interactions of these receptor elements with IL-8, we have constructed soluble proteins in which the N-terminal and E3 elements of either CXCR1 or CXCR2 are juxtaposed on a soluble scaffold protein; these are referred to as CROSS-NX1E3X1 and CROSS-NX2E3X2, respectively. Isothermal titration calorimetry (ITC) and NMR spectroscopy were used to compare the IL-8 binding properties of the receptor mimics to those of control proteins containing only the N-terminal or the E3 receptor element. CROSS-NX2E3X2 bound to monomeric IL-8 with the same affinity and induced the same chemical shift changes as the control protein containing only the N-terminal element of CXCR2, indicating that the E3 element of CXCR2 did not contribute to IL-8 binding. In contrast, CROSS-NX1E3X1 bound to IL-8 with ~10-fold increased affinity and induced different chemical shift changes compared to the control protein containing only the N-terminal element of CXCR1, suggesting that the E3 region of CXCR1 was interacting with IL-8. However, a chimeric protein containing the N-terminal region of CXCR1 and the E3 region of CXCR2 (CROSS-NX1E3X2) bound to IL-8 with thermodynamic properties and induced chemical shift changes indistinguishable from those of CROSS-NX1E3X1 and substantially different from those of CROSS-NX2E3X2. These results indicate that the N-terminal and E3 regions of CXCR1 interact synergistically to achieve optimal binding interactions with IL-8. PMID:22242662

Carrageenans as a new source of drugs with metal binding properties.

PubMed

Khotimchenko, Yuri S; Khozhaenko, Elena V; Khotimchenko, Maxim Y; Kolenchenko, Elena A; Kovalev, Valeri V

2010-04-01

Carrageenans are abundant and safe non-starch polysaccharides exerting their biological effects in living organisms. Apart from their known pro-inflammation properties and some pharmacological activity, carrageenans can also strongly bind and hold metal ions. This property can be used for creation of the new drugs for elimination of metals from the body or targeted delivery of these metal ions for healing purposes. Metal binding activity of different carrageenans in aqueous solutions containing Y(3+) or Pb(2+) ions was studied in a batch sorption system. The metal uptake by carrageenans is not affected by the change of the pH within the range from 2.0 to 6.0. The rates and binding capacities of carrageenans regarding metal ions were evaluated. The Langmuir, Freundlich and BET sorption models were applied to describe the isotherms and constants, and the sorption isothermal data could be explained well by the Langmuir equation. The results obtained through the study suggest that kappa-, iota-, and lambda-carrageenans are favorable sorbents. The largest amount of Y(3+) and Pb(2+) ions are bound by iota-carrageenan. Therefore, it can be concluded that this type of polysaccharide is the more appropriate substance for elaboration of the drugs with high selective metal binding properties.

The interplay of nanointerface curvature and calcium binding in weak polyelectrolyte-coated nanoparticles.

PubMed

Nap, Rikkert J; Gonzalez Solveyra, Estefania; Szleifer, Igal

2018-05-01

When engineering nanomaterials for application in biological systems, it is important to understand how multivalent ions, such as calcium, affect the structural and chemical properties of polymer-modified nanoconstructs. In this work, a recently developed molecular theory was employed to study the effect of surface curvature on the calcium-induced collapse of end-tethered weak polyelectrolytes. In particular, we focused on cylindrical and spherical nanoparticles coated with poly(acrylic acid) in the presence of different amounts of Ca2+ ions. We describe the structural changes that grafted polyelectrolytes undergo as a function of calcium concentration, surface curvature, and morphology. The polymer layers collapse in aqueous solutions that contain sufficient amounts of Ca2+ ions. This collapse, due to the formation of calcium bridges, is not only controlled by the calcium ion concentration but also strongly influenced by the curvature of the tethering surface. The transition from a swollen to a collapsed layer as a function of calcium concentration broadens and shifts to lower amounts of calcium ions as a function of the radius of cylindrical and spherical nanoparticles. The results show how the interplay between calcium binding and surface curvature governs the structural and functional properties of the polymer molecules. This would directly impact the fate of weak polyelectrolyte-coated nanoparticles in biological environments, in which calcium levels are tightly regulated. Understanding such interplay would also contribute to the rational design and optimization of smart interfaces with applications in, e.g., salt-sensitive and ion-responsive materials and devices.

Avoiding false positives and optimizing identification of true negatives in estrogen receptor binding and agonist/antagonist assays

EPA Science Inventory

The potential for chemicals to affect endocrine signaling is commonly evaluated via in vitro receptor binding and gene activation, but these assays, especially antagonism assays, have potential artifacts that must be addressed for accurate interpretation. Results are presented fr...

COMPARATIVE DOCKING STUDIES OF THE BINDING OF POLYCYCLIC AROMATIC HYDROCARBONS TO THE ESTROGEN RECEPTOR

EPA Science Inventory

The interactions of several PAHs, and some of their possible metabolites, with the ligand binding domain of the estrogen receptor have been examined using molecular docking and quantum mechanical methods. The geometries of the PAHs were optimized at the Hartree-Fock level and the...

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2

PubMed Central

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties. PMID:27716822

Signaling Properties of Chemerin Receptors CMKLR1, GPR1 and CCRL2.

PubMed

De Henau, Olivier; Degroot, Gaetan-Nagim; Imbault, Virginie; Robert, Virginie; De Poorter, Cédric; Mcheik, Saria; Galés, Céline; Parmentier, Marc; Springael, Jean-Yves

2016-01-01

Chemerin is a small chemotactic protein originally identified as the natural ligand of CMKLR1. More recently, two other receptors, GPR1 and CCRL2, have been reported to bind chemerin but their functional relevance remains poorly understood. In this study, we compared the binding and signaling properties of the three human chemerin receptors and showed differences in mode of chemerin binding and receptor signaling. Chemerin binds to all three receptors with low nanomolar affinities. However, the contribution of the chemerin C-terminus to binding efficiency varies greatly amongst receptors. By using BRET-based biosensors monitoring the activation of various G proteins, we showed that binding of chemerin and the chemerin 9 nonapeptide (149YFPGQFAFS157) to CMKLR1 activates the three Gαi subtypes (Gαi1, Gαi2 and Gαi3) and the two Gαo isoforms (Gαoa and Gαob) with potencies correlated to binding affinities. In contrast, no significant activation of G proteins was detected upon binding of chemerin to GPR1 or CCRL2. Binding of chemerin and the chemerin 9 peptide also induced the recruitment of β-arrestin1 and 2 to CMKLR1 and GPR1, though to various degree, but not to CCRL2. However, the propensity of chemerin 9 to activate β-arrestins relative to chemerin is higher when bound to GPR1. Finally, we showed that binding of chemerin to CMKLR1 and GPR1 promotes also the internalization of the two receptors and the phosphorylation of ERK1/2 MAP kinases, although with a different efficiency, and that phosphorylation of ERK1/2 requires both Gαi/o and β-arrestin2 activation but not β-arrestin1. Collectively, these data support a model in which each chemerin receptor displays selective signaling properties.

Cotton and Protein Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Goheen, Steven C.; Edwards, J. V.; Rayburn, Alfred R.

The adsorbent properties of important wound fluid proteins and cotton cellulose are reviewed. This review focuses on the adsorption of albumin to cotton-based wound dressings and some chemically modified derivatives targeted for chronic wounds. Adsorption of elastase in the presence of albumin was examined as a model to understand the interactive properties of these wound fluid components with cotton fibers. In the chronic non-healing wound, elastase appears to be over-expressed, and it digests tissue and growth factors, interfering with the normal healing process. Albumin is the most prevalent protein in wound fluid, and in highly to moderately exudative wounds, itmore » may bind significantly to the fibers of wound dressings. Thus, the relative binding properties of both elastase and albumin to wound dressing fibers are of interest in the design of more effective wound dressings. The present work examines the binding of albumin to two different derivatives of cotton, and quantifies the elastase binding to the same derivatives following exposure of albumin to the fiber surface. An HPLC adsorption technique was employed coupled with a colorimetric enzyme assay to quantify the relative binding properties of albumin and elastase to cotton. The results of wound protein binding are discussed in relation to the porosity and surface chemistry interactions of cotton and wound proteins. Studies are directed to understanding the implications of protein adsorption phenomena in terms of fiber-protein models that have implications for rationally designing dressings for chronic wounds.« less

Ab initio study of structural, electronic, optical, and vibrational properties of Zn x S y ( x + y = 2 to 5) nanoclusters

NASA Astrophysics Data System (ADS)

Yadav, P. S.; Pandey, D. K.; Agrawal, S.; Agrawal, B. K.

2010-03-01

An ab initio study of the stability, structural, electronic. and optical properties has been performed for 46 zinc sulfide nanoclusters Zn x S y ( x + y = n = 2 to 5). Five out of them are seen to be unstable as their vibrational frequencies are found to be imaginary. A B3LYP-DFT/6-311G(3df) method is employed to optimize the geometries and a TDDFT method is used for the study of the optical properties. The binding energies (BE), HOMO-LUMO gaps and the bond lengths have been obtained for all the clusters. For the ZnS2, ZnS3, and ZnS4 nanoclusters, our stable structures are seen to be different from those obtained earlier by using the effective core potentials. We have also considered the zero point energy (ZPE) corrections ignored by the earlier workers. For a fixed value of n, we designate the most stable structure the one, which has maximum final binding energy per atom. The adiabatic and vertical ionization potentials (IP) and electron affinities (EA), charges on the atoms, dipole moments, optical properties, vibrational frequencies, infrared intensities, relative infrared intensities, and Raman scattering activities have been investigated for the most stable structures. The nanoclusters containing large number of S atoms for each n is found to be most stable. The HOMO-LUMO gap decreases from n = 2-3 and then increases above n = 3. The IP and EA both fluctuate with the cluster size n. The optical absorption is quite weak in visible region but strong in the ultraviolet region in most of the nanoclusters except a few. The optical absorption spectrum or electron energy loss spectrum (EELS) is unique for every nanocluster and may be used to characterize a specific nanocluster. The growth of most stable nanoclusters may be possible in the experiments.

Does TATA matter? A structural exploration of the selectivity determinants in its complexes with TATA box-binding protein.

PubMed Central

Pastor, N; Pardo, L; Weinstein, H

1997-01-01

The binding of the TATA box-binding protein (TBP) to a TATA sequence in DNA is essential for eukaryotic basal transcription. TBP binds in the minor groove of DNA, causing a large distortion of the DNA helix. Given the apparent stereochemical equivalence of AT and TA basepairs in the minor groove, DNA deformability must play a significant role in binding site selection, because not all AT-rich sequences are bound effectively by TBP. To gain insight into the precise role that the properties of the TATA sequence have in determining the specificity of the DNA substrates of TBP, the solution structure and dynamics of seven DNA dodecamers have been studied by using molecular dynamics simulations. The analysis of the structural properties of basepair steps in these TATA sequences suggests a reason for the preference for alternating pyrimidine-purine (YR) sequences, but indicates that these properties cannot be the sole determinant of the sequence specificity of TBP. Rather, recognition depends on the interplay between the inherent deformability of the DNA and steric complementarity at the molecular interface. Images FIGURE 2 PMID:9251783

Protein Allostery and Conformational Dynamics.

PubMed

Guo, Jingjing; Zhou, Huan-Xiang

2016-06-08

The functions of many proteins are regulated through allostery, whereby effector binding at a distal site changes the functional activity (e.g., substrate binding affinity or catalytic efficiency) at the active site. Most allosteric studies have focused on thermodynamic properties, in particular, substrate binding affinity. Changes in substrate binding affinity by allosteric effectors have generally been thought to be mediated by conformational transitions of the proteins or, alternatively, by changes in the broadness of the free energy basin of the protein conformational state without shifting the basin minimum position. When effector binding changes the free energy landscape of a protein in conformational space, the change affects not only thermodynamic properties but also dynamic properties, including the amplitudes of motions on different time scales and rates of conformational transitions. Here we assess the roles of conformational dynamics in allosteric regulation. Two cases are highlighted where NMR spectroscopy and molecular dynamics simulation have been used as complementary approaches to identify residues possibly involved in allosteric communication. Perspectives on contentious issues, for example, the relationship between picosecond-nanosecond local and microsecond-millisecond conformational exchange dynamics, are presented.

Co-Processed Chitin-Mannitol as a New Excipient for Oro-Dispersible Tablets

PubMed Central

Daraghmeh, Nidal; Chowdhry, Babur Z.; Leharne, Stephen A.; Al Omari, Mahmoud M. H.; Badwan, Adnan A.

2015-01-01

This study describes the preparation, characterization and performance of a novel excipient for use in oro-dispersible tablets (ODT). The excipient (Cop–CM) consists of chitin and mannitol. The excipient with optimal physicochemical properties was obtained at a chitin: mannitol ratio of 2:8 (w/w) and produced by roll compaction (RC). Differential scanning calorimetry (DSC), Fourier transform-Infrared (FT-IR), X-ray powder diffraction (XRPD) and scanning electron microscope (SEM) techniques were used to characterize Cop–CM, in addition to characterization of its powder and ODT dosage form. The effect of particle size distribution of Cop–CM was investigated and found to have no significant influence on the overall tablet physical properties. The compressibility parameter (a) for Cop–CM was calculated from a Kawakita plot and found to be higher (0.661) than that of mannitol (0.576) due to the presence of the highly compressible chitin (0.818). Montelukast sodium and domperidone ODTs produced, using Cop–CM, displayed excellent physicochemical properties. The exceptional binding, fast wetting and superdisintegration properties of Cop–CM, in comparison with commercially available co-processed ODT excipients, results in a unique multifunctional base which can successfully be used in the formulation of oro-dispersible and fast immediate release tablets. PMID:25830680

Size-dependent impact of CNTs on dynamic properties of calmodulin

NASA Astrophysics Data System (ADS)

Gao, Jian; Wang, Liming; Kang, Seung-Gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan

2014-10-01

There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca2+ regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca2+ concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca2+-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca2+-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca2+-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca2+ regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca2+ concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01623h

Selective binding of lectins to normal and neoplastic urothelium in rat and mouse bladder carcinogenesis models.

PubMed

Zupančič, Daša; Kreft, Mateja Erdani; Romih, Rok

2014-01-01

Bladder cancer adjuvant intravesical therapy could be optimized by more selective targeting of neoplastic tissue via specific binding of lectins to plasma membrane carbohydrates. Our aim was to establish rat and mouse models of bladder carcinogenesis to investigate in vivo and ex vivo binding of selected lectins to the luminal surface of normal and neoplastic urothelium. Male rats and mice were treated with 0.05 % N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in drinking water and used for ex vivo and in vivo lectin binding experiments. Urinary bladder samples were also used for paraffin embedding, scanning electron microscopy and immunofluorescence labelling of uroplakins. During carcinogenesis, the structure of the urinary bladder luminal surface changed from microridges to microvilli and ropy ridges and the expression of urothelial-specific glycoproteins uroplakins was decreased. Ex vivo and in vivo lectin binding experiments gave comparable results. Jacalin (lectin from Artocarpus integrifolia) exhibited the highest selectivity for neoplastic compared to normal urothelium of rats and mice. The binding of lectin from Amaranthus caudatus decreased in rat model and increased in mouse carcinogenesis model, indicating interspecies variations of plasma membrane glycosylation. Lectin from Datura stramonium showed higher affinity for neoplastic urothelium compared to the normal in rat and mouse model. The BBN-induced animal models of bladder carcinogenesis offer a promising approach for lectin binding experiments and further lectin-mediated targeted drug delivery research. Moreover, in vivo lectin binding experiments are comparable to ex vivo experiments, which should be considered when planning and optimizing future research.

Binding properties of SUMO-interacting motifs (SIMs) in yeast.

PubMed

Jardin, Christophe; Horn, Anselm H C; Sticht, Heinrich

2015-03-01

Small ubiquitin-like modifier (SUMO) conjugation and interaction play an essential role in many cellular processes. A large number of yeast proteins is known to interact non-covalently with SUMO via short SUMO-interacting motifs (SIMs), but the structural details of this interaction are yet poorly characterized. In the present work, sequence analysis of a large dataset of 148 yeast SIMs revealed the existence of a hydrophobic core binding motif and a preference for acidic residues either within or adjacent to the core motif. Thus the sequence properties of yeast SIMs are highly similar to those described for human. Molecular dynamics simulations were performed to investigate the binding preferences for four representative SIM peptides differing in the number and distribution of acidic residues. Furthermore, the relative stability of two previously observed alternative binding orientations (parallel, antiparallel) was assessed. For all SIMs investigated, the antiparallel binding mode remained stable in the simulations and the SIMs were tightly bound via their hydrophobic core residues supplemented by polar interactions of the acidic residues. In contrary, the stability of the parallel binding mode is more dependent on the sequence features of the SIM motif like the number and position of acidic residues or the presence of additional adjacent interaction motifs. This information should be helpful to enhance the prediction of SIMs and their binding properties in different organisms to facilitate the reconstruction of the SUMO interactome.

Plasticity of BRCA2 Function in Homologous Recombination: Genetic Interactions of the PALB2 and DNA Binding Domains

PubMed Central

Siaud, Nicolas; Lam, Isabel; Christ, Nicole; Schlacher, Katharina; Xia, Bing; Jasin, Maria

2011-01-01

The breast cancer suppressor BRCA2 is essential for the maintenance of genomic integrity in mammalian cells through its role in DNA repair by homologous recombination (HR). Human BRCA2 is 3,418 amino acids and is comprised of multiple domains that interact with the RAD51 recombinase and other proteins as well as with DNA. To gain insight into the cellular function of BRCA2 in HR, we created fusions consisting of various BRCA2 domains and also introduced mutations into these domains to disrupt specific protein and DNA interactions. We find that a BRCA2 fusion peptide deleted for the DNA binding domain and active in HR is completely dependent on interaction with the PALB2 tumor suppressor for activity. Conversely, a BRCA2 fusion peptide deleted for the PALB2 binding domain is dependent on an intact DNA binding domain, providing a role for this conserved domain in vivo; mutagenesis suggests that both single-stranded and double-stranded DNA binding activities in the DNA binding domain are required for its activity. Given that PALB2 itself binds DNA, these results suggest alternative mechanisms to deliver RAD51 to DNA. In addition, the BRCA2 C terminus contains both RAD51-dependent and -independent activities which are essential to HR in some contexts. Finally, binding the small peptide DSS1 is essential for activity when its binding domain is present, but not when it is absent. Our results reveal functional redundancy within the BRCA2 protein and emphasize the plasticity of this large protein built for optimal HR function in mammalian cells. The occurrence of disease-causing mutations throughout BRCA2 suggests sub-optimal HR from a variety of domain modulations. PMID:22194698

Batrachotoxin Changes the Properties of the Muscarinic Receptor in Rat Brain and Heart: Possible Interaction(s) between Muscarinic Receptors and Sodium Channels

NASA Astrophysics Data System (ADS)

Cohen-Armon, Malca; Kloog, Yoel; Henis, Yoav I.; Sokolovsky, Mordechai

1985-05-01

The effects of Na+-channel activator batrachotoxin (BTX) on the binding properties of muscarinic receptors in homogenates of rat brain and heart were studied. BTX enhanced the affinity for the binding of the agonists carbamoylcholine and acetylcholine to the muscarinic receptors in brainstem and ventricle, but not in the cerebral cortex. Analysis of the data according to a two-site model for agonist binding indicated that the effect of BTX was to increase the affinity of the agonists to the high-affinity site. Guanyl nucleotides, known to induce interconversion of high-affinity agonist binding sites to the low-affinity state, canceled the effect of BTX on carbamoylcholine and acetylcholine binding. BTX had no effect on the binding of the agonist oxotremorine or on the binding of the antagonist [3H]-N-methyl-4-piperidyl benzilate. The local anesthetics dibucaine and tetracaine antagonized the effect of BTX on the binding of muscarinic agonists at concentrations known to inhibit the activation of Na+ channels by BTX. On the basis of these findings, we propose that in specific tissues the muscarinic receptors may interact with the BTX binding site (Na+ channels).

PatchSurfers: Two methods for local molecular property-based binding ligand prediction.

PubMed

Shin, Woong-Hee; Bures, Mark Gregory; Kihara, Daisuke

2016-01-15

Protein function prediction is an active area of research in computational biology. Function prediction can help biologists make hypotheses for characterization of genes and help interpret biological assays, and thus is a productive area for collaboration between experimental and computational biologists. Among various function prediction methods, predicting binding ligand molecules for a target protein is an important class because ligand binding events for a protein are usually closely intertwined with the proteins' biological function, and also because predicted binding ligands can often be directly tested by biochemical assays. Binding ligand prediction methods can be classified into two types: those which are based on protein-protein (or pocket-pocket) comparison, and those that compare a target pocket directly to ligands. Recently, our group proposed two computational binding ligand prediction methods, Patch-Surfer, which is a pocket-pocket comparison method, and PL-PatchSurfer, which compares a pocket to ligand molecules. The two programs apply surface patch-based descriptions to calculate similarity or complementarity between molecules. A surface patch is characterized by physicochemical properties such as shape, hydrophobicity, and electrostatic potentials. These properties on the surface are represented using three-dimensional Zernike descriptors (3DZD), which are based on a series expansion of a 3 dimensional function. Utilizing 3DZD for describing the physicochemical properties has two main advantages: (1) rotational invariance and (2) fast comparison. Here, we introduce Patch-Surfer and PL-PatchSurfer with an emphasis on PL-PatchSurfer, which is more recently developed. Illustrative examples of PL-PatchSurfer performance on binding ligand prediction as well as virtual drug screening are also provided. Copyright © 2015 Elsevier Inc. All rights reserved.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity.

PubMed

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites.

Physicochemical characteristics of structurally determined metabolite-protein and drug-protein binding events with respect to binding specificity

PubMed Central

Korkuć, Paula; Walther, Dirk

2015-01-01

To better understand and ultimately predict both the metabolic activities as well as the signaling functions of metabolites, a detailed understanding of the physical interactions of metabolites with proteins is highly desirable. Focusing in particular on protein binding specificity vs. promiscuity, we performed a comprehensive analysis of the physicochemical properties of compound-protein binding events as reported in the Protein Data Bank (PDB). We compared the molecular and structural characteristics obtained for metabolites to those of the well-studied interactions of drug compounds with proteins. Promiscuously binding metabolites and drugs are characterized by low molecular weight and high structural flexibility. Unlike reported for drug compounds, low rather than high hydrophobicity appears associated, albeit weakly, with promiscuous binding for the metabolite set investigated in this study. Across several physicochemical properties, drug compounds exhibit characteristic binding propensities that are distinguishable from those associated with metabolites. Prediction of target diversity and compound promiscuity using physicochemical properties was possible at modest accuracy levels only, but was consistently better for drugs than for metabolites. Compound properties capturing structural flexibility and hydrogen-bond formation descriptors proved most informative in PLS-based prediction models. With regard to diversity of enzymatic activities of the respective metabolite target enzymes, the metabolites benzylsuccinate, hypoxanthine, trimethylamine N-oxide, oleoylglycerol, and resorcinol showed very narrow process involvement, while glycine, imidazole, tryptophan, succinate, and glutathione were identified to possess broad enzymatic reaction scopes. Promiscuous metabolites were found to mainly serve as general energy currency compounds, but were identified to also be involved in signaling processes and to appear in diverse organismal systems (digestive and nervous system) suggesting specific molecular and physiological roles of promiscuous metabolites. PMID:26442281

Assessment of amsacrine binding with DNA using UV-visible, circular dichroism and Raman spectroscopic techniques.

PubMed

Jangir, Deepak Kumar; Dey, Sanjay Kumar; Kundu, Suman; Mehrotra, Ranjana

2012-09-03

Proper understanding of the mechanism of binding of drugs to their targets in cell is a fundamental requirement to develop new drug therapy regimen. Amsacrine is a rationally designed anticancer drug, used to treat leukemia and lymphoma. Binding with cellular DNA is a crucial step in its mechanism of cytotoxicity. Despite numerous studies, DNA binding properties of amsacrine are poorly understood. Its reversible binding with DNA does not permit X-ray crystallography or NMR spectroscopic evaluation of amsacrine-DNA complexes. In the present work, interaction of amsacrine with calf thymus DNA is investigated at physiological conditions. UV-visible, FT-Raman and circular dichroism spectroscopic techniques were employed to determine the binding mode, binding constant, sequence specificity and conformational effects of amsacrine binding to native calf thymus DNA. Our results illustrate that amsacrine interacts with DNA by and large through intercalation between base pairs. Binding constant of the amsacrine-DNA complex was found to be K=1.2±0.1×10(4) M(-1) which is indicative of moderate type of binding of amsacrine to DNA. Raman spectroscopic results suggest that amsacrine has a binding preference of intercalation between AT base pairs of DNA. Minor groove binding is also observed in amsacrine-DNA complexes. These results are in good agreement with in silico investigation of amsacrine binding to DNA and thus provide detailed insight into DNA binding properties of amsacrine, which could ultimately, renders its cytotoxic efficacy. Copyright © 2012 Elsevier B.V. All rights reserved.

A Small World of Neuronal Synchrony

PubMed Central

Yu, Shan; Huang, Debin; Singer, Wolf

2008-01-01

A small-world network has been suggested to be an efficient solution for achieving both modular and global processing—a property highly desirable for brain computations. Here, we investigated functional networks of cortical neurons using correlation analysis to identify functional connectivity. To reconstruct the interaction network, we applied the Ising model based on the principle of maximum entropy. This allowed us to assess the interactions by measuring pairwise correlations and to assess the strength of coupling from the degree of synchrony. Visual responses were recorded in visual cortex of anesthetized cats, simultaneously from up to 24 neurons. First, pairwise correlations captured most of the patterns in the population's activity and, therefore, provided a reliable basis for the reconstruction of the interaction networks. Second, and most importantly, the resulting networks had small-world properties; the average path lengths were as short as in simulated random networks, but the clustering coefficients were larger. Neurons differed considerably with respect to the number and strength of interactions, suggesting the existence of “hubs” in the network. Notably, there was no evidence for scale-free properties. These results suggest that cortical networks are optimized for the coexistence of local and global computations: feature detection and feature integration or binding. PMID:18400792

Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry.

PubMed

Ngambenjawong, Chayanon; Pineda, Julio Marco B; Pun, Suzie H

2016-12-21

Peptide cyclization is a strategy used to improve stability and activity of peptides. The most commonly used cyclization method is disulfide bridge formation of cysteine-containing peptides, as is typically found in nature. Over the years, an increasing number of alternative chemistries for peptide cyclization with improved efficiency, kinetics, orthogonality, and stability have been reported. However, there has been less appreciation for the opportunity to fine-tune peptide activity via the diverse chemical entities introduced at the site of linkage by different cyclization strategies. Here, we demonstrate how cyclization optimization of an M2 "anti-inflammatory" macrophage-binding peptide (M2pep) resulted in a significant increase in binding affinity of the optimized analog to M2 macrophages while maintaining binding selectivity compared to M1 "pro-inflammatory" macrophages. In this study, we report synthesis and evaluation of four cyclic M2pep(RY) analogs with diverse cyclization strategies: (1) Asp-[amide]-Lys, (2) azido-Lys-[triazole(copper(I)-catalyzed alkyne-azide cycloaddition (CuAAC))]-propargyl-Gly, (3) Cys-[decafluorobiphenyl (DFBP)]-Cys, and (4) Cys-[decafluorobiphenyl sulfone (DFS)]-Cys, whereby the chemical entity or linker at the linkage site is shown in the square bracket and is between the residues involved in cyclization. These peptides are compared to a disulfide-cyclized M2pep(RY) that we previously reported as a serum-stable, affinity-enhanced analog to the original linear M2pep. DFBP-cyclized M2pep(RY) exhibits the highest binding activity to M2 macrophages with apparent dissociation constant (K D ) about 2.03 μM compared to 36.3 μM for the original disulfide-cyclized M2pep(RY) and 220 μM for the original linear peptide. DFS-cyclized M2pep(RY) also binds more strongly than the original cyclized analog, whereas amide- and triazole-cyclized M2pep(RY) analogs bind less strongly. We verified that DFBP alone has negligible binding to M2 macrophages and the incorporation of diphenylalanine to the original sequence improves binding activity at the expense of solubility and increased toxicity. In conclusion, we report development of cyclic M2pep(RY) analogs with diverse cyclization strategies leading to the discovery of DFBP-cyclized M2pep(RY) with enhanced M2 macrophage-binding activity.

Optimization of Photosensitized Tryptophan Oxidation in the Presence of Dimegin-Polyvinylpyrrolidone-Chitosan Systems.

PubMed

Solovieva, Anna B; Kardumian, Valeria V; Aksenova, Nadezhda A; Belovolova, Lyudmila V; Glushkov, Mikhail V; Bezrukov, Evgeny A; Sukhanov, Roman B; Kotova, Svetlana L; Timashev, Peter S

2018-05-23

By the example of a model process of tryptophan photooxidation in the aqueous medium in the presence of a three-component photosensitizing complex (porphyrin photosensitizer-polyvinylpyrrolidone- chitosan, PPS-PVP-CT) in the temperature range of 20-40 °С, we have demonstrated a possibility of modification of such a process by selecting different molar ratios of the components in the reaction mixture. The actual objective of this selection is the formation of a certain PPS-PVP-CT composition in which PVP macromolecules would coordinate with PPS molecules and at the same time practically block the complex binding of PPS molecules with chitosan macromolecules. Such blocking allows utilization of the bactericidal properties of chitosan to a greater extent, since chitosan is known to depress the PPS photosensitizing activity in PPS-PVP-CT complexes when using those in photodynamic therapy (PDT). The optimal composition of photosensitizing complexes appears to be dependent on the temperature at which the PDT sessions are performed. We have analyzed the correlations of the effective rate constants of tryptophan photooxidation with the photophysical characteristics of the formed complexes.

Structure and Activity of a New Low Molecular Weight Heparin Produced by Enzymatic Ultrafiltration

PubMed Central

FU, LI; ZHANG, FUMING; LI, GUOYUN; ONISHI, AKIHIRO; BHASKAR, UJJWAL; SUN, PEILONG; LINHARDT, ROBERT J.

2014-01-01

The standard process for preparing the low molecular weight heparin (LMWH) tinzaparin, through the partial enzymatic depolymerization of heparin, results in a reduced yield due to the formation of a high content of undesired disaccharides and tetrasaccharides. An enzymatic ultrafiltration reactor for LMWH preparation was developed to overcome this problem. The behavior, of the heparin oligosaccharides and polysaccharides using various membranes and conditions, was investigated to optimize this reactor. A novel product, LMWH-II, was produced from the controlled depolymerization of heparin using heparin lyase II in this optimized ultrafiltration reactor. Enzymatic ultrafiltration provides easy control and high yields (>80%) of LMWH-II. The molecular weight properties of LMWH-II were similar to other commercial LMWHs. The structure of LMWH-II closely matched heparin’s core structural features. Most of the common process artifacts, present in many commercial LWMHs, were eliminated as demonstrated by 1D and 2D nuclear magnetic resonance spectroscopy. The antithrombin III and platelet factor-4 binding affinity of LMWH-II were comparable to commercial LMWHs, as was its in vitro anticoagulant activity. PMID:24634007

Optimal Ge/SiGe nanofin geometries for hole mobility enhancement: Technology limit from atomic simulations

NASA Astrophysics Data System (ADS)

Vedula, Ravi Pramod; Mehrotra, Saumitra; Kubis, Tillmann; Povolotskyi, Michael; Klimeck, Gerhard; Strachan, Alejandro

2015-05-01

We use first principles simulations to engineer Ge nanofins for maximum hole mobility by controlling strain tri-axially through nano-patterning. Large-scale molecular dynamics predict fully relaxed, atomic structures for experimentally achievable nanofins, and orthogonal tight binding is used to obtain the corresponding electronic structure. Hole transport properties are then obtained via a linearized Boltzmann formalism. This approach explicitly accounts for free surfaces and associated strain relaxation as well as strain gradients which are critical for quantitative predictions in nanoscale structures. We show that the transverse strain relaxation resulting from the reduction in the aspect ratio of the fins leads to a significant enhancement in phonon limited hole mobility (7× over unstrained, bulk Ge, and 3.5× over biaxially strained Ge). Maximum enhancement is achieved by reducing the width to be approximately 1.5 times the height and further reduction in width does not result in additional gains. These results indicate significant room for improvement over current-generation Ge nanofins, provide geometrical guidelines to design optimized geometries and insight into the physics behind the significant mobility enhancement.

Discovery of 4-aryl-N-arylcarbonyl-2-aminothiazoles as Hec1/Nek2 inhibitors. Part I: optimization of in vitro potencies and pharmacokinetic properties.

PubMed

Lee, Ying-Shuan E; Chuang, Shih-Hsien; Huang, Lynn Y L; Lai, Chun-Liang; Lin, Yu-Hsiang; Yang, Ju-Ying; Liu, Chia-Wei; Yang, Sheng-chuan; Lin, Her-Sheng; Chang, Chia-chi; Lai, Jun-Yu; Jian, Pei-Shiou; Lam, King; Chang, Jia-Ming; Lau, Johnson Y N; Huang, Jiann-Jyh

2014-05-22

A series of 4-aryl-N-arylcarbonyl-2-aminothiazoles of scaffold 4 was designed and synthesized as Hec1/Nek2 inhibitors. Structural optimization of 4 led to compound 32 bearing C-4' 4-methoxyphenoxy and 4-(o-fluoropyridyl)carbonyl groups that showed low nanomolar in vitro antiproliferative activity (IC50: 16.3-42.7 nM), high intravenous AUC (64.9 μM·h, 2.0 mg/kg) in SD rats, and significant in vivo antitumor activity (T/C = 32%, 20 mg/kg, IV) in mice bearing human MDA-MB-231 xenografts. Cell responses resulting from Hec1/Nek2 inhibition were observed in cells treated with 32, including a reduced level of Hec1 coimmunoprecipitated with Nek2, degradation of Nek2, mitotic abnormalities, and apoptosis. Compound 32 showed selectivity toward cancer cells over normal phenotype cells and was inactive in a [(3)H]astemizole competitive binding assay for hERG liability screening. Therefore, 32 is as a good lead toward the discovery of a preclinical candidate targeting Hec1/Nek2 interaction.

Reversible Intercalation of Fluoride-Anion Receptor Complexes in Graphite

NASA Technical Reports Server (NTRS)

West, William C.; Whitacre, Jay F.; Leifer, Nicole; Greenbaum, Steve; Smart, Marshall; Bugga, Ratnakumar; Blanco, Mario; Narayanan, S. R.

2007-01-01

We have demonstrated a route to reversibly intercalate fluoride-anion receptor complexes in graphite via a nonaqueous electrochemical process. This approach may find application for a rechargeable lithium-fluoride dual-ion intercalating battery with high specific energy. The cell chemistry presented here uses graphite cathodes with LiF dissolved in a nonaqueous solvent through the aid of anion receptors. Cells have been demonstrated with reversible cathode specific capacity of approximately 80 mAh/g at discharge plateaus of upward of 4.8 V, with graphite staging of the intercalant observed via in situ synchrotron X-ray diffraction during charging. Electrochemical impedance spectroscopy and B-11 nuclear magnetic resonance studies suggest that cointercalation of the anion receptor with the fluoride occurs during charging, which likely limits the cathode specific capacity. The anion receptor type dictates the extent of graphite fluorination, and must be further optimized to realize high theoretical fluorination levels. To find these optimal anion receptors, we have designed an ab initio calculations-based scheme aimed at identifying receptors with favorable fluoride binding and release properties.

Alteration of human serum albumin binding properties induced by modifications: A review

NASA Astrophysics Data System (ADS)

Maciążek-Jurczyk, Małgorzata; Szkudlarek, Agnieszka; Chudzik, Mariola; Pożycka, Jadwiga; Sułkowska, Anna

2018-01-01

Albumin, a major transporting protein in the blood, is the main target of modification that affects the binding of drugs to Sudlow's site I and II. These modification of serum protein moderates its physiological function, and works as a biomarker of some diseases. The main goal of the paper was to explain the possible alteration of human serum albumin binding properties induced by modifications such as glycation, oxidation and ageing, their origin, methods of evaluation and positive and negative meaning described by significant researchers.

Identification of ligand efficient, fragment-like hits from an HTS library: structure-based virtual screening and docking investigations of 2H- and 3H-pyrazolo tautomers for Aurora kinase A selectivity.

PubMed

Sarvagalla, Sailu; Singh, Vivek Kumar; Ke, Yi-Yu; Shiao, Hui-Yi; Lin, Wen-Hsing; Hsieh, Hsing-Pang; Hsu, John T A; Coumar, Mohane Selvaraj

2015-01-01

Furanopyrimidine 1 (IC50 = 273 nM, LE = 0.36, LELP = 10.28) was recently identified by high-throughput screening (HTS) of an in-house library (125,000 compounds) as an Aurora kinase inhibitor. Structure-based hit optimization resulted in lead molecules with in vivo efficacy in a mouse tumour xenograft model, but no oral bioavailability. This is attributed to "molecular obesity", a common problem during hit to lead evolution during which degradation of important molecular properties such as molecular weight (MW) and lipophilicity occurs. This could be effectively tackled by the right choice of hit compounds for optimization. In this regard, ligand efficiency (LE) and ligand efficiency dependent lipophilicity (LELP) indices are more often used to choose fragment-like hits for optimization. To identify hits with appropriate LE, we used a MW cut-off <250, and pyrazole structure to filter HTS library. Next, structure-based virtual screening using software (Libdock and Glide) in the Aurora A crystal structure (PDB ID: 3E5A) was carried out, and the top scoring 18 compounds tested for Aurora A enzyme inhibition. This resulted in the identification of a novel tetrahydro-pyrazolo-isoquinoline hit 7 (IC50 = 852 nM, LE = 0.44, LELP = 8.36) with fragment-like properties suitable for further hit optimization. Moreover, hit 7 was found to be selective for Aurora A (Aurora B IC50 = 35,150 nM) and the possible reasons for selectivity investigated by docking two tautomeric forms (2H- and 3H-pyrazole) of 7 in Auroras A and B (PDB ID: 4AF3) crystal structures. This docking study shows that the major 3H-pyrazole tautomer of 7 binds in Aurora A stronger than in Aurora B.

Identification of ligand efficient, fragment-like hits from an HTS library: structure-based virtual screening and docking investigations of 2 H- and 3 H-pyrazolo tautomers for Aurora kinase A selectivity

NASA Astrophysics Data System (ADS)

Sarvagalla, Sailu; Singh, Vivek Kumar; Ke, Yi-Yu; Shiao, Hui-Yi; Lin, Wen-Hsing; Hsieh, Hsing-Pang; Hsu, John T. A.; Coumar, Mohane Selvaraj

2015-01-01

Furanopyrimidine 1 (IC50 = 273 nM, LE = 0.36, LELP = 10.28) was recently identified by high-throughput screening (HTS) of an in-house library (125,000 compounds) as an Aurora kinase inhibitor. Structure-based hit optimization resulted in lead molecules with in vivo efficacy in a mouse tumour xenograft model, but no oral bioavailability. This is attributed to "molecular obesity", a common problem during hit to lead evolution during which degradation of important molecular properties such as molecular weight (MW) and lipophilicity occurs. This could be effectively tackled by the right choice of hit compounds for optimization. In this regard, ligand efficiency (LE) and ligand efficiency dependent lipophilicity (LELP) indices are more often used to choose fragment-like hits for optimization. To identify hits with appropriate LE, we used a MW cut-off <250, and pyrazole structure to filter HTS library. Next, structure-based virtual screening using software (Libdock and Glide) in the Aurora A crystal structure (PDB ID: 3E5A) was carried out, and the top scoring 18 compounds tested for Aurora A enzyme inhibition. This resulted in the identification of a novel tetrahydro-pyrazolo-isoquinoline hit 7 (IC50 = 852 nM, LE = 0.44, LELP = 8.36) with fragment-like properties suitable for further hit optimization. Moreover, hit 7 was found to be selective for Aurora A (Aurora B IC50 = 35,150 nM) and the possible reasons for selectivity investigated by docking two tautomeric forms (2 H- and 3 H-pyrazole) of 7 in Auroras A and B (PDB ID: 4AF3) crystal structures. This docking study shows that the major 3 H-pyrazole tautomer of 7 binds in Aurora A stronger than in Aurora B.

Quality by Design approach for an in situ gelling microemulsion of Lorazepam via intranasal route.

PubMed

Shah, Vidhi; Sharma, Mukesh; Pandya, Radhika; Parikh, Rajesh K; Bharatiya, Bhavesh; Shukla, Atindra; Tsai, Hsieh-Chih

2017-06-01

The present study illustrates the application of the concept of Quality by Design for development, optimization and evaluation of Lorazepam loaded microemulsion containing ion responsive In situ gelator gellan gum and carbopol 934. A novel approach involving interactions between surfactant and polymer was employed to achieve controlled drug release and reduced mucociliary clearance. Microemulsion formulated using preliminary solubility study and pseudo ternary phase diagrams showed significantly improved solubilization capacity of Lorazepam with 54.31±6.07nm droplets size. The effect of oil to surfactant/cosurfactant ratio and concentration of gelling agent on the drug release and viscosity of microemulsion gel (MEG) was evaluated using a 3 2 full factorial design. The gel of optimized formulation (MEG 1 ) showed a drug release up to 6h of 97.32±1.35% of total drug loaded. The change in shear-dependent viscosity for different formulations on interaction with Simulated Nasal Fluid depicts the crucial role of surfactant-polymer interactions on the gelation properties along with calcium ions binding on the polymer chains. It is proposed that the surfactant-polymer interactions in the form of a stoichiometric hydrogen bonding between oxyethylene and carboxylic groups of the polymers used, provides exceptional ME stability and adhesion properties. Compared with the marketed formulation, optimized MEG showed improved pharmacodynamic activity. Ex vivo diffusion studies revealed significantly higher release for MEG compared to microemulsion and drug solution. MEG showed higher flux and permeation across goat nasal mucosa. According to the study, it could be concluded that formulation would successfully provide the rapid onset of action, and decrease the mucociliary clearance due to formation of in situ gelling mucoadhesive system. Copyright © 2017 Elsevier B.V. All rights reserved.

Locking mechanisms in degree-4 vertex origami structures

NASA Astrophysics Data System (ADS)

Fang, Hongbin; Li, Suyi; Xu, Jian; Wang, K. W.

2016-04-01

Origami has emerged as a potential tool for the design of mechanical metamaterials and metastructures whose novel properties originate from their crease patterns. Most of the attention in origami engineering has focused on the wellknown Miura-Ori, a folded tessellation that is flat-foldable for folded sheet and stacked blocks. This study advances the state of the art and expands the research field to investigate generic degree-4 vertex (4-vertex) origami, with a focus on facet-binding. In order to understand how facet-binding attributes to the mechanical properties of 4-vertex origami structures, geometries of the 4-vertex origami cells are analyzed and analytically expressed. Through repeating and stacking 4-vertex cells, origami sheets and stacked origami blocks can be constructed. Geometry analyses discover four mechanisms that will lead to the self-locking of 4-vertex origami cells, sheets, and stacked blocks: in-cell facet-binding, inlayer facet-binding, inter-layer facet binding, and in-layer and inter-layer facet-bindings. These mechanisms and the predicted self-locking phenomena are verified through 3D simulations and prototype experiments. Finally, this paper briefly introduces the unusual mechanical properties caused by the locking of 4-vertex origami structures. The research reported in this paper could foster a new breed of self-locking structures with various engineering applications.

Recognition by nonaromatic and stereochemical subunit-containing polyamides of the four Watson-Crick base pairs in the DNA minor groove.

PubMed

Zhang, Hong-Fei; Wu, Yan-Ling; Jiang, Shi-Kun; Wang, Pu; Sugiyama, Hiroshi; Chen, Xing-Lai; Zhang, Wen; Ji, Yan-Juan; Guo, Chuan-Xin

2012-06-18

In order to develop an optimal subunit as a T-recognition element in hairpin polyamides, 15 novel chirality-modified polyamides containing (R)-α,β-diaminopropionic acid ((R) β α-NH 2), (S)-α,β-diaminopropionic acid ((S) β α-NH 2), (1R,3S)-3-aminocyclopentanecarboxylic acid ((RS) Cp), (1S,3R)-3-amino-cyclopentanecarboxylic acid ((RS) Cp), (1R,3R)-3-aminocyclopentanecarboxylic acid ((RR) Cp) and (1S,3S)-3-amino-cyclopentanecarboxylic acid ((SS) Cp) residues were synthesized. Their binding characteristics to DNA sequences 5'-TGCNCAT-3'/3'-ACGN'GTA-5' (N⋅N'=A⋅T, T⋅A, G⋅C and C⋅G) were systemically studied by surface plasmon resonance (SPR) and molecular simulation (MSim) techniques. SPR showed that polyamide 4, AcIm-(S) β α-NH 2-ImPy-γ-ImPy-β-Py-βDp (β/(S) β α-NH 2 pair), bound to a DNA sequence containing a core binding site of 5'-TGCACAT-3' with a dissociation equilibrium constant (K(D) ) of 4.5×10(-8)  m. This was a tenfold improvement in specificity over 5'-TGCTCAT-3' (K(D) =4.5×10(-7)  M). MSim studies supported the SPR results. More importantly, for the first time, we found that chiral 3-aminocyclopentanecarboxylic acids in polyamides can be employed as base readers with only a small decrease in binding affinity to DNA. In particular, SPR showed that polyamide 9 ((RR) Cp/β pair) had a 15-fold binding preference for 5'-TGCTCAT-3' over 5'-TGCACAT-3'. A large difference in standard free energy change for A⋅T over T⋅A was determined (ΔΔG(o) =5.9 kJ mol(-1) ), as was a twofold decrease in interaction energy by MSim. Moreover, a 1:1 stoichiometry (9 to 5'-TGCTCAT-3'/3'-ACGAGTA-5') was shown by MSim to be optimal for the chiral five-membered cycle to fit the minor groove. Collectively, the study suggests that the (S)-α-amino-β-aminopropionic acid and (1R,3R)-3-aminocyclopentanecarboxylic acid can serve as a T-recognition element, and the stereochemistry and the nature of these subunits significantly influence binding properties in these recognition events. Subunit (1R,3R)-3-aminocyclopentanecarboxylic acid broadens our scope to design novel polyamides. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Reduced Fluorescent Protein Switching Fatigue by Binding-Induced Emissive State Stabilization

PubMed Central

Dedecker, Peter

2017-01-01

Reversibly switchable fluorescent proteins (RSFPs) enable advanced fluorescence imaging, though the performance of this imaging crucially depends on the properties of the labels. We report on the use of an existing small binding peptide, named Enhancer, to modulate the spectroscopic properties of the recently developed rsGreen series of RSFPs. Fusion constructs of Enhancer with rsGreen1 and rsGreenF revealed an increased molecular brightness and pH stability, although expression in living E. coli or HeLa cells resulted in a decrease of the overall emission. Surprisingly, Enhancer binding also increased off-switching speed and resistance to switching fatigue. Further investigation suggested that the RSFPs can interconvert between fast- and slow-switching emissive states, with the overall protein population gradually converting to the slow-switching state through irradiation. The Enhancer modulates the spectroscopic properties of both states, but also preferentially stabilizes the fast-switching state, supporting the increased fatigue resistance. This work demonstrates how the photo-physical properties of RSFPs can be influenced by their binding to other small proteins, which opens up new horizons for applications that may require such modulation. Furthermore, we provide new insights into the photoswitching kinetics that should be of general consideration when developing new RSFPs with improved or different photochromic properties. PMID:28930199

Computational design of environmental sensors for the potent opioid fentanyl

DOE PAGES

Bick, Matthew J.; Greisen, Per J.; Morey, Kevin J.; ...

2017-09-19

Here, we describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We also use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Computational design of environmental sensors for the potent opioid fentanyl

PubMed Central

Morey, Kevin J; Antunes, Mauricio S; La, David; Sankaran, Banumathi; Reymond, Luc; Johnsson, Kai; Medford, June I

2017-01-01

We describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment. PMID:28925919

Computational design of environmental sensors for the potent opioid fentanyl

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bick, Matthew J.; Greisen, Per J.; Morey, Kevin J.

Here, we describe the computational design of proteins that bind the potent analgesic fentanyl. Our approach employs a fast docking algorithm to find shape complementary ligand placement in protein scaffolds, followed by design of the surrounding residues to optimize binding affinity. Co-crystal structures of the highest affinity binder reveal a highly preorganized binding site, and an overall architecture and ligand placement in close agreement with the design model. We also use the designs to generate plant sensors for fentanyl by coupling ligand binding to design stability. The method should be generally useful for detecting toxic hydrophobic compounds in the environment.

Structural and mechanical properties of hydroxyapatite coatings formed by ion-beam assisted deposition

NASA Astrophysics Data System (ADS)

Zykova, A.; Safonov, V.; Dudin, S.; Yakovin, S.; Donkov, N.; Ghaemi, M. H.; Szkodo, M.; Antoszkiewicz, M.; Szyfelbain, M.; Czaban, A.

2018-03-01

The ion-beam assisted deposition (IBAD) is an advanced method capable of producing crystalline coatings at low temperatures. We determined the characteristics of hydroxyapatite Ca10(PO4)6(OH)2 target and coatings formed by IBAD using X-ray photoemission spectroscopy (XPS), X-ray diffraction (XRD), scanning electron microscopy (SEM), atomic force microscopy (AFM) and energy dispersive X-ray (EDX). The composition of the coatings’ cross-section and surface was close to those of the target. The XPS spectra showed that the binding energy values of Ca (2p1/2, 2p3/2), P (2p3/2), and O 1s levels are related to the hydroxyapatite phase. The coatings demonstrate an optimal H/E ratio, and a good resistance to scratch tests.

DFT studies on the Al, B, and P doping of silicene

NASA Astrophysics Data System (ADS)

Hernández Cocoletzi, H.; Castellanos Águila, J. E.

2018-02-01

The search for efficient adsorbents of atoms and molecules has motivated the study of systems in the presence of defects. For this reason, we have investigated theoretically the creation of mono- and di-vacancies on single layer silicene, as well as the Al, B, and P doping of silicene. Using the first-principles method with the generalized gradient approximation in the parameterization of Perdew-Burke-Ernzerhof, we have found that Al, B, and P interact strongly with Si atoms. Besides, when the vacancies are generated, the dangling bonds are saturated in pairs to form new bonds. Optimal geometries, binding energies, density of states (DOS) and charge density are reported. The results suggest that new chemical modifications can be used to modify the electronic properties of single-layer silicene.

Fragment-based drug design.

PubMed

Feyfant, Eric; Cross, Jason B; Paris, Kevin; Tsao, Désirée H H

2011-01-01

Fragment-based drug design (FBDD), which is comprised of both fragment screening and the use of fragment hits to design leads, began more than 15 years ago and has been steadily gaining in popularity and utility. Its origin lies on the fact that the coverage of chemical space and the binding efficiency of hits are directly related to the size of the compounds screened. Nevertheless, FBDD still faces challenges, among them developing fragment screening libraries that ensure optimal coverage of chemical space, physical properties and chemical tractability. Fragment screening also requires sensitive assays, often biophysical in nature, to detect weak binders. In this chapter we will introduce the technologies used to address these challenges and outline the experimental advantages that make FBDD one of the most popular new hit-to-lead process.

Nonlocal Response in Infrared Detector with Semiconducting Carbon Nanotubes and Graphdiyne

PubMed Central

Zheng, Zhe; Fang, Hehai; Liu, Dan; Tan, Zhenjun; Gao, Xin; Hu, Weida; Peng, Hailin; Tong, Lianming

2017-01-01

Abstract Semiconducting single‐walled carbon nanotubes (s‐SWNTs) are regarded as an important candidate for infrared (IR) optical detection due to their excellent intrinsic properties. However, the strong binding energy of excitons in s‐SWNTs seriously impedes the development of s‐SWNTs IR photodetector. This Communication reports an IR photodetector with highly pure s‐SWNTs and γ‐graphdiyne. The heterojunctions between the two materials can efficiently separate the photogenerated excitons. In comparison to device fabricated only with s‐SWNTs, this IR detector shows a uniform response in the whole channel of the device. The response time is demonstrated to be below 1 ms. The optimal responsivity and detectivity approximately reach 0.4 mA W−1 and 5 × 106 cmHz1/2 W−1, respectively. PMID:29270354

Comparison of localized basis and plane-wave basis for density-functional calculations of organic molecules on metals

NASA Astrophysics Data System (ADS)

Lee, Kyuho; Yu, Jaejun; Morikawa, Yoshitada

2007-01-01

Localized pseudoatomic orbitals (PAOs) are mainly optimized and tested for the strong chemical bonds within molecules and solids with their proven accuracy and efficiency, but are prone to significant basis set superposition error (BSSE) for weakly interacting systems. Here we test the accuracy of PAO basis in comparison with the BSSE-free plane-wave basis for the physisorption of pentacene molecule on Au (001) by calculating the binding energy, adsorption height, and energy level alignment. We show that both the large cutoff radius for localized PAOs and the counter-poise correction for BSSE are necessary to obtain well-converged physical properties. Thereby obtained results are as accurate as the plane-wave basis results. The comparison with experiment is given as well.

Scoring functions for protein-protein interactions.

PubMed

Moal, Iain H; Moretti, Rocco; Baker, David; Fernández-Recio, Juan

2013-12-01

The computational evaluation of protein-protein interactions will play an important role in organising the wealth of data being generated by high-throughput initiatives. Here we discuss future applications, report recent developments and identify areas requiring further investigation. Many functions have been developed to quantify the structural and energetic properties of interacting proteins, finding use in interrelated challenges revolving around the relationship between sequence, structure and binding free energy. These include loop modelling, side-chain refinement, docking, multimer assembly, affinity prediction, affinity change upon mutation, hotspots location and interface design. Information derived from models optimised for one of these challenges can be used to benefit the others, and can be unified within the theoretical frameworks of multi-task learning and Pareto-optimal multi-objective learning. Copyright © 2013 Elsevier Ltd. All rights reserved.

Retro-binding thrombin active site inhibitors: identification of an orally active inhibitor of thrombin catalytic activity.

PubMed

Iwanowicz, Edwin J; Kimball, S David; Lin, James; Lau, Wan; Han, W-C; Wang, Tammy C; Roberts, Daniel G M; Schumacher, W A; Ogletree, Martin L; Seiler, Steven M

2002-11-04

A series of retro-binding inhibitors of human alpha-thrombin was prepared to elucidate structure-activity relationships (SAR) and optimize in vivo performance. Compounds 9 and 11, orally active inhibitors of thrombin catalytic activity, were identified to be efficacious in a thrombin-induced lethality model in mice.

N- vs. C-Domain Selectivity of Catalytic Inactivation of Human Angiotensin Converting Enzyme by Lisinopril-Coupled Transition Metal Chelates

PubMed Central

Hocharoen, Lalintip; Joyner, Jeff C.; Cowan, J. A.

2014-01-01

The N- and C-terminal domains of human somatic Angiotensin I Converting Enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates were tested for both reversible binding and irreversible catalytic inactivation of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of the M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and orientation factors (double-filter effect). PMID:24228790

N- versus C-domain selectivity of catalytic inactivation of human angiotensin converting enzyme by lisinopril-coupled transition metal chelates.

PubMed

Hocharoen, Lalintip; Joyner, Jeff C; Cowan, J A

2013-12-27

The N- and C-terminal domains of human somatic angiotensin I converting enzyme (sACE-1) demonstrate distinct physiological functions, with resulting interest in the development of domain-selective inhibitors for specific therapeutic applications. Herein, the activity of lisinopril-coupled transition metal chelates was tested for both reversible binding and irreversible catalytic inactivation of each domain of sACE-1. C/N domain binding selectivity ratios ranged from 1 to 350, while rates of irreversible catalytic inactivation of the N- and C-domains were found to be significantly greater for the N-domain, suggesting a more optimal orientation of M-chelate-lisinopril complexes within the active site of the N-domain of sACE-1. Finally, the combined effect of binding selectivity and inactivation selectivity was assessed for each catalyst (double-filter selectivity factors), and several catalysts were found to cause domain-selective catalytic inactivation. The results of this study demonstrate the ability to optimize the target selectivity of catalytic metallopeptides through both binding and catalytic factors (double-filter effect).

Optimization of Benzoisothiazole dioxide inhibitory activity of the NS5B polymerase of HCV genotype 4 using ligand-steered homological modeling, reaction-driven scaffold-hopping and Enovo workflow.

PubMed

Mahmoud, Amr Hamed; Mohamed Abouzid, Khaled Abouzid; El Ella, Dalal Abd El Rahman Abou; Hamid Ismail, Mohamed Abdel

2011-01-01

Infection caused by hepatitis C virus (HCV) is a significant world health problem for which novel therapies are in urgent demand. The virus is highly prevalent in the Middle East and Africa particularly Egypt with more than 90% of infections due to genotype 4. Nonstructural (NS5B) viral proteins have emerged as an attractive target for HCV antivirals discovery. A potent class of inhibitors having benzisothiazole dioxide scaffold has been identified on this target, however they were mainly active on genotype 1 while exhibiting much lowered activity on other genotypes due to the high degree of mutation of its binding site. Based on this fact, we employed a novel strategy to optimize this class on genotype 4. This strategy depends on using a refined ligand-steered homological model of this genotype to study the mutation binding energies of the binding site amino acid residues, the essential features for interaction and provide a structure-based pharmacophore model that can aid optimization. This model was applied on a focused library which was generated using a reaction-driven scaffold-hopping strategy. The hits retrieved were subjected to Enovo pipeline pilot optimization workflow that employs R-group enumeration, core-constrained protein docking using modified CDOCKER and finally ranking of poses using an accurate molecular mechanics generalized Born with surface area method.

Unusual dynamic properties of water near the ice-binding plane of hyperactive antifreeze protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kuffel, Anna; Czapiewski, Dariusz; Zielkiewicz, Jan, E-mail: jaz@chem.pg.gda.pl

2015-10-07

The dynamical properties of solvation water of hyperactive antifreeze protein from Choristoneura fumiferana (CfAFP) are analyzed and discussed in context of its antifreeze activity. The protein comprises of three well-defined planes and one of them binds to the surface of ice. The dynamical properties of solvation water around each of these planes were analyzed separately; the results are compared with the dynamical properties of solvation water of ice around its two crystallographic planes: basal and prism. Three main conclusions are inferred from our investigations. The first one is that the solvation shell of CfAFP does not seem to be particularlymore » far-ranged, at least not beyond what is usually observed for proteins that do not interact with ice. Therefore, it does not appear to us that the antifreeze activity is enhanced by a long-ranged retardation of water mobility. Also the correlation between the collective mobility of water and the collective mobility of protein atoms highly resembles the one measured for the protein that does not interact with ice. Our second conclusion is that the dynamical properties of solvation water of CfAFP are non-uniform. The dynamics of solvation water of ice-binding plane is, in some respects, different from the dynamics of solvation water of the two remaining planes. The feature that distinguishes the dynamics of solvation water of the three planes is the activation energy of diffusion process. The third conclusion is that—from the three analyzed solvation shells of CfAFP—the dynamical properties of solvation water of the ice-binding plane resemble the most the properties of solvation water of ice; note, however, that these properties still clearly differ from the dynamic properties of solvation water of ice.« less

Phosphatidic acid binding proteins display differential binding as a function of membrane curvature stress and chemical properties.

PubMed

Putta, Priya; Rankenberg, Johanna; Korver, Ruud A; van Wijk, Ringo; Munnik, Teun; Testerink, Christa; Kooijman, Edgar E

2016-11-01

Phosphatidic acid (PA) is a crucial membrane phospholipid involved in de novo lipid synthesis and numerous intracellular signaling cascades. The signaling function of PA is mediated by peripheral membrane proteins that specifically recognize PA. While numerous PA-binding proteins are known, much less is known about what drives specificity of PA-protein binding. Previously, we have described the ionization properties of PA, summarized in the electrostatic-hydrogen bond switch, as one aspect that drives the specific binding of PA by PA-binding proteins. Here we focus on membrane curvature stress induced by phosphatidylethanolamine and show that many PA-binding proteins display enhanced binding as a function of negative curvature stress. This result is corroborated by the observation that positive curvature stress, induced by lyso phosphatidylcholine, abolishes PA binding of target proteins. We show, for the first time, that a novel plant PA-binding protein, Arabidopsis Epsin-like Clathrin Adaptor 1 (ECA1) displays curvature-dependence in its binding to PA. Other established PA targets examined in this study include, the plant proteins TGD2, and PDK1, the yeast proteins Opi1 and Spo20, and, the mammalian protein Raf-1 kinase and the C2 domain of the mammalian phosphatidylserine binding protein Lact as control. Based on our observations, we propose that liposome binding assays are the preferred method to investigate lipid binding compared to the popular lipid overlay assays where membrane environment is lost. The use of complex lipid mixtures is important to elucidate further aspects of PA binding proteins. Copyright © 2016. Published by Elsevier B.V.

A Single Rainbow Trout Cobalamin-binding Protein Stands in for Three Human Binders

PubMed Central

Greibe, Eva; Fedosov, Sergey; Sorensen, Boe S.; Højrup, Peter; Poulsen, Steen S.; Nexo, Ebba

2012-01-01

Cobalamin uptake and transport in mammals are mediated by three cobalamin-binding proteins: haptocorrin, intrinsic factor, and transcobalamin. The nature of cobalamin-binding proteins in lower vertebrates remains to be elucidated. The aim of this study was to characterize the cobalamin-binding proteins of the rainbow trout (Oncorhynchus mykiss) and to compare their properties with those of the three human cobalamin-binding proteins. High cobalamin-binding capacity was found in trout stomach (210 pmol/g), roe (400 pmol/g), roe fluid (390 nmol/liter), and plasma (2500 nmol/liter). In all cases, it appeared to be the same protein based on analysis of partial sequences and immunological responses. The trout cobalamin-binding protein was purified from roe fluid, sequenced, and further characterized. Like haptocorrin, the trout cobalamin-binding protein was stable at low pH and had a high binding affinity for the cobalamin analog cobinamide. Like haptocorrin and transcobalamin, the trout cobalamin-binding protein was present in plasma and recognized ligands with altered nucleotide moiety. Like intrinsic factors, the trout cobalamin-binding protein was present in the stomach and resisted degradation by trypsin and chymotrypsin. It also resembled intrinsic factor in the composition of conserved residues in the primary cobalamin-binding site in the C terminus. The trout cobalamin-binding protein was glycosylated and displayed spectral properties comparable with those of haptocorrin and intrinsic factor. In conclusion, only one soluble cobalamin-binding protein was identified in the rainbow trout, a protein that structurally behaves like an intermediate between the three human cobalamin-binding proteins. PMID:22872637

Spectroscopic and molecular docking studies on the interaction of antiviral drug nevirapine with calf thymus DNA.

PubMed

Moghadam, Neda Hosseinpour; Salehzadeh, Sadegh; Shahabadi, Nahid

2017-09-02

The interaction of calf thymus DNA with nevirapine at physiological pH was studied by using absorption, circular dichroism, viscosity, differential pulse voltammetry, fluorescence techniques, salt effect studies and computational methods. The drug binds to ct-DNA in a groove binding mode, as shown by slight variation in the viscosity of ct-DNA. Furthermore, competitive fluorimetric studies with Hoechst 33258 indicate that nevirapine binds to DNA via groove binding. Moreover, the structure of nevirapine was optimized by DFT calculations and was used for the molecular docking calculations. The molecular docking results suggested that nevirapine prefers to bind on the minor groove of ct-DNA.

Comparison of solute-binding properties of plastic materials used as pharmaceutical product containers.

PubMed

Jenke, Dennis; Couch, Tom; Gillum, Amy

2010-01-01

Material/water equilibrium binding constants (E(b)) were determined for 11 organic solutes and 2 plastic materials commonly used in pharmaceutical product containers (plasticized polyvinyl chloride and polyolefin). In general, solute binding by the plasticized polyvinyl chloride material was greater, by nearly an order of magnitude, than the binding by the polyolefin (on an equal weight basis). The utilization of the binding constants to facilitate container compatibility assessments (e.g., drug loss by container binding) for drug-containing products is discussed.

Identification and therapeutic potential of a vitronectin binding region of meningococcal msf.

PubMed

Hill, Darryl J; Griffiths, Natalie J; Borodina, Elena; Andreae, Clio A; Sessions, Richard B; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen.

Identification and Therapeutic Potential of a Vitronectin Binding Region of Meningococcal Msf

PubMed Central

Hill, Darryl J.; Griffiths, Natalie J.; Borodina, Elena; Andreae, Clio A.; Sessions, Richard B.; Virji, Mumtaz

2015-01-01

The human pathogen Neisseria meningitides (Nm) attains serum resistance via a number of mechanisms, one of which involves binding to the host complement regulator protein vitronectin. We have shown previously that the Meningococcal surface fibril (Msf), a trimeric autotransporter, binds to the activated form of vitronectin (aVn) to increase Nm survival in human serum. In this study, we aimed to identify the aVn-binding region of Msf to assess its potential as an antigen which can elicit antibodies that block aVn binding and/or possess bactericidal properties. Using several recombinant Msf fragments spanning its surface-exposed region, the smallest aVn-binding recombinants were found to span residues 1-86 and 39-124. The use of further deletion constructs and overlapping recombinant Msf fragments suggested that a region of Msf comprising residues 39-82 may be primarily important for aVn binding and that other regions may also be involved but to a lesser extent. Molecular modelling implicated K66 and K68, conserved in all available Msf sequences, to be involved in the interaction. Recombinant fragments which bound to aVn were able to reduce the survival advantage conveyed by aVn-interaction in serum bactericidal assays. Antibodies raised against one such fragment inhibited aVn binding to Msf. In addition, the antibodies enhanced specific killing of Msf-expressing Nm in a dose-dependent manner. Overall, this study identifies an aVn-binding region of Msf, an adhesin known to impart serum resistance properties to the pathogen; and shows that this region of Msf can elicit antibodies with dual properties which reduce pathogen survival within the host and thus has potential as a vaccine antigen. PMID:25826209

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein.

PubMed

Satheshkumar, Angupillai; Elango, Kuppanagounder P

2014-09-15

The spectral techniques such as UV-Vis, (1)H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3×10(8) L mol(-1). Based on the Forster's theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol(-1), which is comparable to our experimental free energy of binding (-49 kJ mol(-1)) obtained from fluorescence study. Copyright © 2014 Elsevier B.V. All rights reserved.

Spectroscopic and molecular docking studies on the charge transfer complex of bovine serum albumin with quinone in aqueous medium and its influence on the ligand binding property of the protein

NASA Astrophysics Data System (ADS)

Satheshkumar, Angupillai; Elango, Kuppanagounder P.

2014-09-01

The spectral techniques such as UV-Vis, 1H NMR and fluorescence and electrochemical experiments have been employed to investigate the interaction between 2-methoxy-3,5,6-trichloro-1,4-benzoquinone (MQ; a water soluble quinone) and bovine serum albumin (BSA) in aqueous medium. The fluorescence of BSA was quenched by MQ via formation of a 1:1 BSA-MQ charge transfer adduct with a formation constant of 3.3 × 108 L mol-1. Based on the Forster’s theory the binding distance between them is calculated as 2.65 nm indicating high probability of binding. For the first time, influence of quinone on the binding property of various types of ligands such as aspirin, ascorbic acid, nicotinimide and sodium stearate has also been investigated. The results indicated that the strong and spontaneous binding existing between BSA and MQ, decreased the intensity of binding of these ligands with BSA. Since Tryptophan (Trp) is the basic residue present in BSA, a comparison between binding property of Trp-MQ adduct with that of BSA-MQ with these ligands has also been attempted. 1H NMR titration study indicated that the Trp forms a charge transfer complex with MQ, which reduces the interaction of Trp with the ligands. Molecular docking study supported the fact that the quinone interacts with the Trp212 unit of the BSA and the free energy change of binding (ΔG) for the BSA-MQ complex was found to be -46 kJ mol-1, which is comparable to our experimental free energy of binding (-49 kJ mol-1) obtained from fluorescence study.

Engineering of the function of diamond-like carbon binding peptides through structural design.

PubMed

Gabryelczyk, Bartosz; Szilvay, Géza R; Singh, Vivek K; Mikkilä, Joona; Kostiainen, Mauri A; Koskinen, Jari; Linder, Markus B

2015-02-09

The use of phage display to select material-specific peptides provides a general route towards modification and functionalization of surfaces and interfaces. However, a rational structural engineering of the peptides for optimal affinity is typically not feasible because of insufficient structure-function understanding. Here, we investigate the influence of multivalency of diamond-like carbon (DLC) binding peptides on binding characteristics. We show that facile linking of peptides together using different lengths of spacers and multivalency leads to a tuning of affinity and kinetics. Notably, increased length of spacers in divalent systems led to significantly increased affinities. Making multimers influenced also kinetic aspects of surface competition. Additionally, the multivalent peptides were applied as surface functionalization components for a colloidal form of DLC. The work suggests the use of a set of linking systems to screen parameters for functional optimization of selected material-specific peptides.

Fragment growing and linking lead to novel nanomolar lactate dehydrogenase inhibitors.

PubMed

Kohlmann, Anna; Zech, Stephan G; Li, Feng; Zhou, Tianjun; Squillace, Rachel M; Commodore, Lois; Greenfield, Matthew T; Lu, Xiaohui; Miller, David P; Huang, Wei-Sheng; Qi, Jiwei; Thomas, R Mathew; Wang, Yihan; Zhang, Sen; Dodd, Rory; Liu, Shuangying; Xu, Rongsong; Xu, Yongjin; Miret, Juan J; Rivera, Victor; Clackson, Tim; Shakespeare, William C; Zhu, Xiaotian; Dalgarno, David C

2013-02-14

Lactate dehydrogenase A (LDH-A) catalyzes the interconversion of lactate and pyruvate in the glycolysis pathway. Cancer cells rely heavily on glycolysis instead of oxidative phosphorylation to generate ATP, a phenomenon known as the Warburg effect. The inhibition of LDH-A by small molecules is therefore of interest for potential cancer treatments. We describe the identification and optimization of LDH-A inhibitors by fragment-based drug discovery. We applied ligand based NMR screening to identify low affinity fragments binding to LDH-A. The dissociation constants (K(d)) and enzyme inhibition (IC(50)) of fragment hits were measured by surface plasmon resonance (SPR) and enzyme assays, respectively. The binding modes of selected fragments were investigated by X-ray crystallography. Fragment growing and linking, followed by chemical optimization, resulted in nanomolar LDH-A inhibitors that demonstrated stoichiometric binding to LDH-A. Selected molecules inhibited lactate production in cells, suggesting target-specific inhibition in cancer cell lines.

Rapid and Facile Microwave-Assisted Surface Chemistry for Functionalized Microarray Slides

PubMed Central

Lee, Jeong Heon; Hyun, Hoon; Cross, Conor J.; Henary, Maged; Nasr, Khaled A.; Oketokoun, Rafiou; Choi, Hak Soo; Frangioni, John V.

2011-01-01

We describe a rapid and facile method for surface functionalization and ligand patterning of glass slides based on microwave-assisted synthesis and a microarraying robot. Our optimized reaction enables surface modification 42-times faster than conventional techniques and includes a carboxylated self-assembled monolayer, polyethylene glycol linkers of varying length, and stable amide bonds to small molecule, peptide, or protein ligands to be screened for binding to living cells. We also describe customized slide racks that permit functionalization of 100 slides at a time to produce a cost-efficient, highly reproducible batch process. Ligand spots can be positioned on the glass slides precisely using a microarraying robot, and spot size adjusted for any desired application. Using this system, we demonstrate live cell binding to a variety of ligands and optimize PEG linker length. Taken together, the technology we describe should enable high-throughput screening of disease-specific ligands that bind to living cells. PMID:23467787

Advancing Drug Discovery through Enhanced Free Energy Calculations.

PubMed

Abel, Robert; Wang, Lingle; Harder, Edward D; Berne, B J; Friesner, Richard A

2017-07-18

A principal goal of drug discovery project is to design molecules that can tightly and selectively bind to the target protein receptor. Accurate prediction of protein-ligand binding free energies is therefore of central importance in computational chemistry and computer aided drug design. Multiple recent improvements in computing power, classical force field accuracy, enhanced sampling methods, and simulation setup have enabled accurate and reliable calculations of protein-ligands binding free energies, and position free energy calculations to play a guiding role in small molecule drug discovery. In this Account, we outline the relevant methodological advances, including the REST2 (Replica Exchange with Solute Temperting) enhanced sampling, the incorporation of REST2 sampling with convential FEP (Free Energy Perturbation) through FEP/REST, the OPLS3 force field, and the advanced simulation setup that constitute our FEP+ approach, followed by the presentation of extensive comparisons with experiment, demonstrating sufficient accuracy in potency prediction (better than 1 kcal/mol) to substantially impact lead optimization campaigns. The limitations of the current FEP+ implementation and best practices in drug discovery applications are also discussed followed by the future methodology development plans to address those limitations. We then report results from a recent drug discovery project, in which several thousand FEP+ calculations were successfully deployed to simultaneously optimize potency, selectivity, and solubility, illustrating the power of the approach to solve challenging drug design problems. The capabilities of free energy calculations to accurately predict potency and selectivity have led to the advance of ongoing drug discovery projects, in challenging situations where alternative approaches would have great difficulties. The ability to effectively carry out projects evaluating tens of thousands, or hundreds of thousands, of proposed drug candidates, is potentially transformative in enabling hard to drug targets to be attacked, and in facilitating the development of superior compounds, in various dimensions, for a wide range of targets. More effective integration of FEP+ calculations into the drug discovery process will ensure that the results are deployed in an optimal fashion for yielding the best possible compounds entering the clinic; this is where the greatest payoff is in the exploitation of computer driven design capabilities. A key conclusion from the work described is the surprisingly robust and accurate results that are attainable within the conventional classical simulation, fixed charge paradigm. No doubt there are individual cases that would benefit from a more sophisticated energy model or dynamical treatment, and properties other than protein-ligand binding energies may be more sensitive to these approximations. We conclude that an inflection point in the ability of MD simulations to impact drug discovery has now been attained, due to the confluence of hardware and software development along with the formulation of "good enough" theoretical methods and models.

Nano-Sized Cyclodextrin-Based Molecularly Imprinted Polymer Adsorbents for Perfluorinated Compounds—A Mini-Review

PubMed Central

Karoyo, Abdalla H.; Wilson, Lee D.

2015-01-01

Recent efforts have been directed towards the design of efficient and contaminant selective remediation technology for the removal of perfluorinated compounds (PFCs) from soils, sediments, and aquatic environments. While there is a general consensus on adsorption-based processes as the most suitable methodology for the removal of PFCs from aquatic environments, challenges exist regarding the optimal materials design of sorbents for selective uptake of PFCs. This article reviews the sorptive uptake of PFCs using cyclodextrin (CD)-based polymer adsorbents with nano- to micron-sized structural attributes. The relationship between synthesis of adsorbent materials and their structure relate to the overall sorption properties. Hence, the adsorptive uptake properties of CD-based molecularly imprinted polymers (CD-MIPs) are reviewed and compared with conventional MIPs. Further comparison is made with non-imprinted polymers (NIPs) that are based on cross-linking of pre-polymer units such as chitosan with epichlorohydrin in the absence of a molecular template. In general, MIPs offer the advantage of selectivity, chemical tunability, high stability and mechanical strength, ease of regeneration, and overall lower cost compared to NIPs. In particular, CD-MIPs offer the added advantage of possessing multiple binding sites with unique physicochemical properties such as tunable surface properties and morphology that may vary considerably. This mini-review provides a rationale for the design of unique polymer adsorbent materials that employ an intrinsic porogen via incorporation of a macrocyclic compound in the polymer framework to afford adsorbent materials with tunable physicochemical properties and unique nanostructure properties. PMID:28347047

Therapeutic Antibodies by Phage Display.

PubMed

Shim, Hyunbo

2016-01-01

Antibody phage display is a major technological platform for the generation of fully human antibodies for therapeutic purposes. The in vitro binder selection by phage display allows researchers to have more extensive control over binding parameters and facilitates the isolation of clinical candidate antibodies with desired binding and/or functional profiles. Since the invention of antibody phage display in late 1980s, significant technological advancements in the design, construction, and selection of the antibody libraries have been made, and several fully human antibodies generated by phage display are currently approved or in various clinical development stages. In this review, the background and details of antibody phage display technology, and representative antibody libraries with natural or synthetic sequence diversity and different construction strategies are described. The generation, optimization, functional and biophysical properties, and preclinical and clinical developments of some of the phage display-derived therapeutic antibodies approved for use in patients or in late-stage clinical trials are also discussed. With evolving novel disease targets and therapeutic strategies, antibody phage display is expected to continue to play a central role in the development of the next generation of therapeutic antibodies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Functional Characterisation of the WW Minimal Domain for Delivering Therapeutic Proteins by Adenovirus Dodecahedron

PubMed Central

Villegas-Méndez, Ana; Fender, Pascal; Garin, Marina I.; Rothe, Romy; Liguori, Lavinia; Marques, Bruno; Lenormand, Jean-Luc

2012-01-01

Protein transduction offers a great therapeutic potential by efficient delivery of biologically active cargo into cells. The Adenovirus Dd (Dodecahedron) has recently been shown to deliver proteins fused to the tandem WW2-3-4 structural domains from the E3 ubiquitin ligase Nedd4. In this study, we conclusively show that Dd is able to efficiently deliver cargo inside living cells, which mainly localize in fast moving endocytic vesicles, supporting active transport along the cytoskeleton. We further improve this delivery system by expressing a panel of 13 WW-GFP mutant forms to characterize their binding properties towards Dd. We identified the domain WW3 and its mutant form WW3_10_13 to be sufficient for optimal binding to Dd. We greatly minimise the interacting WW modules from 20 to 6 kDa without compromising its efficient delivery by Dd. Using these minimal WW domains fused to the tumor suppressor p53 protein, we show efficient cellular uptake and distribution into cancer cells, leading to specific induction of apoptosis in these cells. Taken together, these findings represent a step further towards the development of a Dd-based delivery system for future therapeutic application. PMID:23028993

AIE active multianalyte fluorescent probe for the detection of Cu2+, Ni2+ and Hg2+ ions.

PubMed

Pannipara, Mehboobali; Al-Sehemi, Abdullah G; Irfan, Ahmad; Assiri, Mohammed; Kalam, Abul; Al-Ammari, Yahya S

2018-08-05

A novel pyrazolyl chromene derivative (Probe 1) displaying aggregation induced emission (AIE) properties that capable of sensing of multiple metal ions has been designed and synthesized. The multi analyte probe exhibits selective sensing for Cu 2+ and Ni 2+ ions via fluorescence turn-off mechanism and ratiometric selectivity for Hg 2+ ions in aqueous media. The extent of binding of the probe with sensitive metal ions has been demonstrated. The experimental results were further investigated by computational means by optimizing the ground state geometries of Probe 1 and its various metal complexes for Probe 1-Ni, Probe 1-Hg and Probe 1-Cu using density functional theory (DFT) at B3LYP/6-31+g(d,p) (LANL2DZ) level. On the basis of binding energies, the stability of metal complexes has been studied. In Probe 1-Ni and Probe 1-Cu complexes, charge transfer has been observed from Probe 1 to metal ions revealing ligand to metal charge transfer (LMCT) while in Probe1-Hg complex LMCT as well as intra-molecular charge tranfer (ICT) within Probe 1. Copyright © 2018 Elsevier B.V. All rights reserved.

Direct replacement of antibodies with molecularly imprinted polymer (MIP) nanoparticles in ELISA – development of a novel assay for vancomycin

PubMed Central

Chianella, Iva; Guerreiro, Antonio; Moczko, Ewa; Caygill, J. Sarah; Piletska, Elena V.; Perez De Vargas Sansalvador, Isabel M.; Whitcombe, Michael J.; Piletsky, Sergey A.

2016-01-01

A simple and straightforward technique for coating microplate wells with molecularly imprinted polymer nanoparticles (nanoMIPs) to develop ELISA type assays is presented here for the first time. NanoMIPs were synthesized by a solid phase approach with immobilized vancomycin (template) and characterized using Biacore 3000, dynamic light scattering and electron microscopy. Immobilization, blocking and washing conditions were optimized in microplate format. The detection of vancomycin was achieved in competitive binding experiments with a HRP-vancomycin conjugate. The assay was capable of measuring vancomycin in buffer and in blood plasma within the range 0.001-70 nM with a detection limit of 0.0025 nM (2.5 pM). The sensitivity of the assay was three orders of magnitude better than a previously described ELISA based on antibodies. In these experiments nanoMIPs have shown high affinity and minimal interference from blood plasma components. Immobilized nanoMIPs were stored for 1 month at room temperature without any detrimental effects to their binding properties. The high affinity of nanoMIPs and the lack of a requirement for cold chain logistics make them an attractive alternative to traditional antibodies used in ELISA. PMID:23947402

Size-dependent impact of CNTs on dynamic properties of calmodulin.

PubMed

Gao, Jian; Wang, Liming; Kang, Seung-gu; Zhao, Lina; Ji, Mingjuan; Chen, Chunying; Zhao, Yuliang; Zhou, Ruhong; Li, Jingyuan

2014-11-07

There are growing concerns about the biosafety of nanomaterials such as carbon nanotubes (CNTs) as their applications become more widespread. We report here a theoretical and experimental study of the binding of various sizes of CNTs [CNT (4,4), (5,5), (6,6) and (7,7)] to calmodulin (CaM) protein and, in particular, their impact on the Ca(2+)-dependent dynamic properties of CaM. Our simulations show that all the CNTs can plug into the hydrophobic binding pocket of Ca(2+)-bound CaM with binding affinities comparable with the native substrate M13 peptide. Even though CNT (4,4) shows a similar behavior to the M13 peptide in its dissociation from Ca(2+)-free CaM, wider CNTs still bind firmly to CaM, indicating a potential failure of Ca(2+) regulation. Such a size-dependent impact of CNTs on the dynamic properties of CaM is a result of the excessively strong hydrophobic interactions between the wider CNTs and CaM. These simulation results were confirmed by circular dichroism spectroscopy, which showed that the secondary structures of CaM become insensitive to Ca(2+) concentrations after the addition of CNTs. Our findings indicate that the cytotoxicity of nanoparticles to proteins arises not only from the inhibition of static protein structures (binding pockets), but also from impacts on their dynamic properties.

Cation induced electrochromism in 2,4-dinitrophenylhydrazine (DNPH): Tuning optical properties of aromatic rings

NASA Astrophysics Data System (ADS)

Sanader, Željka; Brunet, Claire; Broyer, Michel; Antoine, Rodolphe; Dugourd, Philippe; Mitrić, Roland; Bonačić-Koutecký, Vlasta

2013-05-01

We have theoretically investigated the influence of protons and noble metal cations on optical properties of 2,4-dinitrophenylhydrazine (DNPH). We show that optical properties of aromatic rings can be tuned by cation-induced electrochromism in DNPH due to binding to specific NO2 groups. Our findings on cation-induced electrochromism in DNPH may open new routes in two different application areas, due to the fact that DNPH can easily bind to biological molecules and surface materials through carbonyl groups.

Optimization of exopolysaccharide production from Pseudomonas stutzeri AS22 and examination of its metal-binding abilities.

PubMed

Maalej, H; Hmidet, N; Boisset, C; Buon, L; Heyraud, A; Nasri, M

2015-02-01

To investigate the effect of culture conditions and medium components on exopolysaccharide (EPS) production by Pseudomonas stutzeri AS22 and to access the EPS performance as a metal-binding exopolysaccharide. The EPS production conditions of Ps. stutzeri AS22 in submerged culture were optimized using two approaches for EPS quantification, and its metal-binding capacity was evaluated using both single and mixed metal ions systems. Maximum EPS level was achieved after 24 h of incubation at 30°C with an initial pH of 8.0, 250 rev min(-1) stirring level and 10% inoculum size. 50 g l(-1) starch, 5 g l(-1) yeast extract, 0.5 g l(-1) NaCl, 1.4 g l(-1) K2 HPO4, 0.4 g l(-1) MgSO4, 0.4 g l(-1) CaCl2 and 1 g l(-1) mannose were found to be the most suitable carbon, nitrogen, mineral and additional carbohydrate sources, respectively. From metal-binding experiments, the crude EPS showed interesting metal adsorption capacity adopting the order Pb > Co > Fe > Cu > Cd. Lead was preferentially biosorbed with a maximal uptake of 460 mg g(-1) crude EPS. Under the optimal culture requirements, EPS level reached 10.2 g l(-1) after 24 h of fermentation, seven times more than the production under initial conditions. According to the metal-binding assay, the crude EPS has potential to be used as a novel biosorbent in the treatment of heavy metals-contaminated water. Our results are interesting in terms of yield as well as efficiency for the potential use of the Ps. stutzeri exopolysaccharide as a metal-absorbent polymer in the bioremediation field. © 2014 The Society for Applied Microbiology.

Antioxidative capacity and binding affinity of the complex of green tea catechin and beta-lactoglobulin glycated by the Maillard reaction.

PubMed

Perusko, Marija; Al-Hanish, Ayah; Mihailovic, Jelena; Minic, Simeon; Trifunovic, Sara; Prodic, Ivana; Cirkovic Velickovic, Tanja

2017-10-01

Major green tea catechin, epigallocatechin-3-gallate (EGCG), binds non-covalently to numerous dietary proteins, including beta-lactoglobulin of cow's milk. The effects of glycation of proteins via Maillard reaction on the binding capacity for polyphenols and the antiradical properties of the formed complexes have not been studied previously. Binding constant of BLG glycated by milk sugar lactose to EGCG was measured by the method of fluorophore quenching. Binding of EGCG was confirmed by CD and FTIR. The antioxidative properties of the complexes were examined by measuring ABTS radical scavenging capacity, superoxide anion scavenging capacity and total reducing power assay. Glycation of BLG does not significantly influence the binding constant of EGCG for the protein. Conformational changes were observed for both native and glycated BLG upon complexation with EGCG. Masking effect of polyphenol complexation on the antioxidative potential of the protein was of the similar degree for both glycated BLG and native BLG. Copyright © 2017 Elsevier Ltd. All rights reserved.

Protein-ligand docking using fitness learning-based artificial bee colony with proximity stimuli.

PubMed

Uehara, Shota; Fujimoto, Kazuhiro J; Tanaka, Shigenori

2015-07-07

Protein-ligand docking is an optimization problem, which aims to identify the binding pose of a ligand with the lowest energy in the active site of a target protein. In this study, we employed a novel optimization algorithm called fitness learning-based artificial bee colony with proximity stimuli (FlABCps) for docking. Simulation results revealed that FlABCps improved the success rate of docking, compared to four state-of-the-art algorithms. The present results also showed superior docking performance of FlABCps, in particular for dealing with highly flexible ligands and proteins with a wide and shallow binding pocket.

Elaborate ligand-based modeling reveal new submicromolar Rho kinase inhibitors

NASA Astrophysics Data System (ADS)

Shahin, Rand; AlQtaishat, Saja; Taha, Mutasem O.

2012-02-01

Rho Kinase (ROCKII) has been recently implicated in several cardiovascular diseases prompting several attempts to discover and optimize new ROCKII inhibitors. Towards this end we explored the pharmacophoric space of 138 ROCKII inhibitors to identify high quality pharmacophores. The pharmacophoric models were subsequently allowed to compete within quantitative structure-activity relationship (QSAR) context. Genetic algorithm and multiple linear regression analysis were employed to select an optimal combination of pharmacophoric models and 2D physicochemical descriptors capable of accessing self-consistent QSAR of optimal predictive potential ( r 77 = 0.84, F = 18.18, r LOO 2 = 0.639, r PRESS 2 against 19 external test inhibitors = 0.494). Two orthogonal pharmacophores emerged in the QSAR equation suggesting the existence of at least two binding modes accessible to ligands within ROCKII binding pocket. Receiver operating characteristic (ROC) curve analyses established the validity of QSAR-selected pharmacophores. Moreover, the successful pharmacophores models were found to be comparable with crystallographically resolved ROCKII binding pocket. We employed the pharmacophoric models and associated QSAR equation to screen the national cancer institute (NCI) list of compounds Eight submicromolar ROCKII inhibitors were identified. The most potent gave IC50 values of 0.7 and 1.0 μM.

Enzymatic conversion of pretreated biomass into fermentable sugars for biorefinery operation

NASA Astrophysics Data System (ADS)

Gao, Dahai

2011-12-01

Depleting petroleum reserves and potential climate change caused by fossil fuel consumption have attracted significant attention towards the use of alternative renewable resources for production of fuels and chemicals. Lignocellulosic biomass provides a plentiful resource for the sustainable production of biofuels and biochemicals and could serve as an important contributor to the world energy portfolio in the near future. Successful biological conversion of lignocellulosic biomass requires an efficient and economical pretreatment method, high glucose/xylose yields during enzymatic hydrolysis and fermentation of both hexose and pentose to ethanol. High enzyme loading is a major economic bottleneck for the commercial processing of pretreated lignocellulosic biomass to produce fermentable sugars. Optimizing the enzyme cocktail for specific types of pretreated biomass allows for a significant reduction in enzyme loading without sacrificing hydrolysis yield. Core glycosyl hydrolases were isolated and purified from various sources to help rationally optimize an enzyme cocktail to digest ammonia fiber expansion (AFEX) treated corn stover. The four core cellulases were endoglucanase I (EG I), cellobiohydrolase I (CBH I), cellobiohydrolase II (CBH II) and beta-Glucosidase (betaG). The two core hemicellulases were an endoxylanase (EX) and a beta-xylosidase (betaX). A diverse set of accessory hemicellulases from bacterial sources was found necessary to enhance the synergistic action of cellulases hydrolysing AFEX pretreated corn stover. High glucose (around 80%) and xylose (around 70%) yields were achieved with a moderate enzyme loading (˜20 mg protein/g glucan) using an in-house developed enzyme cocktail and this cocktail was compared to commercial enzyme. Studying the binding properties of cellulases to lignocellulosic substrates is critical to achieving a fundamental understanding of plant cell wall saccharification. Lignin auto-fluorescence and degradation products formed during pretreatment impede accurate quantification of individual glycosyl hydrolases (GH) binding to pretreated cell walls. A high-throughput Fast Protein Liquid Chromatography (HT-FPLC) based method has been developed to quantify CBH I, CBH II and EG I present in hydrolyzates of untreated, AFEX, and dilute-acid pretreated corn stover. This method can accurately quantify individual enzymes present in complex binary and ternary protein mixtures without interference from plant cell wall derived components. The binding characteristics of CBH I, CBH II and EG I during 48 hours hydrolysis were studied on different cellulose allomorphs: microcrystalline cellulose Avicel (cellulose Ibeta), liquid ammonia treated cellulose (cellulose III), sodium hydroxide treated cellulose (cellulose II) and phosphoric acid swollen amorphous cellulose (AC). The digestibility ranking is AC>cellulose III>cellulose II>cellulose I. However, AC has the highest initial enzyme binding capacity while cellulose III had the lowest. CBH II is less stable during hydrolysis. Time course binding studies were also performed for pretreated biomass. Ammonia Fiber Expansion (AFEX) treated corn stover (CS), dilute acid (ACID) treated CS and ionic liquid (IL) pretreated CS were compared. The results indicate that presence of lignin is responsible for significant unproductive cellulase binding. These results are critical for improving our understanding of enzyme synergism, productive/unproductive enzyme binding and the role of pretreatment on enzyme accessibility to lignocellulosic plant cell walls. The results also assist in engineering novel low unproductive binding enzyme systems and developing economic enzyme recycle options.

Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

DOE Office of Scientific and Technical Information (OSTI.GOV)

McLellan, Jason S.; Correia, Bruno E.; Chen, Man

2012-06-28

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potentmore » neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.« less

Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG)-containing transcripts.

PubMed

Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J; Childs-Disney, Jessica L; Sobczak, Krzysztof; Disney, Matthew D

2012-03-16

Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)(exp)) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5'CAG/3'GAC motif found in r(CAG)(exp) hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)(exp). The compound was identified by first studying the binding of RNA 1 × 1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5'CAG/3'GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate, a small molecule that improves pre-mRNA splicing defects associated with the r(CAG)(exp)-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)(exp) and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)(exp) toxicity. The approach used in these studies, defining the small RNA motifs that bind small molecules with known affinity for nucleic acids and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in the human genomic sequence.

Chemical correction of pre-mRNA splicing defects associated with sequestration of muscleblind-like 1 protein by expanded r(CAG) transcripts

PubMed Central

Kumar, Amit; Parkesh, Raman; Sznajder, Lukasz J.; Childs-Disney, Jessica; Sobczak, Krzysztof; Disney, Matthew D.

2012-01-01

Recently, it was reported that expanded r(CAG) triplet repeats (r(CAG)exp) associated with untreatable neurological diseases cause pre-mRNA mis-splicing likely due to sequestration of muscleblind-like 1 (MBNL1) splicing factor. Bioactive small molecules that bind the 5’CAG/3’GAC motif found in r(CAG)exp hairpin structure were identified by using RNA binding studies and virtual screening/chemical similarity searching. Specifically, a benzylguanidine-containing small molecule was found to improve pre-mRNA alternative splicing of MBNL1-sensitive exons in cells expressing the toxic r(CAG)exp. The compound was identified by first studying the binding of RNA 1×1 nucleotide internal loops to small molecules known to have affinity for nucleic acids. Those studies identified 4',6-diamidino-2-phenylindole (DAPI) as a specific binder to RNAs with the 5’CAG/3’GAC motif. DAPI was then used as a query molecule in a shape- and chemistry alignment-based virtual screen to identify compounds with improved properties, which identified 4-guanidinophenyl 4-guanidinobenzoate as small molecule capable of improving pre-mRNA splicing defects associated with the r(CAG)exp-MBNL1 complex. This compound may facilitate the development of therapeutics to treat diseases caused by r(CAG)exp and could serve as a useful chemical tool to dissect the mechanisms of r(CAG)exp toxicity. The approach used in these studies, defining the small RNA motifs that bind known nucleic acid binders and then using virtual screening to optimize them for bioactivity, may be generally applicable for designing small molecules that target other RNAs in human genomic sequence. PMID:22252896

Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus.

PubMed

McLellan, Jason S; Correia, Bruno E; Chen, Man; Yang, Yongping; Graham, Barney S; Schief, William R; Kwong, Peter D

2011-06-24

Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required. Published by Elsevier Ltd.

Amino acid signature enables proteins to recognize modified tRNA.

PubMed

Spears, Jessica L; Xiao, Xingqing; Hall, Carol K; Agris, Paul F

2014-02-25

Human tRNA(Lys3)UUU is the primer for HIV replication. The HIV-1 nucleocapsid protein, NCp7, facilitates htRNA(Lys3)UUU recruitment from the host cell by binding to and remodeling the tRNA structure. Human tRNA(Lys3)UUU is post-transcriptionally modified, but until recently, the importance of those modifications in tRNA recognition by NCp7 was unknown. Modifications such as the 5-methoxycarbonylmethyl-2-thiouridine at anticodon wobble position-34 and 2-methylthio-N(6)-threonylcarbamoyladenosine, adjacent to the anticodon at position-37, are important to the recognition of htRNA(Lys3)UUU by NCp7. Several short peptides selected from phage display libraries were found to also preferentially recognize these modifications. Evolutionary algorithms (Monte Carlo and self-consistent mean field) and assisted model building with energy refinement were used to optimize the peptide sequence in silico, while fluorescence assays were developed and conducted to verify the in silico results and elucidate a 15-amino acid signature sequence (R-W-Q/N-H-X2-F-Pho-X-G/A-W-R-X2-G, where X can be most amino acids, and Pho is hydrophobic) that recognized the tRNA's fully modified anticodon stem and loop domain, hASL(Lys3)UUU. Peptides of this sequence specifically recognized and bound modified htRNA(Lys3)UUU with an affinity 10-fold higher than that of the starting sequence. Thus, this approach provides an effective means of predicting sequences of RNA binding peptides that have better binding properties. Such peptides can be used in cell and molecular biology as well as biochemistry to explore RNA binding proteins and to inhibit those protein functions.

The effects of linear assembly of two carbazole groups on acid-base and DNA-binding properties of a ruthenium(II) complex.

PubMed

Chen, Xi; Xue, Long-Xin; Ju, Chun-Chuan; Wang, Ke-Zhi

2013-07-01

A novel Ru(II) complex of [Ru(bpy)2(Hbcpip)](ClO4)2 {where bpy=2,2-bipyridine, Hbcpip=2-(4-(9H-3,9'-bicarbazol-9-yl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} is synthesized and characterized. Calf-thymus DNA-binding properties of the complex were studied by UV-vis absorption and luminescence titrations, steady-state emission quenching by [Fe(CN)6](4-), DNA competitive binding with ethidium bromide, thermal denaturation and DNA viscosity measurements. The results indicate that the complex partially intercalated into the DNA with a binding constant of (5.5±1.4)×10(5) M(-1) in buffered 50 mM NaCl. The acid-base properties of the complex were also studied by UV-visible and luminescence spectrophotometric pH titrations, and ground- and excited-state acidity ionization constant values were derived. Copyright © 2013 Elsevier B.V. All rights reserved.

Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (Haliotis discus discus): Insights as a potent player in host antioxidant defense.

PubMed

Herath, H M L P B; Wickramasinghe, P D S U; Bathige, S D N K; Jayasooriya, R G P T; Kim, Gi-Young; Park, Myoung Ae; Kim, Chul; Lee, Jehee

2017-01-01

Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H 2 O 2 , the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H 2 O 2 . Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, Haliotis discus discus. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171-246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu +2 and Cd +2 ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, AbGSR was found to be ubiquitously distributed in different types of abalone tissues. AbGSR mRNA expression was significantly upregulated in response to three immune challenges: Vibrio parahaemolyticus, Listeria monocytogenes, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR. Copyright © 2016 Elsevier Ltd. All rights reserved.

Selection, Identification, and Binding Mechanism Studies of an ssDNA Aptamer Targeted to Different Stages of E. coli O157:H7.

PubMed

Zou, Ying; Duan, Nuo; Wu, Shijia; Shen, Mofei; Wang, Zhouping

2018-06-06

Enterohemorrhagic Escherichia coli O157:H7 ( E. coli O157:H7) is known as an important food-borne pathogen related to public health. In this study, aptamers which could bind to different stages of E. coli O157:H7 (adjustment phase, log phase, and stationary phase) with high affinity and specificity were obtained by the whole cell-SELEX method through 14 selection rounds including three counter-selection rounds. Altogether, 32 sequences were obtained, and nine families were classified to select the optimal aptamer. To analyze affinity and specificity by flow cytometer, an ssDNA aptamer named Apt-5 was picked out as the optimal aptamer that recognizes different stages of E. coli O157:H7 specifically with the K d value of 9.04 ± 2.80 nM. In addition, in order to study the binding mechanism, target bacteria were treated by proteinase K and trypsin, indicating that the specific binding site is not protein on the cell membrane. Furthermore, when we treated E. coli O157:H7 with EDTA, the result showed that the binding site might be lipopolysaccharide (LPS) on the outer membrane of E. coli O157:H7.

Validation of APF as a Urinary Biomarker for Interstitial Cystitis

DTIC Science & Technology

2014-10-01

its C-terminus—in order to determine whether FL-CKAP4 could improve APF binding response over the truncated rCKAP4 used in our preliminary studies...recognized by a CKAP4 antibody, indicating high activity. However, when tested by SPR, FL-CKAP4 did not improve APF binding response over the truncated ...rCKAP4 (Figure 2). We then concentrated our efforts on optimization of the truncated rCKAP4 to generate stronger binding . Using the extracellular domain

Prediction of Brugia malayi antigenic peptides: candidates for synthetic vaccine design against lymphatic filariasis.

PubMed

Gomase, Virendra S; Chitlange, Nikhilkumar R; Changbhale, Smruti S; Kale, Karbhari V

2013-08-01

Brugia malayi is a threadlike nematode cause's swelling of lymphatic organs, condition well known as lymphatic filariasis; till date no invention made to effectively address lymphatic filariasis. In this analysis we a have predicted suitable antigenic peptides from Brugia malayi antigen protein for peptide vaccine design against lymphatic filariasis based on cross protection phenomenon as, an ample immune response can be generated with a single protein subunit. We found MHC class II binding peptides of Brugia malayi antigen protein are important determinant against the diseased condition. The analysis shows Brugia malayi antigen protein having 505 amino acids, which shows 497 nonamers. In this assay, we have predicted MHC-I binding peptides for 8mer_H2_Db (optimal score- 15.966), 9mer_H2_Db (optimal score- 15.595), 10mer_H2_Db (optimal score- 19.405), 11mer_H2_Dballeles (optimal score- 23.801). We also predicted the SVM based MHCII-IAb nonamers, 51-FQQIDPLDA, 442-FAAIACLVH, 206-YLNPFGHQF, 167-WYVIMAACY, 367-YAMIVIRLL, 434- LVITTAANF, 176-LDSYCLWKP, 435-VITTAANFA, 364-WPGYAMIVI (optimal score- 13.963); MHCII-IAd nonamers, 52-QQIDPLDAE, 171-MAACYLDSY, 239-QWRSVILCN, 168-YVIMAACYL, 3-QYLSVHSLS, 322-EILLHAKVV, 417- LGIIASFVS, 396-KAIFLAHFG, 167-WYVIMAACY, 269-LALHCINVI, 93-FINKAAPKQ, 259-NCIIVLKAF, 79- QGVLLIIPR, 22-TILQRSQAI, 63-RGFVYGNVS, 109-NISSLAFET,(optimal score- 16.748); and MHCII-IAg7 nonamers 171-MAACYLDSY, 73-KIVNGAQGV, 259-NCIIVLKAF, 209-PFGHQFSFE, 102-SCDTLLKNI, 25-QRSQAIRIV, 444- AIACLVHLF, 88-SLVNGFINK, 252-FPRHQLLNC, 471-RFVLANDNE, 52-QQIDPLDAE, 469-HRRFVLAND, 457- SNRHYFLAD, 362-KSWPGYAMI, 476-NDNEGEDFE, 370-IVIRLLQAL (optimal score- 19.847) which represents potential binders from Brugia malayi antigen protein. The method integrates prediction of MHC class I binding proteasomal C-terminal cleavage peptides and Eighteen potential antigenic peptides at average propensity 1.063 having highest local hydrophilicity. Thus a small antigen fragment can induce immune response against whole antigen. This approach can be applied for designing subunit and synthetic peptide vaccines.

Structural specificity of chloroquine-hematin binding related to inhibition of hematin polymerization and parasite growth.

PubMed

Vippagunta, S R; Dorn, A; Matile, H; Bhattacharjee, A K; Karle, J M; Ellis, W Y; Ridley, R G; Vennerstrom, J L

1999-11-04

Considerable data now support the hypothesis that chloroquine (CQ)-hematin binding in the parasite food vacuole leads to inhibition of hematin polymerization and parasite death by hematin poisoning. To better understand the structural specificity of CQ-hematin binding, 13 CQ analogues were chosen and their hematin binding affinity, inhibition of hematin polymerization, and inhibition of parasite growth were measured. As determined by isothermal titration calorimetry (ITC), the stoichiometry data and exothermic binding enthalpies indicated that, like CQ, these analogues bind to two or more hematin mu-oxo dimers in a cofacial pi-pi sandwich-type complex. Association constants (K(a)'s) ranged from 0.46 to 2.9 x 10(5) M(-1) compared to 4.0 x 10(5) M(-1) for CQ. Remarkably, we were not able to measure any significant interaction between hematin mu-oxo dimer and 11, the 6-chloro analogue of CQ. This result indicates that the 7-chloro substituent in CQ is a critical structural determinant in its binding affinity to hematin mu-oxo dimer. Molecular modeling experiments reinforce the view that the enthalpically favorable pi-pi interaction observed in the CQ-hematin mu-oxo dimer complex derives from a favorable alignment of the out-of-plane pi-electron density in CQ and hematin mu-oxo dimer at the points of intermolecular contact. For 4-aminoquinolines related to CQ, our data suggest that electron-withdrawing functional groups at the 7-position of the quinoline ring are required for activity against both hematin polymerization and parasite growth and that chlorine substitution at position 7 is optimal. Our results also confirm that the CQ diaminoalkyl side chain, especially the aliphatic tertiary nitrogen atom, is an important structural determinant in CQ drug resistance. For CQ analogues 1-13, the lack of correlation between K(a) and hematin polymerization IC(50) values suggests that other properties of the CQ-hematin mu-oxo dimer complex, rather than its association constant alone, play a role in the inhibition of hematin polymerization. However, there was a modest correlation between inhibition of hematin polymerization and inhibition of parasite growth when hematin polymerization IC(50) values were normalized for hematin mu-oxo dimer binding affinities, adding further evidence that antimalarial 4-aminoquinolines act by this mechanism.

Effects of acute nicotine on hemodynamics and binding of [11C]raclopride to dopamine D2,3 receptors in pig brain.

PubMed

Cumming, Paul; Rosa-Neto, Pedro; Watanabe, Hideaki; Smith, Donald; Bender, Dirk; Clarke, Paul B S; Gjedde, Albert

2003-07-01

Positive reinforcing properties of nicotine and the psychostimulants have been attributed to elevated dopamine release in the basal ganglia. It is well known that the specific binding of [(11)C]raclopride to dopamine D(2,3) receptors in living striatum is reduced by cocaine and amphetamines, revealing increased competition between endogenous dopamine and [(11)C]raclopride for dopamine D(2,3) receptors. However, the sensitivity of [(11)C]raclopride binding to nicotine-induced dopamine release is less well documented. In order to provide the basis for mapping effects of nicotine, we first optimized reference tissue methods for quantifying [(11)C]raclopride binding sites in striatum of living pigs (n = 16). In the same animals, the rate of cerebral blood flow (CBF) was mapped using [(15)O]water. Neither a low dose of nicotine (50 mu kg(-1), iv) nor a high dose of nicotine (500 microg kg(-1), iv) altered CBF in the pig brain, an important condition for calculating the binding of radioligands when using a reference tissue to estimate the free ligand concentration. The methods of Logan and of Lammertsma were compared using the cerebellum or the occipital cortex as reference tissues for calculating the binding potential (pB) of [(11)C]raclolpride in brain. Irrespective of the method used, the mean undrugged baseline pB in striatum (ca. 2.0) was significantly asymmetric, with highest binding in the left caudate and right putamen. Test-retest estimates of pB were stable. Subtraction of Logan pB maps revealed that the low dose of nicotine reduced the pB of [(11)C]raclopride by 10% in a cluster of voxels in the left anteroventral striatum, but this effect did not persist after correction for multiple comparisons. The high dose of nicotine (n = 9) acutely reduced pB by 10% bilaterally in the ventral striatum; 3 h after the high nicotine dose, the reductions had shifted dorsally and caudally into the caudate and putamen. Evidently, nicotine challenge enhances the competition between endogenous dopamine for [(11)C]raclopride binding sites with a complex temporal and spacial pattern in pig brain, initially presenting in the left ventral striatum.

The RNA-binding complex ESCRT-II in Xenopus laevis eggs recognizes purine-rich sequences through its subunit Vps25.

PubMed

Emerman, Amy B; Blower, Michael

2018-06-14

RNA-binding proteins (RBPs) are critical regulators of gene expression. Recent studies have uncovered hundreds of mRNA-binding proteins that do not contain annotated RNA-binding domains and have well-established roles in other cellular processes. Investigation of these nonconventional RBPs is critical for revealing novel RNA-binding domains and may disclose connections between RNA regulation and other aspects of cell biology. Endosomal sorting complex required for transport II (ESCRT-II) is a nonconventional RNA-binding complex that has a canonical role in multivesicular body formation. ESCRT-II previously has been identified as an RNA-binding complex in Drosophila oocytes, but whether its RNA-binding properties extend beyond Drosophila is unknown. In this study, we found that the RNA-binding properties of ESCRT-II are conserved in Xenopus eggs, where ESCRT-II interacted with hundreds of mRNAs. Using a UV-crosslinking approach, we demonstrated that ESCRT-II binds directly to RNA through its subunit Vps25. UV-crosslinking and immunoprecipitation (CLIP)-Seq revealed that Vps25 specifically recognizes a polypurine (i.e. GA-rich) motif in RNA. Using purified components, we could reconstitute the selective Vps25-mediated binding of the polypurine motif in vitro. Our results provide insight into the mechanism by which ESCRT-II selectively binds to mRNAs and also suggest an unexpected link between endosome biology and RNA regulation. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Calcium ion binding properties and the effect of phosphorylation on the intrinsically disordered Starmaker protein.

PubMed

Wojtas, Magdalena; Hołubowicz, Rafał; Poznar, Monika; Maciejewska, Marta; Ożyhar, Andrzej; Dobryszycki, Piotr

2015-10-27

Starmaker (Stm) is an intrinsically disordered protein (IDP) involved in otolith biomineralization in Danio rerio. Stm controls calcium carbonate crystal formation in vivo and in vitro. Phosphorylation of Stm affects its biomineralization properties. This study examined the effects of calcium ions and phosphorylation on the structure of Stm. We have shown that CK2 kinase phosphorylates 25 or 26 residues in Stm. Furthermore, we have demonstrated that Stm's affinity for calcium binding is dependent on its phosphorylation state. Phosphorylated Stm (StmP) has an estimated 30 ± 1 calcium binding sites per protein molecule with a dissociation constant (KD) of 61 ± 4 μM, while the unphosphorylated protein has 28 ± 3 sites and a KD of 210 ± 22 μM. Calcium ion binding induces a compaction of the Stm molecule, causing a significant decrease in its hydrodynamic radius and the formation of a secondary structure. The screening effect of Na(+) ions on calcium binding was also observed. Analysis of the hydrodynamic properties of Stm and StmP showed that Stm and StmP molecules adopt the structure of native coil-like proteins.

Mechanically tunable actin networks using programmable DNA based cross-linkers

NASA Astrophysics Data System (ADS)

Schnauss, Joerg; Lorenz, Jessica; Schuldt, Carsten; Kaes, Josef; Smith, David

Cells employ multiple cross-linkers with very different properties. Studies of the entire phase space, however, were infeasible since they were restricted to naturally occurring cross-linkers. These components cannot be controllably varied and differ in many parameters. We resolve this limitation by forming artificial actin cross-linkers, which can be controllably varied. The basic building block is DNA enabling a well-defined length variation. DNA can be attached to actin binding peptides with known binding affinities. We used bulk rheology to investigate mechanical properties of these networks. We were able to reproduce mechanical features of actin networks cross-linked by fascin by using a short version of our artificial complex with a high binding affinity. Additionally, we were able to resemble findings for the cross-linker alpha-actinin by employing a long cross-linker with a low binding affinity. Between these natural limits we investigated three different cross-linker lengths each with two different binding affinities. With these controlled variations we are able to precisely screen the phase space of cross-linked actin networks by changing only one specific parameter and not the entire set of properties as in the case of naturally occurring cross-linking complexes.

Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The Identity-Location Binding Problem.

PubMed

Howe, Piers D L; Ferguson, Adam

2015-09-01

The binding problem is fundamental to visual perception. It is the problem of associating an object's visual properties with itself and not with some other object. The problem is made particular difficult because different properties of an object, such as its color, shape, size, and motion, are often processed independently, sometimes in different cortical areas. The results of these separate analyses have to be combined before the object can be seen as a single coherent entity as opposed to a collection of unconnected features. Visual bindings are typically initiated and updated in a serial fashion, one object at a time. Here, we show that one type of binding, location-identity bindings, can be updated in parallel. We do this by using two complementary techniques, the simultaneous-sequential paradigm and systems factorial technology. These techniques make different assumptions and rely on different behavioral measures, yet both came to the same conclusion. Copyright © 2014 Cognitive Science Society, Inc.

In vitro evidence for RNA binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses.

PubMed

Pallás, V; Sánchez-Navarro, J A; Díez, J

1999-01-01

The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Caberoy, Nora B.; Zhou, Yixiong; Alvarado, Gabriela

To efficiently elucidate the biological roles of phosphatidylserine (PS), we developed open-reading-frame (ORF) phage display to identify PS-binding proteins. The procedure of phage panning was optimized with a phage clone expressing MFG-E8, a well-known PS-binding protein. Three rounds of phage panning with ORF phage display cDNA library resulted in {approx}300-fold enrichment in PS-binding activity. A total of 17 PS-binding phage clones were identified. Unlike phage display with conventional cDNA libraries, all 17 PS-binding clones were ORFs encoding 13 real proteins. Sequence analysis revealed that all identified PS-specific phage clones had dimeric basic amino acid residues. GST fusion proteins were expressedmore » for 3 PS-binding proteins and verified for their binding activity to PS liposomes, but not phosphatidylcholine liposomes. These results elucidated previously unknown PS-binding proteins and demonstrated that ORF phage display is a versatile technology capable of efficiently identifying binding proteins for non-protein molecules like PS.« less

The effect of the morphology of supported subnanometer Pt clusters on the first and key step of CO 2 photoreduction [Morphology effect of supported subnanometer Pt clusters on first and key step of CO 2 photoreduction

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Chi -Ta; Wood, Brandon C.; Bhethanabotla, Venkat R.

2015-09-04

In this study, using density functional theory calculations, we investigate the influence of size-dependent cluster morphology on the synergistic catalytic properties of anatase TiO 2(101) surfaces decorated with subnanometer Pt clusters. Focusing on the formation of the key precursor in the CO 2 photoreduction reaction (bent CO 2 –), we find that flatter (2D-like) Pt clusters that “wet” the TiO 2 surface offer significantly less benefit than 3D-like Pt clusters. We attribute the differences to three factors. First, the 3D clusters provide a greater number of accessible Pt–TiO 2 interfacial sites with geometries that can aid CO 2 bond bendingmore » and charge transfer processes. Second, binding competition among each Pt–CO 2 bonding interaction mitigates maximum orbital overlaps, leading to insufficient CO 2 binding. Third and also most interestingly, the 3D clusters tend to possess higher structural fluxionality than the flatter clusters, which is shown to correlate positively with CO2 binding strength. The preferred morphology adopted by the clusters depends on several factors, including the cluster size and the presence of oxygen vacancies on the TiO 2 surface; this suggests a strategy for optimizing the synergistic effect between Pt clusters and TiO 2 surfaces for CO 2 photocatalysis. Clusters of ~6–8 atoms should provide the largest benefit, since they retain the desired 3D morphology, yet are small enough to exhibit high structural fluxionality. Electronic structure analysis provides additional insight into the electronic motivations for the enhanced binding of CO 2 on TiO 2-supported 3D Pt clusters, as well as suppressed binding on flattened, 2D-like clusters.« less

The GM2 Glycan Serves as a Functional Coreceptor for Serotype 1 Reovirus

PubMed Central

Liu, Yan; Blaum, Bärbel S.; Reiter, Dirk M.; Feizi, Ten; Dermody, Terence S.; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus. PMID:23236285

The GM2 glycan serves as a functional coreceptor for serotype 1 reovirus.

PubMed

Reiss, Kerstin; Stencel, Jennifer E; Liu, Yan; Blaum, Bärbel S; Reiter, Dirk M; Feizi, Ten; Dermody, Terence S; Stehle, Thilo

2012-01-01

Viral attachment to target cells is the first step in infection and also serves as a determinant of tropism. Like many viruses, mammalian reoviruses bind with low affinity to cell-surface carbohydrate receptors to initiate the infectious process. Reoviruses disseminate with serotype-specific tropism in the host, which may be explained by differential glycan utilization. Although α2,3-linked sialylated oligosaccharides serve as carbohydrate receptors for type 3 reoviruses, neither a specific glycan bound by any reovirus serotype nor the function of glycan binding in type 1 reovirus infection was known. We have identified the oligosaccharide portion of ganglioside GM2 (the GM2 glycan) as a receptor for the attachment protein σ1 of reovirus strain type 1 Lang (T1L) using glycan array screening. The interaction of T1L σ1 with GM2 in solution was confirmed using NMR spectroscopy. We established that GM2 glycan engagement is required for optimal infection of mouse embryonic fibroblasts (MEFs) by T1L. Preincubation with GM2 specifically inhibited type 1 but not type 3 reovirus infection of MEFs. To provide a structural basis for these observations, we defined the mode of receptor recognition by determining the crystal structure of T1L σ1 in complex with the GM2 glycan. GM2 binds in a shallow groove in the globular head domain of T1L σ1. Both terminal sugar moieties of the GM2 glycan, N-acetylneuraminic acid and N-acetylgalactosamine, form contacts with the protein, providing an explanation for the observed specificity for GM2. Viruses with mutations in the glycan-binding domain display diminished hemagglutination capacity, a property dependent on glycan binding, and reduced capacity to infect MEFs. Our results define a novel mode of virus-glycan engagement and provide a mechanistic explanation for the serotype-dependent differences in glycan utilization by reovirus.

Penicilloyl peptides are recognized as T cell antigenic determinants in penicillin allergy.

PubMed

Padovan, E; Bauer, T; Tongio, M M; Kalbacher, H; Weltzien, H U

1997-06-01

Although hapten immune responses have been intensively studied in the mouse, very little is known about hapten determinants involved in human allergic reactions. Penicillins, as chemically reactive compounds of low molecular weight, constitute typical examples of hapten allergens for humans. Penicillins become immunogenic only after covalent binding to carrier proteins and in this form frequently induced IgE-mediated allergic reactions in patients subjected to antibiotic treatment. However, our previous data strongly indicated that penicillins also form part of the epitopes contacting the antigen receptors of beta lactam-specific T cells in allergic individuals. We have therefore investigated the molecular constraints involved in the T cell immune response to penicillin G (Pen G). Designer peptides containing a DRB1*0401-binding motif and covalently modified with Pen G via a lysine epsilon-amino group were found to induce proliferation of Pen G-specific T cell clones. A precise positioning of the hapten molecule on the peptide backbone was required for optimal T cell recognition. Furthermore, we extended these observations from our designer peptides to show that a peptide sequence derived from a natural DRB1*1101-binding peptide modified in vitro with Pen G, also acquired antigenic properties. Our data for the first time provide insight into the manner in which allergenic haptens are recognized by human T cells involved in allergic reactions to drugs and suggest possible mechanisms leading to the onset of these adverse immune responses.

Effects of target binding kinetics on in vivo drug efficacy: koff , kon and rebinding.

PubMed

Vauquelin, Georges

2016-08-01

Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy. 'Micro'-pharmacokinetic mechanisms like drug rebinding and partitioning into the cell membrane may constitute a potential fix. Simulations were based on solving differential equations. Based on a selected range of association and dissociation rate constants, kon and koff , and rebinding potencies of the drugs as variables, their effects on the temporal in vivo occupancy profile of their targets, after one or multiple repetitive dosings, have here been simulated. Most strikingly, the simulations show that, when rebinding is also taken into account, increasing kon may produce closely the same outcome as decreasing koff when dosing is performed in accordance with the therapeutically most relevant constant [Lmax ]/KD ratio paradigm. Also, under certain conditions, rebinding may produce closely the same outcome as invoking slow diffusion of the drug between the plasma compartment and a target-containing 'effect' compartment. Although the present simulations should only be regarded as a 'proof of principle', these findings may help pharmacologists and medicinal chemists to devise ex vivo and in vitro binding kinetic assays that are more relevant and translatable to in vivo settings. © 2016 The British Pharmacological Society.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations

NASA Astrophysics Data System (ADS)

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A.; Ferreira, Rafaela Salgado

2018-05-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations

NASA Astrophysics Data System (ADS)

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A.; Ferreira, Rafaela Salgado

2018-03-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC50 = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

The Crystal Structures of Apo and cAMP-Bound GlxR from Corynebacterium glutamicum Reveal Structural and Dynamic Changes upon cAMP Binding in CRP/FNR Family Transcription Factors

PubMed Central

Townsend, Philip D.; Jungwirth, Britta; Pojer, Florence; Bußmann, Michael; Money, Victoria A.; Cole, Stewart T.; Pühler, Alfred; Tauch, Andreas; Bott, Michael; Cann, Martin J.; Pohl, Ehmke

2014-01-01

The cyclic AMP-dependent transcriptional regulator GlxR from Corynebacterium glutamicum is a member of the super-family of CRP/FNR (cyclic AMP receptor protein/fumarate and nitrate reduction regulator) transcriptional regulators that play central roles in bacterial metabolic regulatory networks. In C. glutamicum, which is widely used for the industrial production of amino acids and serves as a non-pathogenic model organism for members of the Corynebacteriales including Mycobacterium tuberculosis, the GlxR homodimer controls the transcription of a large number of genes involved in carbon metabolism. GlxR therefore represents a key target for understanding the regulation and coordination of C. glutamicum metabolism. Here we investigate cylic AMP and DNA binding of GlxR from C. glutamicum and describe the crystal structures of apo GlxR determined at a resolution of 2.5 Å, and two crystal forms of holo GlxR at resolutions of 2.38 and 1.82 Å, respectively. The detailed structural analysis and comparison of GlxR with CRP reveals that the protein undergoes a distinctive conformational change upon cyclic AMP binding leading to a dimer structure more compatible to DNA-binding. As the two binding sites in the GlxR homodimer are structurally identical dynamic changes upon binding of the first ligand are responsible for the allosteric behavior. The results presented here show how dynamic and structural changes in GlxR lead to optimization of orientation and distance of its two DNA-binding helices for optimal DNA recognition. PMID:25469635

Investigation of the binding mode of a novel cruzain inhibitor by docking, molecular dynamics, ab initio and MM/PBSA calculations.

PubMed

Martins, Luan Carvalho; Torres, Pedro Henrique Monteiro; de Oliveira, Renata Barbosa; Pascutti, Pedro Geraldo; Cino, Elio A; Ferreira, Rafaela Salgado

2018-05-01

Chagas disease remains a major health problem in South America, and throughout the world. The two drugs clinically available for its treatment have limited efficacy and cause serious adverse effects. Cruzain is an established therapeutic target of Trypanosoma cruzi, the protozoan that causes Chagas disease. Our group recently identified a competitive cruzain inhibitor (compound 1) with an IC 50  = 15 µM that is also more synthetically accessible than the previously reported lead, compound 2. Prior studies, however, did not propose a binding mode for compound 1, hindering understanding of the structure-activity relationship and optimization. Here, the cruzain binding mode of compound 1 was investigated using docking, molecular dynamics (MD) simulations with ab initio derived parameters, ab initio calculations, and MM/PBSA. Two ligand protonation states and four binding poses were evaluated. A careful ligand parameterization method was employed to derive more physically meaningful parameters than those obtained by automated tools. The poses of unprotonated 1 were unstable in MD, showing large conformational changes and diffusing away from the binding site, whereas the protonated form showed higher stability and interaction with negatively charged residues Asp161 and Cys25. MM/PBSA also suggested that these two residues contribute favorably to binding of compound 1. By combining results from MD, ab initio calculations, and MM/PBSA, a binding mode of 1 is proposed. The results also provide insights for further optimization of 1, an interesting lead compound for the development of new cruzain inhibitors.

Full space device optimization for solar cells.

PubMed

Baloch, Ahmer A B; Aly, Shahzada P; Hossain, Mohammad I; El-Mellouhi, Fedwa; Tabet, Nouar; Alharbi, Fahhad H

2017-09-20

Advances in computational materials have paved a way to design efficient solar cells by identifying the optimal properties of the device layers. Conventionally, the device optimization has been governed by single or double descriptors for an individual layer; mostly the absorbing layer. However, the performance of the device depends collectively on all the properties of the material and the geometry of each layer in the cell. To address this issue of multi-property optimization and to avoid the paradigm of reoccurring materials in the solar cell field, a full space material-independent optimization approach is developed and presented in this paper. The method is employed to obtain an optimized material data set for maximum efficiency and for targeted functionality for each layer. To ensure the robustness of the method, two cases are studied; namely perovskite solar cells device optimization and cadmium-free CIGS solar cell. The implementation determines the desirable optoelectronic properties of transport mediums and contacts that can maximize the efficiency for both cases. The resulted data sets of material properties can be matched with those in materials databases or by further microscopic material design. Moreover, the presented multi-property optimization framework can be extended to design any solid-state device.

Quercetin-imprinted chromatographic sorbents revisited: optimization of synthesis and rebinding protocols for application to natural resources.

PubMed

Pardo, Antonelle; Mespouille, Laetitia; Blankert, Bertrand; Trouillas, Patrick; Surin, Mathieu; Dubois, Philippe; Duez, Pierre

2014-10-17

Molecularly imprinted polymers (MIPs) based on quercetin and synthesized by either bulk, precipitation or suspension polymerization were characterized in terms of size and shape by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). After a study of rebinding protocols, the optimal materials were evaluated as sorbents for solid-phase extraction (SPE) and high-performance liquid chromatography (HPLC) to confirm the presence of imprinted cavities and to assess their selectivity. Besides quercetin, other structurally related natural compounds, naringenin, daidzein and curcumin, were employed for selectivity tests of MIPs. Although rebinding protocols previously described for such MIPs are typically based on binding, washing and eluting methanol-based solutions, we show that this highly polar solvent leads to weak specific interactions (imprinting factor<1) and poor sorbent properties, most probably because of hydrogen-bonding interferences between the MIP and MeOH. Similar experiments performed in tetrahydrofuran yield to much more improved properties (imprinting factor>2.4). This calls for reviewing most of previously published data on quercetin-MIPs; in proper binding conditions, published MIPs may prove more performing than initially assessed. As expected, tested MIPs exhibited the highest selective rebinding towards quercetin template (imprinting effect, quercetin, 3.41; naringenin, 1.54; daidzein, 1.38; curcumin, 1.67); the differences in selectivity between quercetin analogues were explained by the ligand geometries and H-bonding patterns obtained from quantum-chemical calculations. The evaluation of MIPs under identical analytical conditions allowed investigating the effect of the production method on chromatographic performances. The MIPs in bead materials (for quercetin, peak width, 0.69; number of theoretical plates, 143; symmetry factor, 2.22) provided a significant improvement in chromatographic efficiency over the bulk materials (for quercetin, peak width, 1.25; number of theoretical plates, 115; symmetry factor, 2.92). Using the quercetin-beaded MIP as SPE sorbent, quercetin was selectively extracted from Allium cepa L. extract. The MIP developed in this work therefore appears highly promising for the enrichment and determination of quercetin in natural products. Copyright © 2014 Elsevier B.V. All rights reserved.

( sup 3 H)-DOB(4-bromo-2,5-dimethoxyphenylisopropylamine) and ( sup 3 H) ketanserin label two affinity states of the cloned human 5-hydroxytryptamine2 receptor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Branchek, T.; Adham, N.; Macchi, M.

1990-11-01

The binding properties of the 5-hydroxytryptamine2 (5-HT2) receptor have been the subject of much interest and debate in recent years. The hallucinogenic amphetamine derivative 4-bromo-2,5-dimethoxyphenylisopropylamine (DOB) has been shown to bind to a small number of binding sites with properties very similar to (3H)ketanserin-labeled 5-HT2 receptors, but with much higher agonist affinities. Some researchers have interpreted this as evidence for the existence of a new subtype of 5-HT2 receptor (termed 5-HT2A), whereas others have interpreted these data as indicative of agonist high affinity and agonist low affinity states for the 5-HT2 receptor. In this investigation, a cDNA clone encoding themore » serotonin 5-HT2 receptor was transiently transfected into monkey kidney Cos-7 cells and stably transfected into mouse fibroblast L-M(TK-) cells. In both systems, expression of this single serotonin receptor cDNA led to the appearance of both (3H)DOB and (3H)ketanserin binding sites with properties that matched their binding characteristics in mammalian brain homogenates. Addition of guanosine 5'-(beta, gamma-imido) triphosphate (Gpp(NH)p) to this system caused a rightward shift and steepening of agonist competition curves for (3H) ketanserin binding, converting a two-site binding curve to a single low affinity binding state. Gpp(NH)p addition also caused a 50% decrease in the number of high affinity (3H)DOB binding sites, with no change in the dissociation constant of the remaining high affinity states. These data on a single human 5-HT2 receptor cDNA expressed in two different transfection host cells indicate that (3H)DOB and (3H)ketanserin binding reside on the same gene product, apparently interacting with agonist and antagonist conformations of a single human 5-HT2 receptor protein.« less

Engineering Tocopherol Selectivity in α-TTP: A Combined In Vitro/In Silico Study

PubMed Central

Helbling, Rachel E.; Aeschimann, Walter; Simona, Fabio; Stocker, Achim; Cascella, Michele

2012-01-01

We present a combined in vitro/in silico study to determine the molecular origin of the selectivity of -tocopherol transfer protein (-TTP) towards -tocopherol. Molecular dynamics simulations combined to free energy perturbation calculations predict a binding free energy for -tocopherol to -TTP 8.262.13 kcal mol lower than that of -tocopherol. Our calculations show that -tocopherol binds to -TTP in a significantly distorted geometry as compared to that of the natural ligand. Variations in the hydration of the binding pocket and in the protein structure are found as well. We propose a mutation, A156L, which significantly modifies the selectivity properties of -TTP towards the two tocopherols. In particular, our simulations predict that A156L binds preferentially to -tocopherol, with striking structural similarities to the wild-type--tocopherol complex. The affinity properties are confirmed by differential scanning fluorimetry as well as in vitro competitive binding assays. Our data indicate that residue A156 is at a critical position for determination of the selectivity of -TTP. The engineering of TTP mutants with modulating binding properties can have potential impact at industrial level for easier purification of single tocopherols from vitamin E mixtures coming from natural oils or synthetic processes. Moreover, the identification of a -tocopherol selective TTP offers the possibility to challenge the hypotheses for the evolutionary development of a mechanism for -tocopherol selection in omnivorous animals. PMID:23152872

Redox process is crucial for inhibitory properties of aurintricarboxylic acid against activity of YopH: virulence factor of Yersinia pestis

PubMed Central

Kuban-Jankowska, Alicja; Gorska, Magdalena; Tuszynski, Jack A; Ossowski, Tadeusz; Wozniak, Michal

2015-01-01

YopH is a bacterial protein tyrosine phosphatase, which is essential for the viability and pathogenic virulence of the plague-causing Yersinia sp. bacteria. Inactivation of YopH activity would lead to the loss of bacterial pathogenicity. We have studied the inhibitory properties of aurintricarboxylic acid (ATA) against YopH phosphatase and found that at nanomolar concentrations ATA reversibly decreases the activity of YopH. Computational docking studies indicated that in all binding poses ATA binds in the YopH active site. Molecular dynamics simulations showed that in the predicted binding pose, ATA binds to the essential Cys403 and Arg409 residues in the active site and has a stronger binding affinity than the natural substrate (pTyr). The cyclic voltammetry experiments suggest that ATA reacts remarkably strongly with molecular oxygen. Additionally, the electrochemical reduction of ATA in the presence of a negative potential from −2.0 to 2.5 V generates a current signal, which is observed for hydrogen peroxide. Here we showed that ATA indicates a unique mechanism of YopH inactivation due to a redox process. We proposed that the potent inhibitory properties of ATA are a result of its strong binding in the YopH active site and in situ generation of hydrogen peroxide near catalytic cysteine residue. PMID:26286963

Enriching peptide libraries for binding affinity and specificity through computationally directed library design

PubMed Central

Foight, Glenna Wink; Chen, T. Scott; Richman, Daniel; Keating, Amy E.

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model. PMID:28236241

Enriching Peptide Libraries for Binding Affinity and Specificity Through Computationally Directed Library Design.

PubMed

Foight, Glenna Wink; Chen, T Scott; Richman, Daniel; Keating, Amy E

2017-01-01

Peptide reagents with high affinity or specificity for their target protein interaction partner are of utility for many important applications. Optimization of peptide binding by screening large libraries is a proven and powerful approach. Libraries designed to be enriched in peptide sequences that are predicted to have desired affinity or specificity characteristics are more likely to yield success than random mutagenesis. We present a library optimization method in which the choice of amino acids to encode at each peptide position can be guided by available experimental data or structure-based predictions. We discuss how to use analysis of predicted library performance to inform rounds of library design. Finally, we include protocols for more complex library design procedures that consider the chemical diversity of the amino acids at each peptide position and optimize a library score based on a user-specified input model.

Effect of processing on the detectability of peanut protein by ELISA.

PubMed

Iqbal, Amjad; Ateeq, Nadia

2013-12-01

Chicken IgY was used for the detection and quantification of peanut proteins by indirect competitive ELISA. The method was optimized by using a checker board approach to determine the optimal concentration of coating antigen, primary antibody and secondary antibody. Peanut protein could be detected in foods down to levels of 10 ppm. The effect of physical (heat treatment at 80 °C and 100 °C) and chemical (acid, alkali and reducing sugar) treatments on the IgY binding of peanut proteins was investigated. The optimized assay was relatively sensitive for the roasted peanut proteins. However, the binding ability of chicken IgYs to peanut proteins was found to be significantly altered by denaturation and hydrolysis of proteins. It was also observed that the effect of Millard chemistry on the detectability of peanut protein was less pronounced at high temperatures than at low temperatures. Copyright © 2013 Elsevier Ltd. All rights reserved.

Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of D-amino acid oxidase inhibitors.

PubMed

Orgován, Zoltán; Ferenczy, György G; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M

2018-02-01

Optimization of fragment size D-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

Validation of tautomeric and protomeric binding modes by free energy calculations. A case study for the structure based optimization of d-amino acid oxidase inhibitors

NASA Astrophysics Data System (ADS)

Orgován, Zoltán; Ferenczy, György G.; Steinbrecher, Thomas; Szilágyi, Bence; Bajusz, Dávid; Keserű, György M.

2018-02-01

Optimization of fragment size d-amino acid oxidase (DAAO) inhibitors was investigated using a combination of computational and experimental methods. Retrospective free energy perturbation (FEP) calculations were performed for benzo[d]isoxazole derivatives, a series of known inhibitors with two potential binding modes derived from X-ray structures of other DAAO inhibitors. The good agreement between experimental and computed binding free energies in only one of the hypothesized binding modes strongly support this bioactive conformation. Then, a series of 1-H-indazol-3-ol derivatives formerly not described as DAAO inhibitors was investigated. Binding geometries could be reliably identified by structural similarity to benzo[d]isoxazole and other well characterized series and FEP calculations were performed for several tautomers of the deprotonated and protonated compounds since all these forms are potentially present owing to the experimental pKa values of representative compounds in the series. Deprotonated compounds are proposed to be the most important bound species owing to the significantly better agreement between their calculated and measured affinities compared to the protonated forms. FEP calculations were also used for the prediction of the affinities of compounds not previously tested as DAAO inhibitors and for a comparative structure-activity relationship study of the benzo[d]isoxazole and indazole series. Selected indazole derivatives were synthesized and their measured binding affinity towards DAAO was in good agreement with FEP predictions.

Computational design of enzyme-ligand binding using a combined energy function and deterministic sequence optimization algorithm.

PubMed

Tian, Ye; Huang, Xiaoqiang; Zhu, Yushan

2015-08-01

Enzyme amino-acid sequences at ligand-binding interfaces are evolutionarily optimized for reactions, and the natural conformation of an enzyme-ligand complex must have a low free energy relative to alternative conformations in native-like or non-native sequences. Based on this assumption, a combined energy function was developed for enzyme design and then evaluated by recapitulating native enzyme sequences at ligand-binding interfaces for 10 enzyme-ligand complexes. In this energy function, the electrostatic interaction between polar or charged atoms at buried interfaces is described by an explicitly orientation-dependent hydrogen-bonding potential and a pairwise-decomposable generalized Born model based on the general side chain in the protein design framework. The energy function is augmented with a pairwise surface-area based hydrophobic contribution for nonpolar atom burial. Using this function, on average, 78% of the amino acids at ligand-binding sites were predicted correctly in the minimum-energy sequences, whereas 84% were predicted correctly in the most-similar sequences, which were selected from the top 20 sequences for each enzyme-ligand complex. Hydrogen bonds at the enzyme-ligand binding interfaces in the 10 complexes were usually recovered with the correct geometries. The binding energies calculated using the combined energy function helped to discriminate the active sequences from a pool of alternative sequences that were generated by repeatedly solving a series of mixed-integer linear programming problems for sequence selection with increasing integer cuts.

Negative Factor from SIV Binds to the Catalytic Subunit of the V-ATPase to Internalize CD4 and to Increase Viral Infectivity

PubMed Central

Mandic, Robert; Fackler, Oliver T.; Geyer, Matthias; Linnemann, Thomas; Zheng, Yong-Hui; Peterlin, B. Matija

2001-01-01

The accessory protein negative factor (Nef) from human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV) is required for optimal viral infectivity and the progression to acquired immunodeficiency syndrome (AIDS). Nef interacts with the endocytic machinery, resulting in the down-regulation of cluster of differentiation antigen 4 (CD4) and major histocompatibility complex class I (MHCI) molecules on the surface of infected cells. Mutations in the C-terminal flexible loop of Nef result in a lower rate of internalization by this viral protein. However, no loop-dependent binding of Nef to adaptor protein-2 (AP-2), which is the adaptor protein complex that is required for the internalization of proteins from the plasma membrane, could be demonstrated. In this study we investigated the relevance of different motifs in Nef from SIVmac239 for its internalization, CD4 down-regulation, binding to components of the trafficking machinery, and viral infectivity. Our data suggest that the binding of Nef to the catalytic subunit H of the vacuolar membrane ATPase (V-ATPase) facilitates its internalization. This binding depends on the integrity of the whole flexible loop. Subsequent studies on Nef mutant viruses revealed that the flexible loop is essential for optimal viral infectivity. Therefore, our data demonstrate how Nef contacts the endocytic machinery in the absence of its direct binding to AP-2 and suggest an important role for subunit H of the V-ATPase in viral infectivity. PMID:11179428

The DNA-recognition mode shared by archaeal feast/famine-regulatory proteins revealed by the DNA-binding specificities of TvFL3, FL10, FL11 and Ss-LrpB

PubMed Central

Yokoyama, Katsushi; Nogami, Hideki; Kabasawa, Mamiko; Ebihara, Sonomi; Shimowasa, Ai; Hashimoto, Keiko; Kawashima, Tsuyoshi; Ishijima, Sanae A.; Suzuki, Masashi

2009-01-01

The DNA-binding mode of archaeal feast/famine-regulatory proteins (FFRPs), i.e. paralogs of the Esherichia coli leucine-responsive regulatory protein (Lrp), was studied. Using the method of systematic evolution of ligands by exponential enrichment (SELEX), optimal DNA duplexes for interacting with TvFL3, FL10, FL11 and Ss-LrpB were identified as TACGA[AAT/ATT]TCGTA, GTTCGA[AAT/ATT]TCGAAC, CCGAAA[AAT/ATT]TTTCGG and TTGCAA[AAT/ATT]TTGCAA, respectively, all fitting into the form abcdeWWWedcba. Here W is A or T, and e.g. a and a are bases complementary to each other. Apparent equilibrium binding constants of the FFRPs and various DNA duplexes were determined, thereby confirming the DNA-binding specificities of the FFRPs. It is likely that these FFRPs recognize DNA in essentially the same way, since their DNA-binding specificities were all explained by the same pattern of relationship between amino-acid positions and base positions to form chemical interactions. As predicted from this relationship, when Gly36 of TvFL3 was replaced by Thr, the b base in the optimal DNA duplex changed from A to T, and, when Thr36 of FL10 was replaced by Ser, the b base changed from T to G/A. DNA-binding characteristics of other archaeal FFRPs, Ptr1, Ptr2, Ss-Lrp and LysM, are also consistent with the relationship. PMID:19468044

Stereochemical and conformational study on fenoterol by ECD spectroscopy and TD-DFT calculations.

PubMed

Tedesco, Daniele; Zanasi, Riccardo; Wainer, Irving W; Bertucci, Carlo

2014-03-01

Fenoterol and its derivatives are selective β2-adrenergic receptor (β2-AR) agonists whose stereoselective biological activities have been extensively investigated in the past decade; a complete stereochemical characterization of fenoterol derivatives is therefore crucial for a better understanding of the effects of stereochemistry on β2-AR binding. In the present project, the relationship between chiroptical properties and absolute stereochemistry of the stereoisomers of fenoterol (1) was investigated by experimental ECD spectroscopy and time-dependent density functional theory (TD-DFT). DFT geometry optimizations were carried out at the RI-B97D/TZVP/IEFPCM(MeOH) level and subsequent TD-DFT calculations were performed using the PBE0 hybrid functional. Despite the large pool of equilibrium conformers found for the investigated compounds and the known limitations of the level of theory employed, the computational protocol was able to reproduce the experimental ECD spectra of the stereoisomers of 1. The main contribution to the overall chiroptical properties was found to arise from the absolute configuration of the chiral center in α-position to the resorcinol moiety. Based on this evidence, a thorough conformational analysis was performed on the optimized DFT conformers, which revealed the occurrence of a different equilibrium between conformational patterns for the diastereomers of fenoterol: the (R,R')/(S,S') enantiomeric pair showed a higher population of folded conformations than the (R,S')/(S,R') pair. Copyright © 2013 Elsevier B.V. All rights reserved.

Variational Calculation of the Ground State of Closed-Shell Nuclei Up to $A$ = 40

DOE PAGES

Lonardoni, Diego; Lovato, Alessandro; Pieper, Steven C.; ...

2017-08-31

Variational calculations of ground-state properties of 4He, 16O and 40Ca are carried out employing realistic phenomenological two- and three-nucleon potentials. The trial wave function includes twoand three-body correlations acting on a product of single-particle determinants. Expectation values are evaluated with a cluster expansion for the spin-isospin dependent correlations considering up to five-body cluster terms. The optimal wave function is obtained by minimizing the energy expectation value over a set of up to 20 parameters by means of a nonlinear optimization library. We present results for the binding energy, charge radius, point density, single-nucleon momentum distribution, charge form factor, and Coulombmore » sum rule. We find that the employed three-nucleon interaction becomes repulsive for A ≥ 16. In 16O the inclusion of such a force provides a better description of the properties of the nucleus. In 40Ca instead, the repulsive behavior of the three-body interaction fails to reproduce experimental data for the charge radius and the charge form factor. We find that the high-momentum region of the momentum distributions, determined by the short-range terms of nuclear correlations, exhibit a universal behavior independent of the particular nucleus. The comparison of the Coulomb sum rules for 4He, 16O, and 40Ca reported in this work will help elucidate in-medium modifications of the nucleon form factors.« less

Analysis and Optimization of the Production Process of Cooked Sausage Meat Matrices

NASA Astrophysics Data System (ADS)

Diez, L.; Rauh, C.; Delgado, A.

2010-09-01

In the production of cooked sausages a critical step for product quality is the cutting process, where the comminuting and mixing of meat, fat, ice and spices are carried out. These processes take usually place in bowl cutters, which main control parameters are the working time, knife geometry (shape and sharpness) and rotational velocities of the knives and the bowl. The choice of the geometry and sharpness of the knives influences not only the meat matrix properties (mechanical, rheological, etc.) and, as a consequence, the sensory value of the sausages (size of connective tissue particles, water binding, etc.), but also the energetic demand for the production. However, the cutting process proves to be understood only fragmentarily due to the complex colloid chemical and mechanical behavior of the product. This is documented on the one hand by numerous knife types on the market, extremely empirical approach during determination of geometry and process parameters in practice as well as, on the other hand, by contradictory statements and explanation approaches of observed phenomena present in literature. The present contribution applies numerical simulations to analyze thermo fluid mechanical phenomena, e.g. shear stresses, during the cutting process of the non-Newtonian meat matrix. Combining these results with selected experimental investigations from literature, e.g. sensory properties, knife geometry, velocity of the knife and bowl, improvements of the cutting and mixing process are proposed using cognitive algorithms (Artificial neural networks) aiming at an optimization regarding energy and time demand and product quality.

Emergent properties of nuclei from ab initio coupled-cluster calculations

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hagen, G.; Hjorth-Jensen, M.; Jansen, G. R.

Emergent properties such as nuclear saturation and deformation, and the effects on shell structure due to the proximity of the scattering continuum and particle decay channels are fascinating phenomena in atomic nuclei. In recent years, ab initio approaches to nuclei have taken the first steps towards tackling the computational challenge of describing these phenomena from Hamiltonians with microscopic degrees of freedom. Our endeavor is now possible due to ideas from effective field theories, novel optimization strategies for nuclear interactions, ab initio methods exhibiting a soft scaling with mass number, and ever-increasing computational power. We review some of the recent accomplishments. We also present new results. The recently optimized chiral interaction NNLOmore » $${}_{{\\rm{sat}}}$$ is shown to provide an accurate description of both charge radii and binding energies in selected light- and medium-mass nuclei up to 56Ni. We derive an efficient scheme for including continuum effects in coupled-cluster computations of nuclei based on chiral nucleon–nucleon and three-nucleon forces, and present new results for unbound states in the neutron-rich isotopes of oxygen and calcium. Finally, the coupling to the continuum impacts the energies of the $${J}^{\\pi }=1/{2}^{-},3/{2}^{-},7/{2}^{-},3/{2}^{+}$$ states in $${}^{\\mathrm{17,23,25}}$$O, and—contrary to naive shell-model expectations—the level ordering of the $${J}^{\\pi }=3/{2}^{+},5/{2}^{+},9/{2}^{+}$$ states in $${}^{\\mathrm{53,55,61}}$$Ca.« less

Emergent properties of nuclei from ab initio coupled-cluster calculations

DOE PAGES

Hagen, G.; Hjorth-Jensen, M.; Jansen, G. R.; ...

2016-05-17

Emergent properties such as nuclear saturation and deformation, and the effects on shell structure due to the proximity of the scattering continuum and particle decay channels are fascinating phenomena in atomic nuclei. In recent years, ab initio approaches to nuclei have taken the first steps towards tackling the computational challenge of describing these phenomena from Hamiltonians with microscopic degrees of freedom. Our endeavor is now possible due to ideas from effective field theories, novel optimization strategies for nuclear interactions, ab initio methods exhibiting a soft scaling with mass number, and ever-increasing computational power. We review some of the recent accomplishments. We also present new results. The recently optimized chiral interaction NNLOmore » $${}_{{\\rm{sat}}}$$ is shown to provide an accurate description of both charge radii and binding energies in selected light- and medium-mass nuclei up to 56Ni. We derive an efficient scheme for including continuum effects in coupled-cluster computations of nuclei based on chiral nucleon–nucleon and three-nucleon forces, and present new results for unbound states in the neutron-rich isotopes of oxygen and calcium. Finally, the coupling to the continuum impacts the energies of the $${J}^{\\pi }=1/{2}^{-},3/{2}^{-},7/{2}^{-},3/{2}^{+}$$ states in $${}^{\\mathrm{17,23,25}}$$O, and—contrary to naive shell-model expectations—the level ordering of the $${J}^{\\pi }=3/{2}^{+},5/{2}^{+},9/{2}^{+}$$ states in $${}^{\\mathrm{53,55,61}}$$Ca.« less

Holistic assessment of covalently-labelled core-shell polymeric nanoparticles with fluorescent contrast agents towards theranostic applications

PubMed Central

Gustafson, Tiffany P.; Lim, Young H.; Flores, Jeniree A.; Heo, Gyu Seong; Zhang, Fuwu; Zhang, Shiyi; Samarajeewa, Sandani; Raymond, Jeffery E.; Wooley, Karen L.

2014-01-01

The successful development of degradable polymeric nanostructures as optical probes for use in nanotheranostic applications requires the intelligent design of materials such that their surface response, degradation, drug delivery and imaging properties are all optimized. In the case of imaging, optimization must result in materials that allow differentiation between unbound optical contrast agents and labeled polymeric materials as they undergo degradation. In this study, we have shown that use of traditional electrophoretic gel-plate assays for determination of the purity of dye-conjugated degradable nanoparticles is limited, due to polymer degradation characteristics. To overcome these limitations, we have outlined a holistic approach to evaluating dye-and peptide-polymer nanoparticle conjugation by utilizing steady-state fluorescence, anisotropy, and emission and anisotropy life-time decay profiles, through which nanoparticle-dye binding can be assessed independent of perturbations, such as those presented during the execution of electrolyte gel-based assays. This approach has been demonstrated to provide an overall understanding of the spectral signature-structure-function relationship, ascertaining key information on interactions between the fluorophore, polymer and solvent components that have a direct and measurable impact on the emissive properties of the optical probe. The use of these powerful techniques provides feedback that can be utilized to improve nanotheranostics by evaluating dye emissivity in degradable nanotheranostic systems, which has become increasingly important as modern platforms transition to architectures intentionally reliant on degradation and built-in environmental responses. PMID:24392760

Molecular dynamic simulations on the structures and properties of epsilon-CL-20(0 0 1)/F 2314 PBX.

PubMed

Xu, Xiaojuan; Xiao, Jijun; Huang, Hui; Li, Jinshan; Xiao, Heming

2010-03-15

Molecular dynamical (MD) simulations with the COMPASS force field were employed to investigate the influences of temperature (T), the concentration of F(2314) binder (W%), and crystal defects on the mechanical properties, binding energy (E(bind)), and detonation properties of epsilon-CL-20(001)/F(2314) PBX (polymer bonded explosives). T was found to have some influences on the mechanical properties, and the PBX at 298 K was considered with better mechanical properties. By radial distribution function g(r) analysis the three types of hydrogen bonds, H...O, H...F, and H...Cl were predicted as the main interaction formats between F(2314) and epsilon-CL-20, and the strength of these interactions changed with temperature changing. The isotropic properties of the PBX increased with W% increasing, but each modulus and E(bind) did not monotonously vary with W% increasing. The detonation properties of the PBX decreased with the increasing W%, and the PBX with 4.69% F(2314) was regarded with good detonation properties. The existence of crystal defects (vacancy or adulteration) might increase the elasticity but destabilize the system to some extent, and the mechanical properties of PBX were chiefly determined by the main body explosive. The above information was thought guidable for practical formulation design of PBX. (c) 2009 Elsevier B.V. All rights reserved.

Mechanics of Platelet-Matrix Composites across Scales: Theory, Multiscale Modeling, and 3D Fabrication

NASA Astrophysics Data System (ADS)

Sakhavand, Navid

Many natural and biomimetic composites - such as nacre, silk and clay-polymer - exhibit a remarkable balance of strength, toughness, and/or stiffness, which call for a universal measure to quantify this outstanding feature given the platelet-matrix structure and material characteristics of the constituents. Analogously, there is an urgent need to quantify the mechanics of emerging electronic and photonic systems such as stacked heterostructures, which are composed of strong in-plane bonding networks but weak interplanar bonding matrices. In this regard, development of a universal composition-structure-property map for natural platelet-matrix composites, and stacked heterostructures opens up new doors for designing materials with superior mechanical performance. In this dissertation, a multiscale bottom-up approach is adopted to analyze and predict the mechanical properties of platelet-matrix composites. Design guidelines are provided by developing universally valid (across different length scales) diagrams for science-based engineering of numerous natural and synthetic platelet-matrix composites and stacked heterostructures while significantly broadening the spectrum of strategies for fabricating new composites with specific and optimized mechanical properties. First, molecular dynamics simulations are utilized to unravel the fundamental underlying physics and chemistry of the binding nature at the atomic-level interface of organic-inorganic composites. Polymer-cementitious composites are considered as case studies to understand bonding mechanism at the nanoscale and open up new venues for potential mechanical enhancement at the macro-scale. Next, sophisticated mathematical derivations based on elasticity and plasticity theories are presented to describe pre-crack (intrinsic) mechanical performance of platelet-matrix composites at the microscale. These derivations lead to developing a unified framework to construct series of universal composition-structure-property maps that decode the interplay between various geometries and inherent material features, encapsulated in a few dimensionless parameters. Finally, after crack mechanical properties (extrinsic) of platelet-matrix composites until ultimate failure of the material at the macroscale is investigated via combinatorial finite element simulations. The effect of different composition-structure-property parameters on mechanical properties synergies are depicted via 2D and 3D maps. 3D-printed specimens are fabricated and tested against the theoretical prediction. The combination of the presented diagrams and guidelines paves the path toward platelet-matrix composites and stacked-heterostructures with superior and optimized mechanical properties.

Rate Constants and Mechanisms of Protein–Ligand Binding

PubMed Central

Pang, Xiaodong; Zhou, Huan-Xiang

2017-01-01

Whereas protein–ligand binding affinities have long-established prominence, binding rate constants and binding mechanisms have gained increasing attention in recent years. Both new computational methods and new experimental techniques have been developed to characterize the latter properties. It is now realized that binding mechanisms, like binding rate constants, can and should be quantitatively determined. In this review, we summarize studies and synthesize ideas on several topics in the hope of providing a coherent picture of and physical insight into binding kinetics. The topics include microscopic formulation of the kinetic problem and its reduction to simple rate equations; computation of binding rate constants; quantitative determination of binding mechanisms; and elucidation of physical factors that control binding rate constants and mechanisms. PMID:28375732

Two-photon probes for in vivo multicolor microscopy of the structure and signals of brain cells.

PubMed

Ricard, Clément; Arroyo, Erica D; He, Cynthia X; Portera-Cailliau, Carlos; Lepousez, Gabriel; Canepari, Marco; Fiole, Daniel

2018-05-11

Imaging the brain of living laboratory animals at a microscopic scale can be achieved by two-photon microscopy thanks to the high penetrability and low phototoxicity of the excitation wavelengths used. However, knowledge of the two-photon spectral properties of the myriad fluorescent probes is generally scarce and, for many, non-existent. In addition, the use of different measurement units in published reports further hinders the design of a comprehensive imaging experiment. In this review, we compile and homogenize the two-photon spectral properties of 280 fluorescent probes. We provide practical data, including the wavelengths for optimal two-photon excitation, the peak values of two-photon action cross section or molecular brightness, and the emission ranges. Beyond the spectroscopic description of these fluorophores, we discuss their binding to biological targets. This specificity allows in vivo imaging of cells, their processes, and even organelles and other subcellular structures in the brain. In addition to probes that monitor endogenous cell metabolism, studies of healthy and diseased brain benefit from the specific binding of certain probes to pathology-specific features, ranging from amyloid-β plaques to the autofluorescence of certain antibiotics. A special focus is placed on functional in vivo imaging using two-photon probes that sense specific ions or membrane potential, and that may be combined with optogenetic actuators. Being closely linked to their use, we examine the different routes of intravital delivery of these fluorescent probes according to the target. Finally, we discuss different approaches, strategies, and prerequisites for two-photon multicolor experiments in the brains of living laboratory animals.

Superposition and alignment of labeled point clouds.

PubMed

Fober, Thomas; Glinca, Serghei; Klebe, Gerhard; Hüllermeier, Eyke

2011-01-01

Geometric objects are often represented approximately in terms of a finite set of points in three-dimensional euclidean space. In this paper, we extend this representation to what we call labeled point clouds. A labeled point cloud is a finite set of points, where each point is not only associated with a position in three-dimensional space, but also with a discrete class label that represents a specific property. This type of model is especially suitable for modeling biomolecules such as proteins and protein binding sites, where a label may represent an atom type or a physico-chemical property. Proceeding from this representation, we address the question of how to compare two labeled points clouds in terms of their similarity. Using fuzzy modeling techniques, we develop a suitable similarity measure as well as an efficient evolutionary algorithm to compute it. Moreover, we consider the problem of establishing an alignment of the structures in the sense of a one-to-one correspondence between their basic constituents. From a biological point of view, alignments of this kind are of great interest, since mutually corresponding molecular constituents offer important information about evolution and heredity, and can also serve as a means to explain a degree of similarity. In this paper, we therefore develop a method for computing pairwise or multiple alignments of labeled point clouds. To this end, we proceed from an optimal superposition of the corresponding point clouds and construct an alignment which is as much as possible in agreement with the neighborhood structure established by this superposition. We apply our methods to the structural analysis of protein binding sites.

Fluorescent DNA-templated silver nanoclusters

NASA Astrophysics Data System (ADS)

Lin, Ruoqian

Because of the ultra-small size and biocompatibility of silver nanoclusters, they have attracted much research interest for their applications in biolabeling. Among the many ways of synthesizing silver nanoclusters, DNA templated method is particularly attractive---the high tunability of DNA sequences provides another degree of freedom for controlling the chemical and photophysical properties. However, systematic studies about how DNA sequences and concentrations are controlling the photophysical properties are still lacking. The aim of this thesis is to investigate the binding mechanisms of silver clusters binding and single stranded DNAs. Here in this thesis, we report synthesis and characterization of DNA-templated silver nanoclusters and provide a systematic interrogation of the effects of DNA concentrations and sequences, including lengths and secondary structures. We performed a series of syntheses utilizing five different sequences to explore the optimal synthesis condition. By characterizing samples with UV-vis and fluorescence spectroscopy, we achieved the most proper reactants ratio and synthesis conditions. Two of them were chosen for further concentration dependence studies and sequence dependence studies. We found that cytosine-rich sequences are more likely to produce silver nanoclusters with stronger fluorescence signals; however, sequences with hairpin secondary structures are more capable in stabilizing silver nanoclusters. In addition, the fluorescence peak emission intensities and wavelengths of the DNA templated silver clusters have sequence dependent fingerprints. This potentially can be applied to sequence sensing in the future. However all the current conclusions are not warranted; there is still difficulty in formulating general rules in DNA strand design and silver nanocluster production. Further investigation of more sequences could solve these questions in the future.

New Kohn-Sham density functional based on microscopic nuclear and neutron matter equations of state

NASA Astrophysics Data System (ADS)

Baldo, M.; Robledo, L. M.; Schuck, P.; Viñas, X.

2013-06-01

A new version of the Barcelona-Catania-Paris energy functional is applied to a study of nuclear masses and other properties. The functional is largely based on calculated ab initio nuclear and neutron matter equations of state. Compared to typical Skyrme functionals having 10-12 parameters apart from spin-orbit and pairing terms, the new functional has only 2 or 3 adjusted parameters, fine tuning the nuclear matter binding energy and fixing the surface energy of finite nuclei. An energy rms value of 1.58 MeV is obtained from a fit of these three parameters to the 579 measured masses reported in the Audi and Wapstra [Nucl. Phys. ANUPABL0375-947410.1016/j.nuclphysa.2003.11.003 729, 337 (2003)] compilation. This rms value compares favorably with the one obtained using other successful mean field theories, which range from 1.5 to 3.0 MeV for optimized Skyrme functionals and 0.7 to 3.0 for the Gogny functionals. The other properties that have been calculated and compared to experiment are nuclear radii, the giant monopole resonance, and spontaneous fission lifetimes.

Multi-Scale Computational Enzymology: Enhancing Our Understanding of Enzymatic Catalysis

PubMed Central

Gherib, Rami; Dokainish, Hisham M.; Gauld, James W.

2014-01-01

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific enzymatic properties. Small or large density functional theory models allow the comparison of a plethora of mechanistic reactive species and divergent catalytic pathways. Molecular docking can model different substrate conformations embedded within enzyme active sites and determine those with optimal binding affinities. Molecular dynamics simulations provide insights into the dynamics and roles of active site components as well as the interactions between substrate and enzymes. Hybrid quantum mechanical/molecular mechanical (QM/MM) can model reactions in active sites while considering steric and electrostatic contributions provided by the surrounding environment. Using previous studies done within our group, on OvoA, EgtB, ThrRS, LuxS and MsrA enzymatic systems, we will review how these methods can be used either independently or cooperatively to get insights into enzymatic catalysis. PMID:24384841

The fragile X mental retardation protein has nucleic acid chaperone properties

PubMed Central

Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W.; Darlix, Jean-Luc

2004-01-01

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA–RNA interactions and thus on the structural status of mRNAs. PMID:15096575

The fragile X mental retardation protein has nucleic acid chaperone properties.

PubMed

Gabus, Caroline; Mazroui, Rachid; Tremblay, Sandra; Khandjian, Edouard W; Darlix, Jean-Luc

2004-01-01

The fragile X syndrome is the most common cause of inherited mental retardation resulting from the absence of the fragile X mental retardation protein (FMRP). FMRP contains two K-homology (KH) domains and one RGG box that are landmarks characteristic of RNA-binding proteins. In agreement with this, FMRP associates with messenger ribonucleoparticles (mRNPs) within actively translating ribosomes, and is thought to regulate translation of target mRNAs, including its own transcript. To investigate whether FMRP might chaperone nucleic acid folding and hybridization, we analysed the annealing and strand exchange activities of DNA oligonucleotides and the enhancement of ribozyme-directed RNA substrate cleavage by FMRP and deleted variants relative to canonical nucleic acid chaperones, such as the cellular YB-1/p50 protein and the retroviral nucleocapsid protein HIV-1 NCp7. FMRP was found to possess all the properties of a potent nucleic acid chaperone, requiring the KH motifs and RGG box for optimal activity. These findings suggest that FMRP may regulate translation by acting on RNA-RNA interactions and thus on the structural status of mRNAs.

First-Principles Study of Carbon and Vacancy Structures in Niobium

DOE PAGES

Ford, Denise C.; Zapol, Peter; Cooley, Lance D.

2015-04-03

The interstitial chemical impurities hydrogen, oxygen, nitrogen, and carbon are important for niobium metal production, and particularly for the optimization of niobium SRF technology. These atoms are present in refined sheets and can be absorbed into niobium during processing treatments, resulting in changes to the residual resistance and the performance of SRF cavities. A first-principles approach is taken to study the properties of carbon in niobium, and the results are compared and contrasted with the properties of the other interstitial impurities. The results indicate that C will likely form precipitates or atmospheres around defects rather than strongly bound complexes withmore » other impurities. Based on the analysis of carbon and hydrogen near niobium lattice vacancies and small vacancy chains and clusters, the formation of extended carbon chains and hydrocarbons is not likely to occur. Association of carbon with hydrogen atoms can, however, occur through the strain fields created by interstitial binding of the impurity atoms. In conclusion, calculated electronic densities of states indicate that interstitial C may have a similar effect as interstitial O on the superconducting transition temperature of Nb.« less

The oligomerization state determines regulatory properties and inhibitor sensitivity of type 4 cAMP-specific phosphodiesterases.

PubMed

Richter, Wito; Conti, Marco

2004-07-16

PDE4 splice variants are classified into long and short forms depending on the presence or absence of two unique N-terminal domains termed upstream conserved regions 1 and 2 (UCR1 and -2). We have shown previously that the UCR module mediates dimerization of PDE4 long forms, whereas short forms, which lack UCR1, behave as monomers. In the present study, we demonstrate that dimerization is an essential structural element that determines the regulatory properties and inhibitor sensitivities of PDE4 enzymes. Comparing the properties of the dimeric wild type PDE4D3 with several monomeric mutant PDE4D3 constructs revealed that disruption of dimerization ablates the activation of PDE4 long forms by either protein kinase A phosphorylation or phosphatidic acid binding. Moreover, the analysis of heterodimers consisting of a catalytically active and a catalytically inactive PDE4D3 subunit indicates that protein kinase A phosphorylation of both subunits is essential to fully activate PDE4 enzymes. In addition to affecting enzyme regulation, disruption of dimerization reduces the sensitivity of the enzymes toward the prototypical PDE4 inhibitor rolipram. Parallel binding assays indicated that this shift in rolipram sensitivity is likely mediated by a decrease in the number of inhibitor binding sites in the high affinity rolipram binding state. Thus, although dimerization is not a requirement for high affinity rolipram binding, it functions to stabilize PDE4 long forms in their high affinity rolipram binding conformation. Taken together, our data indicate that dimerization defines the properties of PDE4 enzymes and suggest a common structural and functional organization for all PDEs.

First principles study of structural, electronic and magnetic properties of SnGe n (0, ±1) ( n = 1–17) clusters

NASA Astrophysics Data System (ADS)

Djaadi, Soumaia; Eddine Aiadi, Kamal; Mahtout, Sofiane

2018-04-01

The structures, relative stability and magnetic properties of pure Ge n +1, neutral cationic and anionic SnGe n (n = 1–17) clusters have been investigated by using the first principles density functional theory implemented in SIESTA packages. We find that with the increasing of cluster size, the Ge n +1 and SnGe n (0, ±1) clusters tend to adopt compact structures. It has been also found that the Sn atom occupied a peripheral position for SnGe n clusters when n < 12 and occupied a core position for n > 12. The structural and electronic properties such as optimized geometries, fragmentation energy, binding energy per atom, HOMO–LUMO gaps and second-order differences in energy of the pure Ge n +1 and SnGe n clusters in their ground state are calculated and analyzed. All isomers of neutral SnGe n clusters are generally nonmagnetic except for n = 1 and 4, where the total spin magnetic moments is 2μ b. The total (DOS) and partial density of states of these clusters have been calculated to understand the origin of peculiar magnetic properties. The cluster size dependence of vertical ionization potentials, vertical electronic affinities, chemical hardness, adiabatic electron affinities and adiabatic ionization potentials have been calculated and discussed.

Parallel screening of drug-like natural compounds using Caco-2 cell permeability QSAR model with applicability domain, lipophilic ligand efficiency index and shape property: A case study of HIV-1 reverse transcriptase inhibitors

NASA Astrophysics Data System (ADS)

Patel, Rikin D.; Kumar, Sivakumar Prasanth; Patel, Chirag N.; Shankar, Shetty Shilpa; Pandya, Himanshu A.; Solanki, Hitesh A.

2017-10-01

The traditional drug design strategy centrally focuses on optimizing binding affinity with the receptor target and evaluates pharmacokinetic properties at a later stage which causes high rate of attrition in clinical trials. Alternatively, parallel screening allows evaluation of these properties and affinity simultaneously. In a case study to identify leads from natural compounds with experimental HIV-1 reverse transcriptase (RT) inhibition, we integrated various computational approaches including Caco-2 cell permeability QSAR model with applicability domain (AD) to recognize drug-like natural compounds, molecular docking to study HIV-1 RT interactions and shape similarity analysis with known crystal inhibitors having characteristic butterfly-like model. Further, the lipophilic properties of the compounds refined from the process with best scores were examined using lipophilic ligand efficiency (LLE) index. Seven natural compound hits viz. baicalien, (+)-calanolide A, mniopetal F, fagaronine chloride, 3,5,8-trihydroxy-4-quinolone methyl ether derivative, nitidine chloride and palmatine, were prioritized based on LLE score which demonstrated Caco-2 well absorption labeling, encompassment in AD structural coverage, better receptor affinity, shape adaptation and permissible AlogP value. We showed that this integrative approach is successful in lead exploration of natural compounds targeted against HIV-1 RT enzyme.

FT-IR, FT-Raman and NMR characterization of 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate and investigation of its reactive and optoelectronic properties by molecular dynamics simulations and DFT calculations

NASA Astrophysics Data System (ADS)

Menon, Vidya V.; Fazal, Edakot; Mary, Y. Sheena; Panicker, C. Yohannan; Armaković, Stevan; Armaković, Sanja J.; Nagarajan, Subban; Van Alsenoy, C.

2017-01-01

The FT-IR and FT-Raman spectra of the synthesized compound, 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate is recorded and analyzed. Optimized molecular structure, wave numbers, corresponding assignments regarding 2-isopropyl-5-methylcyclohexyl quinoline-2-carboxylate has become screened tentatively as well as hypothetically using Gaussian09 program package. Natural bonding orbital assessment has been completed with a reason to clarify charge transfer or conjugative interaction, the intra-molecular re-hybridization and delocalization of electron density within the molecule. The NMR spectral assessment had been made choosing structure property relationship by chemical shifts along with the magnetic shielding effects regarding the title compound. The first and second hyperpolarizabilities were calculated. The calculated first order hyperpolarizability is commensurate with the documented worth of very similar derivatives and could be an interesting object for more experiments on nonlinear optics. Local reactivity properties have been investigated using average local ionization energies and Fukui functions. Investigation of optoelectronic properties encompassed calculations of reorganization energies and hopping rates of charge carriers within the framework of Marcus semi-empiric approach. The docked ligand title compound forms a stable complex with CDK inhibitors and gives a binding affinity value of -9.7 kcal/mol and molecular docking results suggest that the compound might exhibit inhibitory activity against CDK inhibitors.

Biochemical and functional characterization of an albumin protein belonging to the hemopexin superfamily from Lens culinaris seeds.

PubMed

Scarafoni, Alessio; Gualtieri, Elisa; Barbiroli, Alberto; Carpen, Aristodemo; Negri, Armando; Duranti, Marcello

2011-09-14

The present paper reports the purification and biochemical characterization of an albumin identified in mature lentil seeds with high sequence similarity to pea PA2. These proteins are found in many edible seeds and are considered potentially detrimental for human health due to the potential allergenicity and lectin-like activity. Thus, the description of their possible presence in food and the assessment of the molecular properties are relevant. The M(r), pI, and N-terminal sequence of this protein have been determined. The work included the study of (i) the binding properties to hemine to assess the presence of hemopexin structural domains and (ii) the binding properties of the protein to thiamin. In addition, the structural changes induced by heating have been evaluated by means of spectroscopic techniques. Denaturation temperature has also been determined. The present work provides new insights about the structural molecular features and the ligand-binding properties and dynamics of this kind of seed albumin.

Resolving dual binding conformations of cellulosome cohesin-dockerin complexes using single-molecule force spectroscopy.

PubMed

Jobst, Markus A; Milles, Lukas F; Schoeler, Constantin; Ott, Wolfgang; Fried, Daniel B; Bayer, Edward A; Gaub, Hermann E; Nash, Michael A

2015-10-31

Receptor-ligand pairs are ordinarily thought to interact through a lock and key mechanism, where a unique molecular conformation is formed upon binding. Contrary to this paradigm, cellulosomal cohesin-dockerin (Coh-Doc) pairs are believed to interact through redundant dual binding modes consisting of two distinct conformations. Here, we combined site-directed mutagenesis and single-molecule force spectroscopy (SMFS) to study the unbinding of Coh:Doc complexes under force. We designed Doc mutations to knock out each binding mode, and compared their single-molecule unfolding patterns as they were dissociated from Coh using an atomic force microscope (AFM) cantilever. Although average bulk measurements were unable to resolve the differences in Doc binding modes due to the similarity of the interactions, with a single-molecule method we were able to discriminate the two modes based on distinct differences in their mechanical properties. We conclude that under native conditions wild-type Doc from Clostridium thermocellum exocellulase Cel48S populates both binding modes with similar probabilities. Given the vast number of Doc domains with predicted dual binding modes across multiple bacterial species, our approach opens up new possibilities for understanding assembly and catalytic properties of a broad range of multi-enzyme complexes.

Large-scale binding ligand prediction by improved patch-based method Patch-Surfer2.0

PubMed Central

Zhu, Xiaolei; Xiong, Yi; Kihara, Daisuke

2015-01-01

Motivation: Ligand binding is a key aspect of the function of many proteins. Thus, binding ligand prediction provides important insight in understanding the biological function of proteins. Binding ligand prediction is also useful for drug design and examining potential drug side effects. Results: We present a computational method named Patch-Surfer2.0, which predicts binding ligands for a protein pocket. By representing and comparing pockets at the level of small local surface patches that characterize physicochemical properties of the local regions, the method can identify binding pockets of the same ligand even if they do not share globally similar shapes. Properties of local patches are represented by an efficient mathematical representation, 3D Zernike Descriptor. Patch-Surfer2.0 has significant technical improvements over our previous prototype, which includes a new feature that captures approximate patch position with a geodesic distance histogram. Moreover, we constructed a large comprehensive database of ligand binding pockets that will be searched against by a query. The benchmark shows better performance of Patch-Surfer2.0 over existing methods. Availability and implementation: http://kiharalab.org/patchsurfer2.0/ Contact: dkihara@purdue.edu Supplementary information: Supplementary data are available at Bioinformatics online. PMID:25359888

Improved prediction of MHC class I and class II epitopes using a novel Gibbs sampling approach.

PubMed

Nielsen, Morten; Lundegaard, Claus; Worning, Peder; Hvid, Christina Sylvester; Lamberth, Kasper; Buus, Søren; Brunak, Søren; Lund, Ole

2004-06-12

Prediction of which peptides will bind a specific major histocompatibility complex (MHC) constitutes an important step in identifying potential T-cell epitopes suitable as vaccine candidates. MHC class II binding peptides have a broad length distribution complicating such predictions. Thus, identifying the correct alignment is a crucial part of identifying the core of an MHC class II binding motif. In this context, we wish to describe a novel Gibbs motif sampler method ideally suited for recognizing such weak sequence motifs. The method is based on the Gibbs sampling method, and it incorporates novel features optimized for the task of recognizing the binding motif of MHC classes I and II. The method locates the binding motif in a set of sequences and characterizes the motif in terms of a weight-matrix. Subsequently, the weight-matrix can be applied to identifying effectively potential MHC binding peptides and to guiding the process of rational vaccine design. We apply the motif sampler method to the complex problem of MHC class II binding. The input to the method is amino acid peptide sequences extracted from the public databases of SYFPEITHI and MHCPEP and known to bind to the MHC class II complex HLA-DR4(B1*0401). Prior identification of information-rich (anchor) positions in the binding motif is shown to improve the predictive performance of the Gibbs sampler. Similarly, a consensus solution obtained from an ensemble average over suboptimal solutions is shown to outperform the use of a single optimal solution. In a large-scale benchmark calculation, the performance is quantified using relative operating characteristics curve (ROC) plots and we make a detailed comparison of the performance with that of both the TEPITOPE method and a weight-matrix derived using the conventional alignment algorithm of ClustalW. The calculation demonstrates that the predictive performance of the Gibbs sampler is higher than that of ClustalW and in most cases also higher than that of the TEPITOPE method.

Using physics-based pose predictions and free energy perturbation calculations to predict binding poses and relative binding affinities for FXR ligands in the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Athanasiou, Christina; Vasilakaki, Sofia; Dellis, Dimitris; Cournia, Zoe

2018-01-01

Computer-aided drug design has become an integral part of drug discovery and development in the pharmaceutical and biotechnology industry, and is nowadays extensively used in the lead identification and lead optimization phases. The drug design data resource (D3R) organizes challenges against blinded experimental data to prospectively test computational methodologies as an opportunity for improved methods and algorithms to emerge. We participated in Grand Challenge 2 to predict the crystallographic poses of 36 Farnesoid X Receptor (FXR)-bound ligands and the relative binding affinities for two designated subsets of 18 and 15 FXR-bound ligands. Here, we present our methodology for pose and affinity predictions and its evaluation after the release of the experimental data. For predicting the crystallographic poses, we used docking and physics-based pose prediction methods guided by the binding poses of native ligands. For FXR ligands with known chemotypes in the PDB, we accurately predicted their binding modes, while for those with unknown chemotypes the predictions were more challenging. Our group ranked #1st (based on the median RMSD) out of 46 groups, which submitted complete entries for the binding pose prediction challenge. For the relative binding affinity prediction challenge, we performed free energy perturbation (FEP) calculations coupled with molecular dynamics (MD) simulations. FEP/MD calculations displayed a high success rate in identifying compounds with better or worse binding affinity than the reference (parent) compound. Our studies suggest that when ligands with chemical precedent are available in the literature, binding pose predictions using docking and physics-based methods are reliable; however, predictions are challenging for ligands with completely unknown chemotypes. We also show that FEP/MD calculations hold predictive value and can nowadays be used in a high throughput mode in a lead optimization project provided that crystal structures of sufficiently high quality are available.

A robust methodology to subclassify pseudokinases based on their nucleotide-binding properties

PubMed Central

Murphy, James M.; Zhang, Qingwei; Young, Samuel N.; Reese, Michael L.; Bailey, Fiona P.; Eyers, Patrick A.; Ungureanu, Daniela; Hammaren, Henrik; Silvennoinen, Olli; Varghese, Leila N.; Chen, Kelan; Tripaydonis, Anne; Jura, Natalia; Fukuda, Koichi; Qin, Jun; Nimchuk, Zachary; Mudgett, Mary Beth; Elowe, Sabine; Gee, Christine L.; Liu, Ling; Daly, Roger J.; Manning, Gerard; Babon, Jeffrey J.; Lucet, Isabelle S.

2017-01-01

Protein kinase-like domains that lack conserved residues known to catalyse phosphoryl transfer, termed pseudokinases, have emerged as important signalling domains across all kingdoms of life. Although predicted to function principally as catalysis-independent protein-interaction modules, several pseudokinase domains have been attributed unexpected catalytic functions, often amid controversy. We established a thermal-shift assay as a benchmark technique to define the nucleotide-binding properties of kinase-like domains. Unlike in vitro kinase assays, this assay is insensitive to the presence of minor quantities of contaminating kinases that may otherwise lead to incorrect attribution of catalytic functions to pseudokinases. We demonstrated the utility of this method by classifying 31 diverse pseudokinase domains into four groups: devoid of detectable nucleotide or cation binding; cation-independent nucleotide binding; cation binding; and nucleotide binding enhanced by cations. Whereas nine pseudokinases bound ATP in a divalent cation-dependent manner, over half of those examined did not detectably bind nucleotides, illustrating that pseudokinase domains predominantly function as non-catalytic protein-interaction modules within signalling networks and that only a small subset is potentially catalytically active. We propose that henceforth the thermal-shift assay be adopted as the standard technique for establishing the nucleotide-binding and catalytic potential of kinase-like domains. PMID:24107129

Template-based de novo design for type II kinase inhibitors and its extented application to acetylcholinesterase inhibitors.

PubMed

Su, Bo-Han; Huang, Yi-Syuan; Chang, Chia-Yun; Tu, Yi-Shu; Tseng, Yufeng J

2013-10-31

There is a compelling need to discover type II inhibitors targeting the unique DFG-out inactive kinase conformation since they are likely to possess greater potency and selectivity relative to traditional type I inhibitors. Using a known inhibitor, such as a currently available and approved drug or inhibitor, as a template to design new drugs via computational de novo design is helpful when working with known ligand-receptor interactions. This study proposes a new template-based de novo design protocol to discover new inhibitors that preserve and also optimize the binding interactions of the type II kinase template. First, sorafenib (Nexavar) and nilotinib (Tasigna), two type II inhibitors with different ligand-receptor interactions, were selected as the template compounds. The five-step protocol can reassemble each drug from a large fragment library. Our procedure demonstrates that the selected template compounds can be successfully reassembled while the key ligand-receptor interactions are preserved. Furthermore, to demonstrate that the algorithm is able to construct more potent compounds, we considered kinase inhibitors and other protein dataset, acetylcholinesterase (AChE) inhibitors. The de novo optimization was initiated using a template compound possessing a less than optimal activity from a series of aminoisoquinoline and TAK-285 inhibiting type II kinases, and E2020 derivatives inhibiting AChE respectively. Three compounds with greater potency than the template compound were discovered that were also included in the original congeneric series. This template-based lead optimization protocol with the fragment library can help to design compounds with preferred binding interactions of known inhibitors automatically and further optimize the compounds in the binding pockets.

Selection of specific protein binders for pre-defined targets from an optimized library of artificial helicoidal repeat proteins (alphaRep).

PubMed

Guellouz, Asma; Valerio-Lepiniec, Marie; Urvoas, Agathe; Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a "filtration" procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×10(9) independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties.

The Target Residence Time of Antihistamines Determines Their Antagonism of the G Protein-Coupled Histamine H1 Receptor

PubMed Central

Bosma, Reggie; Witt, Gesa; Vaas, Lea A. I.; Josimovic, Ivana; Gribbon, Philip; Vischer, Henry F.; Gul, Sheraz; Leurs, Rob

2017-01-01

The pharmacodynamics of drug-candidates is often optimized by metrics that describe target binding (Kd or Ki value) or target modulation (IC50). However, these metrics are determined at equilibrium conditions, and consequently information regarding the onset and offset of target engagement and modulation is lost. Drug-target residence time is a measure for the lifetime of the drug-target complex, which has recently been receiving considerable interest, as target residence time is shown to have prognostic value for the in vivo efficacy of several drugs. In this study, we have investigated the relation between the increased residence time of antihistamines at the histamine H1 receptor (H1R) and the duration of effective target-inhibition by these antagonists. Hela cells, endogenously expressing low levels of the H1R, were incubated with a series of antihistamines and dissociation was initiated by washing away the unbound antihistamines. Using a calcium-sensitive fluorescent dye and a label free, dynamic mass redistribution based assay, functional recovery of the H1R responsiveness was measured by stimulating the cells with histamine over time, and the recovery was quantified as the receptor recovery time. Using these assays, we determined that the receptor recovery time for a set of antihistamines differed more than 40-fold and was highly correlated to their H1R residence times, as determined with competitive radioligand binding experiments to the H1R in a cell homogenate. Thus, the receptor recovery time is proposed as a cell-based and physiologically relevant metric for the lead optimization of G protein-coupled receptor antagonists, like the H1R antagonists. Both, label-free or real-time, classical signaling assays allow an efficient and physiologically relevant determination of kinetic properties of drug molecules. PMID:29033838

In silico investigation of lavandulyl flavonoids for the development of potent fatty acid synthase-inhibitory prototypes.

PubMed

Oh, Joonseok; Liu, Haining; Park, Hyun Bong; Ferreira, Daneel; Jeong, Gil-Saeng; Hamann, Mark T; Doerksen, Robert J; Na, MinKyun

2017-01-01

Inhibition of fatty acid synthase (FAS) is regarded as a sensible therapeutic strategy for the development of optimal anti-cancer agents. Flavonoids exhibit potent anti-neoplastic properties. The MeOH extract of Sophora flavescens was subjected to chromatographic analyses such as VLC and HPLC for the purification of active flavonoids. The DP4 chemical-shift analysis protocol was employed to investigate the elusive chirality of the lavandulyl moiety of the purified polyphenols. Induced Fit docking protocols and per-residue analyses were utilized to scrutinize structural prerequisites for hampering FAS activity. The FAS-inhibitory activity of the purified flavonoids was assessed via the incorporation of [ 3 H] acetyl-CoA into palmitate. Six flavonoids, including lavandulyl flavanones, were purified and evaluated for FAS inhibition. The lavandulyl flavanone sophoraflavanone G (2) exhibited the highest potency (IC 50 of 6.7±0.2μM), which was more potent than the positive controls. Extensive molecular docking studies revealed the structural requirements for blocking FAS. Per-residue interaction analysis demonstrated that the lavandulyl functional group in the active flavonoids (1-3 and 5) significantly contributed to increasing their binding affinity towards the target enzyme. This research suggests a basis for the in silico design of a lavandulyl flavonoid-based architecture showing anti-cancer effects via enhancement of the binding potential to FAS. FAS inhibition by flavonoids and their derivatives may offer significant potential as an approach to lower the risk of various cancer diseases and related fatalities. In silico technologies with available FAS crystal structures may be of significant use in optimizing preliminary leads. Copyright © 2016 Elsevier B.V. All rights reserved.

Novel Bispecific PSMA/GRPr Targeting Radioligands with Optimized Pharmacokinetics for Improved PET Imaging of Prostate Cancer.

PubMed

Liolios, C; Schäfer, M; Haberkorn, U; Eder, M; Kopka, K

2016-03-16

A new series of bispecific radioligands (BRLs) targeting prostate-specific membrane antigen (PSMA) and gastrin releasing peptide receptor (GRPr), both expressed on prostate cancer cells, was developed. Their design was based on the bombesin (BN) analogue, H2N-PEG2-[D-Tyr(6),β-Ala(11),Thi(13),Nle(14)]BN(6-14), which binds to GRPr with high affinity and specificity, and the peptidomimetic urea-based pseudoirreversible inhibitor of PSMA, Glu-ureido-Lys. The two pharmacophores were coupled through copper(I)-catalyzed azide-alkyne cycloaddition to the bis(tetrafluorophenyl) ester of the chelating agent HBED-CC via amino acid linkers made of positively charged His (H) and negatively charged Glu (E): -(HE)n- (n = 0-3). The BRLs were labeled with (68)Ga, and their preliminary pharmacological properties were evaluated in vitro (competitive and time kinetic binding assays) on prostate cancer (PC-3, LNCaP) and rat pancreatic (AR42J) cell lines and in vivo by biodistribution and small animal PET imaging studies in both normal and tumor-bearing mice. The IC50/Ki values determined for all BRLs essentially matched those of the respective monomers. The maximal cellular uptake of the BLRs was observed between 20 and 30 min. The BRLs showed a synergistic ability in vivo by targeting both PSMA (LNCaP) and GRPr (PC-3) positive tumors, whereas the charged -(HE)n- (n = 1-3) linkers significantly reduced the kidney and spleen uptake. The bispecific (PSMA and GRPr) targeting ability and optimized pharmacokinetics of the compounds developed in this study could lead to their future application in clinical practice as more sensitive radiotracers for noninvasive imaging of prostate cancer (PCa) by PET/CT and PET/MRI.

In silico investigation of lavandulyl flavonoids for the development of potent fatty acid synthase-inhibitory prototypes

PubMed Central

Oh, Joonseok; Liu, Haining; Park, Hyun Bong; Ferreira, Daneel; Jeong, Gil-Saeng; Hamann, Mark T.; Doerksen, Robert J.; Na, MinKyun

2016-01-01

Background Inhibition of fatty acid synthase (FAS) is regarded as a sensible therapeutic strategy for the development of optimal anti-cancer agents. Flavonoids exhibit potent anti-neoplastic properties. Methods The MeOH extract of Sophora flavescens was subjected to chromatographic analyses such as VLC and HPLC for the purification of active flavonoids. The DP4 chemical-shift analysis protocol was employed to investigate the elusive chirality of the lavandulyl moiety of the purified polyphenols. Induced Fit docking protocols and per-residue analyses were utilized to scrutinize structural prerequisites for hampering FAS activity. The FAS-inhibitory activity of the purified flavonoids was assessed via the incorporation of [3H] acetyl-CoA into palmitate. Results Six flavonoids, including lavandulyl flavanones, were purified and evaluated for FAS inhibition. The lavandulyl flavanone sophoraflavanone G (2) exhibited the highest potency (IC50 of 6.7 ± 0.2 μM), which was more potent than the positive controls. Extensive molecular docking studies revealed the structural requirements for blocking FAS. Per-residue interaction analysis demonstrated that the lavandulyl functional group in the active flavonoids (1–3 and 5) significantly contributed to increasing their binding affinity towards the target enzyme. Conclusion This research suggests a basis for the in silico design of a lavandulyl flavonoid-based architecture showing anti-cancer effects via enhancement of the binding potential to FAS. General significance FAS inhibition by flavonoids and their derivatives may offer significant potential as an approach to lower the risk of various cancer diseases and related fatalities. In silico technologies with available FAS crystal structures may be of significant use in optimizing preliminary leads. PMID:27531709

Selection of Specific Protein Binders for Pre-Defined Targets from an Optimized Library of Artificial Helicoidal Repeat Proteins (alphaRep)

PubMed Central

Chevrel, Anne; Graille, Marc; Fourati-Kammoun, Zaineb; Desmadril, Michel; van Tilbeurgh, Herman; Minard, Philippe

2013-01-01

We previously designed a new family of artificial proteins named αRep based on a subgroup of thermostable helicoidal HEAT-like repeats. We have now assembled a large optimized αRep library. In this library, the side chains at each variable position are not fully randomized but instead encoded by a distribution of codons based on the natural frequency of side chains of the natural repeats family. The library construction is based on a polymerization of micro-genes and therefore results in a distribution of proteins with a variable number of repeats. We improved the library construction process using a “filtration” procedure to retain only fully coding modules that were recombined to recreate sequence diversity. The final library named Lib2.1 contains 1.7×109 independent clones. Here, we used phage display to select, from the previously described library or from the new library, new specific αRep proteins binding to four different non-related predefined protein targets. Specific binders were selected in each case. The results show that binders with various sizes are selected including relatively long sequences, with up to 7 repeats. ITC-measured affinities vary with Kd values ranging from micromolar to nanomolar ranges. The formation of complexes is associated with a significant thermal stabilization of the bound target protein. The crystal structures of two complexes between αRep and their cognate targets were solved and show that the new interfaces are established by the variable surfaces of the repeated modules, as well by the variable N-cap residues. These results suggest that αRep library is a new and versatile source of tight and specific binding proteins with favorable biophysical properties. PMID:24014183

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE PAGES

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.; ...

2014-12-31

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Subfamily-specific adaptations in the structures of two penicillin-binding proteins from Mycobacterium tuberculosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prigozhin, Daniil M.; Krieger, Inna V.; Huizar, John P.

Beta-lactam antibiotics target penicillin-binding proteins including several enzyme classes essential for bacterial cell-wall homeostasis. To better understand the functional and inhibitor-binding specificities of penicillin-binding proteins from the pathogen, Mycobacterium tuberculosis, we carried out structural and phylogenetic analysis of two predicted D,D-carboxypeptidases, Rv2911 and Rv3330. Optimization of Rv2911 for crystallization using directed evolution and the GFP folding reporter method yielded a soluble quadruple mutant. Structures of optimized Rv2911 bound to phenylmethylsulfonyl fluoride and Rv3330 bound to meropenem show that, in contrast to the nonspecific inhibitor, meropenem forms an extended interaction with the enzyme along a conserved surface. Phylogenetic analysis shows thatmore » Rv2911 and Rv3330 belong to different clades that emerged in Actinobacteria and are not represented in model organisms such as Escherichia coli and Bacillus subtilis. Clade-specific adaptations allow these enzymes to fulfill distinct physiological roles despite strict conservation of core catalytic residues. The characteristic differences include potential protein-protein interaction surfaces and specificity-determining residues surrounding the catalytic site. Overall, these structural insights lay the groundwork to develop improved beta-lactam therapeutics for tuberculosis.« less

Photoregulating RNA digestion using azobenzene linked dumbbell antisense oligodeoxynucleotides.

PubMed

Wu, Li; He, Yujian; Tang, Xinjing

2015-06-17

Introduction of 4,4'-bis(hydroxymethyl)-azobenzene (azo) to dumbbell hairpin oligonucleotides at the loop position was able to reversibly control the stability of the whole hairpin structure via UV or visible light irradiation. Here, we designed and synthesized a series of azobenzene linked dumbbell antisense oligodeoxynucleotides (asODNs) containing two terminal hairpins that are composed of an asODN and a short inhibitory sense strand. Thermal melting studies of these azobenzene linked dumbbell asODNs indicated that efficient trans to cis photoisomerization of azobenzene moieties induced large difference in thermal stability (ΔTm = 12.1-21.3 °C). In addition, photomodulation of their RNA binding abilities and RNA digestion by RNase H was investigated. The trans-azobenzene linked asODNs with the optimized base pairs between asODN strands and inhibitory sense strands could only bind few percentage of the target RNA, while it was able to recover their binding to the target RNA and degrade it by RNase H after light irradiation. Upon optimization, it is promising to use these azobenzene linked asODNs for reversible spatial and temporal regulation of antisense activities based on both steric binding and RNA digestion by RNase H.

Quantum Chemical and Docking Insights into Bioavailability Enhancement of Curcumin by Piperine in Pepper.

PubMed

Patil, Vaishali M; Das, Sukanya; Balasubramanian, Krishnan

2016-05-26

We combine quantum chemical and molecular docking techniques to provide new insights into how piperine molecule in various forms of pepper enhances bioavailability of a number of drugs including curcumin in turmeric for which it increases its bioavailability by a 20-fold. We have carried out docking studies of quantum chemically optimized piperine structure binding to curcumin, CYP3A4 in cytochrome P450, p-Glycoprotein and UDP-glucuronosyltransferase (UGT), the enzyme responsible for glucuronosylation, which increases the solubility of curcumin. All of these studies establish that piperine binds to multiple sites on the enzymes and also intercalates with curcumin forming a hydrogen bonded complex with curcumin. The conjugated network of double bonds and the presence of multiple charge centers of piperine offer optimal binding sites for piperine to bind to enzymes such as UDP-GDH, UGT, and CYP3A4. Piperine competes for curcumin's intermolecular hydrogen bonding and its stacking propensity by hydrogen bonding with enolic proton of curcumin. This facilitates its metabolic transport, thereby increasing its bioavailability both through intercalation into curcumin layers through intermolecular hydrogen bonding, and by inhibiting enzymes that cause glucuronosylation of curcumin.

The PP1 binding code: a molecular-lego strategy that governs specificity.

PubMed

Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

2013-01-01

Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

Cu(II) binding by a pH-fractionated fulvic acid

USGS Publications Warehouse

Brown, G.K.; Cabaniss, S.E.; MacCarthy, P.; Leenheer, J.A.

1999-01-01

The relationship between acidity, Cu(II) binding and sorption to XAD resin was examined using Suwannee River fulvic acid (SRFA). The work was based on the hypothesis that fractions of SRFA eluted from an XAD column at various pH's from 1.0 to 12.0 would show systematic variations in acidity and possibly aromaticity which in turn would lead to different Cu(II) binding properties. We measured equilibrium Cu(II) binding to these fractions using Cu2+ ion-selective electrode (ISE) potentiometry at pH 6.0. Several model ligands were also examined, including cyclopentane-1,2,3,4-tetracarboxylic acid (CP-TCA) and tetrahydrofuran-2,3,4,5-tetracarboxylic acid (THF-TCA), the latter binding Cu(II) much more strongly as a consequence of the ether linkage. The SRFA Cu(II) binding properties agreed with previous work at high ionic strength, and binding was enhanced substantially at lower ionic strength, in agreement with Poisson-Boltzmann predictions for small spheres. Determining Cu binding constants (K(i)) by non-linear regression with total ligand concentrations (L(Ti)) taken from previous work, the fractions eluted at varying pH had K(i) similar to the unfractionated SRFA, with a maximum enhancement of 0.50 log units. We conclude that variable-pH elution from XAD does not isolate significantly strong (or weak) Cu(II)-binding components from the SRFA mixture. Copyright (C) 1999 Elsevier Science B.V.

The Disassembly of a Core-Satellite Nanoassembled Substrate for Colorimetric Biomolecular Detection

PubMed Central

Waldeisen, John R.; Wang, Tim; Ross, Benjamin M.; Lee, Luke P.

2012-01-01

The disassembly of a core-satellite nanostructured substrate is presented as a colorimetric biosensor observable under dark field illumination. The fabrication method described herein utilizes thiol-mediated adsorption and streptavidin-biotin binding to self-assemble core-satellite nanostructures with a sacrificial linking peptide. Biosensing functionality is demonstrated with the protease trypsin and the optical properties of the nanoassemblies are characterized. A figure of merit is presented to determine the optimal core and satellite size for visual detection. Nanoassemblies with 50 nm cores and 30 nm or 50 nm satellites are superior as these structures achieve an orange to green color shift greater than 70 nm that is easily discernible by naked eye. This colorimetric substrate may prove to be a favorable alternative to liquid-based colloidal sensors and a useful visual readout mechanism for microfluidic diagnostic assays. PMID:21667984