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

Immobilization of enzymes by bioaffinity layering.

PubMed

Singh, Veena; Sardar, Meryam; Gupta, Munishwar Nath

2013-01-01

Bioaffinity immobilization exploits the affinity of the enzyme to a macro-(affinity ligand). Such a macro-(affinity ligand) could be a lectin, a water-soluble polymer, or a bioconjugate of a water-soluble polymer and the appropriate affinity ligand. Successive layering of the enzyme and the macro-(affinity ligand) on a matrix allows deposition of a large amount of enzyme activity on a small surface. Illustrative protocols show affinity layering of a pectinase and horseradish peroxidase on Concanavalin A-agarose and Concanavalin A-Sephadex matrices, respectively.

A solid-phase combinatorial approach for indoloquinolizidine-peptides with high affinity at D(1) and D(2) dopamine receptors.

PubMed

Molero, Anabel; Vendrell, Marc; Bonaventura, Jordi; Zachmann, Julian; López, Laura; Pardo, Leonardo; Lluis, Carme; Cortés, Antoni; Albericio, Fernando; Casadó, Vicent; Royo, Miriam

2015-06-05

Ligands acting at multiple dopamine receptors hold potential as therapeutic agents for a number of neurodegenerative disorders. Specifically, compounds able to bind at D1R and D2R with high affinity could restore the effects of dopamine depletion and enhance motor activation on degenerated nigrostriatal dopaminergic systems. We have directed our research towards the synthesis and characterisation of heterocycle-peptide hybrids based on the indolo[2,3-a]quinolizidine core. This privileged structure is a water-soluble and synthetically accessible scaffold with affinity for diverse GPCRs. Herein we have prepared a solid-phase combinatorial library of 80 indoloquinolizidine-peptides to identify compounds with enhanced binding affinity at D2R, a receptor that is crucial to re-establish activity on dopamine-depleted degenerated GABAergic neurons. We applied computational tools and high-throughput screening assays to identify 9a{1,3,3} as a ligand for dopamine receptors with nanomolar affinity and agonist activity at D2R. Our results validate the application of indoloquinolizidine-peptide combinatorial libraries to fine-tune the pharmacological profiles of multiple ligands at D1 and D2 dopamine receptors. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Evaluation of affinity and pseudo-affinity adsorption processes for penicillin acylase purification.

PubMed

Fonseca, L P; Cabral, J M

1996-01-01

Affinity ligand (6-Aminopenicillanic acid, Amoxycillin, Ampicillin, Benzylpenicillin and 4-Phenylbutylanzine) of penicillin acylase (EC 3.5.1.11) were attached to hydrophilic gels like Sepharose 4B-CNBr and Minileak 'medium'. Ampicillin and 4-Phenylbutylamine were the affinity ligands that presented the higher concentrations attached to both gels. Penicillin acylase adsorption on these affinity gels was mainly dependent on the activated group of the gel, the affinity ligand attached and the experimental conditions of enzyme adsorption. Under affinity conditions only the ligands Amoxycillin, Ampicillin and 4-Phenylbutylamine, immobilized on Minileak, adsorbed the enzyme from osmotic shock extracts at different pH values. These affinity ligand systems were characterized by low adsorption capacities of penicillin acylase activity (1.2-2.1 IU mL-1 gel) and specific activity (1.5-2.9 IU mg-1 prot). Under pseudo-affinity conditions all the ligands attached both activated to gels (Sepharose 4B-CNBr and Minileak) adsorbed the enzyme. The affinity gels were characterized by higher values of adsorption capacity (3.7 and 55.6 IU mL-1 gel) and adsorbed specific activity (2.0 and 6.1 IU mg-1 prot) than those observed under affinity conditions. The space arm of Minileak gel, shown to be fundamental to enzyme adsorption under affinity conditions, preferentially adsorbed proteins in relation to the enzyme under pseudo-affinity conditions. However, this effect was partially minimized when the gel was derivatized by the affinity ligands at concentrations higher than 6 mumol mL-1 gel. Ampicillin was the affinity ligand that presented the best results for specific adsorption of penicillin acylase under affinity and pseudo-affinity adsorption processes. The Sepharose 4B-CNBr derivatized gel also presented a good adsorption capacity of enzyme activity (26.8 IU mL-1 gel) under pseudo-affinity adsorption processes.

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man*

PubMed Central

Macauley, Matthew S.; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M.; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C.

2015-01-01

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2–6Galβ1–4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. PMID:26507663

Unmasking of CD22 Co-receptor on Germinal Center B-cells Occurs by Alternative Mechanisms in Mouse and Man.

PubMed

Macauley, Matthew S; Kawasaki, Norihito; Peng, Wenjie; Wang, Shui-Hua; He, Yuan; Arlian, Britni M; McBride, Ryan; Kannagi, Reiji; Khoo, Kay-Hooi; Paulson, James C

2015-12-11

CD22 is an inhibitory B-cell co-receptor whose function is modulated by sialic acid (Sia)-bearing glycan ligands. Glycan remodeling in the germinal center (GC) alters CD22 ligands, with as yet no ascribed biological consequence. Here, we show in both mice and humans that loss of high affinity ligands on GC B-cells unmasks the binding site of CD22 relative to naive and memory B-cells, promoting recognition of trans ligands. The conserved modulation of CD22 ligands on GC B-cells is striking because high affinity glycan ligands of CD22 are species-specific. In both species, the high affinity ligand is based on the sequence Siaα2-6Galβ1-4GlcNAc, which terminates N-glycans. The human ligand has N-acetylneuraminic acid (Neu5Ac) as the sialic acid, and the high affinity ligand on naive B-cells contains 6-O-sulfate on the GlcNAc. On human GC B-cells, this sulfate modification is lost, giving rise to lower affinity CD22 ligands. Ligands of CD22 on naive murine B-cells do not contain the 6-O-sulfate modification. Instead, the high affinity ligand for mouse CD22 has N-glycolylneuraminic acid (Neu5Gc) as the sialic acid, which is replaced on GC B-cells with Neu5Ac. Human naive and memory B-cells express sulfated glycans as high affinity CD22 ligands, which are lost on GC B-cells. In mice, Neu5Gc-containing glycans serve as high affinity CD22 ligands that are replaced by Neu5Ac-containing glycans on GC B-cells. Our results demonstrate that loss of high affinity CD22 ligands on GC B-cells occurs in both mice and humans through alternative mechanisms, unmasking CD22 relative to naive and memory B-cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Legume receptors perceive the rhizobial lipochitin oligosaccharide signal molecules by direct binding

PubMed Central

Broghammer, Angelique; Krusell, Lene; Blaise, Mickaël; Sauer, Jørgen; Sullivan, John T.; Maolanon, Nicolai; Vinther, Maria; Lorentzen, Andrea; Madsen, Esben B.; Jensen, Knud J.; Roepstorff, Peter; Thirup, Søren; Ronson, Clive W.; Thygesen, Mikkel B.; Stougaard, Jens

2012-01-01

Lipochitin oligosaccharides called Nod factors function as primary rhizobial signal molecules triggering legumes to develop new plant organs: root nodules that host the bacteria as nitrogen-fixing bacteroids. Here, we show that the Lotus japonicus Nod factor receptor 5 (NFR5) and Nod factor receptor 1 (NFR1) bind Nod factor directly at high-affinity binding sites. Both receptor proteins were posttranslationally processed when expressed as fusion proteins and extracted from purified membrane fractions of Nicotiana benthamiana or Arabidopsis thaliana. The N-terminal signal peptides were cleaved, and NFR1 protein retained its in vitro kinase activity. Processing of NFR5 protein was characterized by determining the N-glycosylation patterns of the ectodomain. Two different glycan structures with identical composition, Man3XylFucGlcNAc4, were identified by mass spectrometry and located at amino acid positions N68 and N198. Receptor–ligand interaction was measured by using ligands that were labeled or immobilized by application of chemoselective chemistry at the anomeric center. High-affinity ligand binding was demonstrated with both solid-phase and free solution techniques. The Kd values obtained for Nod factor binding were in the nanomolar range and comparable to the concentration range sufficient for biological activity. Structure-dependent ligand specificity was shown by using chitin oligosaccharides. Taken together, our results suggest that ligand recognition through direct ligand binding is a key step in the receptor-mediated activation mechanism leading to root nodule development in legumes. PMID:22859506

Quantitative analysis of rat brain alpha 2-receptors discriminated by [3H]clonidine and [3H]rauwolscine.

PubMed

Asakura, M; Tsukamoto, T; Imafuku, J; Matsui, H; Ino, M; Hasegawa, K

1984-10-30

Quantitative analysis of direct ligand binding of both [3H]clonidine and [3H]rauwolscine to the rat cerebral cortex alpha 2-receptors indicates the existence of two affinity states of the same receptor populations. In the presence of Mn2+, the high affinity state of [3H]clonidine binding was increased, whereas the high affinity state of [3H]rauwolscine binding was reduced. By contrast, GTP in micromolar ranges caused a decrease of the agonist high affinity state and an increase of the antagonist high affinity state. The total receptor sites and the respective separate affinities for both radioligands were approximately equal to their control values under all conditions, indicating that Mn2+ and GTP modulate the proportion of the two affinity states of the receptor. These results can be incorporated into a two-step, ternary complex model involving a guanine nucleotide binding protein (N protein) for the agonist and antagonist interaction with the alpha 2-receptor. Furthermore, the effects of GTP on the interaction of both ligands with the two affinity states can be mimicked by EDTA. It is suggested that divalent cations induce the formation of the receptor-N protein binary complex showing high affinity for agonists and low affinity for antagonists.

B61 is a ligand for the ECK receptor protein-tyrosine kinase.

PubMed

Bartley, T D; Hunt, R W; Welcher, A A; Boyle, W J; Parker, V P; Lindberg, R A; Lu, H S; Colombero, A M; Elliott, R L; Guthrie, B A

1994-04-07

A protein ligand for the ECK receptor protein-tyrosine kinase has been isolated by using the extracellular domain (ECK-X) of the receptor as an affinity reagent. Initially, concentrated cell culture supernatants were screened for receptor binding activity using immobilized ECK-X in a surface plasmon resonance detection system. Subsequently, supernatants from selected cell lines were fractionated directly by receptor affinity chromatography, resulting in the single-step purification of B61, a protein previously identified as the product of an early response gene induced by tumour necrosis factor-alpha. We report here that recombinant B61 induces autophosphorylation of ECK in intact cells, consistent with B61 being an authentic ligand for ECK. ECK is a member of a large orphan receptor protein-tyrosine kinase family headed by EPH, and we suggest that ligands for other members of this family will be related to B61, and can be isolated in the same way.

Identification and specificity studies of small-molecule ligands for SH3 protein domains.

PubMed

Inglis, Steven R; Stojkoski, Cvetan; Branson, Kim M; Cawthray, Jacquie F; Fritz, Daniel; Wiadrowski, Emma; Pyke, Simon M; Booker, Grant W

2004-10-21

The Src Homology 3 (SH3) domains are small protein-protein interaction domains that bind proline-rich sequences and mediate a wide range of cell-signaling and other important biological processes. Since deregulated signaling pathways form the basis of many human diseases, the SH3 domains have been attractive targets for novel therapeutics. High-affinity ligands for SH3 domains have been designed; however, these have all been peptide-based and no examples of entirely nonpeptide SH3 ligands have previously been reported. Using the mouse Tec Kinase SH3 domain as a model system for structure-based ligand design, we have identified several simple heterocyclic compounds that selectively bind to the Tec SH3 domain. Using a combination of nuclear magnetic resonance chemical shift perturbation, structure-activity relationships, and site-directed mutagenesis, the binding of these compounds at the proline-rich peptide-binding site has been characterized. The most potent of these, 2-aminoquinoline, bound with Kd = 125 microM and was able to compete for binding with a proline-rich peptide. Synthesis of 6-substituted-2-aminoquinolines resulted in ligands with up to 6-fold improved affinity over 2-aminoquinoline and enhanced specificity for the Tec SH3 domain. Therefore, 2-aminoquinolines may potentially be useful for the development of high affinity small molecule ligands for SH3 domains.

Affinity purification using recombinant PXR as a tool to characterize environmental ligands.

PubMed

Dagnino, Sonia; Bellet, Virginie; Grimaldi, Marina; Riu, Anne; Aït-Aïssa, Sélim; Cavaillès, Vincent; Fenet, Hélène; Balaguer, Patrick

2014-02-01

Many environmental endocrine disrupting compounds act as ligands for nuclear receptors. The human pregnane X receptor (hPXR), for instance, is activated by a variety of environmental ligands such as steroids, pharmaceutical drugs, pesticides, alkylphenols, polychlorinated biphenyls and polybromo diethylethers. Some of us have previously reported the occurrence of hPXR ligands in environmental samples but failed to identify them. The aim of this study was to test whether a PXR-affinity column, in which recombinant hPXR was immobilized on solid support, could help the purification of these chemicals. Using PXR ligands of different affinity (10 nM < EC50 < 10 μM), we demonstrated that the PXR-affinity preferentially column captured ligands with medium to high affinities (EC50 < 1 μM). Furthermore, by using the PXR-affinity column to analyze an environmental sample containing ERα, AhR, AR, and PXR activities, we show that (i) half of the PXR activity of the sample was due to compounds with medium to high affinity for PXR and (ii) PXR shared ligands with ERα, AR, and AhR. These findings demonstrate that the newly developed PXR-affinity column coupled to reporter cell lines represents a valuable tool for the characterization of the nature of PXR active compounds and should therefore guide and facilitate their further analysis. Copyright © 2012 Wiley Periodicals, Inc., a Wiley company.

Mapping protein-protein interactions with phage-displayed combinatorial peptide libraries.

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

Kay, B. K.; Castagnoli, L.; Biosciences Division

This unit describes the process and analysis of affinity selecting bacteriophage M13 from libraries displaying combinatorial peptides fused to either a minor or major capsid protein. Direct affinity selection uses target protein bound to a microtiter plate followed by purification of selected phage by ELISA. Alternatively, there is a bead-based affinity selection method. These methods allow one to readily isolate peptide ligands that bind to a protein target of interest and use the consensus sequence to search proteomic databases for putative interacting proteins.

Utilizing a library of synthetic affinity ligands for the enrichment, depletion and one-step purification of leech proteins.

PubMed

Dong, Dexian; Gui, Yanli; Chen, Dezhao; Li, Rongxiu

2008-01-01

Although the concept of affinity purification using synthetic ligands had been utilized for many years, there are few articles related to this research area, and they focus only on the affinity purification of specific protein by a defined library of synthetic ligands. This study presents the design and construction of a 700-member library of synthetic ligands in detail. We selected 297 ligand columns from a 700-member library of synthetic ligands to screen leech protein extract. Of the 297, 154 columns had an enrichment effect, 83 columns had a depletion effect, 36 columns had a one-step purification effect, and 58 columns had a one-step purification via flowthrough effect. The experimental results achieved by this large library of affinity ligands provide solid convincing data for the theory that affinity chromatography could be used for the enrichment of proteins that are present in low abundance, the depletion of high abundance proteins, and one-step purification of special proteins. 2008 John Wiley & Sons, Ltd

Synthesis of biotinylated glycoconjugates and their use in a novel ELISA for direct comparison of HIV-1 Gp120 recognition of GalCer and related carbohydrate analogues.

PubMed

McReynolds, K D; Hadd, M J; Gervay-Hague, J

1999-01-01

As part of our program directed toward the design and synthesis of high-affinity ligands for the GalCer-binding site on the HIV cell surface glycoprotein, gp120, we required a reliable method for qualitatively assessing relative binding affinities for related analogues. Due to the hydrophilic nature of these synthetic conjugates, difficulties were encountered with typical ELISA methods, which rely upon hydrophobic interactions to anchor the ligand to a microtiter plate. Other types of assays were also problematic due to nonspecific binding of gp120. Therefore, we developed a general method for plating water-soluble ligands on microtiter plates using biotin/NeutrAvidin recognition for adhesion. A water-soluble GalCer analogue was prepared by conjugating psychosine to biotin using a novel tetraethylene glycol linker. In a similar manner, LacCer and GlcCer analogues were prepared and these conjugates were plated into microtiter wells containing NeutrAvidin. Unoccupied sites were blocked using biotin functionalized as a primary amide. Gp120 binding to galactosyl sphingosine, GalSph (19), GlcSph (22), and LacSph (23) conjugates was assessed through incubation with recombinant HRP-gp120. It was determined that LacSph has the strongest interaction with gp120. The binding affinities of GalSph and GlcSph were similar to each other and less strong than LacSph. These data contradict earlier studies where HPTLC showed that LacCer and GlcCer do not significantly bind gp120. They also contradict liposome-based assays that reported psychosine is not recognized by gp120. The extent of plating for each biotinylated molecule was quantified using HRP-biotin, allowing direct comparison of ligand plating efficiencies for the first time. Several other synthetic biotin conjugates were prepared and tested, demonstrating the feasibility of performing ELISA on water-soluble ligands.

Molecular Hybridization of Potent and Selective γ-Hydroxybutyric Acid (GHB) Ligands: Design, Synthesis, Binding Studies, and Molecular Modeling of Novel 3-Hydroxycyclopent-1-enecarboxylic Acid (HOCPCA) and trans-γ-Hydroxycrotonic Acid (T-HCA) Analogs.

PubMed

Krall, Jacob; Jensen, Claus Hatt; Bavo, Francesco; Falk-Petersen, Christina Birkedahl; Haugaard, Anne Stæhr; Vogensen, Stine Byskov; Tian, Yongsong; Nittegaard-Nielsen, Mia; Sigurdardóttir, Sara Björk; Kehler, Jan; Kongstad, Kenneth Thermann; Gloriam, David E; Clausen, Rasmus Prætorius; Harpsøe, Kasper; Wellendorph, Petrine; Frølund, Bente

2017-11-09

γ-Hydroxybutyric acid (GHB) is a neuroactive substance with specific high-affinity binding sites. To facilitate target identification and ligand optimization, we herein report a comprehensive structure-affinity relationship study for novel ligands targeting these binding sites. A molecular hybridization strategy was used based on the conformationally restricted 3-hydroxycyclopent-1-enecarboxylic acid (HOCPCA) and the linear GHB analog trans-4-hydroxycrotonic acid (T-HCA). In general, all structural modifications performed on HOCPCA led to reduced affinity. In contrast, introduction of diaromatic substituents into the 4-position of T-HCA led to high-affinity analogs (medium nanomolar K i ) for the GHB high-affinity binding sites as the most high-affinity analogs reported to date. The SAR data formed the basis for a three-dimensional pharmacophore model for GHB ligands, which identified molecular features important for high-affinity binding, with high predictive validity. These findings will be valuable in the further processes of both target characterization and ligand identification for the high-affinity GHB binding sites.

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.

Integration of biotic ligand models (BLM) and bioaccumulation kinetics into a mechanistic framework for metal uptake in aquatic organisms.

PubMed

Veltman, Karin; Huijbregts, Mark A J; Hendriks, A Jan

2010-07-01

Both biotic ligand models (BLM) and bioaccumulation models aim to quantify metal exposure based on mechanistic knowledge, but key factors included in the description of metal uptake differ between the two approaches. Here, we present a quantitative comparison of both approaches and show that BLM and bioaccumulation kinetics can be merged into a common mechanistic framework for metal uptake in aquatic organisms. Our results show that metal-specific absorption efficiencies calculated from BLM-parameters for freshwater fish are highly comparable, i.e. within a factor of 2.4 for silver, cadmium, copper, and zinc, to bioaccumulation-absorption efficiencies for predominantly marine fish. Conditional affinity constants are significantly related to the metal-specific covalent index. Additionally, the affinity constants of calcium, cadmium, copper, sodium, and zinc are significantly comparable across aquatic species, including molluscs, daphnids, and fish. This suggests that affinity constants can be estimated from the covalent index, and constants can be extrapolated across species. A new model is proposed that integrates the combined effect of metal chemodynamics, as speciation, competition, and ligand affinity, and species characteristics, as size, on metal uptake by aquatic organisms. An important direction for further research is the quantitative comparison of the proposed model with acute toxicity values for organisms belonging to different size classes.

High affinity ligands from in vitro selection: Complex targets

PubMed Central

Morris, Kevin N.; Jensen, Kirk B.; Julin, Carol M.; Weil, Michael; Gold, Larry

1998-01-01

Human red blood cell membranes were used as a model system to determine if the systematic evolution of ligands by exponential enrichment (SELEX) methodology, an in vitro protocol for isolating high-affinity oligonucleotides that bind specifically to virtually any single protein, could be used with a complex mixture of potential targets. Ligands to multiple targets were generated simultaneously during the selection process, and the binding affinities of these ligands for their targets are comparable to those found in similar experiments against pure targets. A secondary selection scheme, deconvolution-SELEX, facilitates rapid isolation of the ligands to targets of special interest within the mixture. SELEX provides high-affinity compounds for multiple targets in a mixture and might allow a means for dissecting complex biological systems. PMID:9501188

Application of the principle of linked functions to ATP-driven ion pumps: kinetics of activation by ATP.

PubMed Central

Reynolds, J A; Johnson, E A; Tanford, C

1985-01-01

If a ligand binds with unequal affinity to two distinct states of a protein, then the equilibrium between the two states becomes a function of the concentration of the ligand. A necessary consequence is that the ligand must also affect the forward and/or reverse rate constants for transition between the two states. For an enzyme or transport protein with such a transition as a slow step in the catalytic cycle, the overall rate also becomes a function of ligand concentration. These conclusions are independent of whether or not the ligand is a direct participant in the reaction. If it is a direct participant, then the kinetic effect arising from the principle of linked functions is distinct from the direct catalytic effect. These principles suffice to account for the biphasic response of the hydrolytic activity of ATP-driven ion pumps to the concentration of ATP, without the need to invoke more than one ATP binding site per catalytic center. PMID:2987939

Differential structural properties of GLP-1 and exendin-4 determine their relative affinity for the GLP-1 receptor N-terminal extracellular domain.

PubMed

Runge, Steffen; Schimmer, Susann; Oschmann, Jan; Schiødt, Christine Bruun; Knudsen, Sanne Möller; Jeppesen, Claus Bekker; Madsen, Kjeld; Lau, Jesper; Thøgersen, Henning; Rudolph, Rainer

2007-05-15

Glucagon-like peptide-1 (GLP-1) and exendin-4 (Ex4) are homologous peptides with established potential for treatment of type 2 diabetes. They bind and activate the pancreatic GLP-1 receptor (GLP-1R) with similar affinity and potency and thereby promote insulin secretion in a glucose-dependent manner. GLP-1R belongs to family B of the seven transmembrane G-protein coupled receptors. The N-terminal extracellular domain (nGLP-1R) is a ligand binding domain with differential affinity for Ex4 and GLP-1: low affinity for GLP-1 and high affinity for exendin-4. The superior affinity of nGLP-1R for Ex4 was previously explained by an additional interaction between nGLP-1R and the C-terminal Trp-cage of Ex4. In this study we have combined biophysical and pharmacological approaches thus relating structural properties of the ligands in solution to their relative binding affinity for nGLP-1R. We used both a tracer competition assay and ligand-induced thermal stabilization of nGLP-1R to measure the relative affinity of full length, truncated, and chimeric ligands for soluble refolded nGLP-1R. The ligands in solution and the conformational consequences of ligand binding to nGLP-1R were characterized by circular dichroism and fluorescence spectroscopy. We found a correlation between the helical content of the free ligands and their relative binding affinity for nGLP-1R, supporting the hypothesis that the ligands are helical at least in the segment that binds to nGLP-1R. The Trp-cage of Ex4 was not necessary to maintain a superior helicity of Ex4 compared to GLP-1. The results suggest that the differential affinity of nGLP-1R is explained almost entirely by divergent residues in the central part of the ligands: Leu10-Gly30 of Ex4 and Val16-Arg36 of GLP-1. In view of our results it appears that the Trp-cage plays only a minor role for the interaction between Ex4 and nGLP-1R and for the differential affinity of nGLP-1R for GLP-1 and Ex4.

Challenges and opportunities in the purification of recombinant tagged proteins.

PubMed

Pina, Ana Sofia; Lowe, Christopher R; Roque, Ana Cecília A

2014-01-01

The purification of recombinant proteins by affinity chromatography is one of the most efficient strategies due to the high recovery yields and purity achieved. However, this is dependent on the availability of specific affinity adsorbents for each particular target protein. The diversity of proteins to be purified augments the complexity and number of specific affinity adsorbents needed, and therefore generic platforms for the purification of recombinant proteins are appealing strategies. This justifies why genetically encoded affinity tags became so popular for recombinant protein purification, as these systems only require specific ligands for the capture of the fusion protein through a pre-defined affinity tag tail. There is a wide range of available affinity pairs "tag-ligand" combining biological or structural affinity ligands with the respective binding tags. This review gives a general overview of the well-established "tag-ligand" systems available for fusion protein purification and also explores current unconventional strategies under development. Copyright © 2013 Elsevier Inc. All rights reserved.

Protein purification by aminosquarylium cyanine dye-affinity chromatography.

PubMed

Silva, M S; Graça, V C; Reis, L V; Santos, P F; Almeida, P; Queiroz, J A; Sousa, F

2013-12-01

The most selective purification method for proteins and other biomolecules is affinity chromatography. This method is based on the unique biological-based specificity of the biomolecule-ligand interaction and commonly uses biological ligands. However, these ligands may present some drawbacks, mainly because of their cost and lability. Dye-affinity chromatography overcomes the limitations of biological ligands and is widely used owing to the low cost of synthetic dyes and to their resistance to biological and chemical degradation. In this work, immobilized aminosquarylium cyanine dyes are used in order to exploit affinity interactions with standard proteins such as lysozyme, α-chymotrypsin and trypsin. These studies evaluate the affinity interactions occurring between the immobilized ligand and the different proteins, as a reflection of the sum of several molecular interactions, namely ionic, hydrophobic and van der Waals, spread throughout the structure, in a defined spatial manner. The results show the possibility of using an aminosquarylium cyanine dye bearing a N-hexyl pendant chain, with a ligand density of 1.8 × 10(-2) mmol of dye/g of chromatographic support, to isolate lysozyme, α-chymotrypsin and trypsin from a mixture. The application of a decreasing ammonium sulfate gradient resulted in the recovery of lysozyme in the flowthrough. On the other hand, α-chymotrypsin and trypsin were retained, involving different interactions with the ligand. In conclusion, this study demonstrates the potential applicability of ligands such as aminosquarylium cyanine dyes for the separation and purification of proteins by affinity chromatography. Copyright © 2013 John Wiley & Sons, Ltd.

PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.

PubMed

Walkup, Ward G; Kennedy, Mary B

2014-06-01

PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ∼90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using ThermoFluor.

PubMed

Matulis, Daumantas; Kranz, James K; Salemme, F Raymond; Todd, Matthew J

2005-04-05

ThermoFluor (a miniaturized high-throughput protein stability assay) was used to analyze the linkage between protein thermal stability and ligand binding. Equilibrium binding ligands increase protein thermal stability by an amount proportional to the concentration and affinity of the ligand. Binding constants (K(b)) were measured by examining the systematic effect of ligand concentration on protein stability. The precise ligand effects depend on the thermodynamics of protein stability: in particular, the unfolding enthalpy. An extension of current theoretical treatments was developed for tight binding inhibitors, where ligand effect on T(m) can also reveal binding stoichiometry. A thermodynamic analysis of carbonic anhydrase by differential scanning calorimetry (DSC) enabled a dissection of the Gibbs free energy of stability into enthalpic and entropic components. Under certain conditions, thermal stability increased by over 30 degrees C; the heat capacity of protein unfolding was estimated from the dependence of calorimetric enthalpy on T(m). The binding affinity of six sulfonamide inhibitors to two isozymes (human type 1 and bovine type 2) was analyzed by both ThermoFluor and isothermal titration calorimetry (ITC), resulting in a good correlation in the rank ordering of ligand affinity. This combined investigation by ThermoFluor, ITC, and DSC provides a detailed picture of the linkage between ligand binding and protein stability. The systematic effect of ligands on stability is shown to be a general tool to measure affinity.

Affinity Interaction between Hexamer Peptide Ligand HWRGWV and Immunoglobulin G Studied by Quartz Crystal Microbalance and Surface Plasmon Resonance

NASA Astrophysics Data System (ADS)

Shen, Fei

Immunoglobulins (Ig), also referred to as antibodies, act as protective agents against pathogens trying to invade an organism. Human immunoglobulin G (hIgG), as the most prominent immunoglobulin presented in serum and other human fluids, has broad applications in fields like immunotherapy and clinical diagnostics. Staphylococcus aureus Protein A and Streptococcus Protein G are the most common affinity ligands for IgG purifaction and detection. However, drawbacks associated with these two protein ligands have motivated searches for alternative affinity ligands. The hexamer peptide ligand HWRGWV identified from a one-bead-one-peptide combinatorial library synthesized on chromatography resins has demonstrated high affinity and specificity to the Fc fragment of hIgG. A chromatography resin with HWRGWV can purify human IgG (hIgG) from complete minimum essential medium (cMEM) with purities and yields as high as 95%, which are comparable to using Protein A as affinity ligand (4). As a short peptide ligand, HWRGWV can be produced at relatively low costs under good manufacturing practices (GMP) conditions, it is highly robust, less immunogenic and allows for milder elution conditions for the bound antibody (3, 5). Although this short peptide ligand has exhibited promising properties for IgG capture and purification, limited information is available on the intrinsic mechanisms of affinity interaction between the peptide ligand and target protein. In this study, the affinity interaction between hIgG and peptide ligand immobilized on solid surfaces was studied by quartz crystal microbalance (QCM) and surface plasmon resonance (SPR). Compared with previous methods employed for the peptide characterization, QCM and SPR can provide direct measurements of equilibrium adsorption isotherms and rates of adsorption, allowing a complete kinetic and thermodynamics analyses of the ligand-target interactions. New methods were developed to modify gold and silica surfaces of QCM and SPR sensors for the immobilization of peptide ligands with low nonspecific binding. The silica surface was first modified by the formation of self-assembling monolayer (SAM) of 3-amino-propyl triethoxy silane as an anchor layer. Short chains of poly(ethylene glycol) (PEG) with Fmoc-protected amino groups at one end and carboxyl groups at the other end were then coupled through the carboxyl terminal to the amino groups on the silane. The short PEG chains served as spacer arms to reduce nonspecific binding to the substrate. The gold surface was modified by a two-component SAM using mixtures of HS(CH 2)11(CH2CH2O)6NH2 and HS(CH2)11(CH2CH2O)3OH. The advantage of using a modified silica surface is its relatively higher stability than the SAM on gold during the peptide functionalization step, however the SPR sensors do not work on silica surfaces. In addition, the modification process of the gold surface is relatively simple compared with that of the silica surface. The peptide immobilization process was optimized with silica surfaces and the best conditions were applied for the immobilization on gold surfaces. The results of surface modifications and peptide immobilizations were characterized by various surface analysis techniques including, ellipsometry, contact angle goniometer, chemical force microscopy (CFM), x-ray photoelectron spectroscopy (XPS) and time of flight secondary ion mass spectroscopy (ToF-SIMS). QCM and SPR results indicated that this peptide ligand HWRGWV immobilized on modified silica or gold surfaces has high affinity and specificity to hIgG binding even in a complex medium such as cMEM. Both thermodynamic and kinetic parameters of affinity interaction were obtained by the analysis of QCM and SPR data. Compared with QCM, SPR is more suitable for quantitative analysis of the protein binding, which is essential for the investigation of thermodynamics and kinetics parameters. The maximum binding capacity (4.15 mg m-2 ) and the dissociation constant (1.83 muM) derived from SPR data are both close to those obtained with chromatography techniques. The association and dissociation rate constants (0.68 m3 mol-1 s-1 and 1.24 s-1 respectively) were acquired for the first time for the affinity binding of IgG on peptide ligand HWRGWV functionalized surface. Although QCM is not as quantitative as SPR, it provides additional information on the status of the adsorbed layers. For instance, the dissipation measurement of QCM indicated that no significant denaturation of adsorbed hIgG occurred during the adsorption process. In addition, it was shown that the peptide ligand immobilized on modified silica surfaces has similar affinity and binding characteristics for IgG adsorption as on modified gold surfaces. In summary, new surface modification strategies were developed to study the affinity interaction between peptide ligands and target biomolecules. The use of Fc-specific binding peptides on QCM and SPR sensors could result in new devices for IgG concentration determination and also have promise as platforms for the development of immunosensors.

Accurate Evaluation Method of Molecular Binding Affinity from Fluctuation Frequency

NASA Astrophysics Data System (ADS)

Hoshino, Tyuji; Iwamoto, Koji; Ode, Hirotaka; Ohdomari, Iwao

2008-05-01

Exact estimation of the molecular binding affinity is significantly important for drug discovery. The energy calculation is a direct method to compute the strength of the interaction between two molecules. This energetic approach is, however, not accurate enough to evaluate a slight difference in binding affinity when distinguishing a prospective substance from dozens of candidates for medicine. Hence more accurate estimation of drug efficacy in a computer is currently demanded. Previously we proposed a concept of estimating molecular binding affinity, focusing on the fluctuation at an interface between two molecules. The aim of this paper is to demonstrate the compatibility between the proposed computational technique and experimental measurements, through several examples for computer simulations of an association of human immunodeficiency virus type-1 (HIV-1) protease and its inhibitor (an example for a drug-enzyme binding), a complexation of an antigen and its antibody (an example for a protein-protein binding), and a combination of estrogen receptor and its ligand chemicals (an example for a ligand-receptor binding). The proposed affinity estimation has proven to be a promising technique in the advanced stage of the discovery and the design of drugs.

Effect of urea on protein-ligand association.

PubMed

Stepanian, Lora; Son, Ikbae; Chalikian, Tigran V

2017-12-01

We combine experimental and theoretical approaches to investigate the influence of a cosolvent on a ligand-protein association event. We apply fluorescence measurements to determining the affinity of the inhibitor tri-N-acetylglucosamine [(GlcNAc) 3 ] for lysozyme at urea concentrations ranging from 0 to 8M. Notwithstanding that, at room temperature and neutral pH, lysozyme retains its native conformation up to the solubility limit of urea, the affinity of (GlcNAc) 3 for the protein steadily decreases as the concentration of urea increases. We analyze the urea dependence of the binding free energy within the framework of a simplified statistical thermodynamics-based model that accounts for the excluded volume effect and direct solute-solvent interactions. The analysis reveals that the detrimental action of urea on the inhibitor-lysozyme binding originates from competition between the free energy contributions of the excluded volume effect and direct solute-solvent interactions. The free energy contribution of direct urea-solute interactions narrowly overcomes the excluded volume contribution thereby resulting in urea weakening the protein-ligand association. More broadly, the successful application of the simple model employed in this work points to the possibility of its use in quantifying the stabilizing/destabilizing action of individual cosolvents on biochemical folding and binding reactions. Copyright © 2016 Elsevier B.V. All rights reserved.

Strong Ligand-Protein Interactions Derived from Diffuse Ligand Interactions with Loose Binding Sites.

PubMed

Marsh, Lorraine

2015-01-01

Many systems in biology rely on binding of ligands to target proteins in a single high-affinity conformation with a favorable ΔG. Alternatively, interactions of ligands with protein regions that allow diffuse binding, distributed over multiple sites and conformations, can exhibit favorable ΔG because of their higher entropy. Diffuse binding may be biologically important for multidrug transporters and carrier proteins. A fine-grained computational method for numerical integration of total binding ΔG arising from diffuse regional interaction of a ligand in multiple conformations using a Markov Chain Monte Carlo (MCMC) approach is presented. This method yields a metric that quantifies the influence on overall ligand affinity of ligand binding to multiple, distinct sites within a protein binding region. This metric is essentially a measure of dispersion in equilibrium ligand binding and depends on both the number of potential sites of interaction and the distribution of their individual predicted affinities. Analysis of test cases indicates that, for some ligand/protein pairs involving transporters and carrier proteins, diffuse binding contributes greatly to total affinity, whereas in other cases the influence is modest. This approach may be useful for studying situations where "nonspecific" interactions contribute to biological function.

Controlled method of reducing electrophoretic mobility of various substances

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1989-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and the like is provided. The method comprises interacting the particles or cells with a polymer-linked affinity compound composed of: a hydrophilic neutral polymer such as polyethylene glycol, and an affinity component consisting of a hydrophobic compound such as a fatty acid ester, an immunocompound such as an antibody or active fragment thereof or simular macromolecule, or other ligands. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and the mobility reduction obtainable is up to 100 percent for particular particles and cells. The present invention is advantageous in that analytical electrophoretic separation can not be achieved for macromolecules, particles, and cells whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions. The present method is also advantageous in that it can be used in a variety of standard laboratory electrophoresis equipment.

Septide and neurokinin A are high-affinity ligands on the NK-1 receptor: evidence from homologous versus heterologous binding analysis.

PubMed

Hastrup, H; Schwartz, T W

1996-12-16

The three main tachykinins, substance P, neurokinin A (NKA), and neurokinin B, are believed to be selective ligands for respectively the NK-1, NK-2 and NK-3 receptors. However, NKA also has actions which cannot be mediated through its normal NK-2 receptor and the synthetic peptide [pGlu6,Pro9]-Substance P9-11--called septide--is known to have tachykinin-like actions despite its apparent lack of binding to any known tachykinin receptor. In the cloned NK-1 receptor expressed in COS-7 cells NKA and septide as expected were poor competitors for radiolabeled substance P. However, by using radiolabeled NKA and septide directly, it was found that both peptides in homologous binding assays as well as in competition against each other in fact bound to the NK-1 receptor with high affinity: Kd values of 0.51 +/- 0.15 nM (NKA) and 0.55 +/- 0.03 nM (septide). It is concluded that NKA and septide are high-affinity ligands for the NK-1 receptor but that they are poor competitors for substance P, which in contrast competes very well for binding with both NKA and septide.

Characterizing low affinity epibatidine binding to α4β2 nicotinic acetylcholine receptors with ligand depletion and nonspecific binding

PubMed Central

2011-01-01

Background Along with high affinity binding of epibatidine (Kd1≈10 pM) to α4β2 nicotinic acetylcholine receptor (nAChR), low affinity binding of epibatidine (Kd2≈1-10 nM) to an independent binding site has been reported. Studying this low affinity binding is important because it might contribute understanding about the structure and synthesis of α4β2 nAChR. The binding behavior of epibatidine and α4β2 AChR raises a question about interpreting binding data from two independent sites with ligand depletion and nonspecific binding, both of which can affect equilibrium binding of [3H]epibatidine and α4β2 nAChR. If modeled incorrectly, ligand depletion and nonspecific binding lead to inaccurate estimates of binding constants. Fitting total equilibrium binding as a function of total ligand accurately characterizes a single site with ligand depletion and nonspecific binding. The goal of this study was to determine whether this approach is sufficient with two independent high and low affinity sites. Results Computer simulations of binding revealed complexities beyond fitting total binding for characterizing the second, low affinity site of α4β2 nAChR. First, distinguishing low-affinity specific binding from nonspecific binding was a potential problem with saturation data. Varying the maximum concentration of [3H]epibatidine, simultaneously fitting independently measured nonspecific binding, and varying α4β2 nAChR concentration were effective remedies. Second, ligand depletion helped identify the low affinity site when nonspecific binding was significant in saturation or competition data, contrary to a common belief that ligand depletion always is detrimental. Third, measuring nonspecific binding without α4β2 nAChR distinguished better between nonspecific binding and low-affinity specific binding under some circumstances of competitive binding than did presuming nonspecific binding to be residual [3H]epibatidine binding after adding a large concentration of cold competitor. Fourth, nonspecific binding of a heterologous competitor changed estimates of high and low inhibition constants but did not change the ratio of those estimates. Conclusions Investigating the low affinity site of α4β2 nAChR with equilibrium binding when ligand depletion and nonspecific binding are present likely needs special attention to experimental design and data interpretation beyond fitting total binding data. Manipulation of maximum ligand and receptor concentrations and intentionally increasing ligand depletion are potentially helpful approaches. PMID:22112852

Rapid characterization of a novel taspine derivative-HMQ1611 binding to EGFR by a cell membrane chromatography method.

PubMed

Du, Hui; Lv, Nan; Wang, Sicen; He, Langchong

2013-05-01

A new high-expression endothelial growth factor receptor (EGFR) cell membrane chromatography (CMC) method was applied to recognize the ligands acting on EGFR specifically, and investigate the affinity of gefitinib/HMQ1611 to EGFR. In the self and direct competitive assay, gefitinib/HMQ1611 was used as a competitor in the mobile phase to evaluate the effect of the competitor's concentrations on the retention of the ligands, respectively, and the competition between gefitinib and HMQ1611 binding to EGFR was also been examined. The retention behavior indicated that gefitinib had one type of binding sites on the EGFR, and the equilibrium dissociation constant (K(D)) was (9.11 ± 1.89) × 10(-6) M; HMQ1611 had two major binding regions on the EGFR, and the K(D) values obtained from the model were (2.39 ± 0.33) × 10(-7) and (3.87 ± 0.93) × 10(-5) M for HMQ1611 at the high- and low-affinity sites, respectively. The competition between gefitinib and HMQ1611 occurred at the low-affinity sites on the EGFR. The low-affinity sites were of higher concentrations and contributed to a much larger part of retention of HMQ1611. The results suggested that gefitinib and HMQ1611 competed for the common binding sites on the EGFR, no matter the ligand was used as an analyte or a competitor.

Molecular Determinants of Epidermal Growth Factor Binding: A Molecular Dynamics Study

PubMed Central

Sanders, Jeffrey M.; Wampole, Matthew E.; Thakur, Mathew L.; Wickstrom, Eric

2013-01-01

The epidermal growth factor receptor (EGFR) is a member of the receptor tyrosine kinase family that plays a role in multiple cellular processes. Activation of EGFR requires binding of a ligand on the extracellular domain to promote conformational changes leading to dimerization and transphosphorylation of intracellular kinase domains. Seven ligands are known to bind EGFR with affinities ranging from sub-nanomolar to near micromolar dissociation constants. In the case of EGFR, distinct conformational states assumed upon binding a ligand is thought to be a determining factor in activation of a downstream signaling network. Previous biochemical studies suggest the existence of both low affinity and high affinity EGFR ligands. While these studies have identified functional effects of ligand binding, high-resolution structural data are lacking. To gain a better understanding of the molecular basis of EGFR binding affinities, we docked each EGFR ligand to the putative active state extracellular domain dimer and 25.0 ns molecular dynamics simulations were performed. MM-PBSA/GBSA are efficient computational approaches to approximate free energies of protein-protein interactions and decompose the free energy at the amino acid level. We applied these methods to the last 6.0 ns of each ligand-receptor simulation. MM-PBSA calculations were able to successfully rank all seven of the EGFR ligands based on the two affinity classes: EGF>HB-EGF>TGF-α>BTC>EPR>EPG>AR. Results from energy decomposition identified several interactions that are common among binding ligands. These findings reveal that while several residues are conserved among the EGFR ligand family, no single set of residues determines the affinity class. Instead we found heterogeneous sets of interactions that were driven primarily by electrostatic and Van der Waals forces. These results not only illustrate the complexity of EGFR dynamics but also pave the way for structure-based design of therapeutics targeting EGF ligands or the receptor itself. PMID:23382875

Dextran as a Generally Applicable Multivalent Scaffold for Improving Immunoglobulin-Binding Affinities of Peptide and Peptidomimetic Ligands

PubMed Central

2015-01-01

Molecules able to bind the antigen-binding sites of antibodies are of interest in medicine and immunology. Since most antibodies are bivalent, higher affinity recognition can be achieved through avidity effects in which a construct containing two or more copies of the ligand engages both arms of the immunoglobulin simultaneously. This can be achieved routinely by immobilizing antibody ligands at high density on solid surfaces, such as ELISA plates, but there is surprisingly little literature on scaffolds that routinely support bivalent binding of antibody ligands in solution, particularly for the important case of human IgG antibodies. Here we show that the simple strategy of linking two antigens with a polyethylene glycol (PEG) spacer long enough to span the two arms of an antibody results in higher affinity binding in some, but not all, cases. However, we found that the creation of multimeric constructs in which several antibody ligands are displayed on a dextran polymer reliably provides much higher affinity binding than is observed with the monomer in all cases tested. Since these dextran conjugates are simple to construct, they provide a general and convenient strategy to transform modest affinity antibody ligands into high affinity probes. An additional advantage is that the antibody ligands occupy only a small number of the reactive sites on the dextran, so that molecular cargo can be attached easily, creating molecules capable of delivering this cargo to cells displaying antigen-specific receptors. PMID:25073654

Directed evolution of PDZ variants to generate high-affinity detection reagents.

PubMed

Ferrer, Marc; Maiolo, Jim; Kratz, Patricia; Jackowski, Jessica L; Murphy, Dennis J; Delagrave, Simon; Inglese, James

2005-04-01

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.

Reliable Determinations of Protein-Ligand Interactions by Direct ESI-MS Measurements. Are We There Yet?

NASA Astrophysics Data System (ADS)

Kitova, Elena N.; El-Hawiet, Amr; Schnier, Paul D.; Klassen, John S.

2012-03-01

The association-dissociation of noncovalent interactions between protein and ligands, such as other proteins, carbohydrates, lipids, DNA, or small molecules, are critical events in many biological processes. The discovery and characterization of these interactions is essential to a complete understanding of biochemical reactions and pathways and to the design of novel therapeutic agents that may be used to treat a variety of diseases and infections. Over the last 20 y, electrospray ionization mass spectrometry (ESI-MS) has emerged as a versatile tool for the identification and quantification of protein-ligand interactions in vitro. Here, we describe the implementation of the direct ESI-MS assay for the determination of protein-ligand binding stoichiometry and affinity. Additionally, we outline common sources of error encountered with these measurements and various strategies to overcome them. Finally, we comment on some of the outstanding challenges associated with the implementation of the assay and highlight new areas where direct ESI-MS measurements are expected to make significant contributions in the future.

Affinity Electrophoresis Using Ligands Attached To Polymers

NASA Technical Reports Server (NTRS)

Van Alstine, James M.; Snyder, Robert S.; Harris, J. M.; Brooks, D. E.

1990-01-01

In new technique, reduction of electrophoretic mobilities by addition of polyethylene glycol to ligands increases electrophoretic separabilities. In immuno-affinity electrophoresis, modification of ligands extends specificity of electrophoretic separation to particles having surface electric-charge structures otherwise making them electrophoretically inseparable. Modification of antibodies by polyethylene glycol greatly reduces ability to aggregate while enhancing ability to affect electrophoretic mobilities of cells. In hydrophobic-affinity electrophoresis, addition of polyethylene glycol reduces tendency toward aggregation of cells or macromolecules.

Importance of ligand reorganization free energy in protein-ligand binding-affinity prediction.

PubMed

Yang, Chao-Yie; Sun, Haiying; Chen, Jianyong; Nikolovska-Coleska, Zaneta; Wang, Shaomeng

2009-09-30

Accurate prediction of the binding affinities of small-molecule ligands to their biological targets is fundamental for structure-based drug design but remains a very challenging task. In this paper, we have performed computational studies to predict the binding models of 31 small-molecule Smac (the second mitochondria-derived activator of caspase) mimetics to their target, the XIAP (X-linked inhibitor of apoptosis) protein, and their binding affinities. Our results showed that computational docking was able to reliably predict the binding models, as confirmed by experimentally determined crystal structures of some Smac mimetics complexed with XIAP. However, all the computational methods we have tested, including an empirical scoring function, two knowledge-based scoring functions, and MM-GBSA (molecular mechanics and generalized Born surface area), yield poor to modest prediction for binding affinities. The linear correlation coefficient (r(2)) value between the predicted affinities and the experimentally determined affinities was found to be between 0.21 and 0.36. Inclusion of ensemble protein-ligand conformations obtained from molecular dynamic simulations did not significantly improve the prediction. However, major improvement was achieved when the free-energy change for ligands between their free- and bound-states, or "ligand-reorganization free energy", was included in the MM-GBSA calculation, and the r(2) value increased from 0.36 to 0.66. The prediction was validated using 10 additional Smac mimetics designed and evaluated by an independent group. This study demonstrates that ligand reorganization free energy plays an important role in the overall binding free energy between Smac mimetics and XIAP. This term should be evaluated for other ligand-protein systems and included in the development of new scoring functions. To our best knowledge, this is the first computational study to demonstrate the importance of ligand reorganization free energy for the prediction of protein-ligand binding free energy.

Controlled method of reducing electrophoretic mobility of macromolecules, particles, or cells

NASA Technical Reports Server (NTRS)

Vanalstine, James M. (Inventor)

1992-01-01

A method of reducing electrophoretic mobility of macromolecules, particles, cells, and other substances is provided which comprises interacting in a conventional electrophoretic separating procedure, the substances with a polymer-linked affinity compound comprised of a hydrophilic neutral polymer such as polyethylene glycol bound to a second component such as a hydrophobic compound, an immunocompound such as an antibody or antibody active fragment, or a ligand such as a hormone, drug, antigen, or a hapten. The reduction of electrophoretic mobility achieved is directly proportional to the concentration of the polymer-linked affinity compound employed, and such reduction can comprise up to 100 percent for particular particles and cells. The present invention is advantageous in that electrophoretic separation can now be achieved for substances whose native surface charge structure had prevented them from being separated by normal electrophoretic means. Depending on the affinity component utilized, separation can be achieved on the basis of the specific/irreversible, specific/reversible, semi-specific/reversible, relatively nonspecific/reversible, or relatively nonspecific/irreversible ligand-substance interactions.

Direct detection of ligand binding to Sepharose-immobilised protein using saturation transfer double difference (STDD) NMR spectroscopy

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

Haselhorst, Thomas; Muenster-Kuehnel, Anja K.; Oschlies, Melanie

2007-08-10

We report an easy and direct application of 'Saturation Transfer Double Difference' (STDD) NMR spectroscopy to identify ligands that bind to a Sepharose-immobilised target protein. The model protein, cytidine 5'-monophosphate sialic acid (CMP-Sia) synthetase, was expressed as a Strep-Tag II fusion protein and immobilised on Strep-Tactin Sepharose. STD NMR experiments of the protein-enriched Sepharose matrix in the presence of a binding ligand (cytidine 5'-triphosphate, CTP) and a non-binding ligand ({alpha}/{beta}-glucose) clearly show that CTP binds to the immobilised enzyme, whereas glucose has no affinity. This approach has three major advantages: (a) only low quantities of protein are required, (b) nomore » specialised NMR technology or the application of additional data analysis by non-routine methods is required, and (c) easy multiple use of the immobilised protein is available.« less

Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations

NASA Astrophysics Data System (ADS)

Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

2016-09-01

We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

Binding-affinity predictions of HSP90 in the D3R Grand Challenge 2015 with docking, MM/GBSA, QM/MM, and free-energy simulations.

PubMed

Misini Ignjatović, Majda; Caldararu, Octav; Dong, Geng; Muñoz-Gutierrez, Camila; Adasme-Carreño, Francisco; Ryde, Ulf

2016-09-01

We have estimated the binding affinity of three sets of ligands of the heat-shock protein 90 in the D3R grand challenge blind test competition. We have employed four different methods, based on five different crystal structures: first, we docked the ligands to the proteins with induced-fit docking with the Glide software and calculated binding affinities with three energy functions. Second, the docked structures were minimised in a continuum solvent and binding affinities were calculated with the MM/GBSA method (molecular mechanics combined with generalised Born and solvent-accessible surface area solvation). Third, the docked structures were re-optimised by combined quantum mechanics and molecular mechanics (QM/MM) calculations. Then, interaction energies were calculated with quantum mechanical calculations employing 970-1160 atoms in a continuum solvent, combined with energy corrections for dispersion, zero-point energy and entropy, ligand distortion, ligand solvation, and an increase of the basis set to quadruple-zeta quality. Fourth, relative binding affinities were estimated by free-energy simulations, using the multi-state Bennett acceptance-ratio approach. Unfortunately, the results were varying and rather poor, with only one calculation giving a correlation to the experimental affinities larger than 0.7, and with no consistent difference in the quality of the predictions from the various methods. For one set of ligands, the results could be strongly improved (after experimental data were revealed) if it was recognised that one of the ligands displaced one or two water molecules. For the other two sets, the problem is probably that the ligands bind in different modes than in the crystal structures employed or that the conformation of the ligand-binding site or the whole protein changes.

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

PubMed

Sun, Ying-Chieh; Hsu, Wen-Chi; Hsu, Chia-Jen; Chang, Chia-Ming; Cheng, Kai-Hsiang

2015-11-01

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

DNA Mismatch Binding and Antiproliferative Activity of Rhodium Metalloinsertors

PubMed Central

Ernst, Russell J.; Song, Hang; Barton, Jacqueline K.

2009-01-01

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 104 to 108 M−1 for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo. PMID:19175313

EGF receptor ligands: recent advances.

PubMed

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

Dye-ligand affinity systems.

PubMed

Denizli, A; Pişkin, E

2001-10-30

Dye-ligands have been considered as one of the important alternatives to natural counterparts for specific affinity chromatography. Dye-ligands are able to bind most types of proteins, in some cases in a remarkably specific manner. They are commercially available, inexpensive, and can easily be immobilized, especially on matrices bearing hydroxyl groups. Although dyes are all synthetic in nature, they are still classified as affinity ligands because they interact with the active sites of many proteins mimicking the structure of the substrates, cofactors, or binding agents for those proteins. A number of textile dyes, known as reactive dyes, have been used for protein purification. Most of these reactive dyes consist of a chromophore (either azo dyes, anthraquinone, or phathalocyanine), linked to a reactive group (often a mono- or dichlorotriazine ring). The interaction between the dye ligand and proteins can be by complex combination of electrostatic, hydrophobic, hydrogen bonding. Selection of the supporting matrix is the first important consideration in dye-affinity systems. There are several methods for immobilization of dye molecules onto the support matrix, in which usually several intermediate steps are followed. Both the adsorption and elution steps should carefully be optimized/designed for a successful separation. Dye-affinity systems in the form of spherical sorbents or as affinity membranes have been used in protein separation.

Targeting Anti-Cancer Active Compounds: Affinity-Based Chromatographic Assays

PubMed Central

de Moraes, Marcela Cristina; Cardoso, Carmen Lucia; Seidl, Claudia; Moaddel, Ruin; Cass, Quezia Bezerra

2016-01-01

Affinity-based chromatography assays encompass the use of solid supports containing immobilized biological targets to monitor binding events in the isolation , identification and/or characterization of bioactive compounds. This powerful bioanalytical technique allows the screening of potential binders through fast analyses that can be directly performed using isolated substances or complex matrices. An overview of the recent researches in frontal and zonal affinity-based chromatography screening assays, which has been used as a tool in the identification and characterization of new anti-cancer agents, is discussed. In addition, a critical evaluation of the recently emerged ligands fishing assays in complex mixtures is also discussed. PMID:27306095

A general and fast scoring function for protein-ligand interactions: a simplified potential approach.

PubMed

Muegge, I; Martin, Y C

1999-03-11

A fast, simplified potential-based approach is presented that estimates the protein-ligand binding affinity based on the given 3D structure of a protein-ligand complex. This general, knowledge-based approach exploits structural information of known protein-ligand complexes extracted from the Brookhaven Protein Data Bank and converts it into distance-dependent Helmholtz free interaction energies of protein-ligand atom pairs (potentials of mean force, PMF). The definition of an appropriate reference state and the introduction of a correction term accounting for the volume taken by the ligand were found to be crucial for deriving the relevant interaction potentials that treat solvation and entropic contributions implicitly. A significant correlation between experimental binding affinities and computed score was found for sets of diverse protein-ligand complexes and for sets of different ligands bound to the same target. For 77 protein-ligand complexes taken from the Brookhaven Protein Data Bank, the calculated score showed a standard deviation from observed binding affinities of 1.8 log Ki units and an R2 value of 0.61. The best results were obtained for the subset of 16 serine protease complexes with a standard deviation of 1.0 log Ki unit and an R2 value of 0.86. A set of 33 inhibitors modeled into a crystal structure of HIV-1 protease yielded a standard deviation of 0.8 log Ki units from measured inhibition constants and an R2 value of 0.74. In contrast to empirical scoring functions that show similar or sometimes better correlation with observed binding affinities, our method does not involve deriving specific parameters that fit the observed binding affinities of protein-ligand complexes of a given training set. We compared the performance of the PMF score, Böhm's score (LUDI), and the SMOG score for eight different test sets of protein-ligand complexes. It was found that for the majority of test sets the PMF score performs best. The strength of the new approach presented here lies in its generality as no knowledge about measured binding affinities is needed to derive atomic interaction potentials. The use of the new scoring function in docking studies is outlined.

PLASS: Protein-ligand affinity statistical score a knowledge-based force-field model of interaction derived from the PDB

NASA Astrophysics Data System (ADS)

Ozrin, V. D.; Subbotin, M. V.; Nikitin, S. M.

2004-04-01

We have developed PLASS (Protein-Ligand Affinity Statistical Score), a pair-wise potential of mean-force for rapid estimation of the binding affinity of a ligand molecule to a protein active site. This scoring function is derived from the frequency of occurrence of atom-type pairs in crystallographic complexes taken from the Protein Data Bank (PDB). Statistical distributions are converted into distance-dependent contributions to the Gibbs free interaction energy for 10 atomic types using the Boltzmann hypothesis, with only one adjustable parameter. For a representative set of 72 protein-ligand structures, PLASS scores correlate well with the experimentally measured dissociation constants: a correlation coefficient R of 0.82 and RMS error of 2.0 kcal/mol. Such high accuracy results from our novel treatment of the volume correction term, which takes into account the inhomogeneous properties of the protein-ligand complexes. PLASS is able to rank reliably the affinity of complexes which have as much diversity as in the PDB.

Multitarget-directed tricyclic pyridazinones as G protein-coupled receptor ligands and cholinesterase inhibitors.

PubMed

Pau, Amedeo; Catto, Marco; Pinna, Giovanni; Frau, Simona; Murineddu, Gabriele; Asproni, Battistina; Curzu, Maria M; Pisani, Leonardo; Leonetti, Francesco; Loza, Maria Isabel; Brea, José; Pinna, Gérard A; Carotti, Angelo

2015-06-01

By following a multitarget ligand design approach, a library of 47 compounds was prepared, and they were tested as binders of selected G protein-coupled receptors (GPCRs) and inhibitors of acetyl and/or butyryl cholinesterase. The newly designed ligands feature pyridazinone-based tricyclic scaffolds connected through alkyl chains of variable length to proper amine moieties (e.g., substituted piperazines or piperidines) for GPCR and cholinesterase (ChE) molecular recognition. The compounds were tested at three different GPCRs, namely serotoninergic 5-HT1A, adrenergic α1A, and dopaminergic D2 receptors. Our main goal was the discovery of compounds that exhibit, in addition to ChE inhibition, antagonist activity at 5-HT1A because of its involvement in neuronal deficits typical of Alzheimer's and other neurodegenerative diseases. Ligands with nanomolar affinity for the tested GPCRs were discovered, but most of them behaved as dual antagonists of α1A and 5-HT1A receptors. Nevertheless, several compounds displaying this GPCR affinity profile also showed moderate to good inhibition of AChE and BChE, thus deserving further investigations to exploit the therapeutic potential of such unusual biological profiles. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Native Mass Spectrometry in Fragment-Based Drug Discovery.

PubMed

Pedro, Liliana; Quinn, Ronald J

2016-07-28

The advent of native mass spectrometry (MS) in 1990 led to the development of new mass spectrometry instrumentation and methodologies for the analysis of noncovalent protein-ligand complexes. Native MS has matured to become a fast, simple, highly sensitive and automatable technique with well-established utility for fragment-based drug discovery (FBDD). Native MS has the capability to directly detect weak ligand binding to proteins, to determine stoichiometry, relative or absolute binding affinities and specificities. Native MS can be used to delineate ligand-binding sites, to elucidate mechanisms of cooperativity and to study the thermodynamics of binding. This review highlights key attributes of native MS for FBDD campaigns.

Fluorescent-responsive synthetic C1b domains of protein kinase Cδ as reporters of specific high-affinity ligand binding.

PubMed

Ohashi, Nami; Nomura, Wataru; Narumi, Tetsuo; Lewin, Nancy E; Itotani, Kyoko; Blumberg, Peter M; Tamamura, Hirokazu

2011-01-19

Protein kinase C (PKC) is a critical cell signaling pathway involved in many disorders such as cancer and Alzheimer-type dementia. To date, evaluation of PKC ligand binding affinity has been performed by competitive studies against radiolabeled probes that are problematic for high-throughput screening. In the present study, we have developed a fluorescent-based binding assay system for identifying ligands that target the PKC ligand binding domain (C1 domain). An environmentally sensitive fluorescent dye (solvatochromic fluorophore), which has been used in multiple applications to assess protein-binding interactions, was inserted in proximity to the binding pocket of a novel PKCδ C1b domain. These resultant fluorescent-labeled δC1b domain analogues underwent a significant change in fluorescent intensity upon ligand binding, and we further demonstrate that the fluorescent δC1b domain analogues can be used to evaluate ligand binding affinity.

Small potent ligands to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor.

PubMed

Axén, Andreas; Andersson, Hanna; Lindeberg, Gunnar; Rönnholm, Harriet; Kortesmaa, Jarkko; Demaegdt, Heidi; Vauquelin, Georges; Karlén, Anders; Hallberg, Mathias

2007-07-01

Angiotensin IV analogs encompassing aromatic scaffolds replacing parts of the backbone of angiotensin IV have been synthesized and evaluated in biological assays. Several of the ligands displayed high affinities to the insulin-regulated aminopeptidase (IRAP)/AT(4) receptor. Displacement of the C-terminal of angiotensin IV with an o-substituted aryl acetic acid derivative delivered the ligand 4, which exhibited the highest binding affinity (K(i) = 1.9 nM). The high affinity of this ligand provides support to the hypothesis that angiotensin IV adopts a gamma-turn in the C-terminal of its bioactive conformation. Ligand (4) inhibits both human IRAP and aminopeptidase N-activity and induces proliferation of adult neural stem cells at low concentrations. Furthermore, ligand 4 is degraded considerably more slowly in membrane preparations than angiotensin IV. Hence, it might constitute a suitable research tool for biological studies of the (IRAP)/AT(4) receptor.

Structural basis for the ligand-binding specificity of fatty acid-binding proteins (pFABP4 and pFABP5) in gentoo penguin.

PubMed

Lee, Chang Woo; Kim, Jung Eun; Do, Hackwon; Kim, Ryeo-Ok; Lee, Sung Gu; Park, Hyun Ho; Chang, Jeong Ho; Yim, Joung Han; Park, Hyun; Kim, Il-Chan; Lee, Jun Hyuck

2015-09-11

Fatty acid-binding proteins (FABPs) are involved in transporting hydrophobic fatty acids between various aqueous compartments of the cell by directly binding ligands inside their β-barrel cavities. Here, we report the crystal structures of ligand-unbound pFABP4, linoleate-bound pFABP4, and palmitate-bound pFABP5, obtained from gentoo penguin (Pygoscelis papua), at a resolution of 2.1 Å, 2.2 Å, and 2.3 Å, respectively. The pFABP4 and pFABP5 proteins have a canonical β-barrel structure with two short α-helices that form a cap region and fatty acid ligand binding sites in the hydrophobic cavity within the β-barrel structure. Linoleate-bound pFABP4 and palmitate-bound pFABP5 possess different ligand-binding modes and a unique ligand-binding pocket due to several sequence dissimilarities (A76/L78, T30/M32, underlining indicates pFABP4 residues) between the two proteins. Structural comparison revealed significantly different conformational changes in the β3-β4 loop region (residues 57-62) as well as the flipped Phe60 residue of pFABP5 than that in pFABP4 (the corresponding residue is Phe58). A ligand-binding study using fluorophore displacement assays shows that pFABP4 has a relatively strong affinity for linoleate as compared to pFABP5. In contrast, pFABP5 exhibits higher affinity for palmitate than that for pFABP4. In conclusion, our high-resolution structures and ligand-binding studies provide useful insights into the ligand-binding preferences of pFABPs based on key protein-ligand interactions. Copyright © 2015 Elsevier Inc. All rights reserved.

Optimized hydrophobic interactions and hydrogen bonding at the target-ligand interface leads the pathways of drug-designing.

PubMed

Patil, Rohan; Das, Suranjana; Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K

2010-08-16

Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy.

Optimized Hydrophobic Interactions and Hydrogen Bonding at the Target-Ligand Interface Leads the Pathways of Drug-Designing

PubMed Central

Stanley, Ashley; Yadav, Lumbani; Sudhakar, Akulapalli; Varma, Ashok K.

2010-01-01

Background Weak intermolecular interactions such as hydrogen bonding and hydrophobic interactions are key players in stabilizing energetically-favored ligands, in an open conformational environment of protein structures. However, it is still poorly understood how the binding parameters associated with these interactions facilitate a drug-lead to recognize a specific target and improve drugs efficacy. To understand this, comprehensive analysis of hydrophobic interactions, hydrogen bonding and binding affinity have been analyzed at the interface of c-Src and c-Abl kinases and 4-amino substituted 1H-pyrazolo [3, 4-d] pyrimidine compounds. Methodology In-silico docking studies were performed, using Discovery Studio software modules LigandFit, CDOCKER and ZDOCK, to investigate the role of ligand binding affinity at the hydrophobic pocket of c-Src and c-Abl kinase. Hydrophobic and hydrogen bonding interactions of docked molecules were compared using LigPlot program. Furthermore, 3D-QSAR and MFA calculations were scrutinized to quantify the role of weak interactions in binding affinity and drug efficacy. Conclusions The in-silico method has enabled us to reveal that a multi-targeted small molecule binds with low affinity to its respective targets. But its binding affinity can be altered by integrating the conformationally favored functional groups at the active site of the ligand-target interface. Docking studies of 4-amino-substituted molecules at the bioactive cascade of the c-Src and c-Abl have concluded that 3D structural folding at the protein-ligand groove is also a hallmark for molecular recognition of multi-targeted compounds and for predicting their biological activity. The results presented here demonstrate that hydrogen bonding and optimized hydrophobic interactions both stabilize the ligands at the target site, and help alter binding affinity and drug efficacy. PMID:20808434

Influence of various force fields in estimating the binding affinity of acetylcholinesterase inhibitors using fast pulling of ligand scheme

NASA Astrophysics Data System (ADS)

Tam, Nguyen Minh; Vu, Khanh B.; Vu, Van V.; Ngo, Son Tung

2018-06-01

Acetylcholinesterase (AChE) is considered as one of the most favored drug targets for Alzheimer's disease. The effects of different force fields (FFs) on ranking affinity of acetylcholinesterase inhibitors were obtained using the fast pulling of ligand (FPL) method in steered-molecular dynamics (SMD) simulations. GROMOS, AMBER, CHARMM, and OPLS-AA FFs were investigated in this work. The pulling work derived with GROMOS FF has the strongest correlation and smallest error compared with experimental binding affinity. Moreover, the CPU consumption in the calculations using GROMOS FF is the lowest, which could allow us to rank affinity of a large number of AChE ligands.

Effects of N-Substitutions on the Tetrahydroquinoline (THQ) Core of Mixed-Efficacy μ-Opioid Receptor (MOR)/δ-Opioid Receptor (DOR) Ligands.

PubMed

Harland, Aubrie A; Bender, Aaron M; Griggs, Nicholas W; Gao, Chao; Anand, Jessica P; Pogozheva, Irina D; Traynor, John R; Jutkiewicz, Emily M; Mosberg, Henry I

2016-05-26

N-Acetylation of the tetrahydroquinoline (THQ) core of a series of μ-opioid receptor (MOR) agonist/δ-opioid receptor (DOR) antagonist ligands increases DOR affinity, resulting in ligands with balanced MOR and DOR affinities. We report a series of N-substituted THQ analogues that incorporate various carbonyl-containing moieties to maintain DOR affinity and define the steric and electronic requirements of the binding pocket across the opioid receptors. 4h produced in vivo antinociception (ip) for 1 h at 10 mg/kg.

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

Fedynyshyn, J.P.

The opioid binding characteristics of the rat (PAG) and the signal transduction mechanisms of the opioid receptors were examined with in vitro radioligand binding, GTPase, adenylyl cyclase, and inositol phosphate assays. The nonselective ligand {sup 3}H-ethylketocyclazocine (EKC), the {mu} and {delta} selective ligand {sup 3}H-(D-Ala{sup 2}, D-Leu{sup 5}) enkephalin (DADLE), the {mu} selective ligand {sup 3}H-(D-Ala{sup 2}, N-methyl Phe{sup 4}, Glyol{sup 5}) enkephalin (DAGO), and the {delta} selective ligand {sup 3}H-(D-Pen{sup 2}, D-Pen{sup 5}) enkephalin (DPDPE) were separately used as tracer ligands to label opioid binding sites in rat PAG enriched P{sub 2} membrane in competition with unlabeled DADLE, DAGO,more » DPDPE, or the {kappa} selective ligand trans-3,4-dichloro-N-(2-(1-pyrrolidinyl)cyclohexyl)benzeneacetamide, methane sulfonate, hydrate (U50, 488H). Only {mu} selective high affinity opioid binding was observed. No high affinity {delta} or {kappa} selective binding was detected. {sup 3}H-DAGO was used as a tracer ligand to label {mu} selective high affinity opioid binding sites in PAG enriched P{sub 2} membrane in competition with unlabeled {beta}-endorphin, dynorphin A (1-17), BAM-18, methionine enkephalin, dynorphin A (1-8), and leucine enkephalin. Of these endogenous opioid peptides only those with previously reported high affinity {mu} type opioid binding activity competed with {sup 3}H-DAGO for binding sites in rat PAG enriched P{sub 2} membrane with affinities similar to that of unlabeled DAGO.« less

EGF receptor ligands: recent advances

PubMed Central

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

Determination of the affinity of drugs toward serum albumin by measurement of the quenching of the intrinsic tryptophan fluorescence of the protein.

PubMed

Epps, D E; Raub, T J; Caiolfa, V; Chiari, A; Zamai, M

1999-01-01

Binding of new chemical entities to serum proteins is an issue confronting pharmaceutical companies during development of potential therapeutic agents. Most drugs bind to the most abundant plasma protein, human serum albumin (HSA), at two major binding sites. Excepting fluorescence spectroscopy, existing methods for assaying drug binding to serum albumin are insensitive to higher-affinity compounds and can be labour-intensive, time-consuming, and usually require compound-specific assays. This led us to examine alternative ways to measure drug-albumin interaction. One method described here uses fluorescence quenching of the single tryptophan (Trp) residue in HSA excited at 295 nm to measure drug-binding affinity. Unfortunately, many compounds absorb, fluoresce, or both, in this UV wavelength region of the spectrum. Several types of binding phenomenon and spectral interference were identified by use of six structurally unrelated compounds and the equations necessary to make corrections mathematically were derived and applied to calculate binding constants accurately. The general cases were: direct quenching of Trp fluorescence by optically transparent ligands with low or high affinities; binding of optically transparent, non-fluorescent ligands to two specific sites where both sites or only one site result in Trp fluorescence quenching; and chromophores whose absorption either overlaps the Trp emission and quenches by energy transfer or absorbs light at the Trp fluorescence excitation wavelength producing absorptive screening as well as fluorescence quenching. Unless identification of the site specificity of drug binding to serum albumin is desired, quenching of the Trp fluorescence of albumin by titration with ligand is a rapid and facile method for determining the binding affinities of drugs for serum albumin.

Targeting protein-protein interactions with trimeric ligands: high affinity inhibitors of the MAGUK protein family.

PubMed

Nissen, Klaus B; Haugaard-Kedström, Linda M; Wilbek, Theis S; Nielsen, Line S; Åberg, Emma; Kristensen, Anders S; Bach, Anders; Jemth, Per; Strømgaard, Kristian

2015-01-01

PDZ domains in general, and those of PSD-95 in particular, are emerging as promising drug targets for diseases such as ischemic stroke. We have previously shown that dimeric ligands that simultaneously target PDZ1 and PDZ2 of PSD-95 are highly potent inhibitors of PSD-95. However, PSD-95 and the related MAGUK proteins contain three consecutive PDZ domains, hence we envisioned that targeting all three PDZ domains simultaneously would lead to more potent and potentially more specific interactions with the MAGUK proteins. Here we describe the design, synthesis and characterization of a series of trimeric ligands targeting all three PDZ domains of PSD-95 and the related MAGUK proteins, PSD-93, SAP-97 and SAP-102. Using our dimeric ligands targeting the PDZ1-2 tandem as starting point, we designed novel trimeric ligands by introducing a PDZ3-binding peptide moiety via a cysteine-derivatized NPEG linker. The trimeric ligands generally displayed increased affinities compared to the dimeric ligands in fluorescence polarization binding experiments and optimized trimeric ligands showed low nanomolar inhibition towards the four MAGUK proteins, thus being the most potent inhibitors described. Kinetic experiments using stopped-flow spectrometry showed that the increase in affinity is caused by a decrease in the dissociation rate of the trimeric ligand as compared to the dimeric ligands, likely reflecting the lower probability of simultaneous dissociation of all three PDZ ligands. Thus, we have provided novel inhibitors of the MAGUK proteins with exceptionally high affinity, which can be used to further elucidate the therapeutic potential of these proteins.

Characterization of Gly-D-Phe, Gly-L-Leu, and D-Phe as affinity ligands to thermolysin.

PubMed

Yasukawa, Kiyoshi; Kusano, Masayuki; Nakamura, Koji; Inouye, Kuniyo

2006-04-01

In this study, glycyl-D-phenylalanine (Gly-D-Phe), glycyl-L-leucine (Gly-L-Leu), and D-phenylalanine (D-Phe) were characterized for their abilities as affinity ligands to thermolysin. Each of the ligands was immobilized to the resin. The optimum pH for adsorption of thermolysin is 5.0-6.0 for each of the ligands. By the affinity column chromatography in which 2mg thermolysin was applied onto 4 ml volume of the resins at pH 5.5, the adsorption ratios based on casein hydrolysis activity were 100% for each of the ligands. However, the adsorption ratios of the resins containing Gly-L-Leu and D-Phe, unlike that of Gly-D-Phe, were progressively decreased with increasing the amounts of thermolysin applied to the column. Measurement of adsorption isotherms showed that the association constant to thermolysin at pH 5.5 of the resins containing Gly-D-Phe was (3.3+/-0.8)x10(5)M(-1), while those of Gly-L-Leu and D-Phe were approximately ten times less. This result is coincident with the observations of performances in affinity column chromatography. On the other hand, maximum thermolysin binding capacities were almost the same among the resins examined. These results indicate that Gly-D-Phe is more suitable than Gly-L-Leu and D-Phe as an affinity ligand for purification of thermolysin.

Evaluation of protein-ligand affinity prediction using steered molecular dynamics simulations.

PubMed

Okimoto, Noriaki; Suenaga, Atsushi; Taiji, Makoto

2017-11-01

In computational drug design, ranking a series of compound analogs in a manner that is consistent with experimental affinities remains a challenge. In this study, we evaluated the prediction of protein-ligand binding affinities using steered molecular dynamics simulations. First, we investigated the appropriate conditions for accurate predictions in these simulations. A conic harmonic restraint was applied to the system for efficient sampling of work values on the ligand unbinding pathway. We found that pulling velocity significantly influenced affinity predictions, but that the number of collectable trajectories was less influential. We identified the appropriate pulling velocity and collectable trajectories for binding affinity predictions as 1.25 Å/ns and 100, respectively, and these parameters were used to evaluate three target proteins (FK506 binding protein, trypsin, and cyclin-dependent kinase 2). For these proteins using our parameters, the accuracy of affinity prediction was higher and more stable when Jarzynski's equality was employed compared with the second-order cumulant expansion equation of Jarzynski's equality. Our results showed that steered molecular dynamics simulations are effective for predicting the rank order of ligands; thus, they are a potential tool for compound selection in hit-to-lead and lead optimization processes.

Ligand Binding Analysis and Screening by Chemical Denaturation Shift

PubMed Central

Sch n, Arne; Brown, Richard K.; Hutchins, Burleigh M.; Freire, Ernesto

2013-01-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Towards this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Since ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities and the ligand rank order obtained at denaturation temperatures (60°C or higher) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations in which binding changes the cooperativity of the unfolding transition. In this paper we develop the basic analytical equations and provide several experimental examples. PMID:23994566

Ligand binding analysis and screening by chemical denaturation shift.

PubMed

Schön, Arne; Brown, Richard K; Hutchins, Burleigh M; Freire, Ernesto

2013-12-01

The identification of small molecule ligands is an important first step in drug development, especially drugs that target proteins with no intrinsic activity. Toward this goal, it is important to have access to technologies that are able to measure binding affinities for a large number of potential ligands in a fast and accurate way. Because ligand binding stabilizes the protein structure in a manner dependent on concentration and binding affinity, the magnitude of the protein stabilization effect elicited by binding can be used to identify and characterize ligands. For example, the shift in protein denaturation temperature (Tm shift) has become a popular approach to identify potential ligands. However, Tm shifts cannot be readily transformed into binding affinities, and the ligand rank order obtained at denaturation temperatures (≥60°C) does not necessarily coincide with the rank order at physiological temperature. An alternative approach is the use of chemical denaturation, which can be implemented at any temperature. Chemical denaturation shifts allow accurate determination of binding affinities with a surprisingly wide dynamic range (high micromolar to sub nanomolar) and in situations where binding changes the cooperativity of the unfolding transition. In this article, we develop the basic analytical equations and provide several experimental examples. Copyright © 2013 Elsevier Inc. All rights reserved.

Ligand affinity of the 67-kD elastin/laminin binding protein is modulated by the protein's lectin domain: visualization of elastin/laminin-receptor complexes with gold-tagged ligands

PubMed Central

1991-01-01

Video-enhanced microscopy was used to examine the interaction of elastin- or laminin-coated gold particles with elastin binding proteins on the surface of live cells. By visualizing the binding events in real time, it was possible to determine the specificity and avidity of ligand binding as well as to analyze the motion of the receptor-ligand complex in the plane of the plasma membrane. Although it was difficult to interpret the rates of binding and release rigorously because of the possibility for multiple interactions between particles and the cell surface, relative changes in binding have revealed important aspects of the regulation of affinity of ligand-receptor interaction in situ. Both elastin and laminin were found to compete for binding to the cell surface and lactose dramatically decreased the affinity of the receptor(s) for both elastin and laminin. These findings were supported by in vitro studies of the detergent-solubilized receptor. Further, immobilization of the ligand-receptor complexes through binding to the cytoskeleton dramatically decreased the ability of bound particles to leave the receptor. The changes in the kinetics of ligand-coated gold binding to living cells suggest that both laminin and elastin binding is inhibited by lactose and that attachment of receptor to the cytoskeleton increases its affinity for the ligand. PMID:1848864

Targeting autocrine HB-EGF signaling with specific ADAM12 inhibition using recombinant ADAM12 prodomain

NASA Astrophysics Data System (ADS)

Miller, Miles A.; Moss, Marcia L.; Powell, Gary; Petrovich, Robert; Edwards, Lori; Meyer, Aaron S.; Griffith, Linda G.; Lauffenburger, Douglas A.

2015-10-01

Dysregulation of ErbB-family signaling underlies numerous pathologies and has been therapeutically targeted through inhibiting ErbB-receptors themselves or their cognate ligands. For the latter, “decoy” antibodies have been developed to sequester ligands including heparin-binding epidermal growth factor (HB-EGF); however, demonstrating sufficient efficacy has been difficult. Here, we hypothesized that this strategy depends on properties such as ligand-receptor binding affinity, which varies widely across the known ErbB-family ligands. Guided by computational modeling, we found that high-affinity ligands such as HB-EGF are more difficult to target with decoy antibodies compared to low-affinity ligands such as amphiregulin (AREG). To address this issue, we developed an alternative method for inhibiting HB-EGF activity by targeting its cleavage from the cell surface. In a model of the invasive disease endometriosis, we identified A Disintegrin and Metalloproteinase 12 (ADAM12) as a protease implicated in HB-EGF shedding. We designed a specific inhibitor of ADAM12 based on its recombinant prodomain (PA12), which selectively inhibits ADAM12 but not ADAM10 or ADAM17. In endometriotic cells, PA12 significantly reduced HB-EGF shedding and resultant cellular migration. Overall, specific inhibition of ligand shedding represents a possible alternative to decoy antibodies, especially for ligands such as HB-EGF that exhibit high binding affinity and localized signaling.

Synthesis of 3-alkyl naphthalenes as novel estrogen receptor ligands

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

Fang, Jing; Akwabi-Ameyaw, Adwoa; Britton, Jonathan E.

2009-06-24

A series of estrogen receptor ligands based on a 3-alkyl naphthalene scaffold was synthesized using an intramolecular enolate-alkyne cycloaromatization as the key step. Several of these compounds bearing a C6-OH group were shown to be high affinity ligands. All compounds had similar ER{alpha} and ER{beta} binding affinity ranging from micromolar to low nanomolar.

High-throughput screening of dye-ligands for chromatography.

PubMed

Kumar, Sunil; Punekar, Narayan S

2014-01-01

Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

The effect of ligand affinity on integrins' lateral diffusion in cultured cells.

PubMed

Mainali, Dipak; Smith, Emily A

2013-04-01

The role of ligand affinity in altering αPS2CβPS integrins' lateral mobility was studied using single particle tracking (SPT) with ligand-functionalized quantum dots (QDs) and fluorescence recovery after photobleaching (FRAP) with fluorescent protein tagged integrins. Integrins are ubiquitous transmembrane proteins that are vital for numerous cellular functions, including bidirectional signaling and cell anchorage. Wild-type and high ligand affinity mutant (αPS2CβPS-V409D) integrins were studied in S2 cells. As measured by SPT, the integrin mobile fraction decreased by 22% and had a 4× slower diffusion coefficient for αPS2CβPS-V409D compared to wild-type integrins. These differences are partially the result of αPS2CβPS-V409D integrins' increased clustering. For the wild-type integrins, the average of all diffusion coefficients measured by SPT was statistically similar to the ensemble FRAP results. A 75% slower average diffusion coefficient was measured by SPT compared to FRAP for αPS2CβPS-V409D integrins, and this may be the result of SPT measuring only ligand-bound integrins, in contrast all ligand-bound and ligand-unbound integrins are averaged in FRAP measurements. Specific binding of the ligand-functionalized QDs was 99% for integrin expressing cells. The results prove that the ligand binding affinity affects the lateral dynamics of a subset of integrins based on the complementary SPT and FRAP data.

High-affinity PD-1 molecules deliver improved interaction with PD-L1 and PD-L2.

PubMed

Li, Yanyan; Liang, Zhaoduan; Tian, Ye; Cai, Wenxuan; Weng, Zhiming; Chen, Lin; Zhang, Huanling; Bao, Yifeng; Zheng, Hongjun; Zeng, Sihai; Bei, Chunhua; Li, Yi

2018-06-11

The inhibitory checkpoint molecule programmed death (PD)-1 plays a vital role in maintaining immune homeostasis upon binding to its ligands, PD-L1 and PD-L2. Several recent studies have demonstrated that soluble PD-1 (sPD-1) can block the interaction between membrane PD-1 and PD-L1 to enhance the anti-tumor capability of T cells. However, the affinity of natural sPD-1 binding to PD-L1 is too low to permit therapeutic applications. Here a PD-1 variant with ~3,000-fold and ~70-fold affinity increase to bind PD-L1 and PD-L2, respectively, was generated through directed molecular evolution and phage display technology. Structural analysis showed that mutations at amino acid positions 124 and 132 of PD-1 played major roles in enhancing the affinity of PD-1 binding to its ligands. The high-affinity PD-1 mutant could compete with the binding of antibodies specific to PD-L1 or PD-L2 on cancer cells or dendritic cells (DCs), and it could enhance the proliferation and IFN-γ release of activated lymphocytes. These features potentially qualify the high-affinity PD-1 variant as a unique candidate for the development of a new class of PD-1 immune checkpoint blockade therapeutics. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

Design and Synthesis of High Affinity Inhibitors of Plasmodium falciparum and Plasmodium vivax N-Myristoyltransferases Directed by Ligand Efficiency Dependent Lipophilicity (LELP)

PubMed Central

2014-01-01

N-Myristoyltransferase (NMT) is an essential eukaryotic enzyme and an attractive drug target in parasitic infections such as malaria. We have previously reported that 2-(3-(piperidin-4-yloxy)benzo[b]thiophen-2-yl)-5-((1,3,5-trimethyl-1H-pyrazol-4-yl)methyl)-1,3,4-oxadiazole (34c) is a high affinity inhibitor of both Plasmodium falciparum and P. vivax NMT and displays activity in vivo against a rodent malaria model. Here we describe the discovery of 34c through optimization of a previously described series. Development, guided by targeting a ligand efficiency dependent lipophilicity (LELP) score of less than 10, yielded a 100-fold increase in enzyme affinity and a 100-fold drop in lipophilicity with the addition of only two heavy atoms. 34c was found to be equipotent on chloroquine-sensitive and -resistant cell lines and on both blood and liver stage forms of the parasite. These data further validate NMT as an exciting drug target in malaria and support 34c as an attractive tool for further optimization. PMID:24641010

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

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

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

Design and applications of a clamp for Green Fluorescent Protein with picomolar affinity

DOE PAGES

Hansen, Simon; Stüber, Jakob C.; Ernst, Patrick; ...

2017-11-24

Green fluorescent protein (GFP) fusions are pervasively used to study structures and processes. Specific GFP-binders are thus of great utility for detection, immobilization or manipulation of GFP-fused molecules. We determined structures of two designed ankyrin repeat proteins (DARPins), complexed with GFP, which revealed different but overlapping epitopes. Here in this paper we show a structure-guided design strategy that, by truncation and computational reengineering, led to a stable construct where both can bind simultaneously: by linkage of the two binders, fusion constructs were obtained that “wrap around” GFP, have very high affinities of about 10–30 pM, and extremely slow off-rates. Theymore » can be natively produced in E. coli in very large amounts, and show excellent biophysical properties. Their very high stability and affinity, facile site-directed functionalization at introduced unique lysines or cysteines facilitate many applications. As examples, we present them as tight yet reversible immobilization reagents for surface plasmon resonance, as fluorescently labelled monomeric detection reagents in flow cytometry, as pull-down ligands to selectively enrich GFP fusion proteins from cell extracts, and as affinity column ligands for inexpensive large-scale protein purification. We have thus described a general design strategy to create a “clamp” from two different high-affinity repeat proteins, even if their epitopes overlap.« less

In vitro selection of functional nucleic acids

NASA Technical Reports Server (NTRS)

Wilson, D. S.; Szostak, J. W.

1999-01-01

In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

Biomolecular conformational changes and ligand binding: from kinetics to thermodynamics† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc01627a

PubMed Central

Wang, Yong; Martins, João Miguel

2017-01-01

The behaviour of biomolecular systems is governed by their thermodynamic and kinetic properties. It is thus important to be able to calculate, for example, both the affinity and rate of binding and dissociation of a protein–ligand complex, or the populations and exchange rates between distinct conformational states. Because these are typically rare events, calculating these properties from long molecular dynamics simulations remains extremely difficult. Instead, one often adopts a divide-and-conquer strategy in which equilibrium free-energy differences and the fastest state-to-state transition (e.g. ligand association or minor-to-major state conversion) are combined to estimate the slow rate (e.g. ligand dissociation) using a two-state assumption. Here we instead address these problems by using a previously developed method to calculate both the forward and backward rates directly from simulations. We then estimate the thermodynamics from the rates, and validate these values by independent means. We applied the approach to three systems of increasing complexity, including the association and dissociation of benzene to a fully buried cavity inside the L99A mutant variant of T4 lysozyme. In particular, we were able to determine both millisecond association and dissociation rates, and the affinity, of the protein–ligand system by directly observing dozens of rare events in atomic detail. Our approach both sheds light on the precision of methods for calculating kinetics and further provides a generally useful test for the internal consistency of kinetics and thermodynamics. We also expect our route to be useful for obtaining both the kinetics and thermodynamics at the same time in more challenging cases. PMID:29619200

Hooking horseradish peroxidase by using the affinity Langmuir-Blodgett technique for an oriented immobilization

NASA Astrophysics Data System (ADS)

Peng, Ye; Ling-Ling, Hu; Yu-Zhi, Du; Yong-Juan, Xu; Hua-Gang, Ni; Cong, Chen; Xiao-Lin, Lu; Xiao-Jun, Huang

2017-05-01

A novel method of oriented immobilization was presented: affinity Langmuir-Blodgett (LB) technique. Firstly, a long carbon chain was bond to a ligand of Horseradish Peroxidase (HRP). The ligand derivative appears surface activity with the hydrophobic carbon chain oriented to air and the hydrophilic ligand faced to water. Then, this derivative was put onto the water/air surface to assemble a LB film and formed the affinity interaction with the active site of HRP. After that, the affinity LB film with the enzyme was transferred onto the support to obtain the oriented immobilized HRP. The specific activity of HRP immobilized by affinity LB (182.1 ± 14 U/mg) was higher than that by adsorption (40.5 ± 5 U/mg). HRP immobilized by affinity LB could maintain a more native conformation, compared to that by adsorption. This method could be effectively used to immobilize protein with orientation and show widely promising applications in many fields including biosensor and bioreactor.

The Free Energy Landscape of Small Molecule Unbinding

PubMed Central

Huang, Danzhi; Caflisch, Amedeo

2011-01-01

The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM. The conformations of the FKBP/ligand complex saved along multiple trajectories (50 runs at 310 K for each ligand) are grouped according to a set of intermolecular distances into nodes of a network, and the direct transitions between them are the links. The network analysis reveals that the bound state consists of several subbasins, i.e., binding modes characterized by distinct intermolecular hydrogen bonds and hydrophobic contacts. The dissociation kinetics show a simple (i.e., single-exponential) time dependence because the unbinding barrier is much higher than the barriers between subbasins in the bound state. The unbinding transition state is made up of heterogeneous positions and orientations of the ligand in the FKBP active site, which correspond to multiple pathways of dissociation. For the six small ligands of FKBP, the weaker the binding affinity the closer to the bound state (along the intermolecular distance) are the transition state structures, which is a new manifestation of Hammond behavior. Experimental approaches to the study of fragment binding to proteins have limitations in temporal and spatial resolution. Our network analysis of the unbinding simulations of small inhibitors from an enzyme paints a clear picture of the free energy landscape (both thermodynamics and kinetics) of ligand unbinding. PMID:21390201

The free energy landscape of small molecule unbinding.

PubMed

Huang, Danzhi; Caflisch, Amedeo

2011-02-01

The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM. The conformations of the FKBP/ligand complex saved along multiple trajectories (50 runs at 310 K for each ligand) are grouped according to a set of intermolecular distances into nodes of a network, and the direct transitions between them are the links. The network analysis reveals that the bound state consists of several subbasins, i.e., binding modes characterized by distinct intermolecular hydrogen bonds and hydrophobic contacts. The dissociation kinetics show a simple (i.e., single-exponential) time dependence because the unbinding barrier is much higher than the barriers between subbasins in the bound state. The unbinding transition state is made up of heterogeneous positions and orientations of the ligand in the FKBP active site, which correspond to multiple pathways of dissociation. For the six small ligands of FKBP, the weaker the binding affinity the closer to the bound state (along the intermolecular distance) are the transition state structures, which is a new manifestation of Hammond behavior. Experimental approaches to the study of fragment binding to proteins have limitations in temporal and spatial resolution. Our network analysis of the unbinding simulations of small inhibitors from an enzyme paints a clear picture of the free energy landscape (both thermodynamics and kinetics) of ligand unbinding.

Structure-based Understanding of Binding Affinity and Mode ...

EPA Pesticide Factsheets

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interactions and specific hydrogen bonds with the ligand. Here we present a framework for quantitative analysis of the steric and electronic features of the human ERα-ligand complex using three dimensional (3D) protein-ligand interaction description combined with 3D-QSAR approach. An empirical hydrophobicity density field is applied to account for hydrophobic contacts of ligand within the LBP. The obtained 3D-QSAR model revealed that hydrophobic contacts primarily determine binding affinity and govern binding mode with hydrogen bonds. Several residues of the LBP appear to be quite flexible and adopt a spectrum of conformations in various ERα-ligand complexes, in particular His524. The 3D-QSAR was combined with molecular docking based on three receptor conformations to accommodate receptor flexibility. The model indicates that the dynamic character of the LBP allows accommodation and stable binding of structurally diverse ligands, and proper representation of the protein flexibility is critical for reasonable description of binding of the ligands. Our results provide a quantitative and mechanistic understanding of binding affinity and mode of ERα agonists and antagonists that may be applicab

Integration of element specific persistent homology and machine learning for protein-ligand binding affinity prediction.

PubMed

Cang, Zixuan; Wei, Guo-Wei

2018-02-01

Protein-ligand binding is a fundamental biological process that is paramount to many other biological processes, such as signal transduction, metabolic pathways, enzyme construction, cell secretion, and gene expression. Accurate prediction of protein-ligand binding affinities is vital to rational drug design and the understanding of protein-ligand binding and binding induced function. Existing binding affinity prediction methods are inundated with geometric detail and involve excessively high dimensions, which undermines their predictive power for massive binding data. Topology provides the ultimate level of abstraction and thus incurs too much reduction in geometric information. Persistent homology embeds geometric information into topological invariants and bridges the gap between complex geometry and abstract topology. However, it oversimplifies biological information. This work introduces element specific persistent homology (ESPH) or multicomponent persistent homology to retain crucial biological information during topological simplification. The combination of ESPH and machine learning gives rise to a powerful paradigm for macromolecular analysis. Tests on 2 large data sets indicate that the proposed topology-based machine-learning paradigm outperforms other existing methods in protein-ligand binding affinity predictions. ESPH reveals protein-ligand binding mechanism that can not be attained from other conventional techniques. The present approach reveals that protein-ligand hydrophobic interactions are extended to 40Å  away from the binding site, which has a significant ramification to drug and protein design. Copyright © 2017 John Wiley & Sons, Ltd.

Divergence between human and murine peroxisome proliferator-activated receptor alpha ligand specificities[S

PubMed Central

Oswal, Dhawal P.; Balanarasimha, Madhumitha; Loyer, Jeannette K.; Bedi, Shimpi; Soman, Frances L.; Rider, S. Dean; Hostetler, Heather A.

2013-01-01

Peroxisome proliferator-activated receptor α (PPARα) belongs to the family of ligand-dependent nuclear transcription factors that regulate energy metabolism. Although there exists remarkable overlap in the activities of PPARα across species, studies utilizing exogenous PPARα ligands suggest species differences in binding, activation, and physiological effects. While unsaturated long-chain fatty acids (LCFA) and their thioesters (long-chain fatty acyl-CoA; LCFA-CoA) function as ligands for recombinant mouse PPARα (mPPARα), no such studies have been conducted with full-length human PPARα (hPPARα). The objective of the current study was to determine whether LCFA and LCFA-CoA constitute high-affinity endogenous ligands for hPPARα or whether there exist species differences for ligand specificity and affinity. Both hPPARα and mPPARα bound with high affinity to LCFA-CoA; however, differences were noted in LCFA affinities. A fluorescent LCFA analog was bound strongly only by mPPARα, and naturally occurring saturated LCFA was bound more strongly by hPPARα than mPPARα. Similarly, unsaturated LCFA induced transactivation of both hPPARα and mPPARα, whereas saturated LCFA induced transactivation only in hPPARα-expressing cells. These data identified LCFA and LCFA-CoA as endogenous ligands of hPPARα, demonstrated species differences in binding specificity and activity, and may help delineate the role of PPARα as a nutrient sensor in metabolic regulation. PMID:23797899

Alternative Affinity Ligands for Immunoglobulins.

PubMed

Kruljec, Nika; Bratkovič, Tomaž

2017-08-16

The demand for recombinant therapeutic antibodies and Fc-fusion proteins is expected to increase in the years to come. Hence, extensive efforts are concentrated on improving the downstream processing. In particular, the development of better-affinity chromatography matrices, supporting robust time- and cost-effective antibody purification, is warranted. With the advances in molecular design and high-throughput screening approaches from chemical and biological combinatorial libraries, novel affinity ligands representing alternatives to bacterial immunoglobulin (Ig)-binding proteins have entered the scene. Here, we review the design, development, and properties of diverse classes of alternative antibody-binding ligands, ranging from engineered versions of Ig-binding proteins, to artificial binding proteins, peptides, aptamers, and synthetic small-molecular-weight compounds. We also provide examples of applications for the novel affinity matrices in chromatography and beyond.

Molecular view of ligands specificity for CAG repeats in anti-Huntington therapy.

PubMed

Bochicchio, Anna; Rossetti, Giulia; Tabarrini, Oriana; Krauβ, Sybille; Carloni, Paolo

2015-10-13

Huntington's disease is a fatal and devastating neurodegenerative genetic disorder for which there is currently no cure. It is characterized by Huntingtin protein's mRNA transcripts with 36 or more CAG repeats. Inhibiting the formation of pathological complexes between these expanded transcripts and target proteins may be a valuable strategy against the disease. Yet, the rational design of molecules specifically targeting the expanded CAG repeats is limited by the lack of structural information. Here, we use well-tempered metadynamics-based free energy calculations to investigate pose and affinity of two ligands targeting CAG repeats for which affinities have been previously measured. The first consists of two 4-guanidinophenyl rings linked by an ester group. It is the most potent ligand identified so far, with Kd = 60(30) nM. The second consists of a 4-phenyl dihydroimidazole and 4-1H-indole dihydroimidazole connected by a C-C bond (Kd = 700(80) nM). Our calculations reproduce the experimental affinities and uncover the recognition pattern between ligands' and their RNA target. They also provide a molecular basis for the markedly different affinity of the two ligands for CAG repeats as observed experimentally. These findings may pave the way for a structure-based hit-to-lead optimization to further improve ligand selectivity toward CAG repeat-containing mRNAs.

Direct comparison of linear and macrocyclic compound libraries as a source of protein ligands.

PubMed

Gao, Yu; Kodadek, Thomas

2015-03-09

There has been much discussion of the potential desirability of macrocyclic molecules for the development of tool compounds and drug leads. But there is little experimental data comparing otherwise equivalent macrocyclic and linear compound libraries as a source of protein ligands. In this Letter, we probe this point in the context of peptoid libraries. Bead-displayed libraries of macrocyclic and linear peptoids containing four variable positions and 0-2 fixed residues, to vary the ring size, were screened against streptavidin and the affinity of every hit for the target was measured. The data show that macrocyclization is advantageous, but only when the ring contains 17 atoms, not 20 or 23 atoms. This technology will be useful for conducting direct comparisons between many different types of chemical libraries to determine their relative utility as a source of protein ligands.

Performance of HADDOCK and a simple contact-based protein-ligand binding affinity predictor in the D3R Grand Challenge 2

NASA Astrophysics Data System (ADS)

Kurkcuoglu, Zeynep; Koukos, Panagiotis I.; Citro, Nevia; Trellet, Mikael E.; Rodrigues, J. P. G. L. M.; Moreira, Irina S.; Roel-Touris, Jorge; Melquiond, Adrien S. J.; Geng, Cunliang; Schaarschmidt, Jörg; Xue, Li C.; Vangone, Anna; Bonvin, A. M. J. J.

2018-01-01

We present the performance of HADDOCK, our information-driven docking software, in the second edition of the D3R Grand Challenge. In this blind experiment, participants were requested to predict the structures and binding affinities of complexes between the Farnesoid X nuclear receptor and 102 different ligands. The models obtained in Stage1 with HADDOCK and ligand-specific protocol show an average ligand RMSD of 5.1 Å from the crystal structure. Only 6/35 targets were within 2.5 Å RMSD from the reference, which prompted us to investigate the limiting factors and revise our protocol for Stage2. The choice of the receptor conformation appeared to have the strongest influence on the results. Our Stage2 models were of higher quality (13 out of 35 were within 2.5 Å), with an average RMSD of 4.1 Å. The docking protocol was applied to all 102 ligands to generate poses for binding affinity prediction. We developed a modified version of our contact-based binding affinity predictor PRODIGY, using the number of interatomic contacts classified by their type and the intermolecular electrostatic energy. This simple structure-based binding affinity predictor shows a Kendall's Tau correlation of 0.37 in ranking the ligands (7th best out of 77 methods, 5th/25 groups). Those results were obtained from the average prediction over the top10 poses, irrespective of their similarity/correctness, underscoring the robustness of our simple predictor. This results in an enrichment factor of 2.5 compared to a random predictor for ranking ligands within the top 25%, making it a promising approach to identify lead compounds in virtual screening.

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice

PubMed Central

Matsubara, Naoko; Imamura, Akihiro; Yonemizu, Tatsuya; Akatsu, Chizuru; Yang, Hongrui; Ueki, Akiharu; Watanabe, Natsuki; Abdu-Allah, Hajjaj; Numoto, Nobutaka; Takematsu, Hiromu; Kitazume, Shinobu; Tedder, Thomas F.; Marth, Jamey D.; Ito, Nobutoshi; Ando, Hiromune; Ishida, Hideharu; Kiso, Makoto; Tsubata, Takeshi

2018-01-01

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation. PMID:29725338

CD22-Binding Synthetic Sialosides Regulate B Lymphocyte Proliferation Through CD22 Ligand-Dependent and Independent Pathways, and Enhance Antibody Production in Mice.

PubMed

Matsubara, Naoko; Imamura, Akihiro; Yonemizu, Tatsuya; Akatsu, Chizuru; Yang, Hongrui; Ueki, Akiharu; Watanabe, Natsuki; Abdu-Allah, Hajjaj; Numoto, Nobutaka; Takematsu, Hiromu; Kitazume, Shinobu; Tedder, Thomas F; Marth, Jamey D; Ito, Nobutoshi; Ando, Hiromune; Ishida, Hideharu; Kiso, Makoto; Tsubata, Takeshi

2018-01-01

Sialic acid-binding immunoglobulin-like lectins (Siglecs) are expressed in various immune cells and most of them carry signaling functions. High-affinity synthetic sialoside ligands have been developed for various Siglecs. Therapeutic potentials of the nanoparticles and compounds that contain multiple numbers of these sialosides and other reagents such as toxins and antigens have been demonstrated. However, whether immune responses can be regulated by monomeric sialoside ligands has not yet been known. CD22 (also known as Siglec-2) is an inhibitory molecule preferentially expressed in B lymphocytes (B cells) and is constitutively bound and functionally regulated by α2,6 sialic acids expressed on the same cell (cis-ligands). Here, we developed synthetic sialosides GSC718 and GSC839 that bind to CD22 with high affinity (IC 50 ~100 nM), and inhibit ligand binding of CD22. When B cells are activated by B cell antigen receptor (BCR) ligation, both GSC718 and GSC839 downregulate proliferation of B cells, and this regulation requires both CD22 and α2,6 sialic acids. This result suggests that these sialosides regulate BCR ligation-induced B cell activation by reversing endogenous ligand-mediated regulation of CD22. By contrast, GSC718 and GSC839 augment B cell proliferation induced by TLR ligands or CD40 ligation, and this augmentation requires CD22 but not α2,6 sialic acids. Thus, these sialosides appear to enhance B cell activation by directly suppressing the inhibitory function of CD22 independently of endogenous ligand-mediated regulation. Moreover, GSC839 augments B cell proliferation that depends on both BCR ligation and CD40 ligation as is the case for in vivo B cell responses to antigens, and enhanced antibody production to the extent comparable to CpG oligonuleotides or a small amount of alum. Although these known adjuvants induce production of the inflammatory cytokines or accumulation of inflammatory cells, CD22-binding sialosides do not. Thus, synthetic sialosides that bind to CD22 with high-affinity modulate B cell activation through endogenous ligand-dependent and independent pathways, and carry an adjuvant activity without inducing inflammation.

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

Investigation of metal ligand affinities of atom transfer radical polymerization catalysts with a quadrupole ion trap.

PubMed

di Lena, Fabio; Matyjaszewski, Krzysztof

2009-11-07

An electrospray ionization mass spectrometer equipped with a quadrupole ion trap as the mass analyzer provided a powerful tool for the investigation of metal ligand affinities of catalysts for atom transfer radical polymerization. It allowed, in particular, (i) the identification, in a library of ligands, of the most stable, and thus active, copper catalysts; (ii) the assessment of the effects of the reaction medium on the relative stabilities of the catalyst complexes; and (iii) the evaluation of the influence of the nature of the ligand on both the complex halogenophilicity and the metal-ligand stabilities in the gas-phase.

Aptamer-based downstream processing of his-tagged proteins utilizing magnetic beads.

PubMed

Kökpinar, Öznur; Walter, Johanna-Gabriela; Shoham, Yuval; Stahl, Frank; Scheper, Thomas

2011-10-01

Aptamers are synthetic nucleic acid-based high affinity ligands that are able to capture their corresponding target via molecular recognition. Here, aptamer-based affinity purification for His-tagged proteins was developed. Two different aptamers directed against the His-tag were immobilized on magnetic beads covalently. The resulting aptamer-modified magnetic beads were characterized and successfully applied for purification of different His-tagged proteins from complex E. coli cell lysates. Purification effects comparable to conventional immobilized metal affinity chromatography were achieved in one single purification step. Moreover, we have investigated the possibility to regenerate and reuse the aptamer-modified magnetic beads and have shown their long-term stability over a period of 6 months. Copyright © 2011 Wiley Periodicals, Inc.

Group Additivity in Ligand Binding Affinity: An Alternative Approach to Ligand Efficiency.

PubMed

Reynolds, Charles H; Reynolds, Ryan C

2017-12-26

Group additivity is a concept that has been successfully applied to a variety of thermochemical and kinetic properties. This includes drug discovery, where functional group additivity is often assumed in ligand binding. Ligand efficiency can be recast as a special case of group additivity where ΔG/HA is the group equivalent (HA is the number of non-hydrogen atoms in a ligand). Analysis of a large data set of protein-ligand binding affinities (K i ) for diverse targets shows that in general ligand binding is distinctly nonlinear. It is possible to create a group equivalent scheme for ligand binding, but only in the context of closely related proteins, at least with regard to size. This finding has broad implications for drug design from both experimental and computational points of view. It also offers a path forward for a more general scheme to assess the efficiency of ligand binding.

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

DOE PAGES

Gao, Zhiwen; Gao, Yanfei

2016-05-14

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Beyond Helper Phage: Using "Helper Cells" to Select Peptide Affinity Ligands.

PubMed

Phipps, M Lisa; Lillo, Antoinetta M; Shou, Yulin; Schmidt, Emily N; Paavola, Chad D; Naranjo, Leslie; Bemdich, Sara; Swanson, Basil I; Bradbury, Andrew R M; Martinez, Jennifer S

2016-01-01

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The "helper cell" packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report on the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.

Why do receptor–ligand bonds in cell adhesion cluster into discrete focal-adhesion sites?

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

Gao, Zhiwen; Gao, Yanfei

We report that cell adhesion often exhibits the clustering of the receptor–ligand bonds into discrete focal-adhesion sites near the contact edge, thus resembling a rosette shape or a contracting membrane anchored by a small number of peripheral forces. The ligands on the extracellular matrix are immobile, and the receptors in the cell plasma membrane consist of two types: high-affinity integrins (that bond to the substrate ligands and are immobile) and low-affinity integrins (that are mobile and not bonded to the ligands). Thus the adhesion energy density is proportional to the high-affinity integrin density. This paper provides a mechanistic explanation formore » the clustering/assembling of the receptor–ligand bonds from two main points: (1) the cellular contractile force leads to the density evolution of these two types of integrins, and results into a large high-affinity integrin density near the contact edge and (2) the front of a propagating crack into a decreasing toughness field will be unstable and wavy. From this fracture mechanics perspective, the chemomechanical equilibrium is reached when a small number of patches with large receptor–ligand bond density are anticipated to form at the cell periphery, as opposed to a uniform distribution of bonds on the entire interface. Finally, cohesive fracture simulations show that the de-adhesion force can be significantly enhanced by this nonuniform bond density field, but the de-adhesion force anisotropy due to the substrate elastic anisotropy is significantly reduced.« less

Convergence of Domain Architecture, Structure, and Ligand Affinity in Animal and Plant RNA-Binding Proteins.

PubMed

Dias, Raquel; Manny, Austin; Kolaczkowski, Oralia; Kolaczkowski, Bryan

2017-06-01

Reconstruction of ancestral protein sequences using phylogenetic methods is a powerful technique for directly examining the evolution of molecular function. Although ancestral sequence reconstruction (ASR) is itself very efficient, downstream functional, and structural studies necessary to characterize when and how changes in molecular function occurred are often costly and time-consuming, currently limiting ASR studies to examining a relatively small number of discrete functional shifts. As a result, we have very little direct information about how molecular function evolves across large protein families. Here we develop an approach combining ASR with structure and function prediction to efficiently examine the evolution of ligand affinity across a large family of double-stranded RNA binding proteins (DRBs) spanning animals and plants. We find that the characteristic domain architecture of DRBs-consisting of 2-3 tandem double-stranded RNA binding motifs (dsrms)-arose independently in early animal and plant lineages. The affinity with which individual dsrms bind double-stranded RNA appears to have increased and decreased often across both animal and plant phylogenies, primarily through convergent structural mechanisms involving RNA-contact residues within the β1-β2 loop and a small region of α2. These studies provide some of the first direct information about how protein function evolves across large gene families and suggest that changes in molecular function may occur often and unassociated with major phylogenetic events, such as gene or domain duplications. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

HIGH-AFFINITY T CELL RECEPTOR DIFFERENTIATES COGNATE PEPTIDE-MHC AND ALTERED PEPTIDE LIGANDS WITH DISTINCT KINETICS AND THERMODYNAMICS

PubMed Central

Persaud, Stephen P.; Donermeyer, David L.; Weber, K. Scott; Kranz, David M.; Allen, Paul M.

2010-01-01

Interactions between the T cell receptor and cognate peptide-MHC are crucial initiating events in the adaptive immune response. These binding events are highly specific yet occur with micromolar affinity. Even weaker interactions between TCR and self-pMHC complexes play critical regulatory roles in T cell development, maintenance and coagonist activity. Due to their low affinity, the kinetics and thermodynamics of such weak interactions are difficult to study. In this work, we used M15, a high-affinity TCR engineered from the 3.L2 TCR system, to study the binding properties, thermodynamics, and specificity of two altered peptide ligands (APLs). Our affinity measurements of the high-affinity TCR support the view that the wild type TCR binds these APLs in the millimolar affinity range, and hence very low affinities can still elicit biological functions. Finally, single methylene differences among the APLs gave rise to strikingly different binding thermodynamics. These minor changes in the pMHC antigen were associated with significant and unpredictable changes in both the entropy and enthalpy of the reaction. As the identical TCR was analyzed with several structurally similar ligands, the distinct thermodynamic binding profiles provide a mechanistic perspective on how exquisite antigen specificity is achieved by the T cell receptor. PMID:20334923

Barley as a green factory for the production of functional Flt3 ligand.

PubMed

Erlendsson, Lýdur S; Muench, Marcus O; Hellman, Ulf; Hrafnkelsdóttir, Soffía M; Jonsson, Anders; Balmer, Yves; Mäntylä, Einar; Orvar, Björn L

2010-02-01

Biologically active recombinant human Flt3 ligand was expressed and isolated from transgenic barley seeds. Its expression is controlled by a tissue specific promoter that confines accumulation of the recombinant protein to the endosperm tissue of the seed. The recombinant Flt3 ligand variant expressed in the seeds contains an HQ-tag for affinity purification on immobilized metal ion affinity chromatography (IMAC) resin. The tagged protein was purified from seed extracts to near homogeneity using sequential chromatography on IMAC affinity resin and cation exchange resin. We also show that the recombinant Flt3 ligand protein undergoes posttranslational modifications: it is a glycoprotein containing alpha-1,3-fucose and alpha-1,2-xylose. The HQ-tagged Flt3 ligand variant exhibits comparable biological activity to commercial Flt3 ligand. This is the first report showing expression and accumulation of recombinant human growth factor in barley seeds with a yield of active protein similar to a bacterial expression system. The present results demonstrate that plant molecular farming is a viable approach for the bioproduction of human-derived growth factors.

Boosting Affinity by Correct Ligand Preorganization for the S2 Pocket of Thrombin: A Study by Isothermal Titration Calorimetry, Molecular Dynamics, and High-Resolution Crystal Structures.

PubMed

Rühmann, Eggert H; Rupp, Melinda; Betz, Michael; Heine, Andreas; Klebe, Gerhard

2016-02-04

Structural preorganization to fix bioactive conformations at protein binding sites is a popular strategy to enhance binding affinity during late-stage optimization. The rationale for this enhancement relates to entropic advantages assigned to rigidified versus flexible ligands. We analyzed a narrow series of peptidomimetics binding to thrombin. The individual ligands exhibit at P2 a conformationally flexible glycine, more restricted alanine, N-methylglycine, N-methylhomoalanine, and largely rigidified proline moiety. Overall, affinity was found to increase by a factor of 1000, explained partly by an entropic advantage. All ligands adopt the same binding mode with small deviations. The residual mobility of the bound ligands is decreased across the series, and a protein side chain differs in its order/disorder behavior along with changes in the surface-water network pattern established across the newly generated protein-ligand surfaces. The enthalpy/entropy inventory displays a rather complex picture and emphasizes that thermodynamics can only be compared in terms of relative differences within a structurally similar ligand series. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Characterization of opiates, neuroleptics, and synthetic analogs at ORL1 and opioid receptors

PubMed Central

Zaveri, Nurulain; Polgar, Willma E.; Olsen, Cris M.; Kelson, Andrew B.; Grundt, Peter; Lewis, John W.; Toll, Lawrence

2013-01-01

Nociceptin/orphanin FQ (N/OFQ) was recently identified as the endogenous ligand for the opioid-receptor like (ORL1) receptor. Although the ORL1 receptor shows sequence homology with the opioid receptors, the nociceptin/ORL1 ligand–receptor system has very distinct pharmacological actions compared to the opioid receptor system. Recently, several small-molecule ORL1 receptor ligands were reported by pharmaceutical companies. Most of these ligands had close structural similarities with known neuroleptics and opiates. In this study, we screened several available neuroleptics and opiates for their binding affinity and functional activity at ORL1 and the opioid receptors. We also synthesized several analogs of known opiates with modified piperidine N-substituents in order to characterize the ORL1 receptor ligand binding pocket. Substitution with the large, lipophilic cyclooctylmethyl moiety increased ORL1 receptor affinity and decreased μ receptor affinity and efficacy in the fentanyl series of ligands but had a different effect in the oripavine class of opiate ligands. Our results indicate that opiates and neuroleptics may be good starting points for ORL1 receptor ligand design, and the selectivity may be modulated by appropriate structural modifications. PMID:11779034

The major birch allergen, Bet v 1, shows affinity for a broad spectrum of physiological ligands.

PubMed

Mogensen, Jesper E; Wimmer, Reinhard; Larsen, Jørgen N; Spangfort, Michael D; Otzen, Daniel E

2002-06-28

Bet v 1 is a 17-kDa protein abundantly present in the pollen of the White birch tree and is the primary cause of birch pollen allergy in humans. Its three-dimensional structure is remarkable in that a solvent-accessible cavity traverses the core of the molecule. The biological function of Bet v 1 is unknown, although it is homologous to a family of pathogenesis-related proteins in plants. In this study we first show that Bet v 1 in the native state is able to bind the fluorescent probe 8-anilino-1-naphthalenesulfonic acid (ANS). ANS binds to Bet v 1 with 1:1 stoichiometry, and NMR data indicate that binding takes place in the cavity. Using an ANS displacement assay, we then identify a range of physiologically relevant ligands, including fatty acids, flavonoids, and cytokinins, which generally bind with low micromolar affinity. The ability of these ligands to displace ANS suggests that they also bind in the cavity, although the exact binding sites seem to vary among different ligands. The cytokinins, for example, seem to bind at a separate site close to ANS, because they increase the fluorescence of the ANS. Bet v 1 complex. Also, the fluorescent sterol dehydroergosterol binds to Bet v 1 as demonstrated by direct titrations. This study provides the first qualitative and quantitative data on the ligand binding properties of this important pollen allergen. Our findings indicate that ligand binding is important for the biological function of Bet v 1.

Molecular Recognition of 6′-N-5-Hexynoate Kanamyin A and RNA 1×1 Internal Loops Containing CA Mismatches

PubMed Central

Tran, Tuan; Disney, Matthew D.

2011-01-01

In our previous study to identify the RNA internal loops that bind an aminoglycoside derivative, we determined that 6′-N-5-hexynoate kanamycin A prefers to bind 1×1 nucleotide internal loops containing C•A mismatches. In this present study, the molecular recognition between a variety of RNAs that are mutated around the C•A loop and the ligand was investigated. Studies show that both loop nucleotides and loop closing pairs affect binding affinity. Most interestingly, it was shown that there is a correlation between the thermodynamic stability of the C•A internal loops and ligand affinity. Specifically, C•A loops that had relatively high or low stability bound the ligand most weakly whereas loops with intermediate stability bound the ligand most tightly. In contrast, there is no correlation between the likelihood that a loop forms a C-A+ pair at lower pH and ligand affinity. It was also found that a 1×1 nucleotide C•A loop that bound to the ligand with the highest affinity is identical to the consensus site in RNAs that are edited by adenosine deaminases acting on RNA type 2 (ADAR2). These studies provide a detailed investigation of factors affecting small molecule recognition of internal loops containing C•A mismatches, which are present in a variety of RNAs that cause disease. PMID:21207945

Tryptophan tags and de novo designed complementary affinity ligands for the expression and purification of recombinant proteins.

PubMed

Pina, Ana Sofia; Carvalho, Sara; Dias, Ana Margarida G C; Guilherme, Márcia; Pereira, Alice S; Caraça, Luciana T; Coroadinha, Ana Sofia; Lowe, Christopher R; Roque, A Cecília A

2016-11-11

A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml -1 and 0.48mgml -1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 10 6 M -1 affinity constants and Qmax values of 19.11±2.60ugg -1 and 79.39ugg -1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents. Copyright © 2016 Elsevier B.V. All rights reserved.

Predicting Displaceable Water Sites Using Mixed-Solvent Molecular Dynamics.

PubMed

Graham, Sarah E; Smith, Richard D; Carlson, Heather A

2018-02-26

Water molecules are an important factor in protein-ligand binding. Upon binding of a ligand with a protein's surface, waters can either be displaced by the ligand or may be conserved and possibly bridge interactions between the protein and ligand. Depending on the specific interactions made by the ligand, displacing waters can yield a gain in binding affinity. The extent to which binding affinity may increase is difficult to predict, as the favorable displacement of a water molecule is dependent on the site-specific interactions made by the water and the potential ligand. Several methods have been developed to predict the location of water sites on a protein's surface, but the majority of methods are not able to take into account both protein dynamics and the interactions made by specific functional groups. Mixed-solvent molecular dynamics (MixMD) is a cosolvent simulation technique that explicitly accounts for the interaction of both water and small molecule probes with a protein's surface, allowing for their direct competition. This method has previously been shown to identify both active and allosteric sites on a protein's surface. Using a test set of eight systems, we have developed a method using MixMD to identify conserved and displaceable water sites. Conserved sites can be determined by an occupancy-based metric to identify sites which are consistently occupied by water even in the presence of probe molecules. Conversely, displaceable water sites can be found by considering the sites which preferentially bind probe molecules. Furthermore, the inclusion of six probe types allows the MixMD method to predict which functional groups are capable of displacing which water sites. The MixMD method consistently identifies sites which are likely to be nondisplaceable and predicts the favorable displacement of water sites that are known to be displaced upon ligand binding.

Residues W320 and Y328 within the binding site of the μ-opioid receptor influence opiate ligand bias.

PubMed

Hothersall, J Daniel; Torella, Rubben; Humphreys, Sian; Hooley, Monique; Brown, Alastair; McMurray, Gordon; Nickolls, Sarah A

2017-05-15

The development of G protein-biased agonists for the μ-opioid receptor (MOR) offers a clear drug discovery rationale for improved analgesia and reduced side-effects of opiate pharmacotherapy. However, our understanding of the molecular mechanisms governing ligand bias is limited, which hinders our ability to rationally design biased compounds. We have investigated the role of MOR binding site residues W320 and Y328 in controlling bias, by receptor mutagenesis. The pharmacology of a panel of ligands in a cAMP and a β-arrestin2 assay were compared between the wildtype and mutated receptors, with bias factors calculated by operational analysis using ΔΔlog(τ/K A ) values. [ 3 H]diprenorphine competition binding was used to estimate affinity changes. Introducing the mutations W320A and Y328F caused changes in pathway bias, with different patterns of change between ligands. For example, DAMGO increased relative β-arrestin2 activity at the W320A mutant, whilst its β-arrestin2 response was completely lost at Y328F. In contrast, endomorphin-1 gained activity with Y328F but lost activity at W320A, in both pathways. For endomorphin-2 there was a directional shift from cAMP bias at the wildtype towards more β-arrestin2 bias at W320A. We also observe clear uncoupling between mutation-driven changes in function and binding affinity. These findings suggest that the mutations influenced the balance of pathway activation in a ligand-specific manner, thus identifying residues in the MOR binding pocket that govern ligand bias. This increases our understanding of how ligand/receptor binding interactions can be translated into agonist-specific pathway activation. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Discovery of novel Tetrahydrobenzo[b]thiophene and pyrrole based scaffolds as potent and selective CB2 receptor ligands: The structural elements controlling binding affinity, selectivity and functionality.

PubMed

Osman, Noha A; Ligresti, Alessia; Klein, Christian D; Allarà, Marco; Rabbito, Alessandro; Di Marzo, Vincenzo; Abouzid, Khaled A; Abadi, Ashraf H

2016-10-21

CB2-based therapeutics show strong potential in the treatment of diverse diseases such as inflammation, multiple sclerosis, pain, immune-related disorders, osteoporosis and cancer, without eliciting the typical neurobehavioral side effects of CB1 ligands. For this reason, research activities are currently directed towards the development of CB2 selective ligands. Herein, the synthesis of novel heterocyclic-based CB2 selective compounds is reported. A set of 2,5-dialkyl-1-phenyl-1H-pyrrole-3-carboxamides, 5-subtituted-2-(acylamino)/(2-sulphonylamino)-thiophene-3-carboxylates and 2-(acylamino)/(2-sulphonylamino)-tetrahydrobenzo[b]thiophene-3-carboxylates were synthesized. Biological results revealed compounds with remarkably high CB2 binding affinity and CB2/CB1 subtype selectivity. Compound 19a and 19b from the pyrrole series exhibited the highest CB2 receptor affinity (Ki = 7.59 and 6.15 nM, respectively), as well as the highest CB2/CB1 subtype selectivity (∼70 and ∼200-fold, respectively). In addition, compound 6b from the tetrahydrobenzo[b]thiophene series presented the most potent and selective CB2 ligand in this series (Ki = 2.15 nM and CB2 subtype selectivity of almost 500-fold over CB1). Compound 6b showed a full agonism, while compounds 19a and 19b acted as inverse agonists when tested in an adenylate cyclase assay. The present findings thus pave the way to the design and optimization of heterocyclic-based scaffolds with lipophilic carboxamide and/or retroamide substituent that can be exploited as potential CB2 receptor activity modulators. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Malachite green mediates homodimerization of antibody VL domains to form a fluorescent ternary complex with singular symmetric interfaces

PubMed Central

Szent-Gyorgyi, Chris; Stanfield, Robyn L.; Andreko, Susan; Dempsey, Alison; Ahmed, Mushtaq; Capek, Sara; Waggoner, Alan; Wilson, Ian A.; Bruchez, Marcel P.

2013-01-01

We report that a symmetric small molecule ligand mediates the assembly of antibody light chain variable domains (VLs) into a correspondent symmetric ternary complex with novel interfaces. The L5* Fluorogen Activating Protein (FAP) is a VL domain that binds malachite green dye (MG) to activate intense fluorescence. Crystallography of liganded L5* reveals a 2:1 protein:ligand complex with inclusive C2 symmetry, where MG is almost entirely encapsulated between an antiparallel arrangement of the two VL domains. Unliganded L5* VL domains crystallize as a similar antiparallel VL/VL homodimer. The complementarity determining regions (CDRs) are spatially oriented to form novel VL/VL and VL/ligand interfaces that tightly constrain a propeller conformer of MG. Binding equilibrium analysis suggests highly cooperative assembly to form a very stable VL/MG/VL complex, such that MG behaves as a strong chemical inducer of dimerization. Fusion of two VL domains into a single protein tightens MG binding over 1,000-fold to low picomolar affinity without altering the large binding enthalpy, suggesting that bonding interactions with ligand and restriction of domain movements make independent contributions to binding. Fluorescence activation of a symmetrical fluorogen provides a selection mechanism for the isolation and directed evolution of ternary complexes where unnatural symmetric binding interfaces are favored over canonical antibody interfaces. As exemplified by L5*, these self-reporting complexes may be useful as modulators of protein association or as high affinity protein tags and capture reagents. PMID:23978698

Probing ligand recognition of the opioid pan antagonist AT-076 at nociceptin, kappa, mu, and delta opioid receptors through structure-activity relationships.

PubMed

Journigan, V Blair; Polgar, Willma E; Tuan, Edward W; Lu, James; Daga, Pankaj R; Zaveri, Nurulain T

2017-10-16

Few opioid ligands binding to the three classic opioid receptor subtypes, mu, kappa and delta, have high affinity at the fourth opioid receptor, the nociceptin/orphanin FQ receptor (NOP). We recently reported the discovery of AT-076 (1), (R)-7-hydroxy-N-((S)-1-(4-(3-hydroxyphenyl)piperidin-1-yl)-3-methylbutan-2-yl)-1,2,3,4-tetrahydroisoquinoline-3-carboxamide, a pan antagonist with nanomolar affinity for all four subtypes. Since AT-076 binds with high affinity at all four subtypes, we conducted a structure-activity relationship (SAR) study to probe ligand recognition features important for pan opioid receptor activity, using chemical modifications of key pharmacophoric groups. SAR analysis of the resulting analogs suggests that for the NOP receptor, the entire AT-076 scaffold is crucial for high binding affinity, but the binding mode is likely different from that of NOP antagonists C-24 and SB-612111 bound in the NOP crystal structure. On the other hand, modifications of the 3-hydroxyphenyl pharmacophore, but not the 7-hydroxy Tic pharmacophore, are better tolerated at kappa and mu receptors and yield very high affinity multifunctional (e.g. 12) or highly selective (e.g. 16) kappa ligands. With the availability of the opioid receptor crystal structures, our SAR analysis of the common chemotype of AT-076 suggests rational approaches to modulate binding selectivity, enabling the design of multifunctional or selective opioid ligands from such scaffolds.

Ligand binding affinity and changes in the lateral diffusion of receptor for advanced glycation endproducts (RAGE).

PubMed

Syed, Aleem; Zhu, Qiaochu; Smith, Emily A

2016-12-01

The effect of ligand on the lateral diffusion of receptor for advanced glycation endproducts (RAGE), a receptor involved in numerous pathological conditions, remains unknown. Single particle tracking experiments that use quantum dots specifically bound to hemagglutinin (HA)-tagged RAGE (HA-RAGE) are reported to elucidate the effect of ligand binding on HA-RAGE diffusion in GM07373 cell membranes. The ligand used in these studies is methylglyoxal modified-bovine serum albumin (MGO-BSA) containing advanced glycation end products modifications. The binding affinity between soluble RAGE and MGO-BSA increases by 1.8 to 9.7-fold as the percent primary amine modification increases from 24 to 74% and with increasing negative charge on the MGO-BSA. Ligand incubation affects the HA-RAGE diffusion coefficient, the radius of confinement, and duration of confinement. There is, however, no correlation between MGO-BSA ligand binding affinity with soluble RAGE and the extent of the changes in HA-RAGE lateral diffusion. The ligand induced changes to HA-RAGE lateral diffusion do not occur when cholesterol is depleted from the cell membrane, indicating the mechanism for ligand-induced changes to HA-RAGE diffusion is cholesterol dependent. The results presented here serve as a first step in unraveling how ligand influences RAGE lateral diffusion. Copyright © 2016. Published by Elsevier B.V.

Mutation in Fas Ligand Impairs Maturation of Thymocytes Bearing Moderate Affinity T Cell Receptors

PubMed Central

Boursalian, Tamar E.; Fink, Pamela J.

2003-01-01

Fas ligand, best known as a death-inducer, is also a costimulatory molecule required for maximal proliferation of mature antigen-specific CD4+ and CD8+ T cells. We now extend the role of Fas ligand by showing that it can also influence thymocyte development. T cell maturation in some, but not all, strains of TCR transgenic mice is severely impaired in thymocytes expressing mutant Fas ligand incapable of interacting with Fas. Mutant Fas ligand inhibits neither negative selection nor death by neglect. Instead, it appears to modulate positive selection of thymocytes expressing both class I– and class II–restricted T cell receptors of moderate affinity for their positively selecting ligands. Fas ligand is therefore an inducer of death, a costimulator of peripheral T cell activation, and an accessory molecule in positive selection. PMID:12860933

Computational design of an endo-1,4-[beta]-xylanase ligand binding site

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

Morin, Andrew; Kaufmann, Kristian W.; Fortenberry, Carie

2012-09-05

The field of computational protein design has experienced important recent success. However, the de novo computational design of high-affinity protein-ligand interfaces is still largely an open challenge. Using the Rosetta program, we attempted the in silico design of a high-affinity protein interface to a small peptide ligand. We chose the thermophilic endo-1,4-{beta}-xylanase from Nonomuraea flexuosa as the protein scaffold on which to perform our designs. Over the course of the study, 12 proteins derived from this scaffold were produced and assayed for binding to the target ligand. Unfortunately, none of the designed proteins displayed evidence of high-affinity binding. Structural characterizationmore » of four designed proteins revealed that although the predicted structure of the protein model was highly accurate, this structural accuracy did not translate into accurate prediction of binding affinity. Crystallographic analyses indicate that the lack of binding affinity is possibly due to unaccounted for protein dynamics in the 'thumb' region of our design scaffold intrinsic to the family 11 {beta}-xylanase fold. Further computational analysis revealed two specific, single amino acid substitutions responsible for an observed change in backbone conformation, and decreased dynamic stability of the catalytic cleft. These findings offer new insight into the dynamic and structural determinants of the {beta}-xylanase proteins.« less

Prediction of kinase-inhibitor binding affinity using energetic parameters

PubMed Central

Usha, Singaravelu; Selvaraj, Samuel

2016-01-01

The combination of physicochemical properties and energetic parameters derived from protein-ligand complexes play a vital role in determining the biological activity of a molecule. In the present work, protein-ligand interaction energy along with logP values was used to predict the experimental log (IC50) values of 25 different kinase-inhibitors using multiple regressions which gave a correlation coefficient of 0.93. The regression equation obtained was tested on 93 kinase-inhibitor complexes and an average deviation of 0.92 from the experimental log IC50 values was shown. The same set of descriptors was used to predict binding affinities for a test set of five individual kinase families, with correlation values > 0.9. We show that the protein-ligand interaction energies and partition coefficient values form the major deterministic factors for binding affinity of the ligand for its receptor. PMID:28149052

Development and validation of an affinity chromatography step using a peptide ligand for cGMP production of factor VIII.

PubMed

Kelley, Brian D; Tannatt, Molly; Magnusson, Robert; Hagelberg, Sigrid; Booth, James

2004-08-05

An affinity chromatography step was developed for purification of recombinant B-Domain Deleted Factor VIII (BDDrFVIII) using a peptide ligand selected from a phage display library. The peptide library had variegated residues, contained both within a disulfide bond-constrained ring and flanking the ring. The peptide ligand binds to BDDrFVIII with a dissociation constant of approximately 1 microM both in free solution and when immobilized on a chromatographic resin. The peptide is chemically synthesized and the affinity resin is produced by coupling the peptide to an agarose matrix preactivated with N-hydroxysuccinimide. Coupling conditions were optimized to give consistent and complete ligand incorporation and validated with a robustness study that tested various combinations of processing limits. The peptide affinity chromatographic operation employs conditions very similar to an immunoaffinity chromatography step currently in use for BDDrFVIII manufacture. The process step provides excellent recovery of BDDrFVIII from a complex feed stream and reduces host cell protein and DNA by 3-4 logs. Process validation studies established resin reuse over 26 cycles without changes in product recovery or purity. A robustness study using a factorial design was performed and showed that the step was insensitive to small changes in process conditions that represent normal variation in commercial manufacturing. A scaled-down model of the process step was qualified and used for virus removal studies. A validation package addressing the safety of the leached peptide included leaching rate measurements under process conditions, testing of peptide levels in product pools, demonstration of robust removal downstream by spiking studies, end product testing, and toxicological profiling of the ligand. The peptide ligand affinity step was scaled up for cGMP production of BDDrFVIII for clinical trials.

Estimation of affinities of ligands in mixtures via magnetic recovery of target-ligand complexes and chromatographic analyses: chemometrics and an experimental model

PubMed Central

2011-01-01

Abstract Background The combinatorial library strategy of using multiple candidate ligands in mixtures as library members is ideal in terms of cost and efficiency, but needs special screening methods to estimate the affinities of candidate ligands in such mixtures. Herein, a new method to screen candidate ligands present in unknown molar quantities in mixtures was investigated. Results The proposed method involves preparing a processed-mixture-for-screening (PMFS) with each mixture sample and an exogenous reference ligand, initiating competitive binding among ligands from the PMFS to a target immobilized on magnetic particles, recovering target-ligand complexes in equilibrium by magnetic force, extracting and concentrating bound ligands, and analyzing ligands in the PMFS and the concentrated extract by chromatography. The relative affinity of each candidate ligand to its reference ligand is estimated via an approximation equation assuming (a) the candidate ligand and its reference ligand bind to the same site(s) on the target, (b) their chromatographic peak areas are over five times their intercepts of linear response but within their linear ranges, (c) their binding ratios are below 10%. These prerequisites are met by optimizing primarily the quantity of the target used and the PMFS composition ratio. The new method was tested using the competitive binding of biotin derivatives from mixtures to streptavidin immobilized on magnetic particles as a model. Each mixture sample containing a limited number of candidate biotin derivatives with moderate differences in their molar quantities were prepared via parallel-combinatorial-synthesis (PCS) without purification, or via the pooling of individual compounds. Some purified biotin derivatives were used as reference ligands. This method showed resistance to variations in chromatographic quantification sensitivity and concentration ratios; optimized conditions to validate the approximation equation could be applied to different mixture samples. Relative affinities of candidate biotin derivatives with unknown molar quantities in each mixture sample were consistent with those estimated by a homogenous method using their purified counterparts as samples. Conclusions This new method is robust and effective for each mixture possessing a limited number of candidate ligands whose molar quantities have moderate differences, and its integration with PCS has promise to routinely practice the mixture-based library strategy. PMID:21545719

Computational Approaches for Decoding Select Odorant-Olfactory Receptor Interactions Using Mini-Virtual Screening

PubMed Central

Harini, K.; Sowdhamini, Ramanathan

2015-01-01

Olfactory receptors (ORs) belong to the class A G-Protein Coupled Receptor superfamily of proteins. Unlike G-Protein Coupled Receptors, ORs exhibit a combinatorial response to odors/ligands. ORs display an affinity towards a range of odor molecules rather than binding to a specific set of ligands and conversely a single odorant molecule may bind to a number of olfactory receptors with varying affinities. The diversity in odor recognition is linked to the highly variable transmembrane domains of these receptors. The purpose of this study is to decode the odor-olfactory receptor interactions using in silico docking studies. In this study, a ligand (odor molecules) dataset of 125 molecules was used to carry out in silico docking using the GLIDE docking tool (SCHRODINGER Inc Pvt LTD). Previous studies, with smaller datasets of ligands, have shown that orthologous olfactory receptors respond to similarly-tuned ligands, but are dramatically different in their efficacy and potency. Ligand docking results were applied on homologous pairs (with varying sequence identity) of ORs from human and mouse genomes and ligand binding residues and the ligand profile differed among such related olfactory receptor sequences. This study revealed that homologous sequences with high sequence identity need not bind to the same/ similar ligand with a given affinity. A ligand profile has been obtained for each of the 20 receptors in this analysis which will be useful for expression and mutation studies on these receptors. PMID:26221959

Preparation and characterization of fluorophenylboronic acid-functionalized affinity monolithic columns for the selective enrichment of cis-diol-containing biomolecules.

PubMed

Li, Qianjin; Liu, Zhen

2015-01-01

Boronate affinity monolithic columns have been developed into an important means for the selective recognition and capture of cis-diol-containing biomolecules, such as glycoproteins, nucleosides and saccharides. The ligands of boronic acids are playing an important role in boronate affinity monolithic columns. Although several boronate affinity monoliths with high affinity toward cis-diol-containing biomolecules have been reported, only few publications are focused on their detailed procedures for preparation and characterization. This chapter describes in detail the preparation and characterization of a boronate affinity monolithic column applying 2,4-difluoro-3-formyl-phenylboronic acid (DFFPBA) as a ligand. The DFFPBA-functionalized monolithic column not only exhibited an ultrahigh boronate affinity toward cis-diol-containing biomolecules, but also showed great potential for the selective enrichment of cis-diol-containing biomolecules in real samples.

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

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

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Beyond helper phage: Using "helper cells" to select peptide affinity ligands

DOE PAGES

Phipps, Mary Lisa; Lillo, Antoinetta M.; Shou, Yulin; ...

2016-09-14

Peptides are important affinity ligands for microscopy, biosensing, and targeted delivery. However, because they can have low affinity for their targets, their selection from large naïve libraries can be challenging. When selecting peptidic ligands from display libraries, it is important to: 1) ensure efficient display; 2) maximize the ability to select high affinity ligands; and 3) minimize the effect of the display context on binding. The “helper cell” packaging system has been described as a tool to produce filamentous phage particles based on phagemid constructs with varying display levels, while remaining free of helper phage contamination. Here we report onmore » the first use of this system for peptide display, including the systematic characterization and optimization of helper cells, their inefficient use in antibody display and their use in creating and selecting from a set of phage display peptide libraries. Our libraries were analyzed with unprecedented precision by standard or deep sequencing, and shown to be superior in quality than commercial gold standards. Using our helper cell libraries, we have obtained ligands recognizing Yersinia pestis surface antigen F1V and L-glutamine-binding periplasmic protein QBP. In the latter case, unlike any of the peptide library selections described so far, we used a combination of phage and yeast display to select intriguing peptide ligands. Here, based on the success of our selections we believe that peptide libraries obtained with helper cells are not only suitable, but preferable to traditional phage display libraries for selection of peptidic ligands.« less

Synthesis and binding affinity of new 1,4-disubstituted triazoles as potential dopamine D(3) receptor ligands.

PubMed

Insua, Ignacio; Alvarado, Mario; Masaguer, Christian F; Iglesias, Alba; Brea, José; Loza, María I; Carro, Laura

2013-10-15

A series of new 1,4-disubstituted triazoles was prepared from appropriate arylacetylenes and aminoalkylazides using click chemistry methodology. These compounds were evaluated as potential ligands on several subtypes of dopamine receptors in in vitro competition assays, showing high affinity for dopamine D3 receptors, lower affinity for D2 and D4, and no affinity for the D1 receptors. Compound 18 displayed the highest affinity at the D3 receptor with a Ki value of 2.7 nM, selectivity over D2 (70-fold) and D4 (200-fold), and behaviour as a competitive antagonist in the low nanomolar range. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structure-Based Design of N-Substituted 1-Hydroxy-4-sulfamoyl-2-naphthoates as Selective Inhibitors of the Mcl-1 Oncoprotein

PubMed Central

Lanning, Maryanna E.; Yu, Wenbo; Yap, Jeremy L.; Chauhan, Jay; Chen, Lijia; Whiting, Ellis; Pidugu, Lakshmi S.; Atkinson, Tyler; Bailey, Hala; Li, Willy; Roth, Braden M.; Hynicka, Lauren; Chesko, Kirsty; Toth, Eric A.; Shapiro, Paul; MacKerell, Alexander D.; Wilder, Paul T.; Fletcher, Steven

2016-01-01

Structure-based drug design was utilized to develop novel, 1-hydroxy-2-naphthoate-based small-molecule inhibitors of Mcl-1. Ligand design was driven by exploiting a salt bridge with R263 and interactions with the p2 and p3 pockets of the protein. Significantly, target molecules were accessed in just two synthetic steps, suggesting further optimization will require minimal synthetic effort. Molecular modeling using the Site-Identification by Ligand Competitive Saturation (SILCS) approach was used to qualitatively direct ligand design as well as develop quantitative models for inhibitor binding affinity to Mcl-1 and the Bcl-2 relative Bcl-xL as well as for the specificity of binding to the two proteins. Results indicated hydrophobic interactions with the p2 pockets dominate the affinity of the most favourable binding ligand (3bl: Ki = 31 nM). Compounds were up to 20-fold selective for Mcl-1 over Bcl-xL. Selectivity of the inhibitors was driven by interactions with the deeper p2 pocket in Mcl-1 versus Bcl-xL. The SILCS-based SAR of the present compounds represents the foundation for the development of Mcl-1 specific inhibitors with the potential to treat a wide range of solid tumours and hematological cancers, including acute myeloid leukaemia. PMID:26985630

Bifunctional fusion proteins of calmodulin and protein A as affinity ligands in protein purification and in the study of protein-protein interactions.

PubMed

Hentz, N G; Daunert, S

1996-11-15

An affinity chromatography system is described that incorporates a genetically designed bifunctional affinity ligand. The utility of the system in protein purification and in the study of protein-protein interactions is demonstrated by using the interaction between protein A and the heat shock protein DnaK as a model system. The bifunctional affinity ligand was developed by genetically fusing calmodulin (CaM) to protein A (ProtA). The dual functionality of protein A-calmodulin (ProtA-CaM) stems from the molecular recognition properties of the two components of the fusion protein. In particular, CaM serves as the anchoring component by virtue of its binding properties toward phenothiazine. Thus, the ProtA-CaM can be immobilized on a solid support containing phenothiazine from the C-terminal domain of the fusion protein. Protein A is at the N-terminal domain of the fusion protein and serves as the affinity site for DnaK. While DnaK binds specifically to the protein A domain of the bifunctional ligand, it is released upon addition of ATP and under very mild conditions (pH 7.0). In addition to obtaining highly purified DnaK, this system is very rugged in terms of its performance. The proteinaceous bifunctional affinity ligand can be easily removed by addition of EGTA, and fresh ProtA-CaM can be easily reloaded onto the column. This allows for a facile regeneration of the affinity column because the phenothiazine-silica support matrix is stable for long periods of time under a variety of conditions. This study also demonstrates that calmodulin fusions can provide a new approach to study protein-protein interactions. Indeed, the ProtA-CaM fusion protein identified DnaK as a cellular component that interacts with protein A from among the thousands of proteins present in Escherichia coli.

Quantitation of Membrane-Ligand Interactions Using Backscattering Interferometry

PubMed Central

Baksh, Michael M.; Kussrow, Amanda K.; Mileni, Mauro; Finn, M.G.; Bornhop, Darryl J.

2011-01-01

Though membrane-associated proteins are ubiquitous within all living organisms and represent the majority of drug targets, a general method for direct, label-free measurement of ligand binding to native membranes has not been reported. Here we show backscattering interferometry (BSI) to be a viable technique for quantifying ligand-receptor binding affinities in a variety of membrane environments. By detecting minute changes in the refractive index of a solution, BSI allows binding interactions of proteins with their ligands to be measured at picomolar concentrations. Equilibrium binding constants in the micromolar to picomolar range were obtained for small- and large-molecule interactions in both synthetic- and cell-derived membranes without the use of labels or supporting substrates. The simple and low-cost hardware, high sensitivity, and label-free nature of BSI should make it readily applicable to the study of many membrane-associated proteins of biochemical and pharmacological interest. PMID:21399645

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.

Investigations into the binding affinities of different human 5-HT4 receptor splice variants.

PubMed

Irving, Helen R; Tochon-Danguy, Nathalie; Chinkwo, Kenneth A; Li, Jian G; Grabbe, Carmen; Shapiro, Marina; Pouton, Colin W; Coupar, Ian M

2010-01-01

This study examined whether the drug-receptor-binding sites of 5 selected human 5-HT(4) receptor splice variants [h5-HT4(a), h5-HT4(b), h5-HT4(c), h5-HT4(d) and h5-HT4(g)] display preferential affinities towards agonists. The agonists selected on the basis of chemical diversity and clinical relevance were: 5-HT4 benzamides, renzapride, zacopride and prucalopride; the benzimidazolones, DAU 6236 and BIMU 1; the aromatic ketone, RS67333, and the indole carbazimidamide tegaserod. The rank order of affinities ranging across the splice variants was: tegaserod (pKi: 7.38-7.91) > or = Y-36912 (pKi: 7.03-7.85) = BIMU 1 (pKi: 6.92-7.78) > or = DAU 6236 (pKi: 6.79-7.99) > or = 5-HT (pKi: 5.82-7.29) > or = 5-MeOT (pKi: 5.64-6.83) > or = renzapride (pKi: 4.85-5.56). We obtained affinity values for the 5-HT4(b), (d) and (g) variants for RS67333 (pKi: 7:48-8.29), prucalopride (pKi: 6.86-7.37) and zacopride (pKi: 5.88-7.0). These results indicate that the ligands interact with the same conserved site in each splice variant. Some splice variants have a higher affinity for certain agonists and the direction of selectivity followed a common trend of lowest affinity at the (d) variant. However, this trend was not evident in functional experiments. Our findings suggest that it may be possible to design splice variant selective ligands, which may be of relevance for experimental drugs but may be difficult to develop clinically. 2010 S. Karger AG, Basel.

High-throughput and multiplexed regeneration buffer scouting for affinity-based interactions.

PubMed

Geuijen, Karin P M; Schasfoort, Richard B; Wijffels, Rene H; Eppink, Michel H M

2014-06-01

Affinity-based analyses on biosensors depend partly on regeneration between measurements. Regeneration is performed with a buffer that efficiently breaks all interactions between ligand and analyte while maintaining the active binding site of the ligand. We demonstrated a regeneration buffer scouting using the combination of a continuous flow microspotter with a surface plasmon resonance imaging platform to simultaneously test 48 different regeneration buffers on a single biosensor. Optimal regeneration conditions are found within hours and consume little amounts of buffers, analyte, and ligand. This workflow can be applied to any ligand that is coupled through amine, thiol, or streptavidin immobilization. Copyright © 2014 Elsevier Inc. All rights reserved.

Evaluation of water displacement energetics in protein binding sites with grid cell theory.

PubMed

Gerogiokas, G; Southey, M W Y; Mazanetz, M P; Heifetz, A; Hefeitz, A; Bodkin, M; Law, R J; Michel, J

2015-04-07

Excess free energies, enthalpies and entropies of water in protein binding sites were computed via classical simulations and Grid Cell Theory (GCT) analyses for three pairs of congeneric ligands in complex with the proteins scytalone dehydratase, p38α MAP kinase and EGFR kinase respectively. Comparative analysis is of interest since the binding modes for each ligand pair differ in the displacement of one binding site water molecule, but significant variations in relative binding affinities are observed. Protocols that vary in their use of restraints on protein and ligand atoms were compared to determine the influence of protein-ligand flexibility on computed water structure and energetics, and to assess protocols for routine analyses of protein-ligand complexes. The GCT-derived binding affinities correctly reproduce experimental trends, but the magnitude of the predicted changes in binding affinities is exaggerated with respect to results from a previous Monte Carlo Free Energy Perturbation study. Breakdown of the GCT water free energies into enthalpic and entropic components indicates that enthalpy changes dominate the observed variations in energetics. In EGFR kinase GCT analyses revealed that replacement of a pyrimidine by a cyanopyridine perturbs water energetics up three hydration shells away from the ligand.

Negative Cooperativity in the EGF Receptor

PubMed Central

Pike, Linda J.

2012-01-01

Scatchard analyses of the binding of EGF to its receptor yield concave up Scatchard plots, indicative of some type of heterogenity in ligand binding affinity. This was typically interpreted as being due to the presence of two independent binding site–one of high affinity representing ≤10% of the receptor population and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGF receptor that show symmetric binding of the two ligands. A new approach to the analysis of 125I-EGF binding data combined with the structure of the singly-occupied Drosophila EGF receptor have now shown that this heterogeneity is due to the presence of negative cooperativity in the EGF receptor. Concerns that negative cooperativity precludes ligand-induced dimerization of the EGF receptor confuse the concepts of linkage cooperativity. Linkage refers to the effect of ligand on the assembly of dimers while cooperativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly but shows negative cooperativity within the dimer. PMID:22260659

Affinity modulation of small-molecule ligands by borrowing endogenous protein surfaces

PubMed Central

Briesewitz, Roger; Ray, Gregory T.; Wandless, Thomas J.; Crabtree, Gerald R.

1999-01-01

A general strategy is described for improving the binding properties of small-molecule ligands to protein targets. A bifunctional molecule is created by chemically linking a ligand of interest to another small molecule that binds tightly to a second protein. When the ligand of interest is presented to the target protein by the second protein, additional protein–protein interactions outside of the ligand-binding sites serve either to increase or decrease the affinity of the binding event. We have applied this approach to an intractable target, the SH2 domain, and demonstrate a 3-fold enhancement over the natural peptide. This approach provides a way to modulate the potency and specificity of biologically active compounds. PMID:10051576

A twice-as-smart synthetic G-quartet: PyroTASQ is both a smart quadruplex ligand and a smart fluorescent probe.

PubMed

Laguerre, Aurélien; Stefan, Loic; Larrouy, Manuel; Genest, David; Novotna, Jana; Pirrotta, Marc; Monchaud, David

2014-09-03

Recent and unambiguous evidences of the formation of DNA and RNA G-quadruplexes in cells has provided solid support for these structures to be considered as valuable targets in oncology. Beyond this, they have lent further credence to the anticancer strategies relying on small molecules that selectively target these higher-order DNA/RNA architectures, referred to as G-quadruplex ligands. They have also shed bright light on the necessity of designing multitasking ligands, displaying not only enticing quadruplex interacting properties (affinity, structural selectivity) but also additional features that make them usable for detecting quadruplexes in living cells, notably for determining whether, when, and where these structures fold and unfold during the cell cycle and also for better assessing the consequences of their stabilization by external agents. Herein, we report a brand new design of such multitasking ligands, whose structure experiences a quadruplex-promoted conformational switch that triggers not only its quadruplex affinity (i.e., smart ligands, which display high affinity and selectivity for DNA/RNA quadruplexes) but also its fluorescence (i.e., smart probes, which behave as selective light-up fluorescent reporters on the basis of a fluorogenic electron redistribution). The first prototype of such multifunctional ligands, termed PyroTASQ, represents a brand new generation of quadruplex ligands that can be referred to as "twice-as-smart" quadruplex ligands.

Physics-based scoring of protein-ligand interactions: explicit polarizability, quantum mechanics and free energies.

PubMed

Bryce, Richard A

2011-04-01

The ability to accurately predict the interaction of a ligand with its receptor is a key limitation in computer-aided drug design approaches such as virtual screening and de novo design. In this article, we examine current strategies for a physics-based approach to scoring of protein-ligand affinity, as well as outlining recent developments in force fields and quantum chemical techniques. We also consider advances in the development and application of simulation-based free energy methods to study protein-ligand interactions. Fuelled by recent advances in computational algorithms and hardware, there is the opportunity for increased integration of physics-based scoring approaches at earlier stages in computationally guided drug discovery. Specifically, we envisage increased use of implicit solvent models and simulation-based scoring methods as tools for computing the affinities of large virtual ligand libraries. Approaches based on end point simulations and reference potentials allow the application of more advanced potential energy functions to prediction of protein-ligand binding affinities. Comprehensive evaluation of polarizable force fields and quantum mechanical (QM)/molecular mechanical and QM methods in scoring of protein-ligand interactions is required, particularly in their ability to address challenging targets such as metalloproteins and other proteins that make highly polar interactions. Finally, we anticipate increasingly quantitative free energy perturbation and thermodynamic integration methods that are practical for optimization of hits obtained from screened ligand libraries.

Structure-based CoMFA as a predictive model - CYP2C9 inhibitors as a test case.

PubMed

Yasuo, Kazuya; Yamaotsu, Noriyuki; Gouda, Hiroaki; Tsujishita, Hideki; Hirono, Shuichi

2009-04-01

In this study, we tried to establish a general scheme to create a model that could predict the affinity of small compounds to their target proteins. This scheme consists of a search for ligand-binding sites on a protein, a generation of bound conformations (poses) of ligands in each of the sites by docking, identifications of the correct poses of each ligand by consensus scoring and MM-PBSA analysis, and a construction of a CoMFA model with the obtained poses to predict the affinity of the ligands. By using a crystal structure of CYP 2C9 and the twenty known CYP inhibitors as a test case, we obtained a CoMFA model with a good statistics, which suggested that the classification of the binding sites as well as the predicted bound poses of the ligands should be reasonable enough. The scheme described here would give a method to predict the affinity of small compounds with a reasonable accuracy, which is expected to heighten the value of computational chemistry in the drug design process.

Label-free quantitative 1H NMR spectroscopy to study low-affinity ligand–protein interactions in solution: A contribution to the mechanism of polyphenol-mediated astringency

PubMed Central

Delius, Judith; Frank, Oliver

2017-01-01

Nuclear magnetic resonance (NMR) spectroscopy is well-established in assessing the binding affinity between low molecular weight ligands and proteins. However, conventional NMR-based binding assays are often limited to small proteins of high purity and may require elaborate isotopic labeling of one of the potential binding partners. As protein–polyphenol complexation is assumed to be a key event in polyphenol-mediated oral astringency, here we introduce a label-free, ligand-focused 1H NMR titration assay to estimate binding affinities and characterize soluble complex formation between proteins and low molecular weight polyphenols. The method makes use of the effects of NMR line broadening due to protein–ligand interactions and quantitation of the non-bound ligand at varying protein concentrations by quantitative 1H NMR spectroscopy (qHNMR) using electronic reference to access in vivo concentration (ERETIC 2). This technique is applied to assess the interaction kinetics of selected astringent tasting polyphenols and purified mucin, a major lubricating glycoprotein of human saliva, as well as human whole saliva. The protein affinity values (BC50) obtained are subsequently correlated with the intrinsic mouth-puckering, astringent oral sensation imparted by these compounds. The quantitative NMR method is further exploited to study the effect of carboxymethyl cellulose, a candidate “anti-astringent” protein binding antagonist, on the polyphenol–protein interaction. Consequently, the NMR approach presented here proves to be a versatile tool to study the interactions between proteins and low-affinity ligands in solution and may find promising applications in the discovery of bioactives. PMID:28886151

New Synthesis and Tritium Labeling of a Selective Ligand for Studying High-affinity γ-Hydroxybutyrate (GHB) Binding Sites

PubMed Central

Vogensen, Stine B.; Marek, Aleš; Bay, Tina; Wellendorph, Petrine; Kehler, Jan; Bundgaard, Christoffer; Frølund, Bente; Pedersen, Martin H.F.; Clausen, Rasmus P.

2013-01-01

3-Hydroxycyclopent-1-enecarboxylic acid (HOCPCA, 1) is a potent ligand for the high-affinity GHB binding sites in the CNS. An improved synthesis of 1 together with a very efficient synthesis of [3H]-1 is described. The radiosynthesis employs in situ generated lithium trimethoxyborotritide. Screening of 1 against different CNS targets establishes a high selectivity and we demonstrate in vivo brain penetration. In vitro characterization of [3H]-1 binding shows high specificity to the high-affinity GHB binding sites. PMID:24053696

Affinity chromatographic purification of tetrodotoxin by use of tetrodotoxin-binding high molecular weight substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands.

PubMed

Shiomi, K; Yamaguchi, S; Shimakura, K; Nagashima, Y; Yamamori, K; Matsui, T

1993-12-01

A purification method for tetrodotoxin (TTX), based on affinity chromatography using the TTX-binding high mol. wt substances in the body fluid of shore crab (Hemigrapsus sanguineus) as ligands, was developed. This method was particularly useful for analysis of TTX in biological samples with low concentrations of TTX. The affinity gel prepared was highly specific for TTX, having no ability to bind 4-epi-TTX and anhydro-TTX as well as saxitoxin.

Analysis of the Expression of Peptide–Major Histocompatibility Complexes Using High Affinity Soluble Divalent T Cell Receptors

PubMed Central

O'Herrin, Sean M.; Lebowitz, Michael S.; Bieler, Joan G.; al-Ramadi, Basel K.; Utz, Ursula; Bothwell, Alfred L.M.; Schneck, Jonathan P.

1997-01-01

Understanding the regulation of cell surface expression of specific peptide–major histocompatibility complex (MHC) complexes is hindered by the lack of direct quantitative analyses of specific peptide–MHC complexes. We have developed a direct quantitative biochemical approach by engineering soluble divalent T cell receptor analogues (TCR–Ig) that have high affinity for their cognate peptide–MHC ligands. The generality of this approach was demonstrated by specific staining of peptide-pulsed cells with two different TCR–Ig complexes: one specific for the murine alloantigen 2C, and one specific for a viral peptide from human T lymphocyte virus–1 presented by human histocompatibility leukocyte antigens–A2. Further, using 2C TCR– Ig, a more detailed analysis of the interaction with cognate peptide–MHC complexes revealed several interesting findings. Soluble divalent 2C TCR–Ig detected significant changes in the level of specific antigenic–peptide MHC cell surface expression in cells treated with γ-interferon (γ-IFN). Interestingly, the effects of γ-IFN on expression of specific peptide–MHC complexes recognized by 2C TCR–Ig were distinct from its effects on total H-2 Ld expression; thus, lower doses of γ-IFN were required to increase expression of cell surface class I MHC complexes than were required for upregulation of expression of specific peptide–MHC complexes. Analysis of the binding of 2C TCR–Ig for specific peptide–MHC ligands unexpectedly revealed that the affinity of the 2C TCR–Ig for the naturally occurring alloreactive, putatively, negatively selecting, complex, dEV-8–H-2 Kbm3, is very low, weaker than 71 μM. The affinity of the 2C TCR for the other naturally occurring, negatively selecting, alloreactive complex, p2Ca–H-2 Ld, is ∼1000-fold higher. Thus, negatively selecting peptide–MHC complexes do not necessarily have intrinsically high affinity for cognate TCR. These results, uniquely revealed by this analysis, indicate the importance of using high affinity biologically relevant cognates, such as soluble divalent TCR, in furthering our understanding of immune responses. PMID:9334373

The identification of high-affinity G protein-coupled receptor ligands from large combinatorial libraries using multicolor quantum dot-labeled cell-based screening

PubMed Central

Fu, Junjie; Lee, Timothy; Qi, Xin

2014-01-01

G protein-coupled receptors (GPCRs), which are involved in virtually every biological process, constitute the largest family of transmembrane receptors. Many top-selling and newly approved drugs target GPCRs. In this review, we aim to recapitulate efforts and progress in combinatorial library-assisted GPCR ligand discovery, particularly focusing on one-bead-one-compound library synthesis and quantum dot-labeled cell-based assays, which both effectively enhance the rapid identification of GPCR ligands with higher affinity and specificity. PMID:24941874

Labeling by ( sup 3 H)1,3-di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by sigma ligands

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

Rothman, R.B.; Reid, A.; Mahboubi, A.

1991-02-01

Equilibrium binding studies with the sigma receptor ligand ({sup 3}H)1,3-di(2-tolyl)guanidine (({sup 3}H)DTG) demonstrated two high affinity binding sites in membranes prepared from guinea pig brain. The apparent Kd values of DTG for sites 1 and 2 were 11.9 and 37.6 nM, respectively. The corresponding Bmax values were 1045 and 1423 fmol/mg of protein. Site 1 had high affinity for (+)-pentazocine, haloperidol, (R)-(+)-PPP, carbepentane, and other sigma ligands, suggesting a similarity with the dextromethorphan/sigma 1 binding site described by Musacchio et al. (Life Sci. 45:1721-1732 (1989)). Site 2 had high affinity for DTG and haloperidol (Ki = 36.1 nM) and lowmore » affinity for most other sigma ligands. Kinetic experiments demonstrated that ({sup 3}H)DTG dissociated in a biphasic manner from both site 1 and site 2. DTG and haloperidol increased the dissociation rate of ({sup 3}H)DTG from site 1 and site 2, demonstrating the presence of pseudoallosteric interactions. Inorganic calcium channel blockers such as Cd2+ selectively increased the dissociation rate of ({sup 3}H)DTG from site 2, suggesting an association of this binding site with calcium channels.« less

A Dual-Specific Targeting Approach Based on the Simultaneous Recognition of Duplex and Quadruplex Motifs.

PubMed

Nguyen, Thi Quynh Ngoc; Lim, Kah Wai; Phan, Anh Tuân

2017-09-20

Small-molecule ligands targeting nucleic acids have been explored as potential therapeutic agents. Duplex groove-binding ligands have been shown to recognize DNA in a sequence-specific manner. On the other hand, quadruplex-binding ligands exhibit high selectivity between quadruplex and duplex, but show limited discrimination between different quadruplex structures. Here we propose a dual-specific approach through the simultaneous application of duplex- and quadruplex-binders. We demonstrated that a quadruplex-specific ligand and a duplex-specific ligand can simultaneously interact at two separate binding sites of a quadruplex-duplex hybrid harbouring both quadruplex and duplex structural elements. Such a dual-specific targeting strategy would combine the sequence specificity of duplex-binders and the strong binding affinity of quadruplex-binders, potentially allowing the specific targeting of unique quadruplex structures. Future research can be directed towards the development of conjugated compounds targeting specific genomic quadruplex-duplex sites, for which the linker would be highly context-dependent in terms of length and flexibility, as well as the attachment points onto both ligands.

Camelid VHH affinity ligands enable separation of closely related biopharmaceuticals

PubMed Central

Pabst, Timothy M.; Wendeler, Michaela; Wang, Xiangyang; Bezemer, Sandra; Hermans, Pim

2016-01-01

Abstract Interest in new and diverse classes of molecules such as recombinant toxins, enzymes, and blood factors continues to grow for use a biotherapeutics. Compared to monoclonal antibodies, these novel drugs typically lack a commercially available affinity chromatography option, which leads to greater process complexity, longer development timelines, and poor platformability. To date, for both monoclonal antibodies and novel molecules, affinity chromatography has been mostly reserved for separation of process‐related impurities such as host cell proteins and DNA. Reports of affinity purification of closely related product variants and modified forms are much rarer. In this work we describe custom affinity chromatography development using camelid VHH antibody fragments as "tunable" immunoaffinity ligands for separation of product‐related impurities. One example demonstrates high selectivity for a recombinant immunotoxin where no binding was observed for an undesired deamidated species. Also discussed is affinity purification of a coagulation factor through specific recognition of the gamma‐carboxylglutamic acid domain. PMID:27677057

Host-Guest Complexes with Protein-Ligand-Like Affinities: Computational Analysis and Design

PubMed Central

Moghaddam, Sarvin; Inoue, Yoshihisa

2009-01-01

It has recently been discovered that guests combining a nonpolar core with cationic substituents bind cucurbit[7]uril (CB[7]) in water with ultra-high affinities. The present study uses the Mining Minima algorithm to study the physics of these extraordinary associations and to computationally test a new series of CB[7] ligands designed to bind with similarly high affinity. The calculations reproduce key experimental observations regarding the affinities of ferrocene-based guests with CB[7] and β-cyclodextrin and provide a coherent view of the roles of electrostatics and configurational entropy as determinants of affinity in these systems. The newly designed series of compounds is based on a bicyclo[2.2.2]octane core, which is similar in size and polarity to the ferrocene core of the existing series. Mining Minima predicts that these new compounds will, like the ferrocenes, bind CB[7] with extremely high affinities. PMID:19133781

Characterization of rodent liver and kidney AVP receptors: pharmacologic evidence for species differences.

PubMed

Tahara, A; Tsukada, J; Ishii, N; Tomura, Y; Wada, K; Kusayama, T; Yatsu, T; Uchida, W; Tanaka, A

1999-10-22

Radioligand binding studies with [3H]vasopressin (AVP) were used to determine the affinities of AVP receptor agonists and antagonists for mouse liver and kidney plasma membrane preparations. Both membrane preparations exhibited one class of high-affinity binding site. AVP ligand binding inhibition studies confirmed that mouse liver binding sites belong to the V1A subtype while kidney binding sites belong to the V2 receptor subtype. The affinity of each ligand for mouse V1A receptors was very similar to that for rat V1A receptors, showing differences in Ki values of less than 3-fold. In contrast, several peptide (d(CH2)5Tyr(Me)AVP) and nonpeptide (OPC-21268 and SR 49059) ligands had different affinities for mouse and rat kidney V2 receptors, with differences in Ki values ranging from 14- to 17-fold. These results indicate that mouse and rat kidney V2 receptors show significant pharmacologic differences.

Development of melanoma-targeted polymer micelles by conjugation of a Melanocortin 1 Receptor (MC1R) specific ligand

PubMed Central

Barkey, Natalie M.; Tafreshi, Narges K.; Josan, Jatinder S.; De Silva, Channa R.; Sill, Kevin N.; Hruby, Victor J.; Gillies, Robert J.; Morse, David L.; Vagner, Josef

2012-01-01

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Due to its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to over-express MC1R, MC4R or MC5R. Of these, compound 1 (4-phenylbutyryl-His-Dphe-Arg-Trp-NH2) exhibited high (0.2 nM) binding affinity for MC1R, and low (high nM) affinities for MC4R and MC5R. Subsequently functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted-polymer, as well as the targeted micelle formulation, also resulted in constructs with low nM binding affinity. PMID:22011200

Development of melanoma-targeted polymer micelles by conjugation of a melanocortin 1 receptor (MC1R) specific ligand.

PubMed

Barkey, Natalie M; Tafreshi, Narges K; Josan, Jatinder S; De Silva, Channa R; Sill, Kevin N; Hruby, Victor J; Gillies, Robert J; Morse, David L; Vagner, Josef

2011-12-08

The incidence of malignant melanoma is rising faster than that of any other cancer in the United States. Because of its high expression on the surface of melanomas, MC1R has been investigated as a target for selective imaging and therapeutic agents against melanoma. Eight ligands were screened against cell lines engineered to overexpress MC1R, MC4R, or MC5R. Of these, compound 1 (4-phenylbutyryl-His-dPhe-Arg-Trp-NH(2)) exhibited high (0.2 nM) binding affinity for MC1R and low (high nanomolar) affinities for MC4R and MC5R. Functionalization of the ligand at the C-terminus with an alkyne for use in Cu-catalyzed click chemistry was shown not to affect the binding affinity. Finally, formation of the targeted polymer, as well as the targeted micelle formulation, also resulted in constructs with low nanomolar binding affinity.

Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target.

PubMed

Brown, Dean G; Brown, Giles A; Centrella, Paolo; Certel, Kaan; Cooke, Robert M; Cuozzo, John W; Dekker, Niek; Dumelin, Christoph E; Ferguson, Andrew; Fiez-Vandal, Cédric; Geschwindner, Stefan; Guié, Marie-Aude; Habeshian, Sevan; Keefe, Anthony D; Schlenker, Oliver; Sigel, Eric A; Snijder, Arjan; Soutter, Holly T; Sundström, Linda; Troast, Dawn M; Wiggin, Giselle; Zhang, Jing; Zhang, Ying; Clark, Matthew A

2018-06-01

The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our >100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets.

Concepts in receptor optimization: targeting the RGD peptide.

PubMed

Chen, Wei; Chang, Chia-en; Gilson, Michael K

2006-04-12

Synthetic receptors have a wide range of potential applications, but it has been difficult to design low molecular weight receptors that bind ligands with high, "proteinlike" affinities. This study uses novel computational methods to understand why it is hard to design a high-affinity receptor and to explore the limits of affinity, with the bioactive peptide RGD as a model ligand. The M2 modeling method is found to yield excellent agreement with experiment for a known RGD receptor and then is used to analyze a series of receptors generated in silico with a de novo design algorithm. Forces driving binding are found to be systematically opposed by proportionate repulsions due to desolvation and entropy. In particular, strong correlations are found between Coulombic attractions and the electrostatic desolvation penalty and between the mean energy change on binding and the cost in configurational entropy. These correlations help explain why it is hard to achieve high affinity. The change in surface area upon binding is found to correlate poorly with affinity within this series. Measures of receptor efficiency are formulated that summarize how effectively a receptor uses surface area, total energy, and Coulombic energy to achieve affinity. Analysis of the computed efficiencies suggests that a low molecular weight receptor can achieve proteinlike affinity. It is also found that macrocyclization of a receptor can, unexpectedly, increase the entropy cost of binding because the macrocyclic structure further restricts ligand motion.

Interactions between Human Liver Fatty Acid Binding Protein and Peroxisome Proliferator Activated Receptor Selective Drugs

PubMed Central

Velkov, Tony

2013-01-01

Fatty acid binding proteins (FABPs) act as intracellular shuttles for fatty acids as well as lipophilic xenobiotics to the nucleus, where these ligands are released to a group of nuclear receptors called the peroxisome proliferator activated receptors (PPARs). PPAR mediated gene activation is ultimately involved in maintenance of cellular homeostasis through the transcriptional regulation of metabolic enzymes and transporters that target the activating ligand. Here we show that liver- (L-) FABP displays a high binding affinity for PPAR subtype selective drugs. NMR chemical shift perturbation mapping and proteolytic protection experiments show that the binding of the PPAR subtype selective drugs produces conformational changes that stabilize the portal region of L-FABP. NMR chemical shift perturbation studies also revealed that L-FABP can form a complex with the PPAR ligand binding domain (LBD) of PPARα. This protein-protein interaction may represent a mechanism for facilitating the activation of PPAR transcriptional activity via the direct channeling of ligands between the binding pocket of L-FABP and the PPARαLBD. The role of L-FABP in the delivery of ligands directly to PPARα via this channeling mechanism has important implications for regulatory pathways that mediate xenobiotic responses and host protection in tissues such as the small intestine and the liver where L-FABP is highly expressed. PMID:23476633

The measured and calculated affinity of methyl and methoxy substituted benzoquinones for the QA site of bacterial reaction centers

PubMed Central

Zheng, Zhong; Dutton, P. Leslie; Gunner, M. R.

2010-01-01

Quinones play important roles in mitochondrial and photosynthetic energy conversion acting as intramembrane, mobile electron and proton carriers between catalytic sites in various electron transfer proteins. They display different affinity, selectivity, functionality and exchange dynamics in different binding sites. The computational analysis of quinone binding sheds light on the requirements for quinone affinity and specificity. The affinities of ten oxidized, neutral benzoquinones (BQs) were measured for the high affinity QA site in the detergent solubilized Rhodobacter sphaeroides bacterial photosynthetic reaction center. Multi-Conformation Continuum Electrostatics (MCCE) was then used to calculate their relative binding free energies by Grand Canonical Monte Carlo sampling with a rigid protein backbone, flexible ligand and side chain positions and protonation states. Van der Waals and torsion energies, Poisson-Boltzmann continuum electrostatics and accessible surface area dependent ligand-solvent interactions are considered. An initial, single cycle of GROMACS backbone optimization improves the match with experiment as do coupled ligand and side chain motions. The calculations match experiment with an RMSD of 2.29 and a slope of 1.28. The affinities are dominated by favorable protein-ligand van der Waals rather than electrostatic interactions. Each quinone appears in a closely clustered set of positions. Methyl and methoxy groups move into the same positions as found for the native quinone. Difficulties putting methyls into methoxy sites are observed. Calculations using an SAS dependent implicit van der Waals interaction smoothed out small clashes, providing a better match to experiment with a RMSD of 0.77 and a slope of 0.97. PMID:20607696

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

NASA Astrophysics Data System (ADS)

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-03-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071-40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands.

Acylated heptapeptide binds albumin with high affinity and application as tag furnishes long-acting peptides

NASA Astrophysics Data System (ADS)

Zorzi, Alessandro; Middendorp, Simon J.; Wilbs, Jonas; Deyle, Kaycie; Heinis, Christian

2017-07-01

The rapid renal clearance of peptides in vivo limits this attractive platform for the treatment of a broad range of diseases that require prolonged drug half-lives. An intriguing approach for extending peptide circulation times works through a `piggy-back' strategy in which peptides bind via a ligand to the long-lived serum protein albumin. In accordance with this strategy, we developed an easily synthesized albumin-binding ligand based on a peptide-fatty acid chimera that has a high affinity for human albumin (Kd=39 nM). This ligand prolongs the elimination half-life of cyclic peptides in rats 25-fold to over seven hours. Conjugation to a peptide factor XII inhibitor developed for anti-thrombotic therapy extends the half-life from 13 minutes to over five hours, inhibiting coagulation for eight hours in rabbits. This high-affinity albumin ligand could potentially extend the half-life of peptides in human to several days, substantially broadening the application range of peptides as therapeutics.

Identification and Characterization of a Suite of Tumor Targeting Peptides for Non-Small Cell Lung Cancer

PubMed Central

McGuire, Michael J.; Gray, Bethany Powell; Li, Shunzi; Cupka, Dorothy; Byers, Lauren Averett; Wu, Lei; Rezaie, Shaghayegh; Liu, Ying-Horng; Pattisapu, Naveen; Issac, James; Oyama, Tsukasa; Diao, Lixia; Heymach, John V.; Xie, Xian-Jin; Minna, John D.; Brown, Kathlynn C.

2014-01-01

Tumor targeting ligands are emerging components in cancer therapies. Widespread use of targeted therapies and molecular imaging is dependent on increasing the number of high affinity, tumor-specific ligands. Towards this goal, we biopanned three phage-displayed peptide libraries on a series of well-defined human non-small cell lung cancer (NSCLC) cell lines, isolating 11 novel peptides. The peptides show distinct binding profiles across 40 NSCLC cell lines and do not bind normal bronchial epithelial cell lines. Binding of specific peptides correlates with onco-genotypes and activation of particular pathways, such as EGFR signaling, suggesting the peptides may serve as surrogate markers. Multimerization of the peptides results in cell binding affinities between 0.0071–40 nM. The peptides home to tumors in vivo and bind to patient tumor samples. This is the first comprehensive biopanning for isolation of high affinity peptidic ligands for a single cancer type and expands the diversity of NSCLC targeting ligands. PMID:24670678

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System.

PubMed

Henseleit, Anja; Pohl, Carolin; Kaltenbach, Hans-Michael; Hettwer, Karina; Simon, Kirsten; Uhlig, Steffen; Haustein, Natalie; Bley, Thomas; Boschke, Elke

2015-01-19

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step.

Kinetic Analyses of Data from a Human Serum Albumin Assay Using the liSPR System

PubMed Central

Henseleit, Anja; Pohl, Carolin; Kaltenbach, Hans-Michael; Hettwer, Karina; Simon, Kirsten; Uhlig, Steffen; Haustein, Natalie; Bley, Thomas; Boschke, Elke

2015-01-01

We used the interaction between human serum albumin (HSA) and a high-affinity antibody to evaluate binding affinity measurements by the bench-top liSPR system (capitalis technology GmbH). HSA was immobilized directly onto a carboxylated sensor layer, and the mechanism of interaction between the antibody and HSA was investigated. The bivalence and heterogeneity of the antibody caused a complex binding mechanism. Three different interaction models (1:1 binding, heterogeneous analyte, bivalent analyte) were compared, and the bivalent analyte model best fit the curves obtained from the assay. This model describes the interaction of a bivalent analyte with one or two ligands (A + L ↔ LA + L ↔ LLA). The apparent binding affinity for this model measured 37 pM for the first reaction step, and 20 pM for the second step. PMID:25607476

D3R Grand Challenge 2: blind prediction of protein-ligand poses, affinity rankings, and relative binding free energies

NASA Astrophysics Data System (ADS)

Gaieb, Zied; Liu, Shuai; Gathiaka, Symon; Chiu, Michael; Yang, Huanwang; Shao, Chenghua; Feher, Victoria A.; Walters, W. Patrick; Kuhn, Bernd; Rudolph, Markus G.; Burley, Stephen K.; Gilson, Michael K.; Amaro, Rommie E.

2018-01-01

The Drug Design Data Resource (D3R) ran Grand Challenge 2 (GC2) from September 2016 through February 2017. This challenge was based on a dataset of structures and affinities for the nuclear receptor farnesoid X receptor (FXR), contributed by F. Hoffmann-La Roche. The dataset contained 102 IC50 values, spanning six orders of magnitude, and 36 high-resolution co-crystal structures with representatives of four major ligand classes. Strong global participation was evident, with 49 participants submitting 262 prediction submission packages in total. Procedurally, GC2 mimicked Grand Challenge 2015 (GC2015), with a Stage 1 subchallenge testing ligand pose prediction methods and ranking and scoring methods, and a Stage 2 subchallenge testing only ligand ranking and scoring methods after the release of all blinded co-crystal structures. Two smaller curated sets of 18 and 15 ligands were developed to test alchemical free energy methods. This overview summarizes all aspects of GC2, including the dataset details, challenge procedures, and participant results. We also consider implications for progress in the field, while highlighting methodological areas that merit continued development. Similar to GC2015, the outcome of GC2 underscores the pressing need for methods development in pose prediction, particularly for ligand scaffolds not currently represented in the Protein Data Bank (http://www.pdb.org), and in affinity ranking and scoring of bound ligands.

Affinity-tuning leukocyte integrin for development of safe therapeutics

NASA Astrophysics Data System (ADS)

Park, Spencer

Much attention has been given to the molecular and cellular pathways linking inflammation with cancer and the local tumor environment to identify new target molecules that could lead to improved diagnosis and treatment. Among the many molecular players involved in the complex response, central to the induction of inflammation is intercellular adhesion molecule (ICAM)-1, which is of particular interest for its highly sensitive and localized expression in response to inflammatory signals. ICAM-1, which has been implicated to play a critical role in tumor progression in various types of cancer, has also been linked to cancer metastases, where ICAM-1 facilitates the spread of metastatic cancer cells to secondary sites. This unique expression profile of ICAM-1 throughout solid tumor microenvironment makes ICAM-1 an intriguing molecular target, which holds great potential as an important diagnostic and therapeutic tool. Herein, we have engineered the ligand binding domain, or the inserted (I) domain of a leukocyte integrin, to exhibit a wide range of monovalent affinities to the natural ligand, ICAM-1. Using the resulting I domain variants, we have created drug and gene delivery nanoparticles, as well as targeted immunotherapeutics that have the ability to bind and migrate to inflammatory sites prevalent in tumors and the associated microenvironment. Through the delivery of diagnostic agents, chemotherapeutics, and immunotherapeutics, the following chapters demonstrate that the affinity enhancements achieved by directed evolution bring the affinity of I domains into the range optimal for numerous applications.

Efficient one-cycle affinity selection of binding proteins or peptides specific for a small-molecule using a T7 phage display pool.

PubMed

Takakusagi, Yoichi; Kuramochi, Kouji; Takagi, Manami; Kusayanagi, Tomoe; Manita, Daisuke; Ozawa, Hiroko; Iwakiri, Kanako; Takakusagi, Kaori; Miyano, Yuka; Nakazaki, Atsuo; Kobayashi, Susumu; Sugawara, Fumio; Sakaguchi, Kengo

2008-11-15

Here, we report an efficient one-cycle affinity selection using a natural-protein or random-peptide T7 phage pool for identification of binding proteins or peptides specific for small-molecules. The screening procedure involved a cuvette type 27-MHz quartz-crystal microbalance (QCM) apparatus with introduction of self-assembled monolayer (SAM) for a specific small-molecule immobilization on the gold electrode surface of a sensor chip. Using this apparatus, we attempted an affinity selection of proteins or peptides against synthetic ligand for FK506-binding protein (SLF) or irinotecan (Iri, CPT-11). An affinity selection using SLF-SAM and a natural-protein T7 phage pool successfully detected FK506-binding protein 12 (FKBP12)-displaying T7 phage after an interaction time of only 10 min. Extensive exploration of time-consuming wash and/or elution conditions together with several rounds of selection was not required. Furthermore, in the selection using a 15-mer random-peptide T7 phage pool and subsequent analysis utilizing receptor ligand contact (RELIC) software, a subset of SLF-selected peptides clearly pinpointed several amino-acid residues within the binding site of FKBP12. Likewise, a subset of Iri-selected peptides pinpointed part of the positive amino-acid region of residues from the Iri-binding site of the well-known direct targets, acetylcholinesterase (AChE) and carboxylesterase (CE). Our findings demonstrate the effectiveness of this method and general applicability for a wide range of small-molecules.

Design-Based Peptidomimetic Ligand Discovery to Target HIV TAR RNA Using Comparative Analysis of Different Docking Methods.

PubMed

Fu, Junjie; Xia, Amy; Dai, Yao; Qi, Xin

2016-01-01

Discovering molecules capable of binding to HIV trans-activation responsive region (TAR) RNA thereby disrupting its interaction with Tat protein is an attractive strategy for developing novel antiviral drugs. Computational docking is considered as a useful tool for predicting binding affinity and conducting virtual screening. Although great progress in predicting protein-ligand interactions has been achieved in the past few decades, modeling RNA-ligand interactions is still largely unexplored due to the highly flexible nature of RNA. In this work, we performed molecular docking study with HIV TAR RNA using previously identified cyclic peptide L22 and its analogues with varying affinities toward HIV-1 TAR RNA. Furthermore, sarcosine scan was conducted to generate derivatives of CGP64222, a peptide-peptoid hybrid with inhibitory activity on Tat/TAR RNA interaction. Each compound was docked using CDOCKER, Surflex-Dock and FlexiDock to compare the effectiveness of each method. It was found that FlexiDock energy values correlated well with the experimental Kd values and could be used to predict the affinity of the ligands toward HIV-1 TAR RNA with a superior accuracy. Our results based on comparative analysis of different docking methods in RNA-ligand modeling will facilitate the structure-based discovery of HIV TAR RNA ligands for antiviral therapy.

Into the theory of the partial-filling affinity capillary electrophoresis and the determination of apparent stability constants of analyte-ligand complexes.

PubMed

Ansorge, Martin; Dubský, Pavel; Ušelová, Kateřina

2018-03-01

The partial-filling affinity capillary electrophoresis (pf-ACE) works with a ligand present in a background electrolyte that forms a weak complex with an analyte. In contrast to a more popular mobility-shift affinity capillary electrophoresis, only a short plug of the ligand is introduced into a capillary in the pf-ACE. Both methods can serve for determining apparent stability constants of the formed complexes but this task is hindered in the pf-ACE by the fact that the analyte spends only a part of its migration time in a contact with the ligand. In 1998, Amini and Westerlund published a linearization strategy that allows for extracting an effective mobility of an analyte in the presence of a neutral ligand out of the pf-ACE data. The main purpose of this paper is to show that the original formula is only approximate. We derive a new formula and demonstrate its applicability by means of computer simulations. We further inspect several strategies of data processing in the pf-ACE regarding a risk of an error propagation. This establishes a good practice of determining apparent stability constants of analyte-ligand complexes by means of the pf-ACE. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Specific noncovalent interactions at protein-ligand interface: implications for rational drug design.

PubMed

Zhou, P; Huang, J; Tian, F

2012-01-01

Specific noncovalent interactions that are indicative of attractive, directional intermolecular forces have always been of key interest to medicinal chemists in their search for the "glue" that holds drugs and their targets together. With the rapid increase in the number of solved biomolecular structures as well as the performance enhancement of computer hardware and software in recent years, it is now possible to give more comprehensive insight into the geometrical characteristics and energetic landscape of certain sophisticated noncovalent interactions present at the binding interface of protein receptors and small ligands based on accumulated knowledge gaining from the combination of two quite disparate but complementary approaches: crystallographic data analysis and quantum-mechanical ab initio calculation. In this perspective, we survey massive body of published works relating to structural characterization and theoretical investigation of three kinds of strong, specific, direct, enthalpy-driven intermolecular forces, including hydrogen bond, halogen bond and salt bridge, involved in the formation of protein-ligand complex architecture in order to characterize their biological functions in conferring affinity and specificity for ligand recognition by host protein. In particular, the biomedical implications of raised knowledge are discussed with respect to potential applications in rational drug design.

TCDD, FICZ, and Other High Affinity AhR Ligands Dose-Dependently Determine the Fate of CD4+ T Cell Differentiation.

PubMed

Ehrlich, Allison K; Pennington, Jamie M; Bisson, William H; Kolluri, Siva K; Kerkvliet, Nancy I

2018-02-01

FICZ and TCDD, two high-affinity AhR ligands, are reported to have opposite effects on T cell differentiation with TCDD inducing regulatory T cells and FICZ inducing Th17 cells. This dichotomy has been attributed to ligand-intrinsic differences in AhR activation, although differences in sensitivity to metabolism complicate the issue. TCDD is resistant to AhR-induced metabolism and produces sustained AhR activation following a single dose in the μg/kg range, whereas FICZ is rapidly metabolized and AhR activation is transient. Nonetheless, prior studies comparing FICZ with TCDD have generally used the same 10-50 μg/kg dose range, and thus the two ligands would not equivalently activate AhR. We hypothesized that high-affinity AhR ligands can promote CD4+ T cell differentiation into both Th17 cells and Tregs, with fate depending on the extent and duration of AhR activation. We compared the immunosuppressive effects of TCDD and FICZ, along with two other rapidly metabolized ligands (ITE and 11-Cl-BBQ) in an acute alloresponse mouse model. The dose and timing of administration of each ligand was optimized for TCDD-equivalent Cyp1a1 induction. When optimized, all of the ligands suppressed the alloresponse in conjunction with the induction of Foxp3- Tr1 cells on day 2 and the expansion of natural Foxp3+ Tregs on day 10. In contrast, a low dose of FICZ induced transient expression of Cyp1a1 and did not induce Tregs or suppress the alloresponse but enhanced IL-17 production. Interestingly, low doses of the other ligands, including TCDD, also increased IL-17 production on day 10. These findings support the conclusion that the dose and the duration of AhR activation by high-affinity AhR ligands are the primary factors driving the fate of T cell differentiation. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Affinity labeling of the folate-methotrexate transporter from Leishmania donovani

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

Beck, J.T.; Ullman, B.

1989-08-22

An affinity labeling technique has been developed to identify the folate-methotrexate transporter of Leishmania donovani promastigotes using activated derivatives of the ligands. These activated derivatives were synthesized by incubating folate and methotrexate with a 10-fold excess of 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDC) for 10 min at ambient temperature in dimethyl sulfoxide. When intact wild-type (DI700) Leishmania donovani or preparations of their membranes were incubated with a 0.4 {mu}M concentration of either activated ({sup 3}H)folate or activated ({sup 3}H)methotrexate, the radiolabeled ligands were covalently incorporated into a polypeptide with a molecular weight of approximately 46,000, as demonstrated by SDS-polyacrylamide gel electrophoresis. No affinity labelingmore » of a 46,000-dalton protein was observed when equimolar concentrations of activated radiolabeled ligands were incubated with intact cells or membranes prepared from a methotrexate-resistant mutant clone of Leishmania donovani, MTXA5, that is genetically defective in folate-methotrexate transport capability. Time course studies indicated that maximal labeling of the 46,000-dalton protein occurred within 5-10 min of incubation of intact cells with activated ligand. These studies provide biochemical evidence that the folate-methotrexate transporter of Leishmania donovani can be identified in crude extracts by an affinity labeling technique and serve as a prerequisite to further analysis of the transport protein by providing a vehicle for subsequent purification of this membrane carrier. Moreover, these investigations suggest that the affinity labeling technique using EDC-activated ligands may be exploitable to analyze other cell surface binding proteins in Leishmania donovani, as well as in other organisms.« less

Harnessing Fluorine–Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue Drugs

PubMed Central

2016-01-01

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a KD = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine–protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C–PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol–1 atom–1). High-resolution crystal structures of the Y220C–ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein–fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells. PMID:27267810

Reproducing Crystal Binding Modes of Ligand Functional Groups using Site-Identification by Ligand Competitive Saturation (SILCS) Simulations

PubMed Central

Raman, E. Prabhu; Yu, Wenbo; Guvench, Olgun; MacKerell, Alexander D.

2011-01-01

The applicability of a computational method, Site Identification by Ligand Competitive Saturation (SILCS), to identify regions on a protein surface with which different types of functional groups on low-molecular weight inhibitors interact is demonstrated. The method involves molecular dynamics (MD) simulations of a protein in an aqueous solution of chemically diverse small molecules from which probability distributions of fragments types, termed FragMaps, are obtained. In the present application, SILCS simulations are performed with an aqueous solution of 1 M benzene and propane to map the affinity pattern of the protein for aromatic and aliphatic functional groups. In addition, water hydrogen and oxygen atoms serve as probes for hydrogen bond donor and acceptor affinity, respectively. The method is tested using a set of 7 proteins for which crystal structures of complexes with several high affinity inhibitors are known. Good agreement is obtained between FragMaps and the positions of chemically similar functional groups in inhibitors as observed in the X-ray crystallographic structures. Quantitative capabilities of the SILCS approach are demonstrated by converting FragMaps to free energies, termed Grid Free Energies (GFE), and showing correlation between the GFE values and experimental binding affinities. For proteins for which ligand decoy sets are available, GFE values are shown to typically score the crystal conformation and conformations similar to it more favorable than decoys. Additionally, SILCS is tested for its ability to capture the subtle differences in ligand affinity across homologous proteins, information which may be of utility towards specificity-guided drug design. Taken together, our results show that SILCS can recapitulate the known location of functional groups of bound inhibitors for a number of proteins, suggesting that the method may be of utility for rational drug design. PMID:21456594

Effect of the Flexible Regions of the Oncoprotein Mouse Double Minute X on Inhibitor Binding Affinity.

PubMed

Qin, Lingyun; Liu, Huili; Chen, Rong; Zhou, Jingjing; Cheng, Xiyao; Chen, Yao; Huang, Yongqi; Su, Zhengding

2017-11-07

The oncoprotein MdmX (mouse double minute X) is highly homologous to Mdm2 (mouse double minute 2) in terms of their amino acid sequences and three-dimensional conformations, but Mdm2 inhibitors exhibit very weak affinity for MdmX, providing an excellent model for exploring how protein conformation distinguishes and alters inhibitor binding. The intrinsic conformation flexibility of proteins plays pivotal roles in determining and predicting the binding properties and the design of inhibitors. Although the molecular dynamics simulation approach enables us to understand protein-ligand interactions, the mechanism underlying how a flexible binding pocket adapts an inhibitor has been less explored experimentally. In this work, we have investigated how the intrinsic flexible regions of the N-terminal domain of MdmX (N-MdmX) affect the affinity of the Mdm2 inhibitor nutlin-3a using protein engineering. Guided by heteronuclear nuclear Overhauser effect measurements, we identified the flexible regions that affect inhibitor binding affinity around the ligand-binding pocket on N-MdmX. A disulfide engineering mutant, N-MdmX C25-C110/C76-C88 , which incorporated two staples to rigidify the ligand-binding pocket, allowed an affinity for nutlin-3a higher than that of wild-type N-MdmX (K d ∼ 0.48 vs K d ∼ 20.3 μM). Therefore, this mutant provides not only an effective protein model for screening and designing of MdmX inhibitors but also a valuable clue for enhancing the intermolecular interactions of the pharmacophores of a ligand with pronounced flexible regions. In addition, our results revealed an allosteric ligand-binding mechanism of N-MdmX in which the ligand initially interacts with a compact core, followed by augmenting intermolecular interactions with intrinsic flexible regions. This strategy should also be applicable to many other protein targets to accelerate drug discovery.

Discovery of Tertiary Sulfonamides as Potent Liver X Receptor Antagonists

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

Zuercher, William J.; Buckholz†, Richard G.; Campobasso, Nino

2010-08-12

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Discovery of tertiary sulfonamides as potent liver X receptor antagonists.

PubMed

Zuercher, William J; Buckholz, Richard G; Campobasso, Nino; Collins, Jon L; Galardi, Cristin M; Gampe, Robert T; Hyatt, Stephen M; Merrihew, Susan L; Moore, John T; Oplinger, Jeffrey A; Reid, Paul R; Spearing, Paul K; Stanley, Thomas B; Stewart, Eugene L; Willson, Timothy M

2010-04-22

Tertiary sulfonamides were identified in a HTS as dual liver X receptor (LXR, NR1H2, and NR1H3) ligands, and the binding affinity of the series was increased through iterative analogue synthesis. A ligand-bound cocrystal structure was determined which elucidated key interactions for high binding affinity. Further characterization of the tertiary sulfonamide series led to the identification of high affinity LXR antagonists. GSK2033 (17) is the first potent cell-active LXR antagonist described to date. 17 may be a useful chemical probe to explore the cell biology of this orphan nuclear receptor.

Recognition of peptide–MHC class I complexes by activating killer immunoglobulin-like receptors

PubMed Central

Stewart, C. Andrew; Laugier-Anfossi, Fanny; Vély, Frédéric; Saulquin, Xavier; Riedmuller, Jenifer; Tisserant, Agnès; Gauthier, Laurent; Romagné, François; Ferracci, Géraldine; Arosa, Fernando A.; Moretta, Alessandro; Sun, Peter D.; Ugolini, Sophie; Vivier, Eric

2005-01-01

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein–Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide–MHC class I complexes on Epstein–Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders. PMID:16141329

Recognition of peptide-MHC class I complexes by activating killer immunoglobulin-like receptors.

PubMed

Stewart, C Andrew; Laugier-Anfossi, Fanny; Vély, Frédéric; Saulquin, Xavier; Riedmuller, Jenifer; Tisserant, Agnès; Gauthier, Laurent; Romagné, François; Ferracci, Géraldine; Arosa, Fernando A; Moretta, Alessandro; Sun, Peter D; Ugolini, Sophie; Vivier, Eric

2005-09-13

Inhibitory receptors for MHC class I molecules increase the threshold of lymphocyte activation. Natural Killer (NK) cells express a large number of such inhibitory receptors, including the human killer Ig-like receptors (KIR). However, activating members of the KIR family have poorly defined ligands and functions. Here we describe the use of activating KIR tetramer reagents as probes to detect their ligands. Infection of cells with Epstein-Barr virus leads to expression of a detectable ligand for the activating receptor KIR2DS1. In this case, KIR2DS1 interacts with up-regulated peptide-MHC class I complexes on Epstein-Barr virus-infected cells in a transporter associated with antigen processing (TAP)-dependent manner. In tetramer-based cellular assays and direct affinity measurements, this interaction with MHC class I is facilitated by a broad spectrum of peptides. KIR2DS1 and its inhibitory homologue, KIR2DL1, share sensitivity to peptide sequence alterations at positions 7 and 8. These results fit a model in which activating and inhibitory receptors recognize the same sets of self-MHC class I molecules, differing only in their binding affinities. Importantly, KIR2DS1 is not always sufficient to trigger NK effector responses when faced with cognate ligand, consistent with fine control during NK cell activation. We discuss how our results for KIR2DS1 and parallel studies on KIR2DS2 relate to the association between activating KIR genes and susceptibility to autoimmune disorders.

An engineered Axl 'decoy receptor' effectively silences the Gas6-Axl signaling axis

DOE PAGES

Kariolis, Mihalis S.; Miao, Yu Rebecca; Jones, Douglas S.; ...

2014-09-21

Aberrant signaling through the Axl receptor tyrosine kinase has been associated with a myriad of human diseases, most notably metastatic cancer, identifying Axl and its ligand Gas6 as important therapeutic targets. Using rational and combinatorial approaches, we engineered an Axl ‘decoy receptor’ that binds Gas6 with high affinity and inhibits its function, offering an alternative approach from drug discovery efforts that directly target Axl. Four mutations within this high affinity Axl variant caused structural alterations in side chains across the Gas6/Axl binding interface, stabilizing a conformational change on Gas6. When reformatted as an Fc-fusion, the engineered decoy receptor bound tomore » Gas6 with femtomolar affinity, an 80-fold improvement compared to the wild-type Axl receptor, allowing effective sequestration of Gas6 and specific abrogation of Axl signaling. Additionally, increased Gas6 binding affinity was critical and correlative with the ability of decoy receptors to potently inhibit metastasis and disease progression in vivo.« less

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.

Influence of differentiation on muscarinic receptors in N1E 115 neuroblastoma cells.

PubMed

Buyse, M A; Lefebvre, R A; Fraeyman, N H

1989-01-01

The effect of inducing morphological differentiation in N1E 115 mouse neuroblastoma cells on the number of muscarinic receptors and the ligand binding affinity was investigated using the lipophylic quinuclidinyl benzylate and the hydrophylic N-methylscopolamine as tritiated ligands. Induction of morphological differentiation was accompanied by a two- to three-fold increase of the number of receptors when assayed in a broken cell preparation; the ligand binding affinity was unaffected by differentiation. Using intact cells, this increase was not paralleled by a similar increase in binding sites accessible for N-methylscopolamine, which binds preferentially to extracellular sites.

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.

Improving binding mode and binding affinity predictions of docking by ligand-based search of protein conformations: evaluation in D3R grand challenge 2015

NASA Astrophysics Data System (ADS)

Xu, Xianjin; Yan, Chengfei; Zou, Xiaoqin

2017-08-01

The growing number of protein-ligand complex structures, particularly the structures of proteins co-bound with different ligands, in the Protein Data Bank helps us tackle two major challenges in molecular docking studies: the protein flexibility and the scoring function. Here, we introduced a systematic strategy by using the information embedded in the known protein-ligand complex structures to improve both binding mode and binding affinity predictions. Specifically, a ligand similarity calculation method was employed to search a receptor structure with a bound ligand sharing high similarity with the query ligand for the docking use. The strategy was applied to the two datasets (HSP90 and MAP4K4) in recent D3R Grand Challenge 2015. In addition, for the HSP90 dataset, a system-specific scoring function (ITScore2_hsp90) was generated by recalibrating our statistical potential-based scoring function (ITScore2) using the known protein-ligand complex structures and the statistical mechanics-based iterative method. For the HSP90 dataset, better performances were achieved for both binding mode and binding affinity predictions comparing with the original ITScore2 and with ensemble docking. For the MAP4K4 dataset, although there were only eight known protein-ligand complex structures, our docking strategy achieved a comparable performance with ensemble docking. Our method for receptor conformational selection and iterative method for the development of system-specific statistical potential-based scoring functions can be easily applied to other protein targets that have a number of protein-ligand complex structures available to improve predictions on binding.

Cognitive impairments of alcoholic cirrhotic patients: correlation with endogenous benzodiazepine receptor ligands and increased affinity of platelet receptors.

PubMed Central

Kapczinski, F; Curran, H V; Przemioslo, R; Williams, R; Fluck, E; Fernandes, C; File, S E

1996-01-01

OBJECTIVES--To determine whether differences in cognitive function between alcoholic and non-alcoholic cirrhotic patients relate to differences in endogenous ligands for the benzodiazepine receptor and/or benzodiazepine binding. METHODS--Seventeen grade-I hepatic encephalopathic patients (nine alcoholic, eight non-alcoholic) were compared with 10 matched controls on plasma concentrations of endogenous ligands for the neuronal benzodiazepine receptor, benzodiazepine binding in platelets, and performance on tests of cognitive function. RESULTS--Both groups of patients were impaired on verbal recall and on reaction time tasks compared with controls; alcoholic patients were also impaired on Reitan's trails test and digit cancellation. Four of the 17 patients had detectable concentrations of endogenous benzodiazepine ligands and they were more impaired than other patients on trails and cancellation tests. The groups did not differ in the density of benzodiazepine platelet receptors, but receptor affinity was higher in alcoholic patients than in controls; furthermore, receptor affinity correlated with the time to complete the cancellation task and with reaction time. CONCLUSION--Alcoholic cirrhotic patients may have enhanced concentrations of ligands for neuronal and peripheral benzodiazepine receptors and these may contribute to cognitive impairments in these patients. PMID:8648337

Low density and high affinity of platelet [3H]paroxetine binding in women with bulimia nervosa.

PubMed

Ekman, Agneta; Sundblad-Elverfors, Charlotta; Landén, Mikael; Eriksson, Tomas; Eriksson, Elias

2006-06-15

Impaired serotonin transmission has been suggested to be implicated in the pathophysiology of bulimia nervosa. As an indirect measure of brain serotonergic activity, the binding of tritiated ligands to platelet serotonin transporters has been studied in bulimia nervosa as well as in other putatively serotonin-related psychiatric disorders. In this study, the density and affinity of platelet serotonin transporters were assessed in 20 women meeting the DSM-IV criteria for bulimia nervosa and in 14 controls without previous or ongoing eating disorder using [(3)H]paroxetine as a ligand. In comparison to controls, women with bulimia nervosa had a significantly reduced number of platelet binding sites (B(max) = 721 +/- 313 vs. 1145 +/- 293 fmol/mg protein) and an increase in the affinity for the ligand demonstrated by a lower dissociaton constant (K(d) = 33 +/- 10 vs. 44 +/- 10 pM). A significant correlation between B(max) and K(d) values was found in patients but not in controls. Our results support the notion that bulimia nervosa is associated with a reduction in platelet serotonin transporter density. In addition, our study is the first to report that this reduced transporter density in women with bulimia nervosa is accompanied by an increase in the affinity of the transporter for the ligand.

DNA aptamer affinity ligands for highly selective purification of human plasma-related proteins from multiple sources.

PubMed

Forier, Cynthia; Boschetti, Egisto; Ouhammouch, Mohamed; Cibiel, Agnès; Ducongé, Frédéric; Nogré, Michel; Tellier, Michel; Bataille, Damien; Bihoreau, Nicolas; Santambien, Patrick; Chtourou, Sami; Perret, Gérald

2017-03-17

Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Allelic variation in KIR2DL3 generates a KIR2DL2-like receptor with increased binding to its HLA-C ligand.

PubMed

Frazier, William R; Steiner, Noriko; Hou, Lihua; Dakshanamurthy, Sivanesan; Hurley, Carolyn Katovich

2013-06-15

Although extensive homology exists between their extracellular domains, NK cell inhibitory receptors killer Ig-like receptor (KIR) 2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared with KIR2DL3*001, a low-affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 toward that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations.

Allelic Variation in KIR2DL3 Generates a KIR2DL2-like Receptor with Increased Binding to Its HLA-C Ligand12

PubMed Central

Frazier, William R.; Steiner, Noriko; Hou, Lihua; Dakshanamurthy, Sivanesan; Hurley, Carolyn Katovich

2013-01-01

Although extensive homology exists between their extracellular domains, natural killer cell inhibitory receptors KIR2DL2*001 and KIR2DL3*001 have previously been shown to differ substantially in their HLA-C binding avidity. To explore the largely uncharacterized impact of allelic diversity, the most common KIR2DL2/3 allelic products in European American and African American populations were evaluated for surface expression and binding affinity to their HLA-C group 1 and 2 ligands. Although no significant differences in the degree of cell membrane localization were detected in a transfected human NKL cell line by flow cytometry, surface plasmon resonance and KIR binding to a panel of HLA allotypes demonstrated that KIR2DL3*005 differed significantly from other KIR2DL3 allelic products in its ability to bind HLA-C. The increased affinity and avidity of KIR2DL3*005 for its ligand was also demonstrated to have a larger impact on the inhibition of IFN-γ production by the human KHYG-1 NK cell line compared to KIR2DL3*001, a low affinity allelic product. Site-directed mutagenesis established that the combination of arginine at residue 11 and glutamic acid at residue 35 in KIR2DL3*005 were critical to the observed phenotype. Although these residues are distal to the KIR/HLA-C interface, molecular modeling suggests that alteration in the interdomain hinge angle of KIR2DL3*005 towards that found in KIR2DL2*001, another strong receptor of the KIR2DL2/3 family, may be the cause of this increased affinity. The regain of inhibitory capacity by KIR2DL3*005 suggests that the rapidly evolving KIR locus may be responding to relatively recent selective pressures placed upon certain human populations. PMID:23686481

Ether modifications to 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503): effects on binding affinity and selectivity for sigma receptors and monoamine transporters.

PubMed

Xu, Rong; Lord, Sarah A; Peterson, Ryan M; Fergason-Cantrell, Emily A; Lever, John R; Lever, Susan Z

2015-01-01

Two series of novel ether analogs of the sigma (σ) receptor ligand 1-[2-(3,4-dimethoxyphenyl)ethyl]-4-(3-phenylpropyl)piperazine (SA4503) have been prepared. In one series, the alkyl portion of the 4-methoxy group was replaced with allyl, propyl, bromoethyl, benzyl, phenethyl, and phenylpropyl moieties. In the second series, the 3,4-dimethoxy was replaced with cyclic methylenedioxy, ethylenedioxy and propylenedioxy groups. These ligands, along with 4-O-des-methyl SA4503, were evaluated for σ1 and σ2 receptor affinity, and compared to SA4503 and several known ether analogs. SA4503 and a subset of ether analogs were also evaluated for dopamine transporter (DAT) and serotonin transporter (SERT) affinity. The highest σ1 receptor affinities, Ki values of 1.75-4.63 nM, were observed for 4-O-des-methyl SA4503, SA4503 and the methylenedioxy analog. As steric bulk increased, σ1 receptor affinity decreased, but only to a point. Allyl, propyl and bromoethyl substitutions gave σ1 receptor Ki values in the 20-30 nM range, while bulkier analogs having phenylalkyl, and Z- and E-iodoallyl, ether substitutions showed higher σ1 affinities, with Ki values in the 13-21 nM range. Most ligands studied exhibited comparable σ1 and σ2 affinities, resulting in little to no subtype selectivity. SA4503, the fluoroethyl analog and the methylenedioxy congener showed modest six- to fourteen-fold selectivity for σ1 sites. DAT and SERT interactions proved much more sensitive than σ receptor interactions to these structural modifications. For example, the benzyl congener (σ1Ki=20.8 nM; σ2Ki=16.4 nM) showed over 100-fold higher DAT affinity (Ki=121 nM) and 6-fold higher SERT affinity (Ki=128nM) than the parent SA4503 (DAT Ki=12650 nM; SERT Ki=760 nM). Thus, ether modifications to the SA4503 scaffold can provide polyfunctional ligands having a broader spectrum of possible pharmacological actions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lactose-containing starburst dendrimers: influence of dendrimer generation and binding-site orientation of receptors (plant/animal lectins and immunoglobulins) on binding properties.

PubMed

André, S; Ortega, P J; Perez, M A; Roy, R; Gabius, H J

1999-11-01

Starburst glycodendrimers offer the potential to serve as high-affinity ligands for clinically relevant sugar receptors. In order to define areas of application, their binding behavior towards sugar receptors with differential binding-site orientation but identical monosaccharide specificity must be evaluated. Using poly(amidoamine) starburst dendrimers of five generations, which contain the p-isothiocyanato derivative of p-aminophenyl-beta-D-lactoside as ligand group, four different types of galactoside-binding proteins were chosen for this purpose, i.e., the (AB)(2)-toxic agglutinin from mistletoe, a human immunoglobulin G fraction, the homodimeric galectin-1 with its two binding sites at opposite ends of the jelly-roll-motif-harboring protein and monomeric galectin-3. Direct solid-phase assays with surface-immobilized glycodendrimers resulted in obvious affinity enhancements by progressive core branching for the plant agglutinin and less pronounced for the antibody and galectin-1. High density of binding of galectin-3 with modest affinity increases only from the level of the 32-mer onwards points to favorable protein-protein interactions of the monomeric lectin and a spherical display of the end groups without a major share of backfolding. When the inhibitory potency of these probes was evaluated as competitor of receptor binding to an immobilized neoglycoprotein or to asialofetuin, a marked selectivity was detected. The 32- and 64-mers were second to none as inhibitors for the plant agglutinin against both ligand-exposing matrices and for galectin-1 on the matrix with a heterogeneous array of interglycoside distances even on the per-sugar basis. In contrast, a neoglycoprotein with the same end group was superior in the case of the antibody and, less pronounced, monomeric galectin-3. Intimate details of topological binding-site presentation and the ligand display on different generations of core assembly are major operative factors which determine the potential of dendrimers for applications as lectin-targeting device, as attested by these observations.

Interactions of ligands with active and inactive conformations of the dopamine D2 receptor.

PubMed

Malmberg, A; Mohell, N; Backlund Höök, B; Johansson, A M; Hacksell, U; Nordvall, G

1998-04-10

The affinities of 19 pharmacologically diverse dopamine D2 receptor ligands were determined for the active and inactive conformations of cloned human dopamine D2 receptors expressed in Ltk cells. The agonist [3H]quinpirole was used to selectively label the guanine nucleotide-binding protein-coupled, active receptor conformation. The antagonist [3H]raclopride, in the presence of the non-hydrolysable GTP-analogue Gpp(NH)p and sodium ions and in the absence of magnesium ions, was used to label the free inactive receptor conformation. The intrinsic activities of the ligands were determined in a forskolin-stimulated cyclic AMP assay using the same cells. An excellent correlation was shown between the affinity ratios (KR/KRG) of the ligands for the two receptor conformations and their intrinsic activity (r=0.96). The ligands included eight structurally related and enantiopure 2-aminotetralin derivatives; the enantiomers of 5-hydroxy-2-(dipropylamino)tetralin, 5-methoxy-2-(dipropylamino)tetralin, 5-fluoro-2-(dipropylamino)tetralin and 2-(dipropylamino)tetralin. The (S)-enantiomers behaved as full agonists in the cyclic AMP assay and displayed a large KR/KRG ratio. The (R)-enantiomers were classified as partial agonists and had lower ratios. The structure-affinity relationships of these compounds at the active and the inactive receptor conformations were analysed separately, and used in conjunction with a homology based receptor model of the dopamine D2 receptor. This led to proposed binding modes for agonists, antagonists and partial agonists in the 2-aminotetralin series. The concepts used in this study should be of value in the design of ligands with predetermined affinity and intrinsic activity.

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015.

PubMed

Deng, Nanjie; Flynn, William F; Xia, Junchao; Vijayan, R S K; Zhang, Baofeng; He, Peng; Mentes, Ahmet; Gallicchio, Emilio; Levy, Ronald M

2016-09-01

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.

Large scale free energy calculations for blind predictions of protein-ligand binding: the D3R Grand Challenge 2015

NASA Astrophysics Data System (ADS)

Deng, Nanjie; Flynn, William F.; Xia, Junchao; Vijayan, R. S. K.; Zhang, Baofeng; He, Peng; Mentes, Ahmet; Gallicchio, Emilio; Levy, Ronald M.

2016-09-01

We describe binding free energy calculations in the D3R Grand Challenge 2015 for blind prediction of the binding affinities of 180 ligands to Hsp90. The present D3R challenge was built around experimental datasets involving Heat shock protein (Hsp) 90, an ATP-dependent molecular chaperone which is an important anticancer drug target. The Hsp90 ATP binding site is known to be a challenging target for accurate calculations of ligand binding affinities because of the ligand-dependent conformational changes in the binding site, the presence of ordered waters and the broad chemical diversity of ligands that can bind at this site. Our primary focus here is to distinguish binders from nonbinders. Large scale absolute binding free energy calculations that cover over 3000 protein-ligand complexes were performed using the BEDAM method starting from docked structures generated by Glide docking. Although the ligand dataset in this study resembles an intermediate to late stage lead optimization project while the BEDAM method is mainly developed for early stage virtual screening of hit molecules, the BEDAM binding free energy scoring has resulted in a moderate enrichment of ligand screening against this challenging drug target. Results show that, using a statistical mechanics based free energy method like BEDAM starting from docked poses offers better enrichment than classical docking scoring functions and rescoring methods like Prime MM-GBSA for the Hsp90 data set in this blind challenge. Importantly, among the three methods tested here, only the mean value of the BEDAM binding free energy scores is able to separate the large group of binders from the small group of nonbinders with a gap of 2.4 kcal/mol. None of the three methods that we have tested provided accurate ranking of the affinities of the 147 active compounds. We discuss the possible sources of errors in the binding free energy calculations. The study suggests that BEDAM can be used strategically to discriminate binders from nonbinders in virtual screening and to more accurately predict the ligand binding modes prior to the more computationally expensive FEP calculations of binding affinity.

Methods for quantifying T cell receptor binding affinities and thermodynamics

PubMed Central

Piepenbrink, Kurt H.; Gloor, Brian E.; Armstrong, Kathryn M.; Baker, Brian M.

2013-01-01

αβ T cell receptors (TCRs) recognize peptide antigens bound and presented by class I or class II major histocompatibility complex (MHC) proteins. Recognition of a peptide/MHC complex is required for initiation and propagation of a cellular immune response, as well as the development and maintenance of the T cell repertoire. Here we discuss methods to quantify the affinities and thermodynamics of interactions between soluble ectodomains of TCRs and their peptide/MHC ligands, focusing on titration calorimetry, surface plasmon resonance, and fluorescence anisotropy. As TCRs typically bind ligand with weak-to-moderate affinities, we focus the discussion on means to enhance the accuracy and precision of low affinity measurements. In addition to further elucidating the biology of the T cell mediated immune response, more reliable low affinity measurements will aid with more probing studies with mutants or altered peptides that can help illuminate the physical underpinnings of how TCRs achieve their remarkable recognition properties. PMID:21609868

Structural Basis of the High Affinity Interaction between the Alphavirus Nonstructural Protein-3 (nsP3) and the SH3 Domain of Amphiphysin-2.

PubMed

Tossavainen, Helena; Aitio, Olli; Hellman, Maarit; Saksela, Kalle; Permi, Perttu

2016-07-29

We show that a peptide from Chikungunya virus nsP3 protein spanning residues 1728-1744 binds the amphiphysin-2 (BIN1) Src homology-3 (SH3) domain with an unusually high affinity (Kd 24 nm). Our NMR solution complex structure together with isothermal titration calorimetry data on several related viral and cellular peptide ligands reveal that this exceptional affinity originates from interactions between multiple basic residues in the target peptide and the extensive negatively charged binding surface of amphiphysin-2 SH3. Remarkably, these arginines show no fixed conformation in the complex structure, indicating that a transient or fluctuating polyelectrostatic interaction accounts for this affinity. Thus, via optimization of such dynamic electrostatic forces, viral peptides have evolved a superior binding affinity for amphiphysin-2 SH3 compared with typical cellular ligands, such as dynamin, thereby enabling hijacking of amphiphysin-2 SH3-regulated host cell processes by these viruses. Moreover, our data show that the previously described consensus sequence PXRPXR for amphiphysin SH3 ligands is inaccurate and instead define it as an extended Class II binding motif PXXPXRpXR, where additional positive charges between the two constant arginine residues can give rise to extraordinary high SH3 binding affinity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Rational design of peptide affinity ligands for the purification of therapeutic enzymes.

PubMed

Trasatti, John P; Woo, James; Ladiwala, Asif; Cramer, Steven; Karande, Pankaj

2018-04-25

Non-mAb biologics represent a growing class of therapeutics under clinical development. Although affinity chromatography is a potentially attractive approach for purification, the development of platform technologies, such as Protein A for mAbs, has been challenging due to the inherent chemical and structural diversity of these molecules. Here, we present our studies on the rapid development of peptide affinity ligands for the purification of biologics using a prototypical enzyme therapeutic in clinical use. Employing a suite of de novo rational and combinatorial design strategies we designed and screened a library of peptides on microarray platforms for their ability to bind to the target with high affinity and selectivity in cell culture fluid. Lead peptides were evaluated on resin in batch conditions and compared with a commercially available resin to evaluate their efficacy. Two lead candidates identified from microarray studies provided high binding capacity to the target while demonstrating high selectivity against culture contaminants and product variants compared to a commercial resin system. These findings provide a proof-of-concept for developing affinity peptide-based bioseparations processes for a target biologic. Peptide affinity ligand design and screening approaches presented in this work can also be easily translated to other biologics of interest. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018. © 2018 American Institute of Chemical Engineers.

Quantifying domain-ligand affinities and specificities by high-throughput holdup assay

PubMed Central

Vincentelli, Renaud; Luck, Katja; Poirson, Juline; Polanowska, Jolanta; Abdat, Julie; Blémont, Marilyne; Turchetto, Jeremy; Iv, François; Ricquier, Kevin; Straub, Marie-Laure; Forster, Anne; Cassonnet, Patricia; Borg, Jean-Paul; Jacob, Yves; Masson, Murielle; Nominé, Yves; Reboul, Jérôme; Wolff, Nicolas; Charbonnier, Sebastian; Travé, Gilles

2015-01-01

Many protein interactions are mediated by small linear motifs interacting specifically with defined families of globular domains. Quantifying the specificity of a motif requires measuring and comparing its binding affinities to all its putative target domains. To this aim, we developed the high-throughput holdup assay, a chromatographic approach that can measure up to a thousand domain-motif equilibrium binding affinities per day. Extracts of overexpressed domains are incubated with peptide-coated resins and subjected to filtration. Binding affinities are deduced from microfluidic capillary electrophoresis of flow-throughs. After benchmarking the approach on 210 PDZ-peptide pairs with known affinities, we determined the affinities of two viral PDZ-binding motifs derived from Human Papillomavirus E6 oncoproteins for 209 PDZ domains covering 79% of the human PDZome. We obtained exquisite sequence-dependent binding profiles, describing quantitatively the PDZome recognition specificity of each motif. This approach, applicable to many categories of domain-ligand interactions, has a wide potential for quantifying the specificities of interactomes. PMID:26053890

A theoretical and experimental approach toward the development of affinity adsorbents for GFP and GFP-fusion proteins purification.

PubMed

Fernandes, Cláudia S M; Pina, Ana Sofia; Dias, Ana M G C; Branco, Ricardo J F; Roque, Ana Cecília Afonso

2014-09-30

The green fluorescent protein (GFP) is widely employed to report on a variety of molecular phenomena, but its selective recovery is hampered by the lack of a low-cost and robust purification alternative. This work reports an integrated approach combining rational design and experimental validation toward the optimization of a small fully-synthetic ligand for GFP purification. A total of 56 affinity ligands based on a first-generation lead structure were rationally designed through molecular modeling protocols. The library of ligands was further synthesized by solid-phase combinatorial methods based on the Ugi reaction and screened against Escherichia coli extracts containing GFP. Ligands A4C2, A5C5 and A5C6 emerged as the new lead structures based on the high estimated theoretical affinity constants and the high GFP binding percentages and enrichment factors. The elution of GFP from these adsorbents was further characterized, where the best compromise between mild elution conditions, yield and purity was found for ligands A5C5 and A5C6. These were tested for purifying a model GFP-fusion protein, where ligand A5C5 yielded higher protein recovery and purity. The molecular interactions between the lead ligands and GFP were further assessed by molecular dynamics simulations, showing a wide range of potential hydrophobic and hydrogen-bond interactions. Copyright © 2014 Elsevier B.V. All rights reserved.

Calculating the sensitivity and robustness of binding free energy calculations to force field parameters

PubMed Central

Rocklin, Gabriel J.; Mobley, David L.; Dill, Ken A.

2013-01-01

Binding free energy calculations offer a thermodynamically rigorous method to compute protein-ligand binding, and they depend on empirical force fields with hundreds of parameters. We examined the sensitivity of computed binding free energies to the ligand’s electrostatic and van der Waals parameters. Dielectric screening and cancellation of effects between ligand-protein and ligand-solvent interactions reduce the parameter sensitivity of binding affinity by 65%, compared with interaction strengths computed in the gas-phase. However, multiple changes to parameters combine additively on average, which can lead to large changes in overall affinity from many small changes to parameters. Using these results, we estimate that random, uncorrelated errors in force field nonbonded parameters must be smaller than 0.02 e per charge, 0.06 Å per radius, and 0.01 kcal/mol per well depth in order to obtain 68% (one standard deviation) confidence that a computed affinity for a moderately-sized lead compound will fall within 1 kcal/mol of the true affinity, if these are the only sources of error considered. PMID:24015114

Direct capture of His₆-tagged proteins using megaporous cryogels developed for metal-ion affinity chromatography.

PubMed

Singh, Naveen Kumar; DSouza, Roy N; Bibi, Noor Shad; Fernández-Lahore, Marcelo

2015-01-01

Immobilized metal-ion affinity chromatography (IMAC) has been developed for the rapid isolation and purification of recombinant proteins. In this chapter, megaporous cryogels were synthesized having metal-ion affinity functionality, and their adsorptive properties were investigated. These cryogels have large pore sizes ranging from 10 to 100 μm with corresponding porosities between 80 and 90%. The synthesized IMAC-cryogel had a total ligand density of 770 μmol/g. Twelve milligram of a His6-tagged protein (NAD(P)H-dependent 2-cyclohexen-1-one-reductase) can be purified from a crude cell extract per gram of IMAC-cryogels. The protein binding capacity is increased with higher degrees of grafting, although a slight decrease in column efficiency may result. This chapter provides methodologies for a rapid single-step purification of recombinant His6-tagged proteins from crude cell extracts using IMAC-cryogels.

Synthesis, characterization and binding affinities of rhenium(I) thiosemicarbazone complexes for the estrogen receptor (α/β).

PubMed

Núñez-Montenegro, Ara; Carballo, Rosa; Vázquez-López, Ezequiel M

2014-11-01

The binding affinities towards estrogen receptors (ERs) α and β of a set of thiosemicarbazone ligands (HL(n)) and their rhenium(I) carbonyl complexes [ReX(HL(n))(CO)3] (X=Cl, Br) were determined by a competitive standard radiometric assay with [(3)H]-estradiol. The ability of the coordinated thiosemicarbazone ligands to undergo deprotonation and the lability of the ReX bond were used as a synthetic strategy to obtain [Re(hpy)(L(n))(CO)3] (hpy=3- or 4-hydroxypyridine). The inclusion of the additional hpy ligand endows the new thiosemicarbazonate complexes with an improved affinity towards the estrogen receptors and, consequently, the values of the inhibition constant (Ki) could be determined for some of them. In general, the values of Ki for both ER subtypes suggest an appreciable selectivity towards ERα. Copyright © 2014 Elsevier Inc. All rights reserved.

3-Arylpiperazinylethyl-1H-pyrrolo[2,3-d]pyrimidine-2,4(3H,7H)-dione derivatives as novel, high-affinity and selective alpha(1)-adrenoceptor ligands.

PubMed

Pittalà, Valeria; Romeo, Giuseppe; Salerno, Loredana; Siracusa, Maria Angela; Modica, Maria; Materia, Luisa; Mereghetti, Ilario; Cagnotto, Alfredo; Mennini, Tiziana; Marucci, Gabriella; Angeli, Piero; Russo, Filippo

2006-01-01

The discovery of a new series of selective and high-affinity alpha(1)-adrenoceptor (alpha(1)-AR) ligands, characterized by a 1H-pyrrolo[2,3-d]-pyrimidine-2,4(3H,7H)-dione system, is described in this paper. Some synthesized compounds, including 20, 22, and 30, displayed affinity in the nanomolar range for alpha(1)-ARs and substantial selectivity with respect to 5-HT(1A) and dopaminergic D(1) and D(2) receptors. Functional assays, performed on selected derivatives, showed antagonistic properties.

An endogenous aryl hydrocarbon receptor ligand acts on dendritic cells and T cells to suppress experimental autoimmune encephalomyelitis

PubMed Central

Quintana, Francisco J.; Murugaiyan, Gopal; Farez, Mauricio F.; Mitsdoerffer, Meike; Tukpah, Ann-Marcia; Burns, Evan J.; Weiner, Howard L.

2010-01-01

The ligand-activated transcription factor aryl hydrocarbon receptor (AHR) participates in the differentiation of FoxP3+ Treg, Tr1 cells, and IL-17–producing T cells (Th17). Most of our understanding on the role of AHR on the FoxP3+ Treg compartment results from studies using the toxic synthetic chemical 2,3,7,8-tetrachlorodibenzo-p-dioxin. Thus, the physiological relevance of AHR signaling on FoxP3+ Treg in vivo is unclear. We studied mice that carry a GFP reporter in the endogenous foxp3 locus and a mutated AHR protein with reduced affinity for its ligands, and found that AHR signaling participates in the differentiation of FoxP3+ Treg in vivo. Moreover, we found that treatment with the endogenous AHR ligand 2-(1′H-indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) given parenterally or orally induces FoxP3+ Treg that suppress experimental autoimmune encephalomyelitis. ITE acts not only on T cells, but also directly on dendritic cells to induce tolerogenic dendritic cells that support FoxP3+ Treg differentiation in a retinoic acid-dependent manner. Thus, our work demonstrates that the endogenous AHR ligand ITE promotes the induction of active immunologic tolerance by direct effects on dendritic and T cells, and identifies nontoxic endogenous AHR ligands as potential unique compounds for the treatment of autoimmune disorders. PMID:21068375

Thiophene-Core Estrogen Receptor Ligands Having Superagonist Activity

PubMed Central

Min, Jian; Wang, Pengcheng; Srinivasan, Sathish; Nwachukwu, Jerome C.; Guo, Pu; Huang, Minjian; Carlson, Kathryn E.; Katzenellenbogen, John A.; Nettles, Kendall W.; Zhou, Hai-Bing

2013-01-01

To probe the importance of the heterocyclic core of estrogen receptor (ER) ligands, we prepared a series of thiophene-core ligands by Suzuki cross-coupling of aryl boronic acids with bromo-thiophenes, and we assessed their receptor binding and cell biological activities. The disposition of the phenol substituents on the thiophene core, at alternate or adjacent sites, and the nature of substituents on these phenols all contribute to binding affinity and subtype selectivity. Most of the bis(hydroxyphenyl)-thiophenes were ERβ selective, whereas the tris(hydroxyphenyl)-thiophenes were ERα selective; analogous furan-core compounds generally have lower affinity and less selectivity. Some diarylthiophenes show distinct superagonist activity in reporter gene assays, giving maximal activities 2–3 times that of estradiol, and modeling suggests that these ligands have a different interaction with a hydrogen-bonding residue in helix-11. Ligand-core modification may be a new strategy for developing ER ligands whose selectivity is based on having transcriptional activity greater than that of estradiol. PMID:23586645

Ligand-induced type II interleukin-4 receptor dimers are sustained by rapid re-association within plasma membrane microcompartments

NASA Astrophysics Data System (ADS)

Richter, David; Moraga, Ignacio; Winkelmann, Hauke; Birkholz, Oliver; Wilmes, Stephan; Schulte, Markos; Kraich, Michael; Kenneweg, Hella; Beutel, Oliver; Selenschik, Philipp; Paterok, Dirk; Gavutis, Martynas; Schmidt, Thomas; Garcia, K. Christopher; Müller, Thomas D.; Piehler, Jacob

2017-07-01

The spatiotemporal organization of cytokine receptors in the plasma membrane is still debated with models ranging from ligand-independent receptor pre-dimerization to ligand-induced receptor dimerization occurring only after receptor uptake into endosomes. Here, we explore the molecular and cellular determinants governing the assembly of the type II interleukin-4 receptor, taking advantage of various agonists binding the receptor subunits with different affinities and rate constants. Quantitative kinetic studies using artificial membranes confirm that receptor dimerization is governed by the two-dimensional ligand-receptor interactions and identify a critical role of the transmembrane domain in receptor dimerization. Single molecule localization microscopy at physiological cell surface expression levels, however, reveals efficient ligand-induced receptor dimerization by all ligands, largely independent of receptor binding affinities, in line with the similar STAT6 activation potencies observed for all IL-4 variants. Detailed spatiotemporal analyses suggest that kinetic trapping of receptor dimers in actin-dependent microcompartments sustains robust receptor dimerization and signalling.

CD22 Ligands on a Natural N-Glycan Scaffold Efficiently Deliver Toxins to B-Lymphoma Cells.

PubMed

Peng, Wenjie; Paulson, James C

2017-09-13

CD22 is a sialic acid-binding immunoglobulin-like lectin (Siglec) that is highly expressed on B-cells and B cell lymphomas, and is a validated target for antibody and nanoparticle based therapeutics. However, cell targeted therapeutics are limited by their complexity, heterogeneity, and difficulties in production. We describe here a chemically defined natural N-linked glycan scaffold that displays high affinity CD22 glycan ligands and outcompetes the natural ligand for the receptor, resulting in single molecule binding to CD22 and endocytosis into cells. Binding affinity is increased by up to 1500-fold compared to the monovalent ligand, while maintaining the selectivity for hCD22 over other Siglecs. Conjugates of these multivalent ligands with auristatin and saporin toxins are efficiently internalized via hCD22 resulting in killing of B-cell lymphoma cells. This single molecule ligand targeting strategy represents an alternative to antibody- and nanoparticle-mediated approaches for delivery of agents to cells expressing CD22 and other Siglecs.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties.

PubMed

Šácha, Pavel; Knedlík, Tomáš; Schimer, Jiří; Tykvart, Jan; Parolek, Jan; Navrátil, Václav; Dvořáková, Petra; Sedlák, František; Ulbrich, Karel; Strohalm, Jiří; Majer, Pavel; Šubr, Vladimír; Konvalinka, Jan

2016-02-12

Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named "iBodies", consist of an HPMA copolymer decorated with low-molecular-weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live-cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

iBodies: Modular Synthetic Antibody Mimetics Based on Hydrophilic Polymers Decorated with Functional Moieties

PubMed Central

Šácha, Pavel; Knedlík, Tomáš; Schimer, Jiří; Tykvart, Jan; Parolek, Jan; Navrátil, Václav; Dvořáková, Petra; Sedlák, František; Ulbrich, Karel; Strohalm, Jiří; Majer, Pavel

2016-01-01

Abstract Antibodies are indispensable tools for biomedicine and anticancer therapy. Nevertheless, their use is compromised by high production costs, limited stability, and difficulty of chemical modification. The design and preparation of synthetic polymer conjugates capable of replacing antibodies in biomedical applications such as ELISA, flow cytometry, immunocytochemistry, and immunoprecipitation is reported. The conjugates, named “iBodies”, consist of an HPMA copolymer decorated with low‐molecular‐weight compounds that function as targeting ligands, affinity anchors, and imaging probes. We prepared specific conjugates targeting several proteins with known ligands and used these iBodies for enzyme inhibition, protein isolation, immobilization, quantification, and live‐cell imaging. Our data indicate that this highly modular and versatile polymer system can be used to produce inexpensive and stable antibody substitutes directed toward virtually any protein of interest with a known ligand. PMID:26749427

In vitro selection of RNA aptamer specific to Salmonella typhimurium.

PubMed

Han, Seung Ryul; Lee, Seong-Wook

2013-06-28

Salmonella is a major foodborne pathogen that causes a variety of human diseases. Development of ligands directly and specifically binding to the Salmonella will be crucial for the rapid detection of, and thus for efficient protection from, the virulent bacteria. In this study, we identified a RNA aptamer-based ligand that can specifically recognize Salmonella Typhimurium through SELEX technology. To this end, we isolated and characterized an RNase-resistant RNA aptamer that bound to the OmpC protein of Salmonella Typhimurium with high specificity and affinity (Kd ~ 20 nM). Of note, the selected aptamer was found to specifically bind to Salmonella Typhimurium, but neither to Gram-positive bacteria (Staphylococcus aureus) nor to other Gram-negative bacteria (Escherichia coli O157:H7). This was evinced by aptamer-immobilized ELISA and aptamer-linked precipitation experiments. This Salmonella species-specific aptamer could be useful as a diagnostic ligand against pathogen-caused foodborne sickness.

In Silico Identification of Novel APRIL Peptide Antagonists and Binding Insights by Molecular Modeling and Immunosorbent Assays.

PubMed

Silva, Joao H M da; Calmon-Hamaty, Flavia; Savino, Wilson; Hahne, Michael; Caffarena, Ernesto R

2015-01-01

The "A proliferation inducing ligand" protein (APRIL) is a cytokine over-expressed in many transformed and tumoral cells acting onto two distinct receptors of the Tumoral Necrosis Factor B cell maturation antigen (BCMA) and the transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI). We herein describe, through a detailed computational approach, the molecular interactions between TACI and its ligands APRIL and another structurally similar protein called B-cell activating factor (BAFF) by means of molecular dynamics. Dynamical analysis suggests R84 and D85 residues from TACI as possible mutation candidates, yielding increased affinity between TACI and APRIL. The association of computational simulations, site directed mutagenesis and peptide design could be a powerful tool, driving to better in vitro experiments. Our results contribute to the elucidation of APRIL signaling and help clarify the effects of blocking interaction between APRIL and its receptors through the use of particular peptides.

The Effect of Hydrogen Bonding in Enhancing the Ionic Affinities of Immobilized Monoprotic Phosphate Ligands

DOE PAGES

Alexandratos, Spiro D.; Zhu, Xiaoping

2017-08-18

Environmental remediation requires ion-selective polymers that operate under a wide range of solution conditions. In one example, removal of trivalent and divalent metal ions from waste streams resulting from mining operations before they enter the environment requires treatment at acidic pH. The monoethyl ester phosphate ligands developed in this report operate from acidic solutions. They have been prepared on polystyrene-bound ethylene glycol, glycerol, and pentaerythritol, and it is found that intra-ligand hydrogen bonding affects their metal ion affinities. The affinity for a set of trivalent (Fe(III), Al(III), La(III), and Lu(III)) and divalent (Pb(II), Cd(II), Cu(II), and Zn(II)) ions is greatermore » than that of corresponding neutral diethyl esters and phosphonic acid. In an earlier study, hydrogen bonding was found important in determining the metal ion affinities of immobilized phosphorylated polyol diethyl ester coordinating ligands; their Fourier transform infrared (FTIR) band shifts indicated that the basicity of the phosphoryl oxygen increased by hydrogen bonding to auxiliary –OH groups on the neighboring polyol. The same mechanism is operative with the monoprotic resins along with hydrogen bonding to the P–OH acid site. This is reflected in the FTIR spectra: the neutral phosphate diethyl ester resins have the P=O band at 1265 cm -1 while the monoethyl ester resins have the band shifted to 1230 cm -1; hydrogen bonding is further indicated by the broadness of this region down to 900 cm -1. Of the polymers studied, monoprotic pentaerythritol has the highest metal ion affinities.« less

The Effect of Hydrogen Bonding in Enhancing the Ionic Affinities of Immobilized Monoprotic Phosphate Ligands

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

Alexandratos, Spiro D.; Zhu, Xiaoping

Environmental remediation requires ion-selective polymers that operate under a wide range of solution conditions. In one example, removal of trivalent and divalent metal ions from waste streams resulting from mining operations before they enter the environment requires treatment at acidic pH. The monoethyl ester phosphate ligands developed in this report operate from acidic solutions. They have been prepared on polystyrene-bound ethylene glycol, glycerol, and pentaerythritol, and it is found that intra-ligand hydrogen bonding affects their metal ion affinities. The affinity for a set of trivalent (Fe(III), Al(III), La(III), and Lu(III)) and divalent (Pb(II), Cd(II), Cu(II), and Zn(II)) ions is greatermore » than that of corresponding neutral diethyl esters and phosphonic acid. In an earlier study, hydrogen bonding was found important in determining the metal ion affinities of immobilized phosphorylated polyol diethyl ester coordinating ligands; their Fourier transform infrared (FTIR) band shifts indicated that the basicity of the phosphoryl oxygen increased by hydrogen bonding to auxiliary –OH groups on the neighboring polyol. The same mechanism is operative with the monoprotic resins along with hydrogen bonding to the P–OH acid site. This is reflected in the FTIR spectra: the neutral phosphate diethyl ester resins have the P=O band at 1265 cm -1 while the monoethyl ester resins have the band shifted to 1230 cm -1; hydrogen bonding is further indicated by the broadness of this region down to 900 cm -1. Of the polymers studied, monoprotic pentaerythritol has the highest metal ion affinities.« less

A comparative study of family-specific protein-ligand complex affinity prediction based on random forest approach

NASA Astrophysics Data System (ADS)

Wang, Yu; Guo, Yanzhi; Kuang, Qifan; Pu, Xuemei; Ji, Yue; Zhang, Zhihang; Li, Menglong

2015-04-01

The assessment of binding affinity between ligands and the target proteins plays an essential role in drug discovery and design process. As an alternative to widely used scoring approaches, machine learning methods have also been proposed for fast prediction of the binding affinity with promising results, but most of them were developed as all-purpose models despite of the specific functions of different protein families, since proteins from different function families always have different structures and physicochemical features. In this study, we proposed a random forest method to predict the protein-ligand binding affinity based on a comprehensive feature set covering protein sequence, binding pocket, ligand structure and intermolecular interaction. Feature processing and compression was respectively implemented for different protein family datasets, which indicates that different features contribute to different models, so individual representation for each protein family is necessary. Three family-specific models were constructed for three important protein target families of HIV-1 protease, trypsin and carbonic anhydrase respectively. As a comparison, two generic models including diverse protein families were also built. The evaluation results show that models on family-specific datasets have the superior performance to those on the generic datasets and the Pearson and Spearman correlation coefficients ( R p and Rs) on the test sets are 0.740, 0.874, 0.735 and 0.697, 0.853, 0.723 for HIV-1 protease, trypsin and carbonic anhydrase respectively. Comparisons with the other methods further demonstrate that individual representation and model construction for each protein family is a more reasonable way in predicting the affinity of one particular protein family.

Simultaneous addition of two ligands: a potential strategy for estimating divalent ion affinities in EF-hand proteins by isothermal titration calorimetry.

PubMed

Henzl, Michael T; Markus, Lindsey A; Davis, Meredith E; McMillan, Andrew T

2013-03-01

Capable of providing a detailed thermodynamic picture of noncovalent association reactions, isothermal titration calorimetry (ITC) has become a popular method for studying protein-ligand interactions. We routinely employ the technique to study divalent ion-binding by two-site EF-hand proteins from the parvalbumin- and polcalcin lineages. The combination of high Ca(2+) affinity and relatively low Mg(2+) affinity, and the attendant complication of parameter correlation, conspire to make the simultaneous extraction of binding constants and -enthalpies for both ions challenging. Although global analysis of multiple ITC experiments can overcome these hurdles, our current experimental protocol includes upwards of 10 titrations - requiring a substantial investment in labor, machine time, and material. This paper explores the potential for using a smaller suite of experiments that includes simultaneous titrations with Ca(2+) and Mg(2+) at different ratios of the two ions. The results obtained for four proteins, differing substantially in their divalent ion-binding properties, suggest that the approach has merit. The Ca(2+)- and Mg(2+)-binding constants afforded by the streamlined analysis are in reasonable agreement with those obtained from the standard analysis protocol. Likewise, the abbreviated analysis provides comparable values for the Ca(2+)-binding enthalpies. However, the streamlined analysis can yield divergent values for the Mg(2+)-binding enthalpies - particularly those for lower affinity sites. This shortcoming can be remedied, in large measure, by including data from a direct Ca(2+) titration in the presence of a high, fixed Mg(2+) concentration. Copyright © 2013. Published by Elsevier Inc.

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.

Ligand-modulated interactions between charged monolayer-protected Au144 (SR)60 gold nanoparticles in physiological saline

NASA Astrophysics Data System (ADS)

Villarreal, Oscar; Chen, Liao; Whetten, Robert; Yacaman, Miguel

2015-03-01

We studied the interactions of functionalized Au144 nanoparticles (NPs) in a near-physiological environment through all-atom molecular dynamics simulations. The AuNPs were coated with a homogeneous selection of 60 thiolates: 11-mercapto-1-undecanesulfonate, 5-mercapto-1-pentanesulfonate, 5-mercapto-1-pentane-amine, 4-mercapto-benzoate or 4-mercapto-benzamide. These ligands were selected to elucidate how the aggregation behavior depends on the ligands' sign of charge, length, and flexibility. Simulating the dynamics of a pair of identical AuNPs in a cell of saline of 150 mM NaCl in addition to 120 Na+/Cl- counter-ions, we computed the aggregation affinities from the potential of mean force as a function of the pair separation. We found that NPs coated with negatively charged, short ligands have the strongest affinities mediated by multiple Na+ counter-ions residing on a plane in-between the pair and forming ``salt bridges'' to both NPs. Positively charged NPs have weaker affinities, as Cl counter-ions form fewer and weaker salt bridges. The longer ligands' large fluctuations disfavor the forming of salt bridges, enable hydrophobic contact between the exposed hydrocarbon chains and interact at greater separations due to the fact that the screening effect is rather incomplete. Supported by the CONACYT, NIH, NSF and TACC.

PREDICTING ER BINDING AFFINITY FOR EDC RANKING AND PRIORITIZATION: A COMPARISON OF THREE MODELS

EPA Science Inventory

A comparative analysis of how three COREPA models for ER binding affinity performed when used to predict potential estrogen receptor (ER) ligands is presented. Models I and II were developed based on training sets of 232 and 279 rat ER binding affinity measurements, respectively....

Multilayer affinity adsorption of albumin on polymer brushes modified membranes in a continuous-flow system.

PubMed

Hu, Meng-Xin; Li, Xiang; Li, Ji-Nian; Huang, Jing-Jing; Ren, Ge-Rui

2018-02-23

Polymer brushes modified surfaces have been widely used for protein immobilization and isolation. Modification of membranes with polymer brushes increases the surface concentration of affinity ligands used for protein binding. Albumin is one of the transporting proteins and shows a high affinity to bile acids. In this work, the modified membranes with cholic acid-containing polymer brushes can be facilely prepared by the immobilization of cholic acid on the poly(2-hydroxyethyl methacrylate) grafted microporous polypropylene membranes (MPPMs) for affinity adsorption of albumin. ATR/FT-IR and X-ray photoelectron spectroscopy were used to characterize the chemical composition of the modified membranes. Water contact angle measurements were used to analyze the hydrophilic/hydrophobic properties of the membrane surface. The modified MPPMs show a high affinity to albumin and have little non-specific adsorption of hemoglobin. The dynamic binding capacity of albumin in the continous-flow system increases with the cycle number and feed rate as the binding degree of cholic acid is moderate. The highest binding capacity of affinity membranes is about 52.49 g/m 2 membrane, which is about 24 times more than the monolayer binding capacity. These results reveal proteins could be captured in multilayers by the polymer brushes containing affinity ligands similar to the polymer brushes containing ion-exchange groups, which open up the potential of the polymer brushes containing affinity ligands in protein or another components separation. And the cholic acid containing polymer brushes modified membranes has the promising potential for albumin separation and purification rapidly from serum or fermented solution in medical diagnosis and bioseparation. Copyright © 2018 Elsevier B.V. All rights reserved.

Application of partition technology to particle electrophoresis

NASA Technical Reports Server (NTRS)

Van Alstine, James M.; Harris, J. Milton; Karr, Laurel J.; Bamberger, Stephan; Matsos, Helen C.; Snyder, Robert S.

1989-01-01

The effects of polymer-ligand concentration on particle electrophoretic mobility and partition in aqueous polymer two-phase systems are investigated. Polymer coating chemistry and affinity ligand synthesis, purification, and analysis are conducted. It is observed that poly (ethylene glycol)-ligands are effective for controlling particle electrophoretic mobility.

Identification of Elf-1 and B61 as high affinity ligands for the receptor tyrosine kinase MDK1.

PubMed

Ciossek, T; Ullrich, A

1997-01-09

Mouse Developmental Kinase 1 (MDK1) is a receptor tyrosine kinase of the eck/eph subfamily expressed in a variety of tissues during early mouse embryogenesis. To obtain further insight into the function of MDK1, we determined identity and localisation of its physiological ligand(s). Staining whole embryos with fusion proteins between the extracellular domain of MDK1 and human secreted alkaline phosphatase revealed areas of high receptor binding in the caudal mesencephalon, the frontal neocortex and the limb buds. This staining was sensitive to treatment with phosphatidylinositol-specific phospholipase C. Using Scatchard analysis, high affinity binding of Elf-1 (1.7 x 10(-10) M) and B61 (2.2 x 10(-10) M) towards MDK1 could be demonstrated. However, the transmembrane ligand Lerk2 displayed no measurable affinity for MDK1. Elf-1 and B61 bind to the three full-length MDK1 isoforms with similar dissociation constants. Slightly lower affinities were observed for the two truncated receptors MDK1-Tl and MDK1-T2. The activation of MDK1 with Elf-1 or B61 leads to the rapid autophosphorylation of MDK1 as well as tyrosine phosphorylation of an unknown 62 kDa phosphoprotein in Rat1 cells. These findings implicate MDK1 in patterning processes during early mouse embryogenesis and suggest MDK1 involvement in early organogenesis and midbrain development.

Asymmetric configurations in a reengineered homodimer reveal multiple subunit communication pathways in protein allostery

PubMed Central

Lanfranco, Maria Fe; Gárate, Fernanda; Engdahl, Ashton J.; Maillard, Rodrigo A.

2017-01-01

Many allosteric proteins form homo-oligomeric complexes to regulate a biological function. In homo-oligomers, subunits establish communication pathways that are modulated by external stimuli like ligand binding. A challenge for dissecting the communication mechanisms in homo-oligomers is identifying intermediate liganded states, which are typically transiently populated. However, their identities provide the most mechanistic information on how ligand-induced signals propagate from bound to empty subunits. Here, we dissected the directionality and magnitude of subunit communication in a reengineered single-chain version of the homodimeric transcription factor cAMP receptor protein. By combining wild-type and mutant subunits in various asymmetric configurations, we revealed a linear relationship between the magnitude of cooperative effects and the number of mutant subunits. We found that a single mutation is sufficient to change the global allosteric behavior of the dimer even when one subunit was wild type. Dimers harboring two mutations with opposite cooperative effects had different allosteric properties depending on the arrangement of the mutations. When the two mutations were placed in the same subunit, the resulting cooperativity was neutral. In contrast, when placed in different subunits, the observed cooperativity was dominated by the mutation with strongest effects over cAMP affinity relative to wild type. These results highlight the distinct roles of intrasubunit interactions and intersubunit communication in allostery. Finally, dimers bound to either one or two cAMP molecules had similar DNA affinities, indicating that both asymmetric and symmetric liganded states activate DNA interactions. These studies have revealed the multiple communication pathways that homo-oligomers employ to transduce signals. PMID:28188293

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.

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

Mutagenesis Analysis Reveals Distinct Amino Acids of the Human Serotonin 5-HT2C Receptor Underlying the Pharmacology of Distinct Ligands.

PubMed

Liu, Yue; Canal, Clinton E; Cordova-Sintjago, Tania C; Zhu, Wanying; Booth, Raymond G

2017-01-18

While exploring the structure-activity relationship of 4-phenyl-2-dimethylaminotetralins (PATs) at serotonin 5-HT 2C receptors, we discovered that relatively minor modification of PAT chemistry impacts function at 5-HT 2C receptors. In HEK293 cells expressing human 5-HT 2C-INI receptors, for example, (-)-trans-3'-Br-PAT and (-)-trans-3'-Cl-PAT are agonists regarding Gα q -inositol phosphate signaling, whereas (-)-trans-3'-CF 3 -PAT is an inverse agonist. To investigate the ligand-receptor interactions that govern this change in function, we performed site-directed mutagenesis of 14 amino acids of the 5-HT 2C receptor based on molecular modeling and reported G protein-coupled receptor crystal structures, followed by molecular pharmacology studies. We found that S3.36, T3.37, and F5.47 in the orthosteric binding pocket are critical for affinity (K i ) of all PATs tested, we also found that F6.44, M6.47, C7.45, and S7.46 are primarily involved in regulating EC/IC 50 functional potencies of PATs. We discovered that when residue S5.43, N6.55, or both are mutated to alanine, (-)-trans-3'-CF 3 -PAT switches from inverse agonist to agonist function, and when N6.55 is mutated to leucine, (-)-trans-3'-Br-PAT switches from agonist to inverse agonist function. Notably, most point-mutations that affected PAT pharmacology did not significantly alter affinity (K D ) of the antagonist radioligand [ 3 H]mesulergine, but every mutation tested negatively impacted serotonin binding. Also, amino acid mutations differentially affected the pharmacology of other commercially available 5-HT 2C ligands tested. Collectively, the data show that functional outcomes shared by different ligands are mediated by different amino acids and that some 5-HT 2C receptor residues important for pharmacology of one ligand are not necessarily important for another ligand.

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting RGD ligands with a build-in handle

PubMed Central

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y.; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C.; Takada, Yoshikazu; Lam, Kit S.

2012-01-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiological processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted-therapy and cancer imaging. In this report, one-bead-one-compound (OBOC) combinatorial libraries containing the RGD motif were designed and screened against K562 myeloid leukemia cells that had been transfected with human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a build-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC50 = 0.68±0.08 μM) to some of the well-known RGD “head-to-tail” cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2 to 8 fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with biotinylated-ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake. PMID:20858725

Stable coordination of the inhibitory Ca2+ ion at MIDAS in integrin CD11b/CD18 by an antibody-derived ligand aspartate: Implications for integrin regulation and structure-based drug design

PubMed Central

Mahalingam, Bhuvaneshwari; Ajroud, Kaouther; Alonso, Jose Luis; Anand, Saurabh; Adair, Brian; Horenstein, Alberto L; Malavasi, Fabio; Xiong, Jian-Ping; Arnaout, M. Amin

2011-01-01

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal-ion-dependent-adhesion site (MIDAS) in the integrin A-domain. This interaction stabilizes the A-domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A-domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking monoclonal antibody (mAb) 107 binds MIDAS of integrin CD11b/CD18 A-domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over Mg2+ at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of Ca2+ at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+. Binding of Fab 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that denticity of the ligand Asp/Glu can modify divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists. PMID:22095715

Structure-based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists.

EPA Science Inventory

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

Structure-Based Understanding of Binding Affinity and Mode of Estrogen Receptor α Agonists and Antagonists

EPA Science Inventory

The flexible hydrophobic ligand binding pocket (LBP) of estrogen receptor α (ERα) allows the binding of a wide variety of endocrine disruptors. Upon ligand binding, the LBP reshapes around the contours of the ligand and stabilizes the complex by complementary hydrophobic interact...

SKF 525-A and cytochrome P-450 ligands inhibit with high affinity the binding of ( sup 3 H)dextromethorphan and. sigma. ligands to guinea pig brain

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

Klein, M.; Canoll, P.D.; Musacchio, J.M.

1991-01-01

The DM{sub 1}/{sigma}{sub 1} site binds dextromethorphan (DM) and {sigma} receptor ligands. The broad binding specificity of this site and its peculiar subcellular distribution prompted us to explore the possibility that this site is a member of the cytochrome P-450 superfamily of enzymes. We tested the effects of the liver microsomal monooxygenase inhibitor SKF 525-A (Proadifen), and other P-450 substrates on the binding of ({sup 3}H)dextromethorphan, ({sup 3}H)3- (3-Hydroxyphenyl) -N- (1-propyl) piperidine and (+)-({sup 3}H)1,3-Di-o-tolyl-guanidine (({sup 3}H)DTG) to the guinea pig brain. SKF 525-A, l-lobeline and GBR-12909 inhibited the binding of the three labeled ligands with nM affinity. Each drugmore » has identical nM K{sub i} values for the high-affinity site labeled by the three ligands. This indicated that they displaced the labeled ligands from the common DM{sub 1}{sigma}{sub 1} site. Debrisoquine and sparteine, prototypical substrates for liver debrisoquine 4-hydroxylase, displayed K{sub i} values of 9-13 and 3-4 {mu}M respectively against the three labeled ligands. These results, the broad specificity of the DM{sub 1}/{sigma}{sub 1} binding site, and its peculiar subcellular distribution, raises the possibility that this binding site is a member of the cytochrome P-450 superfamily of isozymes, rather than a neurotransmitter receptor.« less

Binding of Estrogenic Compounds to Recombinant Estrogen Receptor-α: Application to Environmental Analysis

PubMed Central

Pillon, Arnaud; Boussioux, Anne-Marie; Escande, Aurélie; Aït-Aïssa, Sélim; Gomez, Elena; Fenet, Hélène; Ruff, Marc; Moras, Dino; Vignon, Françoise; Duchesne, Marie-Josèphe; Casellas, Claude; Nicolas, Jean-Claude; Balaguer, Patrick

2005-01-01

Estrogenic activity in environmental samples could be mediated through a wide variety of compounds and by various mechanisms. High-affinity compounds for estrogen receptors (ERs), such as natural or synthetic estrogens, as well as low-affinity compounds such as alkylphenols, phthalates, and polychlorinated biphenyls are present in water and sediment samples. Furthermore, compounds such as polycyclic aromatic hydrocarbons, which do not bind ERs, modulate estrogen activity by means of the aryl hydrocarbon receptor (AhR). In order to characterize compounds that mediate estrogenic activity in river water and sediment samples, we developed a tool based on the ER-αligand-binding domain, which permitted us to estimate contaminating estrogenic compound affinities. We designed a simple transactivation assay in which compounds of high affinity were captured by limited amounts of recombinant ER-αand whose capture led to a selective inhibition of transactivation. This approach allowed us to bring to light that water samples contain estrogenic compounds that display a high affinity for ERs but are present at low concentrations. In sediment samples, on the contrary, we showed that estrogenic compounds possess a low affinity and are present at high concentration. Finally, we used immobilized recombinant ER-αto separate ligands for ER and AhR that are present in river sediments. Immobilized ER-α, which does not retain dioxin-like compounds, enabled us to isolate and concentrate ER ligands to facilitate their further analysis. PMID:15743715

Directed evolution of human T cell receptor CDR2 residues by phage display dramatically enhances affinity for cognate peptide-MHC without increasing apparent cross-reactivity

PubMed Central

Dunn, Steven M.; Rizkallah, Pierre J.; Baston, Emma; Mahon, Tara; Cameron, Brian; Moysey, Ruth; Gao, Feng; Sami, Malkit; Boulter, Jonathan; Li, Yi; Jakobsen, Bent K.

2006-01-01

The mammalian α/β T cell receptor (TCR) repertoire plays a pivotal role in adaptive immunity by recognizing short, processed, peptide antigens bound in the context of a highly diverse family of cell-surface major histocompatibility complexes (pMHCs). Despite the extensive TCR–MHC interaction surface, peptide-independent cross-reactivity of native TCRs is generally avoided through cell-mediated selection of molecules with low inherent affinity for MHC. Here we show that, contrary to expectations, the germ line-encoded complementarity determining regions (CDRs) of human TCRs, namely the CDR2s, which appear to contact only the MHC surface and not the bound peptide, can be engineered to yield soluble low nanomolar affinity ligands that retain a surprisingly high degree of specificity for the cognate pMHC target. Structural investigation of one such CDR2 mutant implicates shape complementarity of the mutant CDR2 contact interfaces as being a key determinant of the increased affinity. Our results suggest that manipulation of germ line CDR2 loops may provide a useful route to the production of high-affinity TCRs with therapeutic and diagnostic potential. PMID:16600963

An enantioselective enzymatic desymmetrization route to hexahydro-4H-furopyranol, a high-affinity ligand for HIV-1 protease inhibitors.

PubMed

Ghosh, Arun K; Sarkar, Anindya

2017-08-16

An enantioselective synthesis of ( 3 a S , 4S , 7 a R )-hexahydro-4 H -furo[2,3- b ]pyran-4-ol, a high-affinity nonpeptide ligand for a variety of potent HIV-1 protease inhibitors is described. The key steps involved a highly enantioselective enzymatic desymmetrization of meso -diacetate, an efficient transacetalization, and a highly diastereoselective reduction of a ketone. This route is amenable to large-scale synthesis using readily available starting materials.

Decahydrobenzoquinolin-5-one sigma receptor ligands: Divergent development of both sigma 1 and sigma 2 receptor selective examples.

PubMed

McLeod, Michael C; Aubé, Jeffrey; Frankowski, Kevin J

2016-12-01

Analogues of the decahydrobenzoquinolin-5-one class of sigma (σ) receptor ligands were used to probe the structure-activity relationship trends for this recently discovered series of σ ligands. In all, 29 representatives were tested for σ and opioid receptor affinity, leading to the identification of compounds possessing improved σ 1 selectivity and, for the first time in this series, examples possessing preferential σ 2 affinity. Several structural features associated with these selectivity trends have been identified. Two analogues of improved selectivity were evaluated in a binding panel of 43 CNS-relevant targets to confirm their sigma receptor preference. Copyright © 2016 Elsevier Ltd. All rights reserved.

Ligand interaction scan: a general method for engineering ligand-sensitive protein alleles.

PubMed

Erster, Oran; Eisenstein, Miriam; Liscovitch, Mordechai

2007-05-01

The ligand interaction scan (LIScan) method is a general procedure for engineering small molecule ligand-regulated forms of a protein that is complementary to other 'reverse' genetic and chemical-genetic methods for drug-target validation. It involves insertional mutagenesis by a chemical-genetic 'switch', comprising a genetically encoded peptide module that binds with high affinity to a small-molecule ligand. We demonstrated the method with TEM-1 beta-lactamase, using a tetracysteine hexapeptide insert and a biarsenical fluorescein ligand (FlAsH).

Molecular mechanism of peroxisome proliferator-activated receptor α activation by WY14643: a new mode of ligand recognition and receptor stabilization.

PubMed

Bernardes, Amanda; Souza, Paulo C T; Muniz, João R C; Ricci, Clarisse G; Ayers, Stephen D; Parekh, Nili M; Godoy, André S; Trivella, Daniela B B; Reinach, Peter; Webb, Paul; Skaf, Munir S; Polikarpov, Igor

2013-08-23

Peroxisome proliferator-activated receptors (PPARs) are members of a superfamily of nuclear transcription factors. They are involved in mediating numerous physiological effects in humans, including glucose and lipid metabolism. PPARα ligands effectively treat dyslipidemia and have significant antiinflammatory and anti-atherosclerotic activities. These effects and their ligand-dependent activity make nuclear receptors obvious targets for drug design. Here, we present the structure of the human PPARα in complex with WY14643, a member of fibrate class of drug, and a widely used PPAR activator. The crystal structure of this complex suggests that WY14643 induces activation of PPARα in an unusual bipartite mechanism involving conventional direct helix 12 stabilization and an alternative mode that involves a second ligand in the pocket. We present structural observations, molecular dynamics and activity assays that support the importance of the second site in WY14643 action. The unique binding mode of WY14643 reveals a new pattern of nuclear receptor ligand recognition and suggests a novel basis for ligand design, offering clues for improving the binding affinity and selectivity of ligand. We show that binding of WY14643 to PPARα was associated with antiinflammatory disease in a human corneal cell model, suggesting possible applications for PPARα ligands. Copyright © 2013 Elsevier Ltd. All rights reserved.

Automated large-scale file preparation, docking, and scoring: evaluation of ITScore and STScore using the 2012 Community Structure-Activity Resource benchmark.

PubMed

Grinter, Sam Z; Yan, Chengfei; Huang, Sheng-You; Jiang, Lin; Zou, Xiaoqin

2013-08-26

In this study, we use the recently released 2012 Community Structure-Activity Resource (CSAR) data set to evaluate two knowledge-based scoring functions, ITScore and STScore, and a simple force-field-based potential (VDWScore). The CSAR data set contains 757 compounds, most with known affinities, and 57 crystal structures. With the help of the script files for docking preparation, we use the full CSAR data set to evaluate the performances of the scoring functions on binding affinity prediction and active/inactive compound discrimination. The CSAR subset that includes crystal structures is used as well, to evaluate the performances of the scoring functions on binding mode and affinity predictions. Within this structure subset, we investigate the importance of accurate ligand and protein conformational sampling and find that the binding affinity predictions are less sensitive to non-native ligand and protein conformations than the binding mode predictions. We also find the full CSAR data set to be more challenging in making binding mode predictions than the subset with structures. The script files used for preparing the CSAR data set for docking, including scripts for canonicalization of the ligand atoms, are offered freely to the academic community.

Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

PubMed Central

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739

Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

PubMed Central

Chung, Wai Keen; Freed, Alexander S.; Holstein, Melissa A.; McCallum, Scott A.; Cramer, Steven M.

2010-01-01

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarse-grained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously undescribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems. PMID:20837551

New Horizons on Molecular Pharmacology Applied to Drug Discovery: When Resonance Overcomes Radioligand Binding.

PubMed

Pernomian, Larissa; Gomes, Mayara Santos; Moreira, Josimar Dornelas; da Silva, Carlos Henrique Tomich de Paula; Rosa, Joaquin Maria Campos; Cardoso, Cristina Ribeiro de Barros

2017-01-01

One of the cornerstones of rational drug development is the measurement of molecular parameters derived from ligand-receptor interaction, which guides therapeutic windows definition. Over the last decades, radioligand binding has provided valuable contributions in this field as key method for such purposes. However, its limitations spurred the development of more exquisite techniques for determining such parameters. For instance, safety risks related to radioactivity waste, expensive and controlled disposal of radioisotopes, radiotracer separation-dependence for affinity analysis, and one-site mathematical models-based fitting of data make radioligand binding a suboptimal approach in providing measures of actual affinity conformations from ligands and G proteincoupled receptors (GPCR). Current advances on high-throughput screening (HTS) assays have markedly extended the options of sparing sensitive ways for monitoring ligand affinity. The advent of the novel bioluminescent donor NanoLuc luciferase (Nluc), engineered from Oplophorus gracilirostris luciferase, allowed fitting bioluminescence resonance energy transfer (BRET) for monitoring ligand binding. Such novel approach named Nluc-based BRET (NanoBRET) binding assay consists of a real-time homogeneous proximity assay that overcomes radioligand binding limitations but ensures the quality in affinity measurements. Here, we cover the main advantages of NanoBRET protocol and the undesirable drawbacks of radioligand binding as molecular methods that span pharmacological toolbox applied to Drug Discovery. Also, we provide a novel perspective for the application of NanoBRET technology in affinity assays for multiple-state binding mechanisms involving oligomerization and/or functional biased selectivity. This new angle was proposed based on specific biophysical criteria required for the real-time homogeneity assigned to the proximity NanoBRET protocol. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

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

Structural and functional characterization of a new recombinant histidine-tagged acyl coenzyme A binding protein (ACBP) from mouse

PubMed Central

Petrescu, Anca D.; Huang, Huan; Hostetler, Heather A.; Schroeder, Friedhelm; Kier, Ann B.

2008-01-01

Acyl-coenzyme A binding protein (ACBP) has been proposed to transport fatty acyl-CoAs intracellularly, facilitating their metabolism. In this study, a new mouse recombinant ACBP was produced by insertion of a histidine (his) tag at the C-terminus to allow efficient purification by Ni-affinity chromatography. The his-tag was inserted at the C-terminus since ACBP is a small molecular size (10 kDa) protein whose structure and activity are sensitive to amino acid substitutions in the N-terminus. The his tag had no or little effect on ACBP structure or ligand binding affinity and specificity. His-ACBP bound the naturally-occurring fluorescent cis-parinaroyl-CoA with very high affinity (Kd=2.15 nM), but exhibited no affinity for non-esterified cis-parinaric acid. To determine if the presence of the C-terminal his tag altered ACBP interactions with other proteins, direct binding to hepatocyte nuclear factor 4α (HNF-4α), a nuclear receptor regulating transcription of genes involved in lipid metabolism, was examined. His-ACBP and HNF-4α were labeled with Cy5 and Cy3, respectively, and direct interaction was determined by a novel fluorescence resonance energy transfer (FRET) binding assay. FRET analysis showed that his-ACBP directly interacted with HNF-4α (intermolecular distance of 73 Å) at high affinity (Kd=64-111 nM) similar to native ACBP. The his-tag also had no effect on ACBPs ability to interact with and stimulate microsomal enzymes utilizing or forming fatty acyl CoA. Thus, C-terminal his-tagged-ACBP maintained very similar structural and functional features of the untagged native protein and can be used in further in vitro experiments that require pure recombinant ACBP. PMID:18178100

Cholera Toxin Inhibitors Studied with High-Performance Liquid Affinity Chromatography: A Robust Method to Evaluate Receptor–Ligand Interactions

PubMed Central

Bergström, Maria; Liu, Shuang; Kiick, Kristi L.; Ohlson, Sten

2009-01-01

Anti-adhesion drugs may be an alternative to antibiotics to control infection of micro-organisms. The well-characterized interaction between cholera toxin and the cellular glycolipid GM1 makes it an attractive model for inhibition studies in general. In this report, we demonstrate a high-performance liquid affinity chromatography approach called weak affinity chromatography to evaluate cholera toxin inhibitors. The cholera toxin B-subunit was covalently coupled to porous silica and a (weak) affinity column was produced. The KD values of galactose and meta-nitrophenyl α-D-galactoside were determined with weak affinity chromatography to be 52 and 1 mM, respectively, which agree well with IC50 values previously reported. To increase inhibition potency multivalent inhibitors have been developed and the interaction with multivalent glycopolypeptides was also evaluated. The affinity of these compounds was found to correlate with the galactoside content but KD values were not obtained because of the inhomogeneous response and slow off-rate from multivalent interactions. Despite the limitations in obtaining direct KD values of the multivalent galactopolypeptides, weak affinity chromatography represents an additional and valuable tool in the evaluation of monovalent as well as multivalent cholera toxin inhibitors. It offers multiple advantages, such as a low sample consumption, high reproducibility and short analysis time, which are often not observed in other methods of analysis. PMID:19152642

Synthesis and biological evaluation of a series of aminoalkyl-tetralones and tetralols as dual dopamine/serotonin ligands.

PubMed

Carro, Laura; Torrado, María; Raviña, Enrique; Masaguer, Christian F; Lage, Sonia; Brea, José; Loza, María I

2014-01-01

A series of novel α-tetralone and α-tetralol derivatives was synthesized, and their binding affinities for 5-HT(2A) and D₂ receptors, the most important targets implicated in the anti-schizophrenia drug action, were evaluated to elucidate how substitutions in the aromatic ring of the pharmacophore affect to the affinity or selectivity for these receptors. The replacement of the H-7 in the tetrahydronaphthalene system by an amino group resulted in privileged 5-HT(2A) affinity of the 6-fluorobenzo[d]isoxazol derivative 36 and the alcohol 25 both showing a pK(i) value for 5-HT(2A) higher than 8.3 and good binding affinities for D₂ receptor leading to a Meltzer's ratio characteristic of an atypical antipsychotic profile. Additionally, a small collection of 3-aminomethyltetralone derivatives was prepared and examined here for their affinities and selectivities as 5-HT(2A)/D₂ dual ligands. Compound 11 shows the best profile with good pKi values for 5-HT(2A) and D₂ receptors leading to a Meltzer's ratio characteristic of a typical antipsychotic behaviour. These three compounds behaved as competitive antagonists of both 5-HT(2A) and D₂ receptors, and might be promising pharmacological tools for the investigation of the dual function of the 5HT(2A)-D₂ ligands. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Synthesis of poly(N-isopropylacrylamide) particles for metal affinity binding of peptides

PubMed Central

Tsai, Hsin-Yi; Lee, Alexander; Peng, Wei; Yates, Matthew Z.

2013-01-01

Temperature-sensitive poly(N-isopropylacrylamide) (PNIPAM) microgel particles with metal affinity ligands were prepared for selective binding of peptides containing the His6-tag (six consecutive histidine residues). The PNIPAM particles were copolymerized with the functional ligand vinylbenzyl iminodiacetic acid (VBIDA) through a two-stage dispersion polymerization using poly(N-vinyl pyrrolidone) (PVP) as a steric stabilizer. The resulting particles were monodisperse in size and colloidally stable over a wide range of temperature and ionic strength due to chemically grafted PVP chains. The particle size was also found to be sensitive to ionic strength and pH of the aqueous environment, likely due to the electrostatic repulsion between ionized VBIDA groups. Divalent nickel ions were chelated to the VBIDA groups, allowing selective metal affinity attachment of a His6-Cys peptide. The peptide was released upon the addition of the competitive ligand imidazole, demonstrating that the peptide attachment to the particles is reversible and selective. PMID:24176889

Synthesis and receptor binding studies of novel 4,4-disubstituted arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as a new class of σ1 ligands.

PubMed

Sadeghzadeh, Masoud; Sheibani, Shahab; Ghandi, Mehdi; Daha, Fariba Johari; Amanlou, Massoud; Arjmand, Mohammad; Hasani Bozcheloie, Abolfazl

2013-06-01

This study presents the synthesis and biological evaluation of a new series of arylalkyl/arylalkylsulfonyl piperazine and piperidine-based derivatives as sigma receptor ligands. It was found that a number of halogen substituted sulfonamides display relatively high and low affinities to σ1 and σ2 receptors, respectively. The σ1 affinities and subtype selectivities of four piperidine derivatives were also found to be generally comparable to those of piperazine analogues. Compared to σ1-Rs compounds with n = 0 and 2, those with n = 1 proved to have optimal length of carbon chain by exhibiting higher affinities. Within this series, the 4-benzyl-1-(3-iodobenzylsulfonyl)piperidine sigma ligand was identified with 96-fold σ1/σ2 selectivity ratio (Kiσ1 = 0.96 ± 0.05 nM and Kiσ2 = 91.8 ± 8.1 nM). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

Detection of Waterborne Viruses Using High Affinity Molecularly Imprinted Polymers.

PubMed

Altintas, Zeynep; Gittens, Micah; Guerreiro, Antonio; Thompson, Katy-Anne; Walker, Jimmy; Piletsky, Sergey; Tothill, Ibtisam E

2015-07-07

Molecularly imprinted polymers (MIPs) are artificial receptor ligands which can recognize and specifically bind to a target molecule. They are more resistant to chemical and biological damage and inactivation than antibodies. Therefore, target specific-MIP nanoparticles are aimed to develop and implemented to biosensors for the detection of biological toxic agents such as viruses, bacteria, and fungi toxins that cause many diseases and death due to the environmental contamination. For the first time, a molecularly imprinted polymer (MIP) targeting the bacteriophage MS2 as the template was investigated using a novel solid-phase synthesis method to obtain the artificial affinity ligand for the detection and removal of waterborne viruses through optical-based sensors. A high affinity between the artificial ligand and the target was found, and a regenerative MIP-based virus detection assay was successfully developed using a new surface plasmon resonance (SPR)-biosensor which provides an alternative technology for the specific detection and removal of waterborne viruses that lead to high disease and death rates all over the world.

Insights into Protein–Ligand Interactions: Mechanisms, Models, and Methods

PubMed Central

Du, Xing; Li, Yi; Xia, Yuan-Ling; Ai, Shi-Meng; Liang, Jing; Sang, Peng; Ji, Xing-Lai; Liu, Shu-Qun

2016-01-01

Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed. PMID:26821017

Ligand and membrane-binding behavior of the phosphatidylinositol transfer proteins PITPα and PITPβ.

PubMed

Baptist, Matilda; Panagabko, Candace; Cockcroft, Shamshad; Atkinson, Jeffrey

2016-12-01

Phosphatidylinositol transfer proteins (PITPs) are believed to be lipid transfer proteins because of their ability to transfer either phosphatidylinositol (PI) or phosphatidylcholine (PC) between membrane compartments, in vitro. However, the detailed mechanism of this transfer process is not fully established. To further understand the transfer mechanism of PITPs we examined the interaction of PITPs with membranes using dual polarization interferometry (DPI), which measures protein binding affinity on a flat immobilized lipid surface. In addition, a fluorescence resonance energy transfer (FRET)-based assay was also employed to monitor how quickly PITPs transfer their ligands to lipid vesicles. DPI analysis revealed that PITPβ had a higher affinity to membranes compared with PITPα. Furthermore, the FRET-based transfer assay revealed that PITPβ has a higher ligand transfer rate compared with PITPα. However, both PITPα and PITPβ demonstrated a preference for highly curved membrane surfaces during ligand transfer. In other words, ligand transfer rate was higher when the accepting vesicles were highly curved.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels

PubMed Central

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L.

2014-01-01

SUMMARY AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine and phenylalanine which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate and serine. AvGluR1 LBD crystal structures reveal a novel scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, while in the alanine, methionine and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl− lowers affinity for these ligands, but not for glutamate, aspartate or for phenylalanine which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure based studies on iGluR-ligand interactions. PMID:23434404

Sensing multiple ligands with single receptor

NASA Astrophysics Data System (ADS)

Singh, Vijay; Nemenman, Ilya

2015-03-01

Cells use surface receptors to measure concentrations of external ligand molecules. Limits on the accuracy of such sensing are well-known for the scenario where concentration of one molecular species is being determined by one receptor [Endres]. However, in more realistic scenarios, a cognate (high-affinity) ligand competes with many non-cognate (low-affinity) ligands for binding to the receptor. We analyze effects of this competition on the accuracy of sensing. We show that maximum-likelihood statistical inference allows determination of concentrations of multiple ligands, cognate and non-cognate, by the same receptor concurrently. While it is unclear if traditional biochemical circuitry downstream of the receptor can implement such inference exactly, we show that an approximate inference can be performed by coupling the receptor to a kinetic proofreading cascade. We characterize the accuracy of such kinetic proofreading sensing in comparison to the exact maximum-likelihood approach. We acknowledge the support from the James S. McDonnell Foundation and the Human Frontier Science Program.

Beyond small molecule SAR – using the dopamine D3 receptor crystal structure to guide drug design

PubMed Central

Keck, Thomas M.; Burzynski, Caitlin; Shi, Lei; Newman, Amy Hauck

2016-01-01

The dopamine D3 receptor is a target of pharmacotherapeutic interest in a variety of neurological disorders including schizophrenia, restless leg syndrome, and drug addiction. The high protein sequence homology between the D3 and D2 receptors has posed a challenge to developing D3 receptor-selective ligands whose behavioral actions can be attributed to D3 receptor engagement, in vivo. However, through primarily small molecule structure-activity relationship (SAR) studies, a variety of chemical scaffolds have been discovered over the past two decades that have resulted in several D3 receptor-selective ligands with high affinity and in vivo activity. Nevertheless, viable clinical candidates remain limited. The recent determination of the high-resolution crystal structure of the D3 receptor has invigorated structure-based drug design, providing refinements to the molecular dynamic models and testable predictions about receptor-ligand interactions. This review will highlight recent preclinical and clinical studies demonstrating potential utility of D3 receptor-selective ligands in the treatment of addiction. In addition, new structure-based rational drug design strategies for D3 receptor-selective ligands that complement traditional small molecule SAR to improve the selectivity and directed efficacy profiles are examined. PMID:24484980

Exploring Thermoresponsive Affinity Agents to Enhance Microdialysis Sampling Efficiency of Proteins

NASA Astrophysics Data System (ADS)

Vasicek, Thaddeus

Affinity agents increase microdialysis protein relative recovery, yet they have not seen widespread use within the microdialysis community due to their additional instrumentation requirements and prohibitive cost. This dissertation describes new affinity agents for microdialysis that require no additional instrumentation to use, have nearly 100% particle recovery, are 7 times more cost efficient than alternatives, and have low specificity enabling their use for a wide variety of proteins. Initially gold nanoparticles were chosen as an affinity ligand support due to their high surface area/volume ratio and colloidal stability. Poly (N-isopropylacrylamide) was immobilized to the gold nanoparticles, which served to sterically stabilize the particles and to act as a generic, reversible protein capture agent. A method was developed to reproducibly vary and quantify poly (N-isopropylacrylamide) graft density from 0.09 to 0.40 ligands/nm2 on gold nanoparticles. During characterization of the polymer coated gold nanoparticles, irreversible particle agglomeration was observed at low polymer graft density in ionic solutions, which prevented further development as a protein capture agent. Poly (N-isopropylacrylamide) nanogels, which have low nonspecific adsorption, low interparticle attractive forces owing to the low curvature of the particle, and a low Hamaker constant, were synthesized to overcome the agglomeration problem. A generic protein affinity ligand cibacron blue, was immobilized to the nanogels, which enabled rapid determination of particle recovery. The perfusion of the nanogels through a microdialysis probe was optimized yielding 100% particle recovery using a combination of a syringe and peristaltic pump. The microdialysis collection efficiency of CCL2, a physiologically relevant cytokine, was increased 3-fold with addition of the nanogel to the microdialysis perfusion fluid. The reduction in instrumentation requirements, low cost, and low specificity obtained with the new affinity agents will lead to increased affinity agent use for microdiaylsis protein sampling.

Elucidation of Ligand-Dependent Modulation of Disorder-Order Transitions in the Oncoprotein MDM2.

PubMed

Bueren-Calabuig, Juan A; Michel, Julien

2015-06-01

Numerous biomolecular interactions involve unstructured protein regions, but how to exploit such interactions to enhance the affinity of a lead molecule in the context of rational drug design remains uncertain. Here clarification was sought for cases where interactions of different ligands with the same disordered protein region yield qualitatively different results. Specifically, conformational ensembles for the disordered lid region of the N-terminal domain of the oncoprotein MDM2 in the presence of different ligands were computed by means of a novel combination of accelerated molecular dynamics, umbrella sampling, and variational free energy profile methodologies. The resulting conformational ensembles for MDM2, free and bound to p53 TAD (17-29) peptide identify lid states compatible with previous NMR measurements. Remarkably, the MDM2 lid region is shown to adopt distinct conformational states in the presence of different small-molecule ligands. Detailed analyses of small-molecule bound ensembles reveal that the ca. 25-fold affinity improvement of the piperidinone family of inhibitors for MDM2 constructs that include the full lid correlates with interactions between ligand hydrophobic groups and the C-terminal lid region that is already partially ordered in apo MDM2. By contrast, Nutlin or benzodiazepinedione inhibitors, that bind with similar affinity to full lid and lid-truncated MDM2 constructs, interact additionally through their solubilizing groups with N-terminal lid residues that are more disordered in apo MDM2.

Titration ELISA as a Method to Determine the Dissociation Constant of Receptor Ligand Interaction.

PubMed

Eble, Johannes A

2018-02-15

The dissociation constant describes the interaction between two partners in the binding equilibrium and is a measure of their affinity. It is a crucial parameter to compare different ligands, e.g., competitive inhibitors, protein isoforms and mutants, for their binding strength to a binding partner. Dissociation constants are determined by plotting concentrations of bound versus free ligand as binding curves. In contrast, titration curves, in which a signal that is proportional to the concentration of bound ligand is plotted against the total concentration of added ligand, are much easier to record. The signal can be detected spectroscopically and by enzyme-linked immunosorbent assay (ELISA). This is exemplified in a protocol for a titration ELISA that measures the binding of the snake venom-derived rhodocetin to its immobilized target domain of α2β1 integrin. Titration ELISAs are versatile and widely used. Any pair of interacting proteins can be used as immobilized receptor and soluble ligand, provided that both proteins are pure, and their concentrations are known. The difficulty so far has been to determine the dissociation constant from a titration curve. In this study, a mathematical function underlying titration curves is introduced. Without any error-prone graphical estimation of a saturation yield, this algorithm allows processing of the raw data (signal intensities at different concentrations of added ligand) directly by mathematical evaluation via non-linear regression. Thus, several titration curves can be recorded simultaneously and transformed into a set of characteristic parameters, among them the dissociation constant and the concentration of binding-active receptor, and they can be evaluated statistically. When combined with this algorithm, titration ELISAs gain the advantage of directly presenting the dissociation constant. Therefore, they may be used more efficiently in the future.

The solvatochromic effects of side chain substitution on the binding interaction of novel tricarbocyanine dyes with human serum albumin.

PubMed

Beckford, Garfield; Owens, Eric; Henary, Maged; Patonay, Gabor

2012-04-15

The effects of solvatochromism on protein-ligand interactions have been studied by absorbance and near-infrared laser induced fluorescence (NIR-LIF) spectroscopy. The utility of three novel classes of cyanine dyes designed for this purpose illustrates that the affinity interactions of ligands at the hydrophobic binding pockets of Human Serum Albumin (HSA) are not only dependent on the overall hydrophobic characteristics of the molecules but are highly influenced by the size of the ligands as well. Whereas changes to the chromophore moiety exhibited slight to moderate changes to the hydrophobic nature of these molecules, substitution at the alkyl indolium side chain has enabled us to vary the binding affinity towards serum albumin. Substitution at the indolium side chain among an ethyl to butyl group results in improved binding characteristics and an almost three-fold increase in affinity constant. In addition, replacement of the ethyl side chain with a phenylpropyl group also yielded unique solvotachromic patterns such as increased hydrophobicity and subsequent biocompatibility with the HSA binding regions. Ligand interaction was however inhibited by steric hindrance associated with the bulky phenyl ring system thus affecting the increased binding that could be realized from the improved hydrophobic nature of the molecules. This characteristic change in binding affinity is of potential interest to developing a methodology which reveals information on the hydrophobic character and steric specificity of the binding cavities. Copyright © 2012 Elsevier B.V. All rights reserved.

The energy and work of a ligand-gated ion channel

PubMed Central

Auerbach, Anthony

2015-01-01

Ligand-gated ion channels are allosteric membrane proteins that isomerize between C(losed) and O(pen) conformations. A difference in affinity for ligands in the two shapes influences the C↔O ‘gating’ equilibrium constant. The energies associated with adult-type mouse neuromuscular nicotinic acetylcholine receptor-channel (AChR) gating have been measured by using single-channel electrophysiology. Without ligands the free energy, enthalpy and entropy of gating are ΔG0=+8.4, ΔH0=+10.9 and ΔS0=+2.4 kcal/mol (−100 mV, 23 °C). Many mutations throughout the protein change ΔG0, including natural ones that cause disease. Agonists and most mutations change approximately independently the ground state energy difference, so it is possible to forecast and engineer AChR responses simply by combining perturbations. The free energy of the low↔high affinity change for the neurotransmitter at each of two functionally-equivalent binding sites is ΔGBACh=−5.1 kcal/mol. ΔGBACh is set mainly by interactions of ACh with just three binding site aromatic groups. For a series of structurally-related agonists there is a correlation between the energies of low- and high-affinity binding, which implies that gating commences with the formation of the low affinity complex. Brief, intermediate states in binding and gating have been detected. Several proposals for the nature of the gating transition state energy landscape and the isomerization mechanism are discussed. PMID:23357172

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 non-chromatographic protein purification strategy using Src 3 homology domains as generalized capture domains.

PubMed

Kim, Heejae; Chen, Wilfred

2016-09-20

Protein purification using inverse phase transition of elastin-like polypeptide (ELP) domains is a useful alternative to chromatography. Genetic fusions of ELP domains to various proteins have the ability to reversibly transition between soluble monomers and micron-sized aggregates and this has been used to selectively purify many ELP fusions. Affinity domains can enhance this technology by using specific protein binding domains to enable ELP mediated affinity capture (EMAC) of proteins of interest (POI) that have been fused to corresponding affinity ligands. In this paper, we highlight the use of Src homology 3 (SH3) domains and corresponding peptide ligands in EMAC that have differential binding affinities towards SH3 for efficient capture and elution of proteins. Furthermore, differences between capture and elution of a monomeric and a multimeric protein were also studied. Copyright © 2016 Elsevier B.V. All rights reserved.

Engineering cofactor and ligand binding in an artificial neuroglobin

NASA Astrophysics Data System (ADS)

Zhang, Lei

HP-7 is one artificial mutated oxygen transport protein, which operates via a mechanism akin to human neuroglobin and cytoglobin. This protein destabilizes one of two heme-ligating histidine residues by coupling histidine side chain ligation with the burial of three charged glutamate residues on the same helix. Replacement of these glutamate residues with alanine, which has a neutral hydrophobicity, slows gaseous ligand binding 22-fold, increases the affinity of the distal histidine ligand by a factor of thirteen, and decreases the binding affinity of carbon monoxide, a nonreactive oxygen analogue, three-fold. Paradoxically, it also decreases heme binding affinity by a factor of three in the reduced state and six in the oxidized state. Application of a two-state binding model, in which an initial pentacoordinate binding event is followed by a protein conformational change to hexacoordinate, provides insight into the mechanism of this seemingly counterintuitive result: the initial pentacoordinate encounter complex is significantly destabilized by the loss of the glutamate side chains, and the increased affinity for the distal histidine only partially compensates. These results point to the importance of considering each oxidation and conformational state in the design of functional artificial proteins. We have also examined the effects these mutations have on function. The K d of the nonnreactive oxygen analogue carbon monoxide (CO) is only decreased three-fold, despite the large increase in distal histidine affinity engendered by the 22-fold decrease in the histidine ligand off-rate. This is a result of the four-fold increase in affinity for CO binding to the pentacoordinate state. Oxygen binds to HP7 with a Kd of 117 µM, while the mutant rapidly oxidizes when exposed to oxygen. EPR analysis of both ferric hemoproteins demonstrates that the mutation increases disorder at the heme binding site. NMR-detected deuterium exchange demonstrates that the mutation causes a large increase in water penetration into the protein core. The inability of the mutant protein may thus either be due to increased water penetration, the large decrease in binding rate caused by the increase in distal histidine affinity, or a combination of the two factors.

A fragment-based approach to the SAMPL3 Challenge

NASA Astrophysics Data System (ADS)

Kulp, John L.; Blumenthal, Seth N.; Wang, Qiang; Bryan, Richard L.; Guarnieri, Frank

2012-05-01

The success of molecular fragment-based design depends critically on the ability to make predictions of binding poses and of affinity ranking for compounds assembled by linking fragments. The SAMPL3 Challenge provides a unique opportunity to evaluate the performance of a state-of-the-art fragment-based design methodology with respect to these requirements. In this article, we present results derived from linking fragments to predict affinity and pose in the SAMPL3 Challenge. The goal is to demonstrate how incorporating different aspects of modeling protein-ligand interactions impact the accuracy of the predictions, including protein dielectric models, charged versus neutral ligands, ΔΔGs solvation energies, and induced conformational stress. The core method is based on annealing of chemical potential in a Grand Canonical Monte Carlo (GC/MC) simulation. By imposing an initially very high chemical potential and then automatically running a sequence of simulations at successively decreasing chemical potentials, the GC/MC simulation efficiently discovers statistical distributions of bound fragment locations and orientations not found reliably without the annealing. This method accounts for configurational entropy, the role of bound water molecules, and results in a prediction of all the locations on the protein that have any affinity for the fragment. Disregarding any of these factors in affinity-rank prediction leads to significantly worse correlation with experimentally-determined free energies of binding. We relate three important conclusions from this challenge as applied to GC/MC: (1) modeling neutral ligands—regardless of the charged state in the active site—produced better affinity ranking than using charged ligands, although, in both cases, the poses were almost exactly overlaid; (2) simulating explicit water molecules in the GC/MC gave better affinity and pose predictions; and (3) applying a ΔΔGs solvation correction further improved the ranking of the neutral ligands. Using the GC/MC method under a variety of parameters in the blinded SAMPL3 Challenge provided important insights to the relevant parameters and boundaries in predicting binding affinities using simulated annealing of chemical potential calculations.

Dynamic Seeding of Perfusing Human Umbilical Vein Endothelial Cells (HUVECs) onto Dual-Function Cell Adhesion Ligands: Arg-Gly-Asp (RGD)-Streptavidin and Biotinylated Fibronectin

PubMed Central

Anamelechi, Charles C.; Clermont, Edward C.; Novak, Matthew T.; Reichert, William M.

2014-01-01

Surfaces decorated with high affinity ligands can be used to facilitate rapid attachment of endothelial cells; however, standard equilibrium cell detachment studies are poorly suited for assessing these initial adhesion events. Here, a dynamic seeding and cell retention method was used to examine the initial attachment of perfusing human umbilical vein endothelial cells (HUVECs) to bare Teflon-AF substrates, substrates pre-adsorbed with fibronectin alone, or substrates co-pre-adsorbed with two dual-function cell-adhesion ligands: biotinylated fibronectin (bFN) and RGD-streptavidin mutant (RGD-SA). Cell attachment was evaluated as a function of cell trypsinization (integrin digestion), surface protein formulation, and cell perfusion rate. Surfaces co-pre-adsorbed with bFN and RGD-SA showed the highest density of attached cells after 8 min of perfusion and the highest percent retention when subjected to shear flow at 60 dynes/cm2 for 2 min. Surfaces with no ligand treatment showed the lowest cell attachment and retention under flow in all cases. HUVECs trypsinized with mild 0.025% trypsin/ethylenediaminetetraacetic acid (EDTA) showed greater cell adhesion after perfusion and higher percent retention after shear flow than those trypsinized using harsher 0.05% trypsin/EDTA. The preferential affinities of the two dual-function ligands for α5β1 and αvβ3 integrins were also examined by surface plasmon resonance (SPR) spectroscopy. The dynamic cell seeding studies confirmed that the dual-function ligand system promotes HUVEC adhesion and retention at short time points when tested using a perfusion assay. SPR studies showed that the two ligands exhibited equal affinity for both α5β1 and αvβ3 integrins but that the combined ligands bound more total integrins than the two ligands tested separately. PMID:19348476

Decoy Strategies: The Structure of TL1A:DcR3 Complex

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

C Zhan; Y Patskovsky; Q Yan

2011-12-31

Decoy Receptor 3 (DcR3), a secreted member of the Tumor Necrosis Factor (TNF) receptor superfamily, neutralizes three different TNF ligands: FasL, LIGHT, and TL1A. Each of these ligands engages unique signaling receptors which direct distinct and critical immune responses. We report the crystal structures of the unliganded DcR3 ectodomain and its complex with TL1A, as well as complementary mutagenesis and biochemical studies. These analyses demonstrate that DcR3 interacts with invariant backbone and side-chain atoms in the membrane-proximal half of TL1A which supports recognition of its three distinct TNF ligands. Additional features serve as antideterminants that preclude interaction with other membersmore » of the TNF superfamily. This mode of interaction is unique among characterized TNF:TNFR family members and provides a mechanistic basis for the broadened specificity required to support the decoy function of DcR3, as well as for the rational manipulation of specificity and affinity of DcR3 and its ligands.« less

GalaxyDock BP2 score: a hybrid scoring function for accurate protein-ligand docking

NASA Astrophysics Data System (ADS)

Baek, Minkyung; Shin, Woong-Hee; Chung, Hwan Won; Seok, Chaok

2017-07-01

Protein-ligand docking is a useful tool for providing atomic-level understanding of protein functions in nature and design principles for artificial ligands or proteins with desired properties. The ability to identify the true binding pose of a ligand to a target protein among numerous possible candidate poses is an essential requirement for successful protein-ligand docking. Many previously developed docking scoring functions were trained to reproduce experimental binding affinities and were also used for scoring binding poses. However, in this study, we developed a new docking scoring function, called GalaxyDock BP2 Score, by directly training the scoring power of binding poses. This function is a hybrid of physics-based, empirical, and knowledge-based score terms that are balanced to strengthen the advantages of each component. The performance of the new scoring function exhibits significant improvement over existing scoring functions in decoy pose discrimination tests. In addition, when the score is used with the GalaxyDock2 protein-ligand docking program, it outperformed other state-of-the-art docking programs in docking tests on the Astex diverse set, the Cross2009 benchmark set, and the Astex non-native set. GalaxyDock BP2 Score and GalaxyDock2 with this score are freely available at http://galaxy.seoklab.org/softwares/galaxydock.html.

Avidin-Based Targeting and Purification of a Protein IX-Modified, Metabolically Biotinylated Adenoviral Vector

PubMed Central

Campos, Samuel K.; Parrott, M. Brandon; Barry, Michael A.

2014-01-01

While genetic modification of adenoviral vectors can produce vectors with modified tropism, incorporation of targeting peptides/proteins into the structural context of the virion can also result in destruction of ligand targeting or virion integrity. To combat this problem, we have developed a versatile targeting system using metabolically biotinylated adenoviral vectors bearing biotinylated fiber proteins. These vectors have been demonstrated to be useful as a platform for avidin-based ligand screening and vector targeting by conjugating biotinylated ligands to the virus using high-affinity tetrameric avidin (Kd = 10−15 M). The biotinylated vector could also be purified by biotin-reversible binding on monomeric avidin (Kd = 10−7 M). In this report, a second metabolically biotinylated adenovirus vector, Ad-IX-BAP, has been engineered by fusing a biotin acceptor peptide (BAP) to the C-terminus of the adenovirus pIX protein. This biotinylated vector displays twice as many biotins and was markedly superior for single-step affinity purification on monomeric avidin resin. However, unlike the fiber-biotinylated vector, Ad-IX-BAP failed to retarget to cells with biotinylated antibodies including anti-CD71 against the transferrin receptor. In contrast, Ad-IX-BAP was retargeted if transferrin, the cognate ligand for CD71, was used as a ligand rather than the anti-CD71. This work demonstrates the utility of metabolic biotinylation as a molecular screening tool to assess the utility of different viral capsid proteins for ligand display and the biology and compatibility of different ligands and receptors for vector targeting applications. These results also demonstrate the utility of the pIX-biotinylated vector as a platform for gentle single-step affinity purification of adenoviral vectors. PMID:15194061

Development of a high specific activity radioligand, /sup 125/I-LSD, and its application to the study of serotonin receptors

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

Kadan, M.J.

/sup 125/I-Labeled receptor ligands can be synthesized with specific activities exceeding 2000 Ci/mmol, making them nearly 70-fold more sensitive in receptor site assays than (mono) tritiated ligands. We have synthesized and characterized /sup 125/I-lysergic acid diethylamide (/sup 125/I-LSD), the first radioiodinated ligand for serotonin receptor studies. The introduction of /sup 125/I at the 2 position of LSD increased both the affinity and selectivity of this compound for serotonin 5-HT/sub 2/ receptors in rat cortex. The high specific activity of /sup 125/I-LSD and its high ratio of specific to nonspecific binding make this ligand especially useful for autoradiographic studies of serotoninmore » receptor distribution. We have found that /sup 125/I-LSD binds with high affinity to a class of serotonin receptors in the CNS of the marine mollusk Aplysia californica.« less

Ligand recognition by RAR and RXR receptors: binding and selectivity.

PubMed

Sussman, Fredy; de Lera, Angel R

2005-10-06

Fundamental biological functions, most notably embriogenesis, cell growth, cell differentiation, and cell apoptosis, are in part regulated by a complex genomic network that starts with the binding (and activation) of retinoids to their cognate receptors, members of the superfamily of nuclear receptors. We have studied ligand recognition of retinoic receptors (RXRalpha and RARgamma) using a molecular-mechanics-based docking method. The protocol used in this work is able to rank the affinity of pairs of ligands for a single retinoid receptor, the highest values corresponding to those that adapt better to the shape of the binding site and generate the optimal set of electrostatic and apolar interactions with the receptor. Moreover, our studies shed light onto some of the energetic contributions to retinoid receptor ligand selectivity. In this regard we show that there is a difference in polarity between the binding site regions that anchor the carboxylate in RAR and RXR, which translates itself into large differences in the energy of interaction of both receptors with the same ligand. We observe that the latter energy change is canceled off by the solvation energy penalty upon binding. This energy compensation is borne out as well by experiments that address the effect of site-directed mutagenesis on ligand binding to RARgamma. The hypothesis that the difference in binding site polarity might be exploited to build RXR-selective ligands is tested with some compounds having a thiazolidinedione anchoring group.

Refined docking as a valuable tool for lead optimization: application to histamine H3 receptor antagonists.

PubMed

Levoin, Nicolas; Calmels, Thierry; Poupardin-Olivier, Olivia; Labeeuw, Olivier; Danvy, Denis; Robert, Philippe; Berrebi-Bertrand, Isabelle; Ganellin, C Robin; Schunack, Walter; Stark, Holger; Capet, Marc

2008-10-01

Drug-discovery projects frequently employ structure-based information through protein modeling and ligand docking, and there is a plethora of reports relating successful use of them in virtual screening. Hit/lead optimization, which represents the next step and the longest for the medicinal chemist, is very rarely considered. This is not surprising because lead optimization is a much more complex task. Here, a homology model of the histamine H(3) receptor was built and tested for its ability to discriminate ligands above a defined threshold of affinity. In addition, drug safety is also evaluated during lead optimization, and "antitargets" are studied. So, we have used the same benchmarking procedure with the HERG channel and CYP2D6 enzyme, for which a minimal affinity is strongly desired. For targets and antitargets, we report here an accuracy as high as at least 70%, for ligands being classified above or below the chosen threshold. Such a good result is beyond what could have been predicted, especially, since our test conditions were particularly stringent. First, we measured the accuracy by means of AUC of ROC plots, i. e. considering both false positive and false negatives. Second, we used as datasets extensive chemical libraries (nearly a thousand ligands for H(3)). All molecules considered were true H(3) receptor ligands with moderate to high affinity (from microM to nM range). Third, the database is issued from concrete SAR (Bioprojet H(3) BF2.649 library) and is not simply constituted by few active ligands buried in a chemical catalogue.

Conformational changes induced in the eukaryotic translation initiation factor eIF4E by a clinically relevant inhibitor, ribavirin triphosphate

PubMed Central

Volpon, Laurent; Osborne, Michael J.; Zahreddine, Hiba; Romeo, Andrea A.; Borden, Katherine L.B.

2013-01-01

The eukaryotic translation initiation factor eIF4E is highly elevated in human cancers including acute myeloid leukemia (AML). A potential anticancer agent, ribavirin, targets eIF4E activity in AML patients corresponding to clinical responses. To date, ribavirin is the only direct inhibitor of eIF4E to reach clinical trials. We showed that ribavirin acts as a competitive inhibitor of the methyl 7-guanosine (m7G) cap, the natural ligand of eIF4E. Here we examine the conformational changes occurring in human eIF4E upon binding the active metabolite of ribavirin, ribavirin triphosphate (RTP). Our NMR data revealed an unexpected concentration dependence on RTP affinity for eIF4E. We observed NMR spectra characteristic of tight binding at low micromolar concentrations (2-5μM eIF4E) but much weaker affinity at more typical NMR concentrations (50-200μM). Comparison of chemical shift perturbation and line broadening suggest that the two eIF4E-RTP complexes differ in the precise positioning of RTP within the cap binding pocket, with the high affinity complex showing more extensive changes to the central β-sheet and dorsal surface of eIF4E, similar to m7G cap. The differences between high and low affinity complexes arise due to concentration dependent aggregation of eIF4E and RTP. Given the intracellular concentrations of eIF4E and RTP and the differential binding toward the W56A eIF4E mutant the high affinity complex is the most physiologically relevant. In summary, these findings demonstrate that RTP binds in the cap-binding site but also suggests new features of this pocket that should be considered in both drug design efforts and reveal new insights into ligand eIF4E recognition. PMID:23583375

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand.

PubMed

Ding, Zhaoyang; Cao, Xuejun

2013-12-17

Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris-HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum.

Affinity precipitation of human serum albumin using a thermo-response polymer with an L-thyroxin ligand

PubMed Central

2013-01-01

Background Affinity precipitation has been reported as a potential technology for the purification of proteins at the early stage of downstream processing. The technology could be achieved using reversible soluble-insoluble polymers coupled with an affinity ligand to purify proteins from large volumes of dilute solution material such as fermentation broths or plasma. In this study, a thermo-response polymer was synthesized using N-methylol acrylamide, N-isopropyl acrylamide and butyl acrylate as monomers. The molecular weight of the polymer measured by the viscosity method was 3.06 × 104 Da and the lower critical solution temperature (LCST) was 28.0°C.The recovery of the polymer above the LCST was over 95.0%. Human serum albumin (HSA) is the most abundant protein in the human serum system, and it has important functions in the human body. High purity HSA is required in pharmaceuticals. Safe and efficient purification is a crucial process during HSA production. Results A thermo-response polymer was synthesized and L-thyroxin immobilized on the polymer as an affinity ligand to enable affinity precipitation of HSA. The LCST of the affinity polymer was 31.0°C and the recovery was 99.6% of its original amount after recycling three times. The optimal adsorption condition was 0.02 M Tris–HCl buffer (pH 7.0) and the HSA adsorption capacity was 14.9 mg/g polymer during affinity precipitation. Circular dichroism spectra and a ForteBio Octet system were used to analyze the interactions between the affinity polymer and HSA during adsorption and desorption. The recovery of total HSA by elution with 1.0 mol/L NaSCN was 93.6%. When the affinity polymer was applied to purification of HSA from human serum, HSA could be purified to single-band purity according to SDS-PAGE. Conclusion A thermo-response polymer was synthesized and L-thyroxin was attached to the polymer. Affinity precipitation was used to purify HSA from human serum. PMID:24341315

Role of Conserved Glycine in Zinc-dependent Medium Chain Dehydrogenase/Reductase Superfamily*

PubMed Central

Tiwari, Manish Kumar; Singh, Raushan Kumar; Singh, Ranjitha; Jeya, Marimuthu; Zhao, Huimin; Lee, Jung-Kul

2012-01-01

The medium-chain dehydrogenase/reductase (MDR) superfamily consists of a large group of enzymes with a broad range of activities. Members of this superfamily are currently the subject of intensive investigation, but many aspects, including the zinc dependence of MDR superfamily proteins, have not yet have been adequately investigated. Using a density functional theory-based screening strategy, we have identified a strictly conserved glycine residue (Gly) in the zinc-dependent MDR superfamily. To elucidate the role of this conserved Gly in MDR, we carried out a comprehensive structural, functional, and computational analysis of four MDR enzymes through a series of studies including site-directed mutagenesis, isothermal titration calorimetry, electron paramagnetic resonance (EPR), quantum mechanics, and molecular mechanics analysis. Gly substitution by other amino acids posed a significant threat to the metal binding affinity and activity of MDR superfamily enzymes. Mutagenesis at the conserved Gly resulted in alterations in the coordination of the catalytic zinc ion, with concomitant changes in metal-ligand bond length, bond angle, and the affinity (Kd) toward the zinc ion. The Gly mutants also showed different spectroscopic properties in EPR compared with those of the wild type, indicating that the binding geometries of the zinc to the zinc binding ligands were changed by the mutation. The present results demonstrate that the conserved Gly in the GHE motif plays a role in maintaining the metal binding affinity and the electronic state of the catalytic zinc ion during catalysis of the MDR superfamily enzymes. PMID:22500022

Functional Analysis of the Citrate Activator CitO from Enterococcus faecalis Implicates a Divalent Metal in Ligand Binding

PubMed Central

Blancato, Víctor S.; Pagliai, Fernando A.; Magni, Christian; Gonzalez, Claudio F.; Lorca, Graciela L.

2016-01-01

The regulator of citrate metabolism, CitO, from Enterococcus faecalis belongs to the FCD family within the GntR superfamily. In the presence of citrate, CitO binds to cis-acting sequences located upstream of the cit promoters inducing the expression of genes involved in citrate utilization. The quantification of the molecular binding affinities, performed by isothermal titration calorimetry (ITC), indicated that CitO has a high affinity for citrate (KD = 1.2 ± 0.2 μM), while it did not recognize other metabolic intermediates. Based on a structural model of CitO where a putative small molecule and a metal binding site were identified, it was hypothesized that the metal ion is required for citrate binding. In agreement with this model, citrate binding to CitO sharply decreased when the protein was incubated with EDTA. This effect was reverted by the addition of Ni2+, and Zn2+ to a lesser extent. Structure-based site-directed mutagenesis was conducted and it was found that changes to alanine in residues Arg97 and His191 resulted in decreased binding affinities for citrate, as determined by EMSA and ITC. Further assays using lacZ fusions confirmed that these residues in CitO are involved in sensing citrate in vivo. These results indicate that the molecular modifications induced by a ligand and a metal binding in the C-terminal domain of CitO are required for optimal DNA binding activity, and consequently, transcriptional activation. PMID:26903980

Synthesis and biological investigation of new equatorial (β) stereoisomers of 3-aminotropane arylamides with atypical antipsychotic profile.

PubMed

Stefanowicz, Jacek; Słowiński, Tomasz; Wróbel, Martyna Zofia; Herold, Franciszek; Gomółka, Anna Edyta; Wesołowska, Anna; Jastrzębska-Więsek, Magdalena; Partyka, Anna; Andres-Mach, Marta; Czuczwar, Stanisław Jerzy; Łuszczki, Jarogniew Jacek; Zagaja, Mirosław; Siwek, Agata; Nowak, Gabriel; Żołnierek, Maria; Bączek, Tomasz; Ulenberg, Szymon; Belka, Mariusz; Turło, Jadwiga

2016-09-15

A series of novel 3β-aminotropane derivatives containing a 2-naphthalene or a 2-quinoline moiety was synthesised and evaluated for their affinity for 5-HT1A, 5-HT2A and D2 receptors. Their affinity for the receptors was in the nanomolar to micromolar range. p-Substitution (6c, 6f, 6i, 6l, 6o), as well as substitution with chlorine atoms (6g, 6h, 6i), led to a significant increase in binding affinity for D2 receptors with compounds 6f (Ki=0.6nM), 6c and 6i (Ki=0.4nM), having the highest binding affinities. m-Substituted derivatives were the most promising ligands in terms of 5-HT2A receptor binding affinity whereas 2-quinoline derivatives (10a, 10b) displayed the highest affinity for 5-HT1AR and were the most selective ligands with Ki=62.7nM and Ki=30.5nM, respectively. Finally, the selected ligands 6b, 6d, 6e, 6g, 6h, 6k, 6n and 6o, with triple binding activity for the D2, 5-HT1A and 5-HT2A receptors, were subjected to in vivo tests, such as those for induced hypothermia, climbing behaviour and the head twitch response, in order to determine their pharmacological profile. The tested ligands presented neither agonist nor antagonist properties for the 5-HT1A receptors in the induced hypothermia and lower lip retraction (LLR) tests. All tested compounds displayed antagonistic activity against 5-HT2A, with 6n and 6o being the most active. Four (6b, 6k, 6n and 6o) out of eight tested compounds could be classified as D2 antagonists. Additionally, evaluation of metabolic stability was performed for selected ligands, and introduction of halogen atoms into the benzene ring of 6h, 6k, 6n and 6o improved their metabolic stability. The project resulted in the selection of the lead compounds 6n and 6o, which had antipsychotic profiles, combining dopamine D2-receptor and 5-HT2A antagonism and metabolic stability. Copyright © 2016 Elsevier Ltd. All rights reserved.

Interaction of ligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides

PubMed Central

Blume, Arthur J.

1978-01-01

This study shows that nucleotides, as well as ions, regulate the opiate receptors of brain. GMP-P(NH)P and Na+ reduce the amount of steady-state specific [3H]dihydromorphine binding and increase the rate of dissociation of the ligand from the opiate receptor. In contrast, Mn2+ decreases the rate of ligand dissociation and antagonizes the ability of Na+ to increase dissociation. The effects of GMP-P(NH)P on steady-state binding and dissociation are not reversed by washing. Only GTP, GDP, ITP, and IMP-P(NH)P, in addition to GMP-P(NH)P, increase the rate of dihydromorphine dissociation. The site of nucleotide action appears to have high affinity: <1 μM GMP-P(NH)P produces half-maximal increases in ligand dissociation. GMP-P(NH)P- and Na+-directed increases in dissociation have also been found for the opiate agonists [3H]etorphine, [3H]Leu-enkephalin, and [3H]Met-enkephalin and the opiate antagonist [3H]naltrexone. Mn2+-directed decreases in dissociation have been found for the agonist [3H]-etorphine and the antagonist [3H]naltrexone. Although the plasma membrane receptors for a number of other neuro-transmitters and hormones are also regulated by guanine nucleotides, the opiate receptors appear unique because only they show nucleotide regulation of both agonist and antagonist binding. PMID:205867

A Lectin Purified from Blood Red Bracket Mushroom, Pycnoporus sanguineus (Agaricomycetidae), Mycelium Displayed Affinity Toward Bovine Transferrin.

PubMed

Albores, Silvana; Moros, Maria; Cerdeiras, Maria Pia; de la Fuente, Jesus Martinez; Grazu, Valeria; Fraguas, Laura Franco

2016-01-01

Fungal lectins constitute excellent ligands for development of affinity adsorbents useful in affinity chromatography. In this work, a lectin was purified from Pycnoporus sanguineus (PSL) mycelium using 3 procedures: by affinity chromatography, using magnetic galactosyl-nanoparticles or galactose coupled to Sepharose, and by ionic exchange chromatography (IEC). The highest lectin yield was achieved by IEC (55%); SDS-PAGE of PSL showed 2 bands with molecular mass of 68.7 and 55.2 kDa and IEC displayed 2 bands at pi 5.5 and 5.2. The lectin agglutinates rat erythrocytes, exhibiting broad specificity toward several monosaccharides, including galactose. The agglutination was also inhibited by the glycoproteins fetal calf fetuin, bovine lactoferrin, bovine transferrin, and horseradish peroxidase. The lectin was then used to synthesize an affinity adsorbent (PSL-Sepharose) and the interaction with glycoproteins was evaluated by analyzing their chromatographic behaviors. The strongest interaction with the PSL-derivative was observed with transferrin, although lower interactions were also displayed toward fetuin and lactoferrin. These results indicate that the purified PSL constitutes an interesting ligand for the design of affinity adsorbents to be used (i.e., in glycoprotein purification).

The Effects of Protein-Ligand Associations on the Subunit Interactions of Phosphofructokinase from B. stearothermophilus†

PubMed Central

Quinlan, R. Jason; Reinhart, Gregory D.

2008-01-01

Differences between the crystal structures of inhibitor-bound and uninihibited forms of phosphofructokinase (PFK) from B. stearothermophilus have led to a structural model for allosteric inhibition by phosphenolpyruvate (PEP) wherein a dimer-dimer interface within the tetrameric enzyme undergoes a quaternary shift. We have developed a labeling and hybridization technique to generate a tetramer with subunits containing two different extrinsic fluorophores simultaneously in known subunit orientations. This construct has been utilized in the examination of the effects of allosteric ligand and substrate binding on the subunit affinities of tetrameric PFK using several biophysical and spectroscopic techniques including 2-photon, dual-channel Fluorescence Correlation Spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is sufficient to reduce the affinity of the active site interface from beyond the limits of experimental detection to nanomolar affinity, while conversely strengthening the interface at which it is bound. The reduced interface affinity is specific to inhibitor-binding, as binding the activator ADP at the same allosteric site causes no reduction in subunit affinity. With inhibitor bound, the weakened subunit affinity has allowed the kinetics of dimer association to be elucidated. PMID:16981693

Continuous microfluidic assortment of interactive ligands (CMAIL)

NASA Astrophysics Data System (ADS)

Hsiao, Yi-Hsing; Huang, Chao-Yang; Hu, Chih-Yung; Wu, Yen-Yu; Wu, Chung-Hsiun; Hsu, Chia-Hsien; Chen, Chihchen

2016-08-01

Finding an interactive ligand-receptor pair is crucial to many applications, including the development of monoclonal antibodies. Biopanning, a commonly used technique for affinity screening, involves a series of washing steps and is lengthy and tedious. Here we present an approach termed continuous microfluidic assortment of interactive ligands, or CMAIL, for the screening and sorting of antigen-binding single-chain variable antibody fragments (scFv) displayed on bacteriophages (phages). Phages carrying native negative charges on their coat proteins were electrophoresed through a hydrogel matrix functionalized with target antigens under two alternating orthogonal electric fields. During the weak horizontal electric field phase, phages were differentially swept laterally depending on their affinity for the antigen, and all phages were electrophoresed down to be collected during the strong vertical electric field phase. Phages of different affinity were spatially separated, allowing the continuous operation. More than 105 CFU (colony forming unit) antigen-interacting phages were isolated with ~100% specificity from a phage library containing 3 × 109 individual members within 40 minutes of sorting using CMAIL. CMAIL is rapid, sensitive, specific, and does not employ washing, elution or magnetic beads. In conclusion, we have developed an efficient and cost-effective method for isolating and sorting affinity reagents involving phage display.

What reaches the antenna? How to calibrate odor flux and ligand-receptor affinities.

PubMed

Andersson, Martin N; Schlyter, Fredrik; Hill, Sharon Rose; Dekker, Teun

2012-06-01

Physiological studies on olfaction frequently ignore the airborne quantities of stimuli reaching the sensory organ. We used a gas chromatography-calibrated photoionization detector to estimate quantities released from standard Pasteur pipette stimulus cartridges during repeated puffing of 27 compounds and verified how lack of quantification could obscure olfactory sensory neuron (OSN) affinities. Chemical structure of the stimulus, solvent, dose, storage condition, puff interval, and puff number all influenced airborne quantities. A model including boiling point and lipophilicity, but excluding vapor pressure, predicted airborne quantities from stimuli in paraffin oil on filter paper. We recorded OSN responses of Drosophila melanogaster, Ips typographus, and Culex quinquefasciatus, to known quantities of airborne stimuli. These demonstrate that inferred OSN tuning width, ligand affinity, and classification can be confounded and require stimulus quantification. Additionally, proper dose-response analysis shows that Drosophila AB3A OSNs are not promiscuous, but highly specific for ethyl hexanoate, with other earlier proposed ligands 10- to 10 000-fold less potent. Finally, we reanalyzed published Drosophila OSN data (DoOR) and demonstrate substantial shifts in affinities after compensation for quantity and puff number. We conclude that consistent experimental protocols are necessary for correct OSN classification and present some simple rules that make calibration, even retroactively, readily possible.

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

Wu, Huixian; Wacker, Daniel; Mileni, Mauro

Opioid receptors mediate the actions of endogenous and exogenous opioids on many physiological processes, including the regulation of pain, respiratory drive, mood, and - in the case of {kappa}-opioid receptor ({kappa}-OR) - dysphoria and psychotomimesis. Here we report the crystal structure of the human {kappa}-OR in complex with the selective antagonist JDTic, arranged in parallel dimers, at 2.9 {angstrom} resolution. The structure reveals important features of the ligand-binding pocket that contribute to the high affinity and subtype selectivity of JDTic for the human {kappa}-OR. Modelling of other important {kappa}-OR-selective ligands, including the morphinan-derived antagonists norbinaltorphimine and 5'-guanidinonaltrindole, and the diterpenemore » agonist salvinorin A analogue RB-64, reveals both common and distinct features for binding these diverse chemotypes. Analysis of site-directed mutagenesis and ligand structure-activity relationships confirms the interactions observed in the crystal structure, thereby providing a molecular explanation for {kappa}-OR subtype selectivity, and essential insights for the design of compounds with new pharmacological properties targeting the human {kappa}-OR.« less

Elimination of a ligand gating site generates a supersensitive olfactory receptor.

PubMed

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I

2016-06-21

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors.

Elimination of a ligand gating site generates a supersensitive olfactory receptor

PubMed Central

Sharma, Kanika; Ahuja, Gaurav; Hussain, Ashiq; Balfanz, Sabine; Baumann, Arnd; Korsching, Sigrun I.

2016-01-01

Olfaction poses one of the most complex ligand-receptor matching problems in biology due to the unparalleled multitude of odor molecules facing a large number of cognate olfactory receptors. We have recently deorphanized an olfactory receptor, TAAR13c, as a specific receptor for the death-associated odor cadaverine. Here we have modeled the cadaverine/TAAR13c interaction, exchanged predicted binding residues by site-directed mutagenesis, and measured the activity of the mutant receptors. Unexpectedly we observed a binding site for cadaverine at the external surface of the receptor, in addition to an internal binding site, whose mutation resulted in complete loss of activity. In stark contrast, elimination of the external binding site generated supersensitive receptors. Modeling suggests this site to act as a gate, limiting access of the ligand to the internal binding site and thereby downregulating the affinity of the native receptor. This constitutes a novel mechanism to fine-tune physiological sensitivity to socially relevant odors. PMID:27323929

The Extracellular Surface of the GLP-1 Receptor Is a Molecular Trigger for Biased Agonism.

PubMed

Wootten, Denise; Reynolds, Christopher A; Smith, Kevin J; Mobarec, Juan C; Koole, Cassandra; Savage, Emilia E; Pabreja, Kavita; Simms, John; Sridhar, Rohan; Furness, Sebastian G B; Liu, Mengjie; Thompson, Philip E; Miller, Laurence J; Christopoulos, Arthur; Sexton, Patrick M

2016-06-16

Ligand-directed signal bias offers opportunities for sculpting molecular events, with the promise of better, safer therapeutics. Critical to the exploitation of signal bias is an understanding of the molecular events coupling ligand binding to intracellular signaling. Activation of class B G protein-coupled receptors is driven by interaction of the peptide N terminus with the receptor core. To understand how this drives signaling, we have used advanced analytical methods that enable separation of effects on pathway-specific signaling from those that modify agonist affinity and mapped the functional consequence of receptor modification onto three-dimensional models of a receptor-ligand complex. This yields molecular insights into the initiation of receptor activation and the mechanistic basis for biased agonism. Our data reveal that peptide agonists can engage different elements of the receptor extracellular face to achieve effector coupling and biased signaling providing a foundation for rational design of biased agonists. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Insight into the Meligethes aeneus voltage-sensitive sodium channel structure and an attempt to select the best pyrethroid ligands.

PubMed

Obrępalska-Stęplowska, Aleksandra; Czerwoniec, Anna; Wieczorek, Przemysław; Wrzesińska, Barbara

2016-01-01

The voltage-sensitive sodium channel (VSSC) is a target for the pharmacological action of pyrethroids which are used in controlling pests, including those of agricultural importance. Among these is the pollen beetle (Meligethes aeneus F.) - the most serious pest of Brassica napus. Owing to the heavy use of pyrethroids, a widespread build-up of resistance has occurred. The main cause of pyrethroid insensitivity in M. aeneus is considered to be an increased oxidative metabolism; however, the additional mechanism of resistance associated with mutations in the VSSC might contribute to this phenomenon. We generated a VSSC 3D model to study the docking affinities of pyrethroids to their target site within the channel. Our goal was to identify the pyrethroids for which docking affinity scores were high and not affected by potential mutations in the VSSC. We found that the docking scores of cypermethrin are hardly influenced by the appearance of point mutations. Additionally, tau-fluvalinate, deltamethrin and bifenthrin are VSSC ligands with high affinity scores. Our docking models suggest that point mutations in the VSSC binding pocket might affect the stability of ligand interactions and change the pattern of ligand docking locations, which might have a potential effect on VSSC gating properties. © 2015 Society of Chemical Industry.

Recent improvements to Binding MOAD: a resource for protein–ligand binding affinities and structures

PubMed Central

Ahmed, Aqeel; Smith, Richard D.; Clark, Jordan J.; Dunbar, James B.; Carlson, Heather A.

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein–ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23 269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. PMID:25378330

Functional Stability of the Human Kappa Opioid Receptor Reconstituted in Nanodiscs Revealed by a Time-Resolved Scintillation Proximity Assay

PubMed Central

Hansen, Randi Westh; Wang, Xiaole; Golab, Agnieszka; Bornert, Olivier; Oswald, Christine; Wagner, Renaud; Martinez, Karen Laurence

2016-01-01

Long-term functional stability of isolated membrane proteins is crucial for many in vitro applications used to elucidate molecular mechanisms, and used for drug screening platforms in modern pharmaceutical industry. Compared to soluble proteins, the understanding at the molecular level of membrane proteins remains a challenge. This is partly due to the difficulty to isolate and simultaneously maintain their structural and functional stability, because of their hydrophobic nature. Here we show, how scintillation proximity assay can be used to analyze time-resolved high-affinity ligand binding to membrane proteins solubilized in various environments. The assay was used to establish conditions that preserved the biological function of isolated human kappa opioid receptor. In detergent solution the receptor lost high-affinity ligand binding to a radiolabelled ligand within minutes at room temperature. After reconstitution in Nanodiscs made of phospholipid bilayer the half-life of high-affinity ligand binding to the majority of receptors increased 70-fold compared to detergent solubilized receptors—a level of stability that is appropriate for further downstream applications. Time-resolved scintillation proximity assay has the potential to screen numerous conditions in parallel to obtain high levels of stable and active membrane proteins, which are intrinsically unstable in detergent solution, and with minimum material consumption. PMID:27035823

Large scale affinity calculations of cyclodextrin host-guest complexes: Understanding the role of reorganization in the molecular recognition process

PubMed Central

Wickstrom, Lauren; He, Peng; Gallicchio, Emilio; Levy, Ronald M.

2013-01-01

Host-guest inclusion complexes are useful models for understanding the structural and energetic aspects of molecular recognition. Due to their small size relative to much larger protein-ligand complexes, converged results can be obtained rapidly for these systems thus offering the opportunity to more reliably study fundamental aspects of the thermodynamics of binding. In this work, we have performed a large scale binding affinity survey of 57 β-cyclodextrin (CD) host guest systems using the binding energy distribution analysis method (BEDAM) with implicit solvation (OPLS-AA/AGBNP2). Converged estimates of the standard binding free energies are obtained for these systems by employing techniques such as parallel Hamitionian replica exchange molecular dynamics, conformational reservoirs and multistate free energy estimators. Good agreement with experimental measurements is obtained in terms of both numerical accuracy and affinity rankings. Overall, average effective binding energies reproduce affinity rank ordering better than the calculated binding affinities, even though calculated binding free energies, which account for effects such as conformational strain and entropy loss upon binding, provide lower root mean square errors when compared to measurements. Interestingly, we find that binding free energies are superior rank order predictors for a large subset containing the most flexible guests. The results indicate that, while challenging, accurate modeling of reorganization effects can lead to ligand design models of superior predictive power for rank ordering relative to models based only on ligand-receptor interaction energies. PMID:25147485

Elucidation of Hydrogen Bonding Patterns in Ligand-Free, Lactose- and Glycerol-Bound Galectin-3C by Neutron Crystallography to Guide Drug Design.

PubMed

Manzoni, Francesco; Wallerstein, Johan; Schrader, Tobias E; Ostermann, Andreas; Coates, Leighton; Akke, Mikael; Blakeley, Matthew P; Oksanen, Esko; Logan, Derek T

2018-05-24

The medically important drug target galectin-3 binds galactose-containing moieties on glycoproteins through an intricate pattern of hydrogen bonds to a largely polar surface-exposed binding site. All successful inhibitors of galectin-3 to date have been based on mono- or disaccharide cores closely resembling natural ligands. A detailed understanding of the H-bonding networks in these natural ligands will provide an improved foundation for the design of novel inhibitors. Neutron crystallography is an ideal technique to reveal the geometry of hydrogen bonds because the positions of hydrogen atoms are directly detected rather than being inferred from the positions of heavier atoms as in X-ray crystallography. We present three neutron crystal structures of the C-terminal carbohydrate recognition domain of galectin-3: the ligand-free form and the complexes with the natural substrate lactose and with glycerol, which mimics important interactions made by lactose. The neutron crystal structures reveal unambiguously the exquisite fine-tuning of the hydrogen bonding pattern in the binding site to the natural disaccharide ligand. The ligand-free structure shows that most of these hydrogen bonds are preserved even when the polar groups of the ligand are replaced by water molecules. The protonation states of all histidine residues in the protein are also revealed and correlate well with NMR observations. The structures give a solid starting point for molecular dynamics simulations and computational estimates of ligand binding affinity that will inform future drug design.

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches

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

Zhang, Liying; Sedykh, Alexander; Tripathi, Ashutosh

2013-10-01

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure–activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα bindingmore » affinity (MTL R{sup 2} = 0.71, STL R{sup 2} = 0.73). For ERβ binding affinity, MTL models were significantly more predictive (R{sup 2} = 0.53, p < 0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation. - Highlights: • This is the largest curated dataset inclusive of ERα and β (the latter is unique). • New methodology that for the first time affords acceptable ERβ models. • A combination of QSAR and docking enables prediction of affinity and function. • The results have potential applications to green chemistry. • Models are publicly available for virtual screening via a web portal.« less

Evaluation of Cu(i) binding to the E2 domain of the amyloid precursor protein - a lesson in quantification of metal binding to proteins via ligand competition.

PubMed

Young, Tessa R; Wedd, Anthony G; Xiao, Zhiguang

2018-01-24

The extracellular domain E2 of the amyloid precursor protein (APP) features a His-rich metal-binding site (denoted as the M1 site). In conjunction with surrounding basic residues, the site participates in interactions with components of the extracellular matrix including heparins, a class of negatively charged polysaccharide molecules of varying length. This work studied the chemistry of Cu(i) binding to APP E2 with the probe ligands Bcs, Bca, Fz and Fs. APP E2 forms a stable Cu(i)-mediated ternary complex with each of these anionic ligands. The complex with Bca was selected for isolation and characterization and was demonstrated, by native ESI-MS analysis, to have the stoichiometry E2 : Cu(i) : Bca = 1 : 1 : 1. Formation of these ternary complexes is specific for the APP E2 domain and requires Cu(i) coordination to the M1 site. Mutation of the M1 site was consistent with the His ligands being part of the E2 ligand set. It is likely that interactions between the negatively charged probe ligands and a positively charged patch on the surface of APP E2 are one aspect of the generation of the stable ternary complexes. Their formation prevented meaningful quantification of the affinity of Cu(i) binding to the M1 site with these probe ligands. However, the ternary complexes are disrupted by heparin, allowing reliable determination of a picomolar Cu(i) affinity for the E2/heparin complex with the Fz or Bca probe ligands. This is the first documented example of the formation of stable ternary complexes between a Cu(i) binding protein and a probe ligand. The ready disruption of the complexes by heparin identified clear 'tell-tale' signs for diagnosis of ternary complex formation and allowed a systematic review of conditions and criteria for reliable determination of affinities for metal binding via ligand competition. This study also provides new insights into a potential correlation of APP functions regulated by copper binding and heparin interaction.

Evidence for an allosteric mechanism of substrate release from membrane-transporter accessory binding proteins.

PubMed

Marinelli, Fabrizio; Kuhlmann, Sonja I; Grell, Ernst; Kunte, Hans-Jörg; Ziegler, Christine; Faraldo-Gómez, José D

2011-12-06

Numerous membrane importers rely on accessory water-soluble proteins to capture their substrates. These substrate-binding proteins (SBP) have a strong affinity for their ligands; yet, substrate release onto the low-affinity membrane transporter must occur for uptake to proceed. It is generally accepted that release is facilitated by the association of SBP and transporter, upon which the SBP adopts a conformation similar to the unliganded state, whose affinity is sufficiently reduced. Despite the appeal of this mechanism, however, direct supporting evidence is lacking. Here, we use experimental and theoretical methods to demonstrate that an allosteric mechanism of enhanced substrate release is indeed plausible. First, we report the atomic-resolution structure of apo TeaA, the SBP of the Na(+)-coupled ectoine TRAP transporter TeaBC from Halomonas elongata DSM2581(T), and compare it with the substrate-bound structure previously reported. Conformational free-energy landscape calculations based upon molecular dynamics simulations are then used to dissect the mechanism that couples ectoine binding to structural change in TeaA. These insights allow us to design a triple mutation that biases TeaA toward apo-like conformations without directly perturbing the binding cleft, thus mimicking the influence of the membrane transporter. Calorimetric measurements demonstrate that the ectoine affinity of the conformationally biased triple mutant is 100-fold weaker than that of the wild type. By contrast, a control mutant predicted to be conformationally unbiased displays wild-type affinity. This work thus demonstrates that substrate release from SBPs onto their membrane transporters can be facilitated by the latter through a mechanism of allosteric modulation of the former.

The use of one-bead one-compound combinatorial library technology to discover high-affinity αvβ3 integrin and cancer targeting arginine-glycine-aspartic acid ligands with a built-in handle.

PubMed

Xiao, Wenwu; Wang, Yan; Lau, Edmond Y; Luo, Juntao; Yao, Nianhuan; Shi, Changying; Meza, Leah; Tseng, Harry; Maeda, Yoshiko; Kumaresan, Pappanaicken; Liu, Ruiwu; Lightstone, Felice C; Takada, Yoshikazu; Lam, Kit S

2010-10-01

The αvβ3 integrin, expressed on the surface of various normal and cancer cells, is involved in numerous physiologic processes such as angiogenesis, apoptosis, and bone resorption. Because this integrin plays a key role in angiogenesis and metastasis of human tumors, αvβ3 integrin ligands are of great interest to advances in targeted therapy and cancer imaging. In this report, one-bead one-compound (OBOC) combinatorial libraries containing the arginine-glycine-aspartic acid (RGD) motif were designed and screened against K562 myeloid leukemia cells that had been transfected with the human αvβ3 integrin gene. Cyclic peptide LXW7 was identified as a leading ligand with a built-in handle that binds specifically to αvβ3 and showed comparable binding affinity (IC(50) = 0.68 ± 0.08 μmol/L) to some of the well-known RGD "head-to-tail" cyclic pentapeptide ligands reported in the literature. The biotinylated form of LXW7 ligand showed similar binding strength as LXW7 against αvβ3 integrin, whereas biotinylated RGD cyclopentapeptide ligands revealed a 2- to 8-fold weaker binding affinity than their free forms. LXW7 was able to bind to both U-87MG glioblastoma and A375M melanoma cell lines, both of which express high levels of αvβ3 integrin. In vivo and ex vivo optical imaging studies with the biotinylated ligand/streptavidin-Cy5.5 complex in nude mice bearing U-87MG or A375M xenografts revealed preferential uptake of biotinylated LXW7 in tumor. When compared with biotinylated RGD cyclopentapeptide ligands, biotinylated LXW7 showed higher tumor uptake but lower liver uptake.

Ligand deconstruction: Why some fragment binding positions are conserved and others are not.

PubMed

Kozakov, Dima; Hall, David R; Jehle, Stefan; Jehle, Sefan; Luo, Lingqi; Ochiana, Stefan O; Jones, Elizabeth V; Pollastri, Michael; Allen, Karen N; Whitty, Adrian; Vajda, Sandor

2015-05-19

Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots--regions of the protein where interactions with a ligand contribute substantial binding free energy--the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand.

The design, synthesis and pharmacological characterization of novel β2-adrenoceptor antagonists

PubMed Central

Hothersall, J Daniel; Black, James; Caddick, Stephen; Vinter, Jeremy G; Tinker, Andrew; Baker, James R

2011-01-01

BACKGROUND AND PURPOSE Selective and potent antagonists for the β2-adrenoceptor are potentially interesting as experimental and clinical tools, and we sought to identify novel ligands with this pharmacology. EXPERIMENTAL APPROACH A range of pharmacological assays was used to assess potency, affinity, selectivity (β2-adrenoceptor vs. β1-adrenoceptor) and efficacy. KEY RESULTS Ten novel compounds were identified but none had as high affinity as the prototypical β2-adrenoceptor blocker ICI-118,551, although one of the novel compounds was more selective for β2-adrenoceptors. Most of the ligands were inverse agonists for β2-adrenoceptor-cAMP signalling, although one (5217377) was a partial agonist and another a neutral antagonist (7929193). None of the ligands were efficacious with regard to β2-adrenoceptor-β-arrestin signalling. The (2S,3S) enantiomers were identified as the most active, although unusually the racemates were the most selective for the β2-adrenoceptors. This was taken as evidence for some unusual enantiospecific behaviour. CONCLUSIONS AND IMPLICATIONS In terms of improving on the pharmacology of the ligand ICI-118,551, one of the compounds was more selective (racemic JB-175), while one was a neutral antagonist (7929193), although none had as high an affinity. The results substantiate the notion that β-blockers do more than simply inhibit receptor activation, and differences between the ligands could provide useful tools to investigate receptor biology. PMID:21323900

Synthesis and structure-activity relationship studies in a series of 2-substituted 1,3-dioxolanes modified at the cationic head.

PubMed

Angeli, P; Brasili, L; Cingolani, M L; Marucci, G; Piergentili, A; Pigini, M; Quaglia, W

1997-04-01

To develop ligands that may be useful in exploring muscarinic receptor heterogeneity, we synthesized a series of analogues of 2,2-diphenyl-[1,3]-dioxolan-4-ylmethyl-dimethylamine oxalate and methiodide bearing a modified cationic head. These compounds, when tested on tissues containing the three subtypes M1, M2, and M3, behaved as muscarinic antagonists whose results showed that different substituents on the quaternary and tertiary nitrogen affect affinity and selectivity in different ways. In particular comparison of the affinities of these ligands with those of the reference compounds points out that compounds bearing an ethyl substituent improve the affinity of the molecule at the three subtypes while compounds bearing a phenethyl substituent are more selective for the M3 sites.

Lessons learned from participating in D3R 2016 Grand Challenge 2: compounds targeting the farnesoid X receptor

NASA Astrophysics Data System (ADS)

Duan, Rui; Xu, Xianjin; Zou, Xiaoqin

2018-01-01

D3R 2016 Grand Challenge 2 focused on predictions of binding modes and affinities for 102 compounds against the farnesoid X receptor (FXR). In this challenge, two distinct methods, a docking-based method and a template-based method, were employed by our team for the binding mode prediction. For the new template-based method, 3D ligand similarities were calculated for each query compound against the ligands in the co-crystal structures of FXR available in Protein Data Bank. The binding mode was predicted based on the co-crystal protein structure containing the ligand with the best ligand similarity score against the query compound. For the FXR dataset, the template-based method achieved a better performance than the docking-based method on the binding mode prediction. For the binding affinity prediction, an in-house knowledge-based scoring function ITScore2 and MM/PBSA approach were employed. Good performance was achieved for MM/PBSA, whereas the performance of ITScore2 was sensitive to ligand composition, e.g. the percentage of carbon atoms in the compounds. The sensitivity to ligand composition could be a clue for the further improvement of our knowledge-based scoring function.

Potential interaction of natural dietary bioactive compounds with COX-2.

PubMed

Maldonado-Rojas, Wilson; Olivero-Verbel, Jesus

2011-09-01

Bioactive natural products present in the diet play an important role in several biological processes, and many have been involved in the alleviation and control of inflammation-related diseases. These actions have been linked to both gene expression modulation of pro-inflammatory enzymes, such as cyclooxygenase 2 (COX-2), and to an action involving a direct inhibitory binding on this protein. In this study, several food-related compounds with known gene regulatory action on inflammation have been examined in silico as COX-2 ligands, utilizing AutoDock Vina, GOLD and Surflex-Dock (SYBYL) as docking protocols. Curcumin and all-trans retinoic acid presented the maximum absolute AutoDock Vina-derived binding affinities (9.3 kcal/mol), but genistein, apigenin, cyanidin, kaempferol, and docosahexaenoic acid, were close to this value. AutoDock Vina affinities and GOLD scores for several known COX-2 inhibitors significatively correlated with reported median inhibitory concentrations (R² = 0.462, P < 0.001 and R² = 0.238, P = 0.029, respectively), supporting the computational reliability of the predictions made by our docking simulations. Moreover, docking analysis insinuate the synergistic action of curcumin on celecoxib-induced inhibition of COX-2 may occur allosterically, as this natural compound docks to a place different from the inhibitor binding site. These results suggest that the anti-inflammatory properties of some food-derived molecules could be the result of their direct binding capabilities to COX-2, and this process can be modeled using protein-ligand docking methodologies. Copyright © 2011 Elsevier Inc. All rights reserved.

Optical diamagnetic biosensor for immunocomplexes on beads

NASA Astrophysics Data System (ADS)

Norina, Svetlana B.

2000-12-01

In the present work, diamagnetic separation parameters for the porous beads are studied using optical video recording microscopy. The possible direct amount determination of single or double macromolecular layers immobilized in the meshes of the porous beads is demonstrated for the concentrations' range used in heterogenic immunotest and the affinity chromatography, where the direct rapid detection of ligands within sorbent particles is known to be the actual task. A gradient diamagnetic biosensor is described as suitable for rapid quantitative detection of single or double macromolecular layers in porous nonmagnetic beads. Measurements of capture traveling time or accumulation radius in gradient magnetic field have shown that it is possible to determine 0.20 mg/ml of macromolecular amount within several seconds. The portative devices were made on the base of the fabre optic technique to detect accumulation radius of collected beads in two gradient magnetic positions: diamagnetic and paramagnetic zones of magnetized wire with 55 μm in diameter and to registrate with a lot of fabre wires having 30 μm in diameters. The successive procedures of the present method can be described by: the obtaining of agarose immuno-beads, the incubation of beads with the ligand sample or the injection of sample through affinity mini-column, the submerging of the loaded beads into the glass cell containing Ni-wire or the narrow gap of magnetic poles; the computational obtaining of immuno- parameters; binding constants, accumulation radius. Several biotechnological applications of the biosensor are presented on sorbent beads, human lymphocytes.

Inter-residue coupling contributes to high-affinity subtype-selective binding of α-bungarotoxin to nicotinic receptors

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

Sine, Steven M.; Huang, Sun; Li, Shu-Xing

2013-09-01

The crystal structure of a pentameric α7 ligand-binding domain chimaera with bound α-btx (α-bungarotoxin) showed that of the five conserved aromatic residues in α7, only Tyr 184 in loop C of the ligand-binding site was required for high-affinity binding. To determine whether the contribution of Tyr 184 depends on local residues, we generated mutations in an α7/5HT 3A (5-hydroxytryptamine type 3A) receptor chimaera, individually and in pairs, and measured 125I-labelled α-btx binding. The results show that mutations of individual residues near Tyr 184 do not affect α-btx affinity, but pairwise mutations decrease affinity in an energetically coupled manner. Kinetic measurementsmore » show that the affinity decreases arise through increases in the α-btx dissociation rate with little change in the association rate. Replacing loop C in α7 with loop C from the α-btx-insensitive α2 or α3 subunits abolishes high-affinity α-btx binding, but preserves acetylcholine-elicited single channel currents. However, in both the α2 and α3 construct, mutating either residue that flanks Tyr 184 to its α7 counterpart restores high-affinity α-btx binding. Analogously, in α7, mutating both residues that flank Tyr 184 to the α2 or α3 counterparts abolishes high-affinity α-btx binding. Thus interaction between Tyr 184 and local residues contributes to high-affinity subtype-selective α-btx binding.« less

Docking simulations suggest that all-trans retinoic acid could bind to retinoid X receptors.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2015-10-01

Retinoid X receptors (RXRs) are ligand-controlled transcription factors which heterodimerize with other nuclear receptors to regulate gene transcriptions associated with crucial biological events. 9-cis retinoic acid (9cRA), which transactivates RXRs, is believed to be an endogenous RXR ligand. All-trans retinoic acid (ATRA) is a natural ligand for retinoic acid receptors (RARs), which heterodimerize with RXRs. Although the concentration of 9cRA in tissues is very low, ATRA is relatively abundant and some reports show that ATRA activates RXRs. We computationally studied the possibility of ATRA binding to RXRs using two different docking methods with our developed programs to assess the binding affinities of naturally occurring retinoids. The simulations showed good correlations to the reported binding affinities of these molecules for RXRs and RARs.

Synthesis and pharmacological characterization of novel xanthine carboxylate amides as A2A adenosine receptor ligands exhibiting bronchospasmolytic activity.

PubMed

Yadav, Rakesh; Bansal, Ranju; Rohilla, Suman; Kachler, Sonja; Klotz, Karl-Norbert

2016-04-01

The carboxylate amides of 8-phenyl-1,3-dimethylxanthine described herein represent a new series of selective ligands of the adenosine A2A receptors exhibiting bronchospasmolytic activity. The effects of location of 8-phenyl substitutions on the adenosine receptor (AR) binding affinities of the newly synthesized xanthines have also been studied. The compounds displayed moderate to potent binding affinities toward various adenosine receptor subtypes when evaluated through radioligand binding studies. However, most of the compounds showed the maximum affinity for the A2A subtype, some with high selectivity versus all other subtypes. Xanthine carboxylate amide 13b with a diethylaminoethylamino moiety at the para-position of the 8-phenylxanthine scaffold was identified as the most potent A2A adenosine receptor ligand with Ki=0.06μM. Similarly potent and highly A2A-selective are the isovanillin derivatives 16a and 16d. In addition, the newly synthesized xanthine derivatives showed good in vivo bronchospasmolytic activity when tested in guinea pigs. Copyright © 2016 Elsevier Inc. All rights reserved.

Comparative analysis the binding affinity of mycophenolic sodium and meprednisone with human serum albumin: Insight by NMR relaxation data and docking simulation.

PubMed

Ma, Xiaoli; He, Jiawei; Yan, Jin; Wang, Qing; Li, Hui

2016-03-25

Mycophenolic sodium is an immunosuppressive agent that is always combined administration with corticosteroid in clinical practice. Considering the distribution and side-effect of the drug may change when co-administrated drug exist, this paper comparatively analyzed the binding ability of mycophenolic sodium and meprednisone toward human serum albumin by nuclear magnetic resonance relaxation data and docking simulation. The nuclear magnetic resonance approach was based on the analysis of proton selective and non-selective relaxation rate enhancement of the ligand in the absence and presence of macromolecules. The contribution of the bound ligand fraction to the observed relaxation rate in relation to protein concentration allowed the calculation of the affinity index. This approach allowed the comparison of the binding affinity of mycophenolic sodium and meprednisone. Molecular modeling was operated to simulate the binding model of ligand and albumin through Autodock 4.2.5. Competitive binding of mycophenolic sodium and meprednisone was further conducted through fluorescence spectroscopy. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Towards the identification of alkaline phosphatase binding ligands in Li-Dan-Hua-Shi pills: A Box-Behnken design optimized affinity selection approach tandem with UHPLC-Q-TOF/MS analysis.

PubMed

Tao, Yi; Huang, Surun; Gu, Xianghui; Li, Weidong; Cai, Baochang

2018-05-30

Alkaline phosphatase conjugated magnetic microspheres were synthesized via amide reaction, and employed as an effective adsorbent in affinity selection of binding ligands followed by UHPLC-Q-TOF/MS analysis. The analytical validity of the developed approach was evaluated under optimized conditions and the following figures of merit were obtained: linearity, 0.01-0.5 g L -1 with good determination coefficients (R 2  = 0.9992); limits of detection (LODs), 0.003 g L -1 ; and limits of quantitation (LOQ), 0.01 g L -1 . The precision (RSD%) of the proposed affinity selection approach was studied based on intra-day (0.8%) and inter-day (1.3%) precisions. Finally, the adsorbent was successfully applied to identification of binding ligands in Li-Dan-Hua-Shi pills and good recoveries were obtained in the range from 96.9 to 99.4% (RSDs 1.6-3.0%). Copyright © 2018 Elsevier B.V. All rights reserved.

Fluoxetine (Prozac) Binding to Serotonin Transporter Is Modulated by Chloride and Conformational Changes

PubMed Central

Tavoulari, Sotiria; Forrest, Lucy R.; Rudnick, Gary

2010-01-01

Serotonin transporter (SERT) is the main target for widely used antidepressant agents. Several of these drugs, including imipramine, citalopram, sertraline, and fluoxetine (Prozac), bound more avidly to SERT in the presence of Cl–. In contrast, Cl– did not enhance cocaine or paroxetine binding. A Cl– binding site recently identified in SERT, and shown to be important for Cl– dependent transport, was also critical for the Cl– dependence of antidepressant affinity. Mutation of the residues contributing to this site eliminated the Cl–-mediated affinity increase for imipramine and fluoxetine. Analysis of ligand docking to a single state of SERT indicated only small differences in the energy of interaction between bound ligands and Cl–. These differences in interaction energy cannot account for the affinity differences observed for Cl– dependence. However, fluoxetine binding led to a conformational change, detected by cysteine accessibility experiments, that was qualitatively different from that induced by cocaine or other ligands. Given the known Cl– requirement for serotonin-induced conformational changes, we propose that Cl– binding facilitates conformational changes required for optimal binding of fluoxetine and other antidepressant drugs. PMID:19641126

High Throughput, Label-free Screening Small Molecule Compound Libraries for Protein-Ligands using Combination of Small Molecule Microarrays and a Special Ellipsometry-based Optical Scanner.

PubMed

Landry, James P; Fei, Yiyan; Zhu, X D

2011-12-01

Small-molecule compounds remain the major source of therapeutic and preventative drugs. Developing new drugs against a protein target often requires screening large collections of compounds with diverse structures for ligands or ligand fragments that exhibit sufficiently affinity and desirable inhibition effect on the target before further optimization and development. Since the number of small molecule compounds is large, high-throughput screening (HTS) methods are needed. Small-molecule microarrays (SMM) on a solid support in combination with a suitable binding assay form a viable HTS platform. We demonstrate that by combining an oblique-incidence reflectivity difference optical scanner with SMM we can screen 10,000 small-molecule compounds on a single glass slide for protein ligands without fluorescence labeling. Furthermore using such a label-free assay platform we can simultaneously acquire binding curves of a solution-phase protein to over 10,000 immobilized compounds, thus enabling full characterization of protein-ligand interactions over a wide range of affinity constants.

Anions mediate ligand binding in Adineta vaga glutamate receptor ion channels.

PubMed

Lomash, Suvendu; Chittori, Sagar; Brown, Patrick; Mayer, Mark L

2013-03-05

AvGluR1, a glutamate receptor ion channel from the primitive eukaryote Adineta vaga, is activated by alanine, cysteine, methionine, and phenylalanine, which produce lectin-sensitive desensitizing responses like those to glutamate, aspartate, and serine. AvGluR1 LBD crystal structures reveal an unusual scheme for binding dissimilar ligands that may be utilized by distantly related odorant/chemosensory receptors. Arginine residues in domain 2 coordinate the γ-carboxyl group of glutamate, whereas in the alanine, methionine, and serine complexes a chloride ion acts as a surrogate ligand, replacing the γ-carboxyl group. Removal of Cl(-) lowers affinity for these ligands but not for glutamate or aspartate nor for phenylalanine, which occludes the anion binding site and binds with low affinity. AvGluR1 LBD crystal structures and sedimentation analysis also provide insights into the evolutionary link between prokaryotic and eukaryotic iGluRs and reveal features unique to both classes, emphasizing the need for additional structure-based studies on iGluR-ligand interactions. Copyright © 2013 Elsevier Ltd. All rights reserved.

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

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

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with boundmore » ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity.« less

Structural basis of subunit selectivity for competitive NMDA receptor antagonists with preference for GluN2A over GluN2B subunits

PubMed Central

Lind, Genevieve E.; Mou, Tung-Chung; Tamborini, Lucia; Pomper, Martin G.; De Micheli, Carlo; Conti, Paola; Pinto, Andrea

2017-01-01

NMDA-type glutamate receptors are ligand-gated ion channels that contribute to excitatory neurotransmission in the central nervous system (CNS). Most NMDA receptors comprise two glycine-binding GluN1 and two glutamate-binding GluN2 subunits (GluN2A–D). We describe highly potent (S)-5-[(R)-2-amino-2-carboxyethyl]-4,5-dihydro-1H-pyrazole-3-carboxylic acid (ACEPC) competitive GluN2 antagonists, of which ST3 has a binding affinity of 52 nM at GluN1/2A and 782 nM at GluN1/2B receptors. This 15-fold preference of ST3 for GluN1/2A over GluN1/2B is improved compared with NVP-AAM077, a widely used GluN2A-selective antagonist, which we show has 11-fold preference for GluN1/2A over GluN1/2B. Crystal structures of the GluN1/2A agonist binding domain (ABD) heterodimer with bound ACEPC antagonists reveal a binding mode in which the ligands occupy a cavity that extends toward the subunit interface between GluN1 and GluN2A ABDs. Mutational analyses show that the GluN2A preference of ST3 is primarily mediated by four nonconserved residues that are not directly contacting the ligand, but positioned within 12 Å of the glutamate binding site. Two of these residues influence the cavity occupied by ST3 in a manner that results in favorable binding to GluN2A, but occludes binding to GluN2B. Thus, we reveal opportunities for the design of subunit-selective competitive NMDA receptor antagonists by identifying a cavity for ligand binding in which variations exist between GluN2A and GluN2B subunits. This structural insight suggests that subunit selectivity of glutamate-site antagonists can be mediated by mechanisms in addition to direct contributions of contact residues to binding affinity. PMID:28760974

Native Electrospray Ionization Mass Spectrometry Reveals Multiple Facets of Aptamer-Ligand Interactions: From Mechanism to Binding Constants.

PubMed

Gülbakan, Basri; Barylyuk, Konstantin; Schneider, Petra; Pillong, Max; Schneider, Gisbert; Zenobi, Renato

2018-06-20

Aptamers are oligonucleotide receptors obtained through an iterative selection process from random-sequence libraries. Though many aptamers for a broad range of targets with high affinity and selectivity have been generated, a lack of high-resolution structural data and the limitations of currently available biophysical tools greatly impede understanding of the mechanisms of aptamer-ligand interactions. Here we demonstrate that an approach based on native electrospray ionization mass spectrometry (ESI-MS) can be successfully applied to characterize aptamer-ligand complexes in all details. We studied an adenosine-binding aptamer (ABA), a l-argininamide-binding aptamer (LABA), and a cocaine-binding aptamer (CBA) and their noncovalent interactions with ligands by native ESI-MS and complemented these measurements by ion mobility spectrometry (IMS), isothermal titration calorimetry (ITC), and circular dichroism (CD) spectroscopy. The ligand selectivity of the aptamers and the respective complex stoichiometry could be determined by the native ESI-MS approach. The ESI-MS data can also help refining the binding model for aptamer-ligand complexes and deliver accurate aptamer-ligand binding affinities for specific and nonspecific binding events. For specific ligands, we found K d1 = 69.7 μM and K d2 = 5.3 μM for ABA (two binding sites); K d1 = 22.04 μM for LABA; and K d1 = 8.5 μM for CBA.

The use of selective adsorbents in capillary electrophoresis-mass spectrometry for analyte preconcentration and microreactions: a powerful three-dimensional tool for multiple chemical and biological applications.

PubMed

Guzman, N A; Stubbs, R J

2001-10-01

Much attention has recently been directed to the development and application of online sample preconcentration and microreactions in capillary electrophoresis using selective adsorbents based on chemical or biological specificity. The basic principle involves two interacting chemical or biological systems with high selectivity and affinity for each other. These molecular interactions in nature usually involve noncovalent and reversible chemical processes. Properly bound to a solid support, an "affinity ligand" can selectively adsorb a "target analyte" found in a simple or complex mixture at a wide range of concentrations. As a result, the isolated analyte is enriched and highly purified. When this affinity technique, allowing noncovalent chemical interactions and biochemical reactions to occur, is coupled on-line to high-resolution capillary electrophoresis and mass spectrometry, a powerful tool of chemical and biological information is created. This paper describes the concept of biological recognition and affinity interaction on-line with high-resolution separation, the fabrication of an "analyte concentrator-microreactor", optimization conditions of adsorption and desorption, the coupling to mass spectrometry, and various applications of clinical and pharmaceutical interest.

Affinity monolith chromatography: A review of principles and recent analytical applications

PubMed Central

Pfaunmiller, Erika L.; Paulemond, Marie Laura; Dupper, Courtney M.; Hage, David S.

2012-01-01

Affinity monolith chromatography (AMC) is a type of liquid chromatography that uses a monolithic support and a biologically-related binding agent as a stationary phase. AMC is a powerful method for the selective separation, analysis or studies of specific target compounds in a sample. This review discusses the basic principles of AMC and recent developments or applications of this method, with particular emphasis being given to work that has appeared in the last five years. Various materials that have been used to prepare columns for AMC are examined, including organic monoliths, silica monoliths, agarose monoliths and cryogels. These supports have been used in AMC for formats that have ranged from traditional columns to disks, microcolumns and capillaries. Many binding agents have also been employed in AMC, such as antibodies, enzymes, proteins, lectins, immobilized metal-ions and dyes. Some applications that have been reported with these binding agents in AMC are bioaffinity chromatography, immunoaffinity chromatography or immunoextraction, immobilized metal-ion affinity chromatography, dye-ligand affinity chromatography, chiral separations and biointeraction studies. Examples are presented from fields that include analytical chemistry, pharmaceutical analysis, clinical testing and biotechnology. Current trends and possible future directions in AMC are also discussed. PMID:23187827

Self-Assembled N-Heterocyclic Carbene-Based Carboxymethylated Dextran Monolayers on Gold as a Tunable Platform for Designing Affinity-Capture Biosensor Surfaces.

PubMed

Li, Zhijun; Munro, Kim; Narouz, Mina R; Lau, Andrew; Hao, Hongxia; Crudden, Cathleen M; Horton, J Hugh

2018-05-30

Sensor surfaces play a predominant role in the development of optical biosensor technologies for the analysis of biomolecular interactions. Thiol-based self-assembled monolayers (SAMs) on gold have been widely used as linker layers for sensor surfaces. However, the degradation of the thiol-gold bond can limit the performance and durability of such surfaces, directly impacting their performance and cost-effectiveness. To this end, a new family of materials based on N-heterocyclic carbenes (NHCs) has emerged as an alternative for surface modification, capable of self-assembling onto a gold surface with higher affinity and superior stability as compared to the thiol-based systems. Here we demonstrate three applications of NHC SAMs supporting a dextran layer as a tunable platform for developing various affinity-capture biosensor surfaces. We describe the development and testing of NHC-based dextran biosensor surfaces modified with each of streptavidin, nitrilotriacetic acid, and recombinant Protein A. These affinity-capture sensor surfaces enable oriented binding of ligands for optimal performance in biomolecular assays. Together, the intrinsic high stability and flexible design of the NHC biosensing platforms show great promise and open up exciting possibilities for future biosensing applications.

Comparing sixteen scoring functions for predicting biological activities of ligands for protein targets.

PubMed

Xu, Weijun; Lucke, Andrew J; Fairlie, David P

2015-04-01

Accurately predicting relative binding affinities and biological potencies for ligands that interact with proteins remains a significant challenge for computational chemists. Most evaluations of docking and scoring algorithms have focused on enhancing ligand affinity for a protein by optimizing docking poses and enrichment factors during virtual screening. However, there is still relatively limited information on the accuracy of commercially available docking and scoring software programs for correctly predicting binding affinities and biological activities of structurally related inhibitors of different enzyme classes. Presented here is a comparative evaluation of eight molecular docking programs (Autodock Vina, Fitted, FlexX, Fred, Glide, GOLD, LibDock, MolDock) using sixteen docking and scoring functions to predict the rank-order activity of different ligand series for six pharmacologically important protein and enzyme targets (Factor Xa, Cdk2 kinase, Aurora A kinase, COX-2, pla2g2a, β Estrogen receptor). Use of Fitted gave an excellent correlation (Pearson 0.86, Spearman 0.91) between predicted and experimental binding only for Cdk2 kinase inhibitors. FlexX and GOLDScore produced good correlations (Pearson>0.6) for hydrophilic targets such as Factor Xa, Cdk2 kinase and Aurora A kinase. By contrast, pla2g2a and COX-2 emerged as difficult targets for scoring functions to predict ligand activities. Although possessing a high hydrophobicity in its binding site, β Estrogen receptor produced reasonable correlations using LibDock (Pearson 0.75, Spearman 0.68). These findings can assist medicinal chemists to better match scoring functions with ligand-target systems for hit-to-lead optimization using computer-aided drug design approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

Crystal structure and ligand affinity of avidin in the complex with 4‧-hydroxyazobenzene-2-carboxylic acid

NASA Astrophysics Data System (ADS)

Strzelczyk, Paweł; Bujacz, Grzegorz

2016-04-01

Avidin is a protein found in egg white that binds numerous organic compounds with high affinity, especially biotin and its derivatives. Due to its extraordinary affinity for its ligands, avidin is extensively used in biotechnology. X-ray crystallography and fluorescence-based biophysical techniques were used to show that avidin binds the dye 4‧-hydroxyazobenzene-2-carboxylic acid (HABA) with a lower affinity than biotin. The apparent dissociation constant determined for the avidin complex with HABA by microscale thermophoresis (MST) is 4.12 μM. The crystal structure of avidin-HABA complex was determined at a resolution of 2.2 Å (PDB entry 5chk). The crystals belong to a hexagonal system, in the space group P6422. In that structure, the hydrazone tautomer of HABA is bound at the bottom part of the central calyx near the polar residues. We show interactions of the dye with avidin and compare them with the previously reported avidin-biotin complex.

Calculation of protein-ligand binding affinities.

PubMed

Gilson, Michael K; Zhou, Huan-Xiang

2007-01-01

Accurate methods of computing the affinity of a small molecule with a protein are needed to speed the discovery of new medications and biological probes. This paper reviews physics-based models of binding, beginning with a summary of the changes in potential energy, solvation energy, and configurational entropy that influence affinity, and a theoretical overview to frame the discussion of specific computational approaches. Important advances are reported in modeling protein-ligand energetics, such as the incorporation of electronic polarization and the use of quantum mechanical methods. Recent calculations suggest that changes in configurational entropy strongly oppose binding and must be included if accurate affinities are to be obtained. The linear interaction energy (LIE) and molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) methods are analyzed, as are free energy pathway methods, which show promise and may be ready for more extensive testing. Ultimately, major improvements in modeling accuracy will likely require advances on multiple fronts, as well as continued validation against experiment.

Enhance the performance of current scoring functions with the aid of 3D protein-ligand interaction fingerprints.

PubMed

Liu, Jie; Su, Minyi; Liu, Zhihai; Li, Jie; Li, Yan; Wang, Renxiao

2017-07-18

In structure-based drug design, binding affinity prediction remains as a challenging goal for current scoring functions. Development of target-biased scoring functions provides a new possibility for tackling this problem, but this approach is also associated with certain technical difficulties. We previously reported the Knowledge-Guided Scoring (KGS) method as an alternative approach (BMC Bioinformatics, 2010, 11, 193-208). The key idea is to compute the binding affinity of a given protein-ligand complex based on the known binding data of an appropriate reference complex, so the error in binding affinity prediction can be reduced effectively. In this study, we have developed an upgraded version, i.e. KGS2, by employing 3D protein-ligand interaction fingerprints in reference selection. KGS2 was evaluated in combination with four scoring functions (X-Score, ChemPLP, ASP, and GoldScore) on five drug targets (HIV-1 protease, carbonic anhydrase 2, beta-secretase 1, beta-trypsin, and checkpoint kinase 1). In the in situ scoring test, considerable improvements were observed in most cases after application of KGS2. Besides, the performance of KGS2 was always better than KGS in all cases. In the more challenging molecular docking test, application of KGS2 also led to improved structure-activity relationship in some cases. KGS2 can be applied as a convenient "add-on" to current scoring functions without the need to re-engineer them, and its application is not limited to certain target proteins as customized scoring functions. As an interpolation method, its accuracy in principle can be improved further with the increasing knowledge of protein-ligand complex structures and binding affinity data. We expect that KGS2 will become a practical tool for enhancing the performance of current scoring functions in binding affinity prediction. The KGS2 software is available upon contacting the authors.

Traceless affinity labeling of endogenous proteins for functional analysis in living cells.

PubMed

Hayashi, Takahiro; Hamachi, Itaru

2012-09-18

Protein labeling and imaging techniques have provided tremendous opportunities to study the structure, function, dynamics, and localization of individual proteins in the complex environment of living cells. Molecular biology-based approaches, such as GFP-fusion tags and monoclonal antibodies, have served as important tools for the visualization of individual proteins in cells. Although these techniques continue to be valuable for live cell imaging, they have a number of limitations that have only been addressed by recent progress in chemistry-based approaches. These chemical approaches benefit greatly from the smaller probe sizes that should result in fewer perturbations to proteins and to biological systems as a whole. Despite the research in this area, so far none of these labeling techniques permit labeling and imaging of selected endogenous proteins in living cells. Researchers have widely used affinity labeling, in which the protein of interest is labeled by a reactive group attached to a ligand, to identify and characterize proteins. Since the first report of affinity labeling in the early 1960s, efforts to fine-tune the chemical structures of both the reactive group and ligand have led to protein labeling with excellent target selectivity in the whole proteome of living cells. Although the chemical probes used for affinity labeling generally inactivate target proteins, this strategy holds promise as a valuable tool for the labeling and imaging of endogenous proteins in living cells and by extension in living animals. In this Account, we summarize traceless affinity labeling, a technique explored mainly in our laboratory. In our overview of the different labeling techniques, we emphasize the challenge of designing chemical probes that allow for dissociation of the affinity module (often a ligand) after the labeling reaction so that the labeled protein retains its native function. This feature distinguishes the traceless labeling approach from the traditional affinity labeling method and allows for real-time monitoring of protein activity. With the high target specificity and biocompatibility of this technique, we have achieved individual labeling and imaging of endogenously expressed proteins in samples of high biological complexity. We also highlight applications in which our current approach enabled the monitoring of important biological events, such as ligand binding, in living cells. These novel chemical labeling techniques are expected to provide a molecular toolbox for studying a wide variety of proteins and beyond in living cells.

Molecular Dynamics Simulations of Protein-Ligand Complexes in Near Physiological Conditions

NASA Astrophysics Data System (ADS)

Wambo, Thierry Oscar

Proteins are important molecules for their key functions. However, under certain circumstances, the function of these proteins needs to be regulated to keep us healthy. Ligands are small molecules often used to modulate the function of proteins. The binding affinity is a quantitative measure of how strong the ligand will modulate the function of the protein: a strong binding affinity will highly impact the performance of the protein. It becomes clear that it is critical to have appropriate techniques to accurately compute the binding affinity. The most difficult task in computer simulations is how to efficiently sample the space spanned by the ligand during the binding process. In this work, we have developed some schemes to compute the binding affinity of a ligand to a protein, and of a metal ion to a protein. Application of these techniques to some complexes yield results in agreement with experimental values. These methods are a brute force approach and make no assumption other than that the complexes are governed by the force field used. Specifically, we computed the free energy of binding between (1) human carbonic anhydrase II and the drug acetazolamide (hcaII-AZM), (2) human carbonic anhydrase II and the zinc ion (hcaII-Zinc), and (3) beta-lactoglobulin and five fatty acids complexes (BLG-FAs). We found the following free energies of binding in unit of kcal/mol: -12.96 +/-2.44 (-15.74) for hcaII-Zinc complex, -5.76+/-0.76 (-5.57) for BLG-OCA , -4.44+/-1.08 (-5.22) for BLG-DKA,-6.89+/-1.25 (-7.24) for BLG-DAO, -8.57+/-0.82 (-8.14) for BLG-MYR, -8.99+/-0.87 (-8.72) for BLG-PLM, and -11.87+/-1.8 (-10.8) for hcaII-AZM. The values inside the parentheses are experimental results. The simulations and quantitative analysis of each system provide interesting insights into the interactions between each entity and helps us to better understand the dynamics of these systems.

Residues within the Transmembrane Domain of the Glucagon-Like Peptide-1 Receptor Involved in Ligand Binding and Receptor Activation: Modelling the Ligand-Bound Receptor

PubMed Central

Coopman, K.; Wallis, R.; Robb, G.; Brown, A. J. H.; Wilkinson, G. F.; Timms, D.

2011-01-01

The C-terminal regions of glucagon-like peptide-1 (GLP-1) bind to the N terminus of the GLP-1 receptor (GLP-1R), facilitating interaction of the ligand N terminus with the receptor transmembrane domain. In contrast, the agonist exendin-4 relies less on the transmembrane domain, and truncated antagonist analogs (e.g. exendin 9–39) may interact solely with the receptor N terminus. Here we used mutagenesis to explore the role of residues highly conserved in the predicted transmembrane helices of mammalian GLP-1Rs and conserved in family B G protein coupled receptors in ligand binding and GLP-1R activation. By iteration using information from the mutagenesis, along with the available crystal structure of the receptor N terminus and a model of the active opsin transmembrane domain, we developed a structural receptor model with GLP-1 bound and used this to better understand consequences of mutations. Mutation at Y152 [transmembrane helix (TM) 1], R190 (TM2), Y235 (TM3), H363 (TM6), and E364 (TM6) produced similar reductions in affinity for GLP-1 and exendin 9–39. In contrast, other mutations either preferentially [K197 (TM2), Q234 (TM3), and W284 (extracellular loop 2)] or solely [D198 (TM2) and R310 (TM5)] reduced GLP-1 affinity. Reduced agonist affinity was always associated with reduced potency. However, reductions in potency exceeded reductions in agonist affinity for K197A, W284A, and R310A, while H363A was uncoupled from cAMP generation, highlighting critical roles of these residues in translating binding to activation. Data show important roles in ligand binding and receptor activation of conserved residues within the transmembrane domain of the GLP-1R. The receptor structural model provides insight into the roles of these residues. PMID:21868452

istar: a web platform for large-scale protein-ligand docking.

PubMed

Li, Hongjian; Leung, Kwong-Sak; Ballester, Pedro J; Wong, Man-Hon

2014-01-01

Protein-ligand docking is a key computational method in the design of starting points for the drug discovery process. We are motivated by the desire to automate large-scale docking using our popular docking engine idock and thus have developed a publicly-accessible web platform called istar. Without tedious software installation, users can submit jobs using our website. Our istar website supports 1) filtering ligands by desired molecular properties and previewing the number of ligands to dock, 2) monitoring job progress in real time, and 3) visualizing ligand conformations and outputting free energy and ligand efficiency predicted by idock, binding affinity predicted by RF-Score, putative hydrogen bonds, and supplier information for easy purchase, three useful features commonly lacked on other online docking platforms like DOCK Blaster or iScreen. We have collected 17,224,424 ligands from the All Clean subset of the ZINC database, and revamped our docking engine idock to version 2.0, further improving docking speed and accuracy, and integrating RF-Score as an alternative rescoring function. To compare idock 2.0 with the state-of-the-art AutoDock Vina 1.1.2, we have carried out a rescoring benchmark and a redocking benchmark on the 2,897 and 343 protein-ligand complexes of PDBbind v2012 refined set and CSAR NRC HiQ Set 24Sept2010 respectively, and an execution time benchmark on 12 diverse proteins and 3,000 ligands of different molecular weight. Results show that, under various scenarios, idock achieves comparable success rates while outperforming AutoDock Vina in terms of docking speed by at least 8.69 times and at most 37.51 times. When evaluated on the PDBbind v2012 core set, our istar platform combining with RF-Score manages to reproduce Pearson's correlation coefficient and Spearman's correlation coefficient of as high as 0.855 and 0.859 respectively between the experimental binding affinity and the predicted binding affinity of the docked conformation. istar is freely available at http://istar.cse.cuhk.edu.hk/idock.

Molecular modeling and dynamics simulations of PNP from Streptococcus agalactiae.

PubMed

Caceres, Rafael Andrade; Saraiva Timmers, Luis Fernando; Dias, Raquel; Basso, Luiz Augusto; Santos, Diogenes Santiago; de Azevedo, Walter Filgueira

2008-05-01

This work describes for the first time a structural model of purine nucleoside phosphorylase from Streptococcus agalactiae (SaPNP). PNP catalyzes the cleavage of N-ribosidic bonds of the purine ribonucleosides and 2-deoxyribonucleosides in the presence of inorganic orthophosphate as a second substrate. This enzyme is a potential target for the development of antibacterial drugs. We modeled the complexes of SaPNP with 15 different ligands in order to determine the structural basis for the specificity of these ligands against SaPNP. The application of a novel empirical scoring function to estimate the affinity of a ligand for a protein was able to identify the ligands with high affinity for PNPs. The analysis of molecular dynamics trajectory for SaPNP indicates that the functionally important motifs have a very stable structure. This new structural model together with a novel empirical scoring function opens the possibility to explorer larger library of compounds in order to identify the new inhibitors for PNPs in virtual screening projects.

Mapping Protein–Protein Interactions of the Resistance-Related Bacterial Zeta Toxin–Epsilon Antitoxin Complex (ε2ζ2) with High Affinity Peptide Ligands Using Fluorescence Polarization

PubMed Central

Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

2016-01-01

Toxin–antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta–Epsilon toxin–antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε2ζ2 complex. Three α helices of Zeta forming the protein–protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε2ζ2 complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay. PMID:27438853

Mapping Protein-Protein Interactions of the Resistance-Related Bacterial Zeta Toxin-Epsilon Antitoxin Complex (ε₂ζ₂) with High Affinity Peptide Ligands Using Fluorescence Polarization.

PubMed

Fernández-Bachiller, María Isabel; Brzozowska, Iwona; Odolczyk, Norbert; Zielenkiewicz, Urszula; Zielenkiewicz, Piotr; Rademann, Jörg

2016-07-16

Toxin-antitoxin systems constitute a native survival strategy of pathogenic bacteria and thus are potential targets of antibiotic drugs. Here, we target the Zeta-Epsilon toxin-antitoxin system, which is responsible for the stable maintenance of certain multiresistance plasmids in Gram-positive bacteria. Peptide ligands were designed on the basis of the ε₂ζ₂ complex. Three α helices of Zeta forming the protein-protein interaction (PPI) site were selected and peptides were designed conserving the residues interacting with Epsilon antitoxin while substituting residues binding intramolecularly to other parts of Zeta. Designed peptides were synthesized with an N-terminal fluoresceinyl-carboxy-residue for binding assays and provided active ligands, which were used to define the hot spots of the ε₂ζ₂ complex. Further shortening and modification of the binding peptides provided ligands with affinities <100 nM, allowing us to determine the most relevant PPIs and implement a robust competition binding assay.

Rate constants of agonist binding to muscarinic receptors in rat brain medulla. Evaluation by competition kinetics

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

Schreiber, G.; Henis, Y.I.; Sokolovsky, M.

The method of competition kinetics, which measures the binding kinetics of an unlabeled ligand through its effect on the binding kinetics of a labeled ligand, was employed to investigate the kinetics of muscarinic agonist binding to rat brain medulla pons homogenates. The agonists studied were acetylcholine, carbamylcholine, and oxotremorine, with N-methyl-4-(TH)piperidyl benzilate employed as the radiolabeled ligand. Our results suggested that the binding of muscarinic agonists to the high affinity sites is characterized by dissociation rate constants higher by 2 orders of magnitude than those of antagonists, with rather similar association rate constants. Our findings also suggest that isomerization ofmore » the muscarinic receptors following ligand binding is significant in the case of antagonists, but not of agonists. Moreover, it is demonstrated that in the medulla pons preparation, agonist-induced interconversion between high and low affinity bindings sites does not occur to an appreciable extent.« less

Simple method to assess stability of immobilized peptide ligands against proteases.

PubMed

Giudicessi, Silvana L; Salum, María L; Saavedra, Soledad L; Martínez-Ceron, María C; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2017-09-01

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin-bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide-ChemMatrix resin. Peptidyl-resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C-terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Tetrahydroquinoline Ring as a Versatile Bioisostere of Tetralin for Melatonin Receptor Ligands.

PubMed

Rivara, Silvia; Scalvini, Laura; Lodola, Alessio; Mor, Marco; Caignard, Daniel-Henri; Delagrange, Philippe; Collina, Simona; Lucini, Valeria; Scaglione, Francesco; Furiassi, Lucia; Mari, Michele; Lucarini, Simone; Bedini, Annalida; Spadoni, Gilberto

2018-04-26

A new family of melatonin receptor ligands, characterized by a tetrahydroquinoline (THQ) scaffold carrying an amide chain in position 3, was devised as conformationally constrained analogs of flexible N-anilinoethylamides previously developed. Molecular superposition models allowed to identify the patterns of substitution conferring high receptor binding affinity and to support the THQ ring as a suitable scaffold for the preparation of melatonin ligands. The biological activity of 3-acylamino-THQs was compared with that of the corresponding tetralin derivatives. The THQ ring proved to be a versatile scaffold for easy feasible MT 1 and MT 2 ligands, which resulted as more polar bioisosteres of their tetralin analogs. Potent partial agonists, with subnanomolar binding affinity for the MT 2 receptor, were obtained, and a new series of THQ derivatives is presented. The putative binding mode of potent THQs and tetralines was discussed on the basis of their conformational equilibria as inferred from molecular dynamics simulations and experimental NMR data.

Structural and Antioxidant Properties of Compounds Obtained from Fe2+ Chelation by Juglone and Two of Its Derivatives: DFT, QTAIM, and NBO Studies

PubMed Central

Tamafo Fouegue, Aymard Didier; Bikélé Mama, Désiré; Nkungli, Nyiang Kennet; Younang, Elie

2016-01-01

The chelating ability of juglone and two of its derivatives towards Fe2+ion and the antioxidant activity (AOA) of the resulting chelates and complexes (in the presence of H2O and CH3OH as ligands) in gas phase is reported via bond dissociation enthalpy, ionization potential, proton dissociation enthalpy, proton affinity, and electron transfer enthalpy. The DFT/B3LYP level of theory associated with the 6-31+G(d,p) and 6-31G(d) Pople-style basis sets on the atoms of the ligands and the central Fe(II), respectively, was used. Negative chelation free energies obtained revealed that juglone derivatives possessing the O-H substituent (L2) have the greatest ability to chelate Fe2+ ion. Apart from 1B, thermodynamic descriptors of the AOA showed that the direct hydrogen atom transfer is the preferred mechanism of the studied molecules. NBO analysis showed that the Fe-ligand bonds are all formed through metal to ligand charge transfer. QTAIM studies revealed that among all the Fe-ligand bonds, the O1-Fe bond of 1A is purely covalent. The aforementioned results show that the ligands can be used to fight against Fe(II) toxicity, thus preserving human health, and fight against the deterioration of industrial products. In addition, most of the complexes studied have shown a better AOA than their corresponding ligands. PMID:27774044

Detachment of affinity-captured bioparticles by elastic deformation of a macroporous hydrogel

PubMed Central

Dainiak, Maria B.; Kumar, Ashok; Galaev, Igor Yu.; Mattiasson, Bo

2006-01-01

Adsorption of bioparticles to affinity surfaces involves polyvalent interactions, complicating greatly the recovery of the adsorbed material. A unique system for the efficient binding and release of different cells and particles is described. Affinity-bound bioparticles and synthetic particles are detached from the macroporous hydrogel matrix, a so-called cryogel, when the cryogel undergoes elastic deformation. The particle detachment upon elastic deformation is believed to be due to breaking of many of the multipoint attachments between the particles and the affinity matrix and the change in the distance between affinity ligands when the matrix is deformed. However, no release of affinity-bound protein occurred upon elastic deformation. The phenomenon of particle detachment upon elastic deformation is believed to be of a generic nature, because it was demonstrated for a variety of bioparticles of different sizes and for synthetic particles, for different ligand–receptor pairs (IgG–protein A, sugar–ConA, metal ion–chelating ligand), and when the deformation was caused by either external forces (mechanical deformation) or internal forces (the shrinkage of thermosensitive, macroporous hydrogel upon an increase in temperature). The elasticity of cryogel monoliths ensures high recovery of captured cells under mild conditions, with highly retained viability. This property, along with their continuous porous structure makes cryogel monoliths very attractive for applications in affinity cell separation. PMID:16418282

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations

PubMed Central

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.

2014-01-01

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally. PMID:24459184

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

PubMed

Kisley, Lydia; Chen, Jixin; Mansur, Andrea P; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Chen, Wen-Hsiang; Dhamane, Sagar; Willson, Richard C; Landes, Christy F

2014-02-11

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography. By quantifying the interactions of single proteins with individual charged ligands, we demonstrate that clusters of charges are necessary to create detectable adsorption sites and that even chemically identical ligands create adsorption sites of varying kinetic properties that depend on steric availability at the interface. Additionally, we relate experimental results to the stochastic theory of chromatography. Simulated elution profiles calculated from the molecular-scale data suggest that, if it were possible to engineer uniform optimal interactions into ion-exchange systems, separation efficiencies could be improved by as much as a factor of five by deliberately exploiting clustered interactions that currently dominate the ion-exchange process only accidentally.

Isolation and purification of wheat germ agglutinin and analysis of its properties

NASA Astrophysics Data System (ADS)

Wang, Han

2017-12-01

In this paper, the wheat germ agglutinin was isolated and purified by affinity chromatography of chicken ovomucoid as ligand. The physicochemical properties were analyzed. The chicken ovomucoid was isolated from egg white and conjugated to affinity chromatography column agarose gel to prepare affinity adsorbent. The crude extract of wheat germ was freezedried by affinity chromatography. The physicochemical properties were analyzed by SDSpolyacrylamide gel electrophoresis and isoelectric focusing electrophoresis. And the relative molecular mass and isoelectric point of wheat germ agglutinin were obtained, and the high efficiency of purification of wheat germ agglutinin was proved by affinity chromatography.

Vitamin D metabolites in captivity? Should we measure free or total 25(OH)D to assess vitamin D status?

PubMed

Bikle, Daniel; Bouillon, Roger; Thadhani, Ravi; Schoenmakers, Inez

2017-10-01

There is general consensus that serum 25(OH)D is the best biochemical marker for nutritional vitamin D status. Whether free 25(OH)D would be a better marker than total 25(OH)D is so far unclear. Free 25(OH)D can either be calculated based on the measurement of the serum concentrations of total 25(OH)D, vitamin D-binding protein (DBP), albumin, and the affinity between 25(OH)D and its binding proteins in physiological situations. Free 25(OH)D can also be measured directly by equilibrium dialysis, ultrafitration or immunoassays. During the vitamin D workshop held in Boston in March 2016, a debate was organized about the measurements and clinical value of free 25(OH)D, and this debate is summarized in the present manuscript. Overall there is consensus that most cells apart from the renal tubular cells are exposed to free rather than to total 25(OH)D. Therefore free 25(OH)D may be highly relevant for the local production and action of 1,25(OH) 2 D. During the debate it became clear that there is a need for standardization of measurements of serum DBP and of direct measurements of free 25(OH)D. There seems to be very limited genetic or racial differences in DBP concentrations or (probably) in the affinity of DBP for its major ligands. Therefore, free 25(OH)D is strongly correlated to total 25(OH)D in most normal populations. Appropriate studies are needed to define the clinical implications of free rather than total 25(OH)D in normal subjects and in disease states. Special attention is needed for such studies in cases of abnormal DBP concentrations or when one could expect changes in its affinity for its ligands. Copyright © 2017. Published by Elsevier Ltd.

Choline+ is a low-affinity ligand for alpha 1-adrenoceptors.

PubMed

Unelius, L; Cannon, B; Nedergaard, J

1994-10-07

The effect of choline+, a commonly used Na+ substitute, on ligand binding to alpha 1-adrenoceptors was investigated. It was found that replacement of 25% of the Na+ in a Krebs-Ringer bicarbonate buffer with choline+ led to a 3-fold decrease in the apparent affinity of [3H]prazosin for its binding site (i.e. the alpha 1-receptor) in a membrane preparation from brown adipose tissue, while no decrease in the total number of binding sites was observed. Similar effects were seen in membrane preparations from liver and brain. In competition experiments, it was found that choline+ could inhibit [3H]prazosin binding; from the inhibition curve, an affinity (Ki) of 31 mM choline+ for the [3H]prazosin-binding site could be calculated. In fully choline(+)-substituted buffers, where the level of [3H]prazosin binding was substantially reduced, both phentolamine and norepinephrine could still compete with [3H]prazosin for its binding site, with virtually unaltered affinity; thus choline+ did not substantially affect the characteristics of those receptors to which it did not bind. Choline+ did not affect the binding characteristics of the beta 1/beta 2 radioligand [3H]CGP-12177; thus, the effect on alpha 1-receptors was not due to general, unspecific effects on the membrane preparations. It is concluded that choline+ possesses characteristics similar to those of a competitive ligand for the alpha 1-adrenoceptor; it has a low affinity but the competitive type of interaction of choline may nonetheless under experimental conditions interfere with agonist interaction with the alpha 1-receptor.

Hierarchy and Assortativity as New Tools for Binding-Affinity Investigation: The Case of the TBA Aptamer-Ligand Complex.

PubMed

Cataldo, Rosella; Alfinito, Eleonora; Reggiani, Lino

2017-12-01

Aptamers are single stranded DNA, RNA, or peptide sequences having the ability to bind several specific targets (proteins, molecules as well as ions). Therefore, aptamer production and selection for therapeutic and diagnostic applications is very challenging. Usually, they are generated in vitro, although computational approaches have been recently developed for the in silico production. Despite these efforts, the mechanism of aptamer-ligand formation is not completely clear, and producing high-affinity aptamers is still quite difficult. This paper aims to develop a computational model able to describe aptamer-ligand affinity. Topological tools, such as the conventional degree distribution, the rank-degree distribution (hierarchy), and the node assortativity are employed. In doing so, the macromolecules tertiary-structures are mapped into appropriate graphs. These graphs reproduce the main topological features of the macromolecules, by preserving the distances between amino acids (nucleotides). Calculations are applied to the thrombin binding aptamer (TBA), and the TBA-thrombin complex produced in the presence of Na + or K + . The topological analysis is able to detect several differences between complexes obtained in the presence of the two cations, as expected by previous investigations. These results support graph analysis as a novel computational tool for testing affinity. Otherwise, starting from the graphs, an electrical network can be obtained by using the specific electrical properties of amino acids and nucleobases. Therefore, a further analysis concerns with the electrical response, revealing that the resistance is sensitively affected by the presence of sodium or potassium, thus suggesting resistance as a useful physical parameter for testing binding affinity.

Familial secondary erythrocytosis due to increased oxygen affinity is caused by destabilization of the T state of hemoglobin Brigham (α2β2Pro100Leu)

PubMed Central

Mollan, Todd L; Abraham, Bindu; Strader, Michael Brad; Jia, Yiping; Lozier, Jay N; Olson, John S; Alayash, Abdu I

2012-01-01

Hemoglobin Brigham (β Pro100 to Leu) was first reported in a patient with familial erythrocytosis. Erythrocytes of an affected individual from the same family contain both HbA and Hb Brigham and exhibit elevated O2 affinity compared with normal cells (P50 = 23 mm Hg vs. 31 mmHg at pH 7.4 at 37°C). O2 affinities measured for hemolysates were sensitive to changes in pH or chloride concentrations, indicating little change in the Bohr and Chloride effects. Hb Brigham was separated from normal HbA by nondenaturing cation exchange liquid chromatography, and the amino acid substitution was verified by mass spectrometry. The properties of Hb Brigham isolated from the patient's blood were then compared with those of recombinant Hb Brigham expressed in Escherichia coli. Kinetic experiments suggest that the rate constants for ligand binding and release in the high (R) and low (T) affinity quaternary states of Hb Brigham are similar to those of native hemoglobin. However, the Brigham mutation decreases the T to R equilibrium constant (L) which accelerates the switch to the R state during ligand binding to deoxy-Hb, increasing the rate of association by approximately twofold, and decelerates the switch during ligand dissociation from HbO2, decreasing the rate approximately twofold. These kinetic data help explain the high O2 affinity characteristics of Hb Brigham and provide further evidence for the importance of the contribution of Pro100 to intersubunit contacts and stabilization of the T quaternary structure. PMID:22821886

Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3.

PubMed

Diehl, Carl; Engström, Olof; Delaine, Tamara; Håkansson, Maria; Genheden, Samuel; Modig, Kristofer; Leffler, Hakon; Ryde, Ulf; Nilsson, Ulf J; Akke, Mikael

2010-10-20

Rational drug design is predicated on knowledge of the three-dimensional structure of the protein-ligand complex and the thermodynamics of ligand binding. Despite the fundamental importance of both enthalpy and entropy in driving ligand binding, the role of conformational entropy is rarely addressed in drug design. In this work, we have probed the conformational entropy and its relative contribution to the free energy of ligand binding to the carbohydrate recognition domain of galectin-3. Using a combination of NMR spectroscopy, isothermal titration calorimetry, and X-ray crystallography, we characterized the binding of three ligands with dissociation constants ranging over 2 orders of magnitude. (15)N and (2)H spin relaxation measurements showed that the protein backbone and side chains respond to ligand binding by increased conformational fluctuations, on average, that differ among the three ligand-bound states. Variability in the response to ligand binding is prominent in the hydrophobic core, where a distal cluster of methyl groups becomes more rigid, whereas methyl groups closer to the binding site become more flexible. The results reveal an intricate interplay between structure and conformational fluctuations in the different complexes that fine-tunes the affinity. The estimated change in conformational entropy is comparable in magnitude to the binding enthalpy, demonstrating that it contributes favorably and significantly to ligand binding. We speculate that the relatively weak inherent protein-carbohydrate interactions and limited hydrophobic effect associated with oligosaccharide binding might have exerted evolutionary pressure on carbohydrate-binding proteins to increase the affinity by means of conformational entropy.

Ligand deconstruction: Why some fragment binding positions are conserved and others are not

PubMed Central

Kozakov, Dima; Hall, David R.; Jehle, Stefan; Luo, Lingqi; Ochiana, Stefan O.; Jones, Elizabeth V.; Pollastri, Michael; Allen, Karen N.; Whitty, Adrian; Vajda, Sandor

2015-01-01

Fragment-based drug discovery (FBDD) relies on the premise that the fragment binding mode will be conserved on subsequent expansion to a larger ligand. However, no general condition has been established to explain when fragment binding modes will be conserved. We show that a remarkably simple condition can be developed in terms of how fragments coincide with binding energy hot spots—regions of the protein where interactions with a ligand contribute substantial binding free energy—the locations of which can easily be determined computationally. Because a substantial fraction of the free energy of ligand binding comes from interacting with the residues in the energetically most important hot spot, a ligand moiety that sufficiently overlaps with this region will retain its location even when other parts of the ligand are removed. This hypothesis is supported by eight case studies. The condition helps identify whether a protein is suitable for FBDD, predicts the size of fragments required for screening, and determines whether a fragment hit can be extended into a higher affinity ligand. Our results show that ligand binding sites can usefully be thought of in terms of an anchor site, which is the top-ranked hot spot and dominates the free energy of binding, surrounded by a number of weaker satellite sites that confer improved affinity and selectivity for a particular ligand and that it is the intrinsic binding potential of the protein surface that determines whether it can serve as a robust binding site for a suitably optimized ligand. PMID:25918377

Molecular simulations of multimodal ligand-protein binding: elucidation of binding sites and correlation with experiments.

PubMed

Freed, Alexander S; Garde, Shekhar; Cramer, Steven M

2011-11-17

Multimodal chromatography, which employs more than one mode of interaction between ligands and proteins, has been shown to have unique selectivity and high efficacy for protein purification. To test the ability of free solution molecular dynamics (MD) simulations in explicit water to identify binding regions on the protein surface and to shed light on the "pseudo affinity" nature of multimodal interactions, we performed MD simulations of a model protein ubiquitin in aqueous solution of free ligands. Comparisons of MD with NMR spectroscopy of ubiquitin mutants in solutions of free ligands show a good agreement between the two with regard to the preferred binding region on the surface of the protein and several binding sites. MD simulations also identify additional binding sites that were not observed in the NMR experiments. "Bound" ligands were found to be sufficiently flexible and to access a number of favorable conformations, suggesting only a moderate loss of ligand entropy in the "pseudo affinity" binding of these multimodal ligands. Analysis of locations of chemical subunits of the ligand on the protein surface indicated that electrostatic interaction units were located on the periphery of the preferred binding region on the protein. The analysis of the electrostatic potential, the hydrophobicity maps, and the binding of both acetate and benzene probes were used to further study the localization of individual ligand moieties. These results suggest that water-mediated electrostatic interactions help the localization and orientation of the MM ligand to the binding region with additional stability provided by nonspecific hydrophobic interactions.

Molecular Dynamics in Mixed Solvents Reveals Protein-Ligand Interactions, Improves Docking, and Allows Accurate Binding Free Energy Predictions.

PubMed

Arcon, Juan Pablo; Defelipe, Lucas A; Modenutti, Carlos P; López, Elias D; Alvarez-Garcia, Daniel; Barril, Xavier; Turjanski, Adrián G; Martí, Marcelo A

2017-04-24

One of the most important biological processes at the molecular level is the formation of protein-ligand complexes. Therefore, determining their structure and underlying key interactions is of paramount relevance and has direct applications in drug development. Because of its low cost relative to its experimental sibling, molecular dynamics (MD) simulations in the presence of different solvent probes mimicking specific types of interactions have been increasingly used to analyze protein binding sites and reveal protein-ligand interaction hot spots. However, a systematic comparison of different probes and their real predictive power from a quantitative and thermodynamic point of view is still missing. In the present work, we have performed MD simulations of 18 different proteins in pure water as well as water mixtures of ethanol, acetamide, acetonitrile and methylammonium acetate, leading to a total of 5.4 μs simulation time. For each system, we determined the corresponding solvent sites, defined as space regions adjacent to the protein surface where the probability of finding a probe atom is higher than that in the bulk solvent. Finally, we compared the identified solvent sites with 121 different protein-ligand complexes and used them to perform molecular docking and ligand binding free energy estimates. Our results show that combining solely water and ethanol sites allows sampling over 70% of all possible protein-ligand interactions, especially those that coincide with ligand-based pharmacophoric points. Most important, we also show how the solvent sites can be used to significantly improve ligand docking in terms of both accuracy and precision, and that accurate predictions of ligand binding free energies, along with relative ranking of ligand affinity, can be performed.

Motifs for molecular recognition exploiting hydrophobic enclosure in protein-ligand binding.

PubMed

Young, Tom; Abel, Robert; Kim, Byungchan; Berne, Bruce J; Friesner, Richard A

2007-01-16

The thermodynamic properties and phase behavior of water in confined regions can vary significantly from that observed in the bulk. This is particularly true for systems in which the confinement is on the molecular-length scale. In this study, we use molecular dynamics simulations and a powerful solvent analysis technique based on inhomogenous solvation theory to investigate the properties of water molecules that solvate the confined regions of protein active sites. Our simulations and analysis indicate that the solvation of protein active sites that are characterized by hydrophobic enclosure and correlated hydrogen bonds induce atypical entropic and enthalpic penalties of hydration. These penalties apparently stabilize the protein-ligand complex with respect to the independently solvated ligand and protein, which leads to enhanced binding affinities. Our analysis elucidates several challenging cases, including the super affinity of the streptavidin-biotin system.

Design of ligands for the nicotinic acetylcholine receptors: the quest for selectivity.

PubMed

Bunnelle, William H; Dart, Michael J; Schrimpf, Michael R

2004-01-01

In the last decade, nicotinic acetylcholine receptors (nAChRs) have emerged as important targets for drug discovery. The therapeutic potential of nicotinic agonists depends substantially on the ability to selectively activate certain receptor subtypes that mediate beneficial effects. The design of such compounds has proceeded in spite of a general shortage of data pertaining to subtype selectivity. Medicinal chemistry efforts have been guided principally by binding affinities to the alpha4beta2 and/or alpha7 subtypes, even though these are not predictive of agonist activity at either subtype. Nevertheless, a diverse family of nAChR ligands has been developed, and several analogs with promising therapeutic potential have now advanced to human clinical trials. This paper provides an overview of the structure-affinity relationships that continue to drive development of new nAChR ligands.

Architecture effects on multivalent interactions by polypeptide-based multivalent ligands

NASA Astrophysics Data System (ADS)

Liu, Shuang

Multivalent interactions are characterized by the simultaneous binding between multiple ligands and multiple binding sites, either in solutions or at interfaces. In biological systems, most multivalent interactions occur between protein receptors and carbohydrate ligands through hydrogen-bonding and hydrophobic interactions. Compared with weak affinity binding between one ligand and one binding site, i.e. monovalent interaction, multivalent interactioins provide greater avidity and specificity, and therefore play unique roles in a broad range of biological activities. Moreover, the studies of multivalent interactions are also essential for producing effective inhibitors and effectors of biological processes that could have important therapeutic applications. Synthetic multivalent ligands have been designed to mimic the biological functions of natural multivalent interactions, and various types of scaffolds have been used to display multiple ligands, including small molecules, linear polymers, dendrimers, nanoparticle surfaces, monolayer surfaces and liposomes. Studies have shown that multivalent interactions can be highly affected by various architectural parameters of these multivalent ligands, including ligand identities, valencies, spacing, ligand densities, nature of linker arms, scaffold length and scaffold conformation. Most of these multivalent ligands are chemically synthesized and have limitations of controlling over sequence and conformation, which is a barrier for mimicking ordered and controlled natural biological systems. Therefore, multivalent ligands with precisely controlled architecture are required for improved structure-function relationship studies. Protein engineering methods with subsequent chemical coupling of ligands provide significant advantages of controlling over backbone conformation and functional group placement, and therefore have been used to synthesize recombinant protein-based materials with desired properties similar to natural protein materials, including structural as well as functional proteins. Therefore, polypeptide-based multivalent scaffolds are used to display ligands to assess the contribution of different architectural parameters to the multivalent binding events. In this work, a family of alanine-rich alpha-helical glycopolypeptides was designed and synthesized by a combination of protein engineering and chemical coupling, to display two types of saccharide ligands for two different multivalent binding systems. The valencies, chain length and spacing between adjacent ligands of these multivalent ligands were designed in order to study architecture effects on multivalent interactions. The polypeptides and their glycoconjugates were characterized via various methods, including SDS-PAGE, NMR, HPLC, amino acid analysis (AAA), MALDI, circular dichroism (CD) and GPC. In the first multivalent binding system, cholera toxin B pentamer (CT B5) was chosen to be the protein receptor due to its well-characterized structure, lack of significant steric interference of binding to multiple binding sites, and requirement of only simple monosaccharide as ligands. Galactopyranoside was incorporated into polypeptide scaffolds through amine-carboxylic acid coupling to the side chains of glutamic acid residues. The inhibition and binding to CT B5 of these glycopolypeptide ligands were evaluated by direct enzyme-linked assay (DELA). As a complement method, weak affinity chromatography (WAC) was also used to evaluate glycopolypeptides binding to a CT B5 immobilized column. The architecture effects on CT B 5 inhibition are discussed. In the second system, cell surface receptor L-selectin was targeted by polypeptide-based multivalent ligands containing disulfated galactopyranoside ligands, due to its important roles in various immunological activities. The effects of glycopolypeptide architectural variables L-selectin shedding were evaluated via ELISA-based assays. These polypeptide-based multivalent ligands are suggested to be useful for elucidating architecture effects on multivalent interactions, manipulating multivalent interactions and the subsequent cellular responses in different systems. These materials have great potential applications in therapeutics and could also provide guidelines for design of multivalent ligands for other protein receptors.

Improved estimation of ligand macromolecule binding affinities by linear response approach using a combination of multi-mode MD simulation and QM/MM methods

NASA Astrophysics Data System (ADS)

Khandelwal, Akash; Balaz, Stefan

2007-01-01

Structure-based predictions of binding affinities of ligands binding to proteins by coordination bonds with transition metals, covalent bonds, and bonds involving charge re-distributions are hindered by the absence of proper force fields. This shortcoming affects all methods which use force-field-based molecular simulation data on complex formation for affinity predictions. One of the most frequently used methods in this category is the Linear Response (LR) approach of Åquist, correlating binding affinities with van der Waals and electrostatic energies, as extended by Jorgensen's inclusion of solvent-accessible surface areas. All these terms represent the differences, upon binding, in the ensemble averages of pertinent quantities, obtained from molecular dynamics (MD) or Monte Carlo simulations of the complex and of single components. Here we report a modification of the LR approach by: (1) the replacement of the two energy terms through the single-point QM/MM energy of the time-averaged complex structure from an MD simulation; and (2) a rigorous consideration of multiple modes (mm) of binding. The first extension alleviates the force-field related problems, while the second extension deals with the ligands exhibiting large-scale motions in the course of an MD simulation. The second modification results in the correlation equation that is nonlinear in optimized coefficients, but does not lead to an increase in the number of optimized coefficients. The application of the resulting mm QM/MM LR approach to the inhibition of zinc-dependent gelatinase B (matrix metalloproteinase 9) by 28 hydroxamate ligands indicates a significant improvement of descriptive and predictive abilities.

Structure-guided engineering of Anticalins with improved binding behavior and biochemical characteristics for application in radio-immuno imaging and/or therapy.

PubMed

Eggenstein, E; Eichinger, A; Kim, H-J; Skerra, A

2014-02-01

Modern strategies in radio-immuno therapy and in vivo imaging require robust, small, and specific ligand-binding proteins. In this context we have previously developed artificial lipocalins, so-called Anticalins, with high binding activity toward rare-earth metal-chelate complexes using combinatorial protein design. Here we describe further improvement of the Anticalin C26 via in vitro affinity maturation to yield CL31, which has a fourfold slower dissociation half-life above 2h. Also, we present the crystallographic analyses of both the initial and the improved Anticalin, providing insight into the molecular mechanism of chelated metal binding and the role of amino acid substitutions during the step-wise affinity maturation. Notably, one of the four structurally variable loops that form the ligand pocket in the lipocalin scaffold undergoes a significant conformational change from C26 to CL31, acting as a lid that closes over the accommodated metal-chelate ligand. A systematic mutational study indicated that further improvement of ligand affinity is difficult to achieve while providing clues on the contribution of relevant side chains in the engineered binding pocket. Unexpectedly, some of the amino acid replacements led to strong increases - more then 10-fold - in the yield of soluble protein from periplasmic secretion in Escherichia coli. Copyright © 2013 Elsevier Inc. All rights reserved.

Application of binding free energy calculations to prediction of binding modes and affinities of MDM2 and MDMX inhibitors.

PubMed

Lee, Hui Sun; Jo, Sunhwan; Lim, Hyun-Suk; Im, Wonpil

2012-07-23

Molecular docking is widely used to obtain binding modes and binding affinities of a molecule to a given target protein. Despite considerable efforts, however, prediction of both properties by docking remains challenging mainly due to protein's structural flexibility and inaccuracy of scoring functions. Here, an integrated approach has been developed to improve the accuracy of binding mode and affinity prediction and tested for small molecule MDM2 and MDMX antagonists. In this approach, initial candidate models selected from docking are subjected to equilibration MD simulations to further filter the models. Free energy perturbation molecular dynamics (FEP/MD) simulations are then applied to the filtered ligand models to enhance the ability in predicting the near-native ligand conformation. The calculated binding free energies for MDM2 complexes are overestimated compared to experimental measurements mainly due to the difficulties in sampling highly flexible apo-MDM2. Nonetheless, the FEP/MD binding free energy calculations are more promising for discriminating binders from nonbinders than docking scores. In particular, the comparison between the MDM2 and MDMX results suggests that apo-MDMX has lower flexibility than apo-MDM2. In addition, the FEP/MD calculations provide detailed information on the different energetic contributions to ligand binding, leading to a better understanding of the sensitivity and specificity of protein-ligand interactions.

Recent improvements to Binding MOAD: a resource for protein-ligand binding affinities and structures.

PubMed

Ahmed, Aqeel; Smith, Richard D; Clark, Jordan J; Dunbar, James B; Carlson, Heather A

2015-01-01

For over 10 years, Binding MOAD (Mother of All Databases; http://www.BindingMOAD.org) has been one of the largest resources for high-quality protein-ligand complexes and associated binding affinity data. Binding MOAD has grown at the rate of 1994 complexes per year, on average. Currently, it contains 23,269 complexes and 8156 binding affinities. Our annual updates curate the data using a semi-automated literature search of the references cited within the PDB file, and we have recently upgraded our website and added new features and functionalities to better serve Binding MOAD users. In order to eliminate the legacy application server of the old platform and to accommodate new changes, the website has been completely rewritten in the LAMP (Linux, Apache, MySQL and PHP) environment. The improved user interface incorporates current third-party plugins for better visualization of protein and ligand molecules, and it provides features like sorting, filtering and filtered downloads. In addition to the field-based searching, Binding MOAD now can be searched by structural queries based on the ligand. In order to remove redundancy, Binding MOAD records are clustered in different families based on 90% sequence identity. The new Binding MOAD, with the upgraded platform, features and functionalities, is now equipped to better serve its users. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Structure–Activity Relationships for a Novel Series of Dopamine D2-like Receptor Ligands Based on N-Substituted 3-Aryl-8-azabicyclo[3.2.1]octan-3-ol

PubMed Central

Paul, Noel M.; Taylor, Michelle; Kumar, Rakesh; Deschamps, Jeffrey R.; Luedtke, Robert R.; Newman, Amy Hauck

2011-01-01

Discovering dopamine D2-like receptor subtype-selective ligands has been a focus of significant investigation. The D2R-selective antagonist 3-[4-(4-chlorophenyl)-4-hydroxypiperidinyl]methylindole (1, L741,626; Ki(D2R/D3R) = 11.2:163 nM) has previously provided a lead template for chemical modification. Herein, analogues have been synthesized where the piperidine was replaced by a tropane ring that reversed the selectivity seen in the parent compound, in human hD2LR- or hD3R-transfected HEK 293 cells (31, Ki(D2R/D3R) = 33.4: 15.5 nM). Further exploration of both N-substituted and aryl ring-substituted analogues resulted in the discovery of several high affinity D2R/D3R ligands with 3-benzofurylmethyl-substituents (e.g., 45, Ki(D2R/D3R) = 1.7:0.34 nM) that induced high affinity not achieved in similarly N-substituted piperidine analogues and significantly (470-fold) improved D3R binding affinity compared to the parent ligand 1. X-ray crystallographic data revealed a distinctive spatial arrangement of pharmacophoric elements in the piperidinol vs tropine analogues, providing clues for the diversity in SAR at the D2 and D3 receptor subtypes. PMID:18774793

Design synthesis and structure-activity relationship of 5-substituted (tetrahydronaphthalen-2yl)methyl with N-phenyl-N-(piperidin-2-yl)propionamide derivatives as opioid ligands.

PubMed

Deekonda, Srinivas; Rankin, David; Davis, Peg; Lai, Josephine; Vanderah, Todd W; Porecca, Frank; Hruby, Victor J

2016-01-15

Here, we report the design, synthesis and structure activity relationship of novel small molecule opioid ligands based on 5-amino substituted (tetrahydronaphthalen-2-yl)methyl moiety with N-phenyl-N-(piperidin-2-yl)propionamide derivatives. We synthesized various molecules including amino, amide and hydroxy substitution on the 5th position of the (tetrahydronaphthalen-2-yl)methyl moiety. In our further designs we replaced the (tetrahydronaphthalen-2-yl)methyl moiety with benzyl and phenethyl moiety. These N-phenyl-N-(piperidin-2-yl)propionamide analogues showed moderate to good binding affinities (850-4 nM) and were selective towards the μ opioid receptor over the δ opioid receptors. From the structure activity relationship studies, we found that a hydroxyl substitution at the 5th position of (tetrahydronapthalen-2yl)methyl group, ligands 19 and 20, showed excellent binding affinities 4 and 5 nM, respectively, and 1000 fold selectivity towards the μ opioid relative to the delta opioid receptor. The ligand 19 showed potent agonist activities 75±21 nM, and 190±42 nM in the GPI and MVD assays. Surprisingly the fluoro analogue 20 showed good agonist activities in MVD assays 170±42 nM, in contrast to its binding affinity results. Copyright © 2015 Elsevier Ltd. All rights reserved.

Protease-Resistant Peptide Ligands from a Knottin Scaffold Library

PubMed Central

Getz, Jennifer A.; Rice, Jeffrey J.; Daugherty, Patrick S.

2011-01-01

Peptides within the knottin family have been shown to possess inherent stability, making them attractive scaffolds for the development of therapeutic and diagnostic agents. Given its remarkable stability to proteases, the cyclic peptide kalata B1 was employed as a scaffold to create a large knottin library displayed on the surface of E. coli. A library exceeding 109 variants was constructed by randomizing seven amino acids within a loop of the kalata B1 scaffold and screened using fluorescence-activated cell sorting to identify peptide ligands specific for the active site of human thrombin. Refolded thrombin binders exhibited high nanomolar affinities in solution, slow dissociation rates, and were able to inhibit thrombin’s enzymatic activity. Importantly, 80% of a knottin-based thrombin inhibitor remained intact after a two hour incubation both with trypsin and with chymotrypsin, demonstrating that modifying the kalata B1 sequence did not compromise its stability properties. In addition, the knottin variant mediated 20-fold enhanced affinity for thrombin, when compared to the same seven residue binding epitope constrained by a single disulfide bond. Our results indicate that peptide libraries derived from the kalata B1 scaffold can yield high affinity protein ligands that retain the remarkable protease resistance associated with the parent scaffold. More generally, this strategy may prove useful in the development of stable peptide ligands suitable for in vivo applications. PMID:21615106

Role of hydrogen bonding in ligand interaction with the N-methyl-D-aspartate receptor ion channel

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

Leeson, P.D.; Carling, R.W.; James, K.

1990-05-01

Displacement of (3H)MK-801 (dizocilpine, 1) binding to rat brain membranes has been used to evaluate the affinities of novel dibenzocycloalkenimines related to 1 for the ion channel binding site (also known as the phencyclidine or PCP receptor) on the N-methyl-D-aspartate (NMDA) subtype of excitory amino acid receptor. In common with many other agents having actions in the central nervous system, these compounds contain a hydrophobic aromatic moiety and a basic nitrogen atom. The conformational rigidity of these ligands provides a unique opportunity to evaluate the importance of specific geometrical properties that influence active-site recognition, in particular the role of themore » nitrogen atom in hydrogen-bonding interactions. The relative affinities (IC50s) of hydrocarbon-substituted analogues of 1 and ring homologated cyclooctenimines illustrate the importance of size-limited hydrophobic binding of both aryl rings and of the quaternary C-5 methyl group. Analysis of the binding of a series of the 10 available structurally rigid dibenzoazabicyclo(x.y.z)alkanes, by using molecular modeling techniques, uncovered a highly significant correlation between affinity and a proposed ligand-active site hydrogen bonding vector (r = 0.950, p less than 0.001). These results are used to generate a pharmacophore of the MK-801 recognition site/PCP receptor, which accounts for the binding of all of the known ligands.« less

RXR function requires binding to an endogenous terpenoid ligand

USDA-ARS?s Scientific Manuscript database

The issue of whether the nuclear receptor RXR must bind to an endogenous, nanomolar affinity ligand in order to perform its natural function is still unsettled (1). On the basis of our previous studies establishing that the Drosophilamelanogaster ortholog of the retinoid X receptor ("ultraspiracle,"...

The roles of two O-donor ligands in the Fe2+-binding and H2O2-sensing by the Fe2+-dependent H2O2 sensor PerR.

PubMed

Ji, Chang-Jun; Yang, Yoon-Mo; Kim, Jung-Hoon; Ryu, Su-Hyun; Youn, Hwan; Lee, Jin-Won

2018-05-10

PerR is a metal-dependent peroxide sensing transcription factor which controls the expression of genes involved in peroxide resistance. The function of Bacillus subtilis PerR is mainly dictated by the regulatory metal ion (Fe 2+ or Mn 2+ ) coordinated by three N-donor ligands (His37, His91, and His93) and two O-donor ligands (Asp85 and Asp104). While H 2 O 2 sensing by PerR is mediated by Fe 2+ -dependent oxidation of N-donor ligand (either His37 or His91), one of the O-donor ligands (Asp104), but not Asp85, has been proposed as the key residue that regulates the sensitivity of PerR to H 2 O 2 . Here we systematically investigated the relative roles of two O-donor ligands of PerR in metal-binding affinity and H 2 O 2 sensitivity in vivo and in vitro. Consistent with the previous report, in vitro the D104E-PerR could not sense low levels of H 2 O 2 in the presence of excess Fe 2+ sufficient for the formation of the Fe 2+ -bound D104E-PerR. However, the expression of PerR-regulated reporter fusion was not repressed by D104E-PerR in the presence of Fe 2+ , suggesting that Fe 2+ is not an effective corepressor for this mutant protein in vivo. Furthermore, in vitro metal titration assays indicate that D104E-PerR has a significantly reduced affinity for Fe 2+ , but not for Mn 2+ , when compared to wild type PerR. These data indicate that the type of O-donor ligand (Asp vs. Glu) at position 104 is an important determinant in providing high Fe 2+ -binding affinity required for the sensing of the physiologically relevant Fe 2+ -levels, in addition to its role in rendering PerR highly sensitive to physiological levels of H 2 O 2 . In comparison, the D85E-PerR did not show a perturbed change in Fe 2+ -binding affinity, however, it displayed a slightly decreased sensitivity to H 2 O 2 both in vivo and in vitro, suggesting that the type of O-donor ligand (Asp vs. Glu) at position 85 may be important for the fine-tuning of H 2 O 2 sensitivity. Copyright © 2018 Elsevier Inc. All rights reserved.

Multiple Functions of Aromatic-Carbohydrate Interactions in a Processive Cellulase Examined with Molecular Simulation*

PubMed Central

Payne, Christina M.; Bomble, Yannick J.; Taylor, Courtney B.; McCabe, Clare; Himmel, Michael E.; Crowley, Michael F.; Beckham, Gregg T.

2011-01-01

Proteins employ aromatic residues for carbohydrate binding in a wide range of biological functions. Glycoside hydrolases, which are ubiquitous in nature, typically exhibit tunnels, clefts, or pockets lined with aromatic residues for processing carbohydrates. Mutation of these aromatic residues often results in significant activity differences on insoluble and soluble substrates. However, the thermodynamic basis and molecular level role of these aromatic residues remain unknown. Here, we calculate the relative ligand binding free energy by mutating tryptophans in the Trichoderma reesei family 6 cellulase (Cel6A) to alanine. Removal of aromatic residues near the catalytic site has little impact on the ligand binding free energy, suggesting that aromatic residues immediately upstream of the active site are not directly involved in binding, but play a role in the glucopyranose ring distortion necessary for catalysis. Removal of aromatic residues at the entrance and exit of the Cel6A tunnel, however, dramatically impacts the binding affinity, suggesting that these residues play a role in chain acquisition and product stabilization, respectively. The roles suggested from differences in binding affinity are confirmed by molecular dynamics and normal mode analysis. Surprisingly, our results illustrate that aromatic-carbohydrate interactions vary dramatically depending on the position in the enzyme tunnel. As aromatic-carbohydrate interactions are present in all carbohydrate-active enzymes, these results have implications for understanding protein structure-function relationships in carbohydrate metabolism and recognition, carbon turnover in nature, and protein engineering strategies for biomass utilization. Generally, these results suggest that nature employs aromatic-carbohydrate interactions with a wide range of binding affinities for diverse functions. PMID:21965672

Quantitative screening of yeast surface-displayed polypeptide libraries by magnetic bead capture.

PubMed

Yeung, Yik A; Wittrup, K Dane

2002-01-01

Magnetic bead capture is demonstrated here to be a feasible alternative for quantitative screening of favorable mutants from a cell-displayed polypeptide library. Flow cytometric sorting with fluorescent probes has been employed previously for high throughput screening for either novel binders or improved mutants. However, many laboratories do not have ready access to this technology as a result of the limited availability and high cost of cytometers, restricting the use of cell-displayed libraries. Using streptavidin-coated magnetic beads and biotinylated ligands, an alternative approach to cell-based library screening for improved mutants was developed. Magnetic bead capture probability of labeled cells is shown to be closely correlated with the surface ligand density. A single-pass enrichment ratio of 9400 +/- 1800-fold, at the expense of 85 +/- 6% binder losses, is achieved from screening a library that contains one antibody-displaying cell (binder) in 1.1 x 10(5) nondisplaying cells. Additionally, kinetic screening for an initial high affinity to low affinity (7.7-fold lower) mutant ratio of 1:95,000, the magnetic bead capture method attains a single-pass enrichment ratio of 600 +/- 200-fold with a 75 +/- 24% probability of loss for the higher affinity mutant. The observed high loss probabilities can be straightforwardly compensated for by library oversampling, given the inherently parallel nature of the screen. Overall, these results demonstrate that magnetic beads are capable of quantitatively screening for novel binders and improved mutants. The described methods are directly analogous to procedures in common use for phage display and should lower the barriers to entry for use of cell surface display libraries.

Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein.

PubMed

Poongavanam, Vasanthanathan; Kongsted, Jacob; Wüstner, Daniel

2016-09-13

Transport of cholesterol derived from hydrolysis of lipoprotein associated cholesteryl esters out of late endosomes depends critically on the function of the Niemann Pick C1 (NPC1) and C2 (NPC2) proteins. Both proteins bind cholesterol but also various other sterols and both with strongly varying affinity. The molecular mechanisms underlying this multiligand specificity are not known. On the basis of the crystal structure of NPC2, we have here investigated structural details of NPC2-sterol interactions using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. We found that an aliphatic side chain in the sterol ligand results in strong binding to NPC2, while side-chain oxidized sterols gave weaker binding. Estradiol and the hydrophobic amine U18666A had the lowest affinity of all tested ligands and at the same time showed the highest flexibility within the NPC2 binding pocket. The binding affinity of all ligands correlated highly with their calculated partitioning coefficient (logP) between octanol/water phases and with the potential of sterols to stabilize the protein backbone. From molecular dynamics simulations, we suggest a general mechanism for NPC2 mediated sterol transfer, in which Phe66, Val96, and Tyr100 act as reversible gate keepers. These residues stabilize the sterol in the binding pose via π-π stacking but move transiently apart during sterol release. A computational mutation analysis revealed that the binding of various ligands depends critically on the same specific amino acid residues within the binding pocket providing shape complementary to sterols, but also on residues in distal regions of the protein.

[Changes in the secondary and tertiary structure of serum albumin in interactions with ligands of various structures].

PubMed

Trinus, F P; Braver-Chernobul'skaia, B S; Luĭk, A I; Boldeskul, A E; Velichko, A N

1984-01-01

High affinity interactions between blood serum albumin and five substances of various chemical structure, exhibiting distinct physiological activity, were accompanied by alterations in the protein tertiary structure, while the albumin secondary structure was involved in conformational transformation after less effective affinity binding.

Prediction of cyclin-dependent kinase 2 inhibitor potency using the fragment molecular orbital method

PubMed Central

2011-01-01

Background The reliable and robust estimation of ligand binding affinity continues to be a challenge in drug design. Many current methods rely on molecular mechanics (MM) calculations which do not fully explain complex molecular interactions. Full quantum mechanical (QM) computation of the electronic state of protein-ligand complexes has recently become possible by the latest advances in the development of linear-scaling QM methods such as the ab initio fragment molecular orbital (FMO) method. This approximate molecular orbital method is sufficiently fast that it can be incorporated into the development cycle during structure-based drug design for the reliable estimation of ligand binding affinity. Additionally, the FMO method can be combined with approximations for entropy and solvation to make it applicable for binding affinity prediction for a broad range of target and chemotypes. Results We applied this method to examine the binding affinity for a series of published cyclin-dependent kinase 2 (CDK2) inhibitors. We calculated the binding affinity for 28 CDK2 inhibitors using the ab initio FMO method based on a number of X-ray crystal structures. The sum of the pair interaction energies (PIE) was calculated and used to explain the gas-phase enthalpic contribution to binding. The correlation of the ligand potencies to the protein-ligand interaction energies gained from FMO was examined and was seen to give a good correlation which outperformed three MM force field based scoring functions used to appoximate the free energy of binding. Although the FMO calculation allows for the enthalpic component of binding interactions to be understood at the quantum level, as it is an in vacuo single point calculation, the entropic component and solvation terms are neglected. For this reason a more accurate and predictive estimate for binding free energy was desired. Therefore, additional terms used to describe the protein-ligand interactions were then calculated to improve the correlation of the FMO derived values to experimental free energies of binding. These terms were used to account for the polar and non-polar solvation of the molecule estimated by the Poisson-Boltzmann equation and the solvent accessible surface area (SASA), respectively, as well as a correction term for ligand entropy. A quantitative structure-activity relationship (QSAR) model obtained by Partial Least Squares projection to latent structures (PLS) analysis of the ligand potencies and the calculated terms showed a strong correlation (r2 = 0.939, q2 = 0.896) for the 14 molecule test set which had a Pearson rank order correlation of 0.97. A training set of a further 14 molecules was well predicted (r2 = 0.842), and could be used to obtain meaningful estimations of the binding free energy. Conclusions Our results show that binding energies calculated with the FMO method correlate well with published data. Analysis of the terms used to derive the FMO energies adds greater understanding to the binding interactions than can be gained by MM methods. Combining this information with additional terms and creating a scaled model to describe the data results in more accurate predictions of ligand potencies than the absolute values obtained by FMO alone. PMID:21219630

Lactosylamidine-based affinity purification for cellulolytic enzymes EG I and CBH I from Hypocrea jecorina and their properties.

PubMed

Ogata, Makoto; Kameshima, Yumiko; Hattori, Takeshi; Michishita, Kousuke; Suzuki, Tomohiro; Kawagishi, Hirokazu; Totani, Kazuhide; Hiratake, Jun; Usui, Taichi

2010-12-10

Selective adsorption and separation of β-glucosidase, endo-acting endo-β-(1→4)-glucanase I (EG I), and exo-acting cellobiohydrolase I (CBH I) were achieved by affinity chromatography with β-lactosylamidine as ligand. A crude cellulase preparation from Hypocrea jecorina served as the source of enzyme. When crude cellulase was applied to the lactosylamidine-based affinity column, β-glucosidase appeared in the unbound fraction. By contrast, EG I and CBH I were retained on the column and then separated from each other by appropriately adjusting the elution conditions. The relative affinities of the enzymes, based on their column elution conditions, were strongly dependent on the ligand. The highly purified EG I and CBH I, obtained by affinity chromatography, were further purified by Mono P and DEAE chromatography, respectively. EG I and CBH I cleave only at the phenolic bond in p-nitrophenyl glycosides with lactose and N-acetyllactosamine (LacNAc). By contrast, both scissile bonds in p-nitrophenyl glycosides with cellobiose were subject to hydrolysis although with important differences in their kinetic parameters. Copyright © 2010 Elsevier Ltd. All rights reserved.

Metal-Chelate Affinity Precipitation with Thermo-Responsive Polymer for Purification of ε-Poly-L-Lysine.

PubMed

Li, Sipeng; Ding, Zhaoyang; Liu, Jifu; Cao, Xuejun

2017-12-01

ε-Poly-L-lysine (ε-PL) is a natural preservative for food processing industry. A thermo-responsive polymer, attached with Cu 2+ or Ni 2+ , was prepared for metal-chelate affinity precipitation for purification of ε-PL. The low critical solution temperatures (LCSTs) of these polymers were close to the room temperature (31.0-35.0 °C). The optimal adsorption conditions were as follows: pH 4.0, 0 mol/L NaCl, ligand density 75.00 μmol/g, and 120 min. The ligand Cu 2+ showed a stronger affinity interaction with ε-PL and the highest adsorption amount reached 251.93 mg/g polymer. The elution recovery of ε-PL could be 98.42% with 0.50 mol/L imidazole (pH = 8.0) as the eluent. The method could purify ε-PL from fermentation broth and the final product was proved as electrophoretic pure by SDS-PAGE. Moreover, these affinity polymers could be recycled after the purification of ε-PL and the recoveries were above 95.00%. Graphical Abstract Scheme for affinity precipitation of ε-PL.

An Unusual Ligand Coordination Gives Rise to a New Family of Rhodium Metalloinsertors with Improved Selectivity and Potency

PubMed Central

2015-01-01

Rhodium metalloinsertors are octahedral complexes that bind DNA mismatches with high affinity and specificity and exhibit unique cell-selective cytotoxicity, targeting mismatch repair (MMR)-deficient cells over MMR-proficient cells. Here we describe a new generation of metalloinsertors with enhanced biological potency and selectivity, in which the complexes show Rh–O coordination. In particular, it has been found that both Δ- and Λ-[Rh(chrysi)(phen)(DPE)]2+ (where chrysi =5,6 chrysenequinone diimmine, phen =1,10-phenanthroline, and DPE = 1,1-di(pyridine-2-yl)ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the mismatch site. We ascribe this difference to the higher pKa of the coordinated immine of the chrysi ligand in these complexes, so that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than cisplatin and N-methyl-N′-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective cytotoxicity for MMR-deficient versus proficient colorectal cancer cells. PMID:25254630

Structural Factors and Mechanisms Underlying the Improved Photodynamic Cell Killing with Silicon Phthalocyanine Photosensitizers Directed to Lysosomes Versus Mitochondria

PubMed Central

Rodriguez, Myriam E.; Zhang, Ping; Azizuddin, Kashif; Delos Santos, Grace B.; Chiu, Song-mao; Xue, Liang-yan; Berlin, Jeffery C.; Peng, Xinzhan; Wu, Hongqiao; Lam, Minh; Nieminen, Anna-Liisa; Kenney, Malcolm E.; Oleinick, Nancy L.

2012-01-01

The phthalocyanine photosensitizer Pc 4 has been shown to bind preferentially to mitochondrial and endoplasmic reticulum membranes. Upon photoirradiation of Pc 4-loaded cells, membrane components, especially Bcl-2, are photodamaged and apoptosis, as indicated by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase, is triggered. A series of analogs of Pc 4 were synthesized, and the results demonstrate that Pcs with the aminopropylsiloxy ligand of Pc 4 or a similar one on one side of the Pc ring and a second large axial ligand on the other side of the ring have unexpected properties, including enhanced cell uptake, greater monomerization resulting in greater intracellular fluorescence and three-fold higher affinity constants for liposomes. The hydroxyl-bearing axial ligands tend to reduce aggregation of the Pc and direct it to lysosomes, resulting in four to six times more killing of cells, as defined by loss of clonogenicity, than with Pc 4. Whereas Pc 4-PDT photodamages Bcl-2 and Bcl-xL, Pc 181-PDT causes much less photodamage to Bcl-2 over the same dose–response range relative to cell killing, with earlier cleavage of Bid and slower caspase-3-dependent apoptosis. Therefore, within this series of photosensitizers, these hydroxyl-bearing axial ligands are less aggregated than is Pc 4, tend to localize to lysosomes and are more effective in overall cell killing than is Pc 4, but induce apoptosis more slowly and by a modified pathway. PMID:19508642

Fluorescent and Lanthanide Labeling for Ligand Screens, Assays, and Imaging

PubMed Central

Josan, Jatinder S.; De Silva, Channa R.; Yoo, Byunghee; Lynch, Ronald M.; Pagel, Mark D.; Vagner, Josef; Hruby, Victor J.

2012-01-01

The use of fluorescent (or luminescent) and metal contrast agents in high-throughput screens, in vitro assays, and molecular imaging procedures has rapidly expanded in recent years. Here we describe the development and utility of high-affinity ligands for cancer theranostics and other in vitro screening studies. In this context, we also illustrate the syntheses and use of heteromultivalent ligands as targeted imaging agents. PMID:21318902

Application of NMR Methods to Identify Detection Reagents for Use in the Development of Robust Nanosensors

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

Cosman, M; Krishnan, V V; Balhorn, R

2004-04-29

Nuclear Magnetic Resonance (NMR) spectroscopy is a powerful technique for studying bi-molecular interactions at the atomic scale. Our NMR lab is involved in the identification of small molecules, or ligands that bind to target protein receptors, such as tetanus (TeNT) and botulinum (BoNT) neurotoxins, anthrax proteins and HLA-DR10 receptors on non-Hodgkin's lymphoma cancer cells. Once low affinity binders are identified, they can be linked together to produce multidentate synthetic high affinity ligands (SHALs) that have very high specificity for their target protein receptors. An important nanotechnology application for SHALs is their use in the development of robust chemical sensors ormore » biochips for the detection of pathogen proteins in environmental samples or body fluids. Here, we describe a recently developed NMR competition assay based on transferred nuclear Overhauser effect spectroscopy (trNOESY) that enables the identification of sets of ligands that bind to the same site, or a different site, on the surface of TeNT fragment C (TetC) than a known ''marker'' ligand, doxorubicin. Using this assay, we can identify the optimal pairs of ligands to be linked together for creating detection reagents, as well as estimate the relative binding constants for ligands competing for the same site.« less

Novel Chemical Ligands to Ebola Virus and Marburg Virus Nucleoproteins Identified by Combining Affinity Mass Spectrometry and Metabolomics Approaches

PubMed Central

Fu, Xu; Wang, Zhihua; Li, Lixin; Dong, Shishang; Li, Zhucui; Jiang, Zhenzuo; Wang, Yuefei; Shui, Wenqing

2016-01-01

The nucleoprotein (NP) of Ebola virus (EBOV) and Marburg virus (MARV) is an essential component of the viral ribonucleoprotein complex and significantly impacts replication and transcription of the viral RNA genome. Although NP is regarded as a promising antiviral druggable target, no chemical ligands have been reported to interact with EBOV NP or MARV NP. We identified two compounds from a traditional Chinese medicine Gancao (licorice root) that can bind both NPs by combining affinity mass spectrometry and metabolomics approaches. These two ligands, 18β-glycyrrhetinic acid and licochalcone A, were verified by defined compound mixture screens and further characterized with individual ligand binding assays. Accompanying biophysical analyses demonstrate that binding of 18β-glycyrrhetinic acid to EBOV NP significantly reduces protein thermal stability, induces formation of large NP oligomers, and disrupts the critical association of viral ssRNA with NP complexes whereas the compound showed no such activity on MARV NP. Our study has revealed the substantial potential of new analytical techniques in ligand discovery from natural herb resources. In addition, identification of a chemical ligand that influences the oligomeric state and RNA-binding function of EBOV NP sheds new light on antiviral drug development. PMID:27403722

Automated docking of ligands to an artificial active site: augmenting crystallographic analysis with computer modeling

NASA Astrophysics Data System (ADS)

Rosenfeld, Robin J.; Goodsell, David S.; Musah, Rabi A.; Morris, Garrett M.; Goodin, David B.; Olson, Arthur J.

2003-08-01

The W191G cavity of cytochrome c peroxidase is useful as a model system for introducing small molecule oxidation in an artificially created cavity. A set of small, cyclic, organic cations was previously shown to bind in the buried, solvent-filled pocket created by the W191G mutation. We docked these ligands and a set of non-binders in the W191G cavity using AutoDock 3.0. For the ligands, we compared docking predictions with experimentally determined binding energies and X-ray crystal structure complexes. For the ligands, predicted binding energies differed from measured values by ± 0.8 kcal/mol. For most ligands, the docking simulation clearly predicted a single binding mode that matched the crystallographic binding mode within 1.0 Å RMSD. For 2 ligands, where the docking procedure yielded an ambiguous result, solutions matching the crystallographic result could be obtained by including an additional crystallographically observed water molecule in the protein model. For the remaining 2 ligands, docking indicated multiple binding modes, consistent with the original electron density, suggesting disordered binding of these ligands. Visual inspection of the atomic affinity grid maps used in docking calculations revealed two patches of high affinity for hydrogen bond donating groups. Multiple solutions are predicted as these two sites compete for polar hydrogens in the ligand during the docking simulation. Ligands could be distinguished, to some extent, from non-binders using a combination of two trends: predicted binding energy and level of clustering. In summary, AutoDock 3.0 appears to be useful in predicting key structural and energetic features of ligand binding in the W191G cavity.

Point mutation increases a form of the NK1 receptor with high affinity for neurokinin A and B and septide

PubMed Central

Ciucci, Alessandra; Palma, Carla; Manzini, Stefano; Werge, Thomas M

1998-01-01

The binding modalities of substance P and neurokinin A on the wild type and Gly166 to-Cys mutant NK1 receptors expressed on CHO cells were investigated in homologous and heterologous binding experiments using both radiolabelled substance P and neurokinin A.On the wild type NK1 receptor NKA displaces radiolabelled substance P with very low apparent affinity, despite its high-affinity binding constant (determined in homologous binding experiments). The Gly166 to-Cys substitution in the NK1 tachykinin receptor greatly enhances the apparent affinity of neurokinin A in competition for radiolabelled substance P, but it does not change the binding constant of neurokinin A. The mutation, thereby, eliminates the discrepancy between the low apparent affinity and the high binding constant of neurokinin A.On the wild type receptor the binding capacity of neurokinin A is significantly smaller than that of substance P. In contrast, the two tachykinins bind to approximately the same number of sites on the mutant receptor.Simultaneous mass action law analysis of binding data in which multiple radioligands were employed in parallel demonstrated that a one-site model was unable to accommodate all the experimental data, whereas a two-site model provided a dramatically better description.These two receptor-sites display equally high affinity for substance P, while neurokinin A strongly discriminates between a high and a low affinity component. The binding affinities of neurokinin A are not affected by the mutation, which instead specifically alters the distribution between receptor sites in favour of a high affinity neurokinin A binding form.The low apparent affinity and binding capacity of neurokinin A on the wild type receptor results from neurokinin A binding with high affinity only to a fraction of the sites labelled by substance P. The mutation increases the proportion of this site, and consequently enhances the apparent affinity and binding capacity of neurokinin A.The binding modalities of septide-like ligands (i.e. neurokinin B, SP(6-11), SP-methyl ester) are affected similarly to neurokinin A and are better resolved into two sites. The mutation leaves the affinity of these ligands for the two receptor forms unchanged, but increases the fraction of high-affinity sites. On the other hand, the binding of non-peptide and peptide antagonists (SR140.333 and FK888) behaved similarly to substance P with a single high affinity site that is unaffected by the mutation.These findings may suggest that the NK1 receptor exists in two different forms with similar affinity for substance P and NK1 antagonists, but with a high and a low affinity for neurokinin A and septide-like ligands. Hence, the Gly166 in the NK1 receptor would seem to control the distribution between a pan-reactive form and a substance P-selective form of the receptor. PMID:9786514

The Multileveled Regulation of the Human Cholinesterase Genes and Their Protein Products

DTIC Science & Technology

1993-09-30

s that are involved in binding and penetration of ligands. In essence , binding affinity consists of ligand penetration in addition to its binding to...J. Cell Biol. 110, 715-719. Rotundo RL, Jasmin BJ, Lee RK, Rossi SG (1992) Compartmentalization of acetylcholinesterase mENA and protein expression

Fluorescence Analysis of Sulfonamide Binding to Carbonic Anhydrase

ERIC Educational Resources Information Center

Wang, Sheila C.; Zamble, Deborah B.

2006-01-01

A practical laboratory experiment is described that illustrates the application of fluorescence resonance energy transfer to the study of protein-ligand binding. The affinities of wild-type and mutant human carbonic anhydrase II for dansylamide were determined by monitoring the increase in ligand fluorescence that occurs due to energy transfer…

Analyzing Ligand Depletion in a Saturation Equilibrium Binding Experiment

ERIC Educational Resources Information Center

Claro, Enrique

2006-01-01

I present a proposal for a laboratory practice to generate and analyze data from a saturation equilibrium binding experiment addressed to advanced undergraduate students. [[superscript 3]H]Quinuclidinyl benzilate is a nonselective muscarinic ligand with very high affinity and very low nonspecific binding to brain membranes, which contain a high…

Exploration of gated ligand binding recognizes an allosteric site for blocking FABP4-protein interaction.

PubMed

Li, Yan; Li, Xiang; Dong, Zigang

2015-12-28

Fatty acid binding protein 4 (FABP4), reversibly binding to fatty acids and other lipids with high affinities, is a potential target for treatment of cancers. The binding site of FABP4 is buried in an interior cavity and thereby ligand binding/unbinding is coupled with opening/closing of FABP4. It is a difficult task both experimentally and computationally to illuminate the entry or exit pathway, especially with the conformational gating. In this report we combine extensive computer simulations, clustering analysis, and the Markov state model to investigate the binding mechanism of FABP4 and troglitazone. Our simulations capture spontaneous binding and unbinding events as well as the conformational transition of FABP4 between the open and closed states. An allosteric binding site on the protein surface is recognized for the development of novel FABP4 inhibitors. The binding affinity is calculated and compared with the experimental value. The kinetic analysis suggests that ligand residence on the protein surface may delay the binding process. Overall, our results provide a comprehensive picture of ligand diffusion on the protein surface, ligand migration into the buried cavity, and the conformational change of FABP4 at an atomic level.

01-ERD-111 - The Development of Synthetic High Affinity Ligands

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

Perkins, J; Balhorn, R; Cosman, M

2004-02-05

The aim of this project was to develop Synthetic High-Affinity Ligands (SHALs), which bind with high affinity and specificity to proteins of interest for national security and cancer therapy applications. The aim of producing synthetic ligands for sensory devices as an alternative to antibody-based detection assays and therapeutic agents is to overcome the drawbacks associated with antibody-based in next-generation sensors and systems. The focus area of the project was the chemical synthesis of the SHALs. The project concentrated on two different protein targets. (a) The C fragment of tetanus and botulinum toxin, potential biowarfare agents. A SHAL for tetanus ormore » botulinum toxin would be incorporated into a sensory device for the toxins. (b) HLA-DR10, a protein found in high abundance on the surface of Non-Hodgkins Lymphoma. A SHAL specific to a tumor marker, labeled with a radionuclide, would enable the targeted delivery of radiation therapy to metastatic disease. The technical approach used to develop a SHAL for each protein target will be described in more detail below. However, in general, the development of a SHAL requires a combination of computational modeling techniques, modern nuclear magnetic resonance spectroscopy (NMR) and synthetic chemistry.« less

Insights from the predicted structural analysis of carborane substituted withaferin A with Indoleamine - 2,3-dioxygenase as a potent inhibitor.

PubMed

Basha, Syed Hussain; Thakur, Abhishek; Samad, Firoz A

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) an immunoregulatory enzyme and emerging as a new therapeutic drug target for the treatment of cancer. Carboranes, an icosahedral arrangement of eleven boron atoms plus one carbon atom with unique pharmacological properties such low toxicity, isosterism with phenyl ring and stability to hydrolysis. On the other hand, carboranes are known to increase the interaction of ligand with non-polar region of the protein provides an excellent platform to explore these carboranes towards designing and development of novel, potent and target specific drug candidates with further enhanced binding affinities. Despite of their many potential applications, molecular modeling studies of carborane-substituted ligands with macromolecules have been rarely reported. Previously, we have demonstrated the promising high binding affinity of Withaferin-A (WA) for IDO. In this present study, we investigated the effect of carborane substitutions on WA compound towards developing novel analogs for target specific IDO inhibition with better potency. Interesting docked poses and molecular interactions for the carborane substituted WA ligands were elucidated. Based on our In-silico studies, carborane substituted at various position of WA has shown enhanced binding affinity towards IDO, worth of considering for further studies.

Insights from the predicted structural analysis of carborane substituted withaferin A with Indoleamine - 2,3-dioxygenase as a potent inhibitor

PubMed Central

Samad, Firoz A

2016-01-01

Indoleamine-2,3-dioxygenase (IDO) an immunoregulatory enzyme and emerging as a new therapeutic drug target for the treatment of cancer. Carboranes, an icosahedral arrangement of eleven boron atoms plus one carbon atom with unique pharmacological properties such low toxicity, isosterism with phenyl ring and stability to hydrolysis. On the other hand, carboranes are known to increase the interaction of ligand with non-polar region of the protein provides an excellent platform to explore these carboranes towards designing and development of novel, potent and target specific drug candidates with further enhanced binding affinities. Despite of their many potential applications, molecular modeling studies of carborane-substituted ligands with macromolecules have been rarely reported. Previously, we have demonstrated the promising high binding affinity of Withaferin-A (WA) for IDO. In this present study, we investigated the effect of carborane substitutions on WA compound towards developing novel analogs for target specific IDO inhibition with better potency. Interesting docked poses and molecular interactions for the carborane substituted WA ligands were elucidated. Based on our In-silico studies, carborane substituted at various position of WA has shown enhanced binding affinity towards IDO, worth of considering for further studies. PMID:28250615

NetMHCpan-4.0: Improved Peptide-MHC Class I Interaction Predictions Integrating Eluted Ligand and Peptide Binding Affinity Data.

PubMed

Jurtz, Vanessa; Paul, Sinu; Andreatta, Massimo; Marcatili, Paolo; Peters, Bjoern; Nielsen, Morten

2017-11-01

Cytotoxic T cells are of central importance in the immune system's response to disease. They recognize defective cells by binding to peptides presented on the cell surface by MHC class I molecules. Peptide binding to MHC molecules is the single most selective step in the Ag-presentation pathway. Therefore, in the quest for T cell epitopes, the prediction of peptide binding to MHC molecules has attracted widespread attention. In the past, predictors of peptide-MHC interactions have primarily been trained on binding affinity data. Recently, an increasing number of MHC-presented peptides identified by mass spectrometry have been reported containing information about peptide-processing steps in the presentation pathway and the length distribution of naturally presented peptides. In this article, we present NetMHCpan-4.0, a method trained on binding affinity and eluted ligand data leveraging the information from both data types. Large-scale benchmarking of the method demonstrates an increase in predictive performance compared with state-of-the-art methods when it comes to identification of naturally processed ligands, cancer neoantigens, and T cell epitopes. Copyright © 2017 by The American Association of Immunologists, Inc.

Cell-specific targeting by heterobivalent ligands.

PubMed

Josan, Jatinder S; Handl, Heather L; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M; Vagner, Josef; Mash, Eugene A; Hruby, Victor J; Gillies, Robert J

2011-07-20

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach--to specifically target combinations of cell-surface receptors using heteromultivalent ligands ("receptor combination approach"). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle(4), dPhe(7)]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20-50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH(2). Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging.

Cell-Specific Targeting by Heterobivalent Ligands

PubMed Central

Josan, Jatinder S.; Handl, Heather L.; Sankaranarayanan, Rajesh; Xu, Liping; Lynch, Ronald M.; Vagner, Josef; Mash, Eugene A.; Hruby, Victor J.; Gillies, Robert J.

2012-01-01

Current cancer therapies exploit either differential metabolism or targeting to specific individual gene products that are overexpressed in aberrant cells. The work described herein proposes an alternative approach—to specifically target combinations of cell-surface receptors using heteromultivalent ligands (“receptor combination approach”). As a proof-of-concept that functionally unrelated receptors can be noncovalently cross-linked with high avidity and specificity, a series of heterobivalent ligands (htBVLs) were constructed from analogues of the melanocortin peptide ligand ([Nle4, DPhe7]-α-MSH) and the cholecystokinin peptide ligand (CCK-8). Binding of these ligands to cells expressing the human Melanocortin-4 receptor and the Cholecystokinin-2 receptor was analyzed. The MSH(7) and CCK(6) were tethered with linkers of varying rigidity and length, constructed from natural and/or synthetic building blocks. Modeling data suggest that a linker length of 20–50 Å is needed to simultaneously bind these two different G-protein coupled receptors (GPCRs). These ligands exhibited up to 24-fold enhancement in binding affinity to cells that expressed both (bivalent binding), compared to cells with only one (monovalent binding) of the cognate receptors. The htBVLs had up to 50-fold higher affinity than that of a monomeric CCK ligand, i.e., Ac-CCK(6)-NH2. Cell-surface targeting of these two cell types with labeled heteromultivalent ligand demonstrated high avidity and specificity, thereby validating the receptor combination approach. This ability to noncovalently cross-link heterologous receptors and target individual cells using a receptor combination approach opens up new possibilities for specific cell targeting in vivo for therapy or imaging. PMID:21639139

Phage display—A powerful technique for immunotherapy

PubMed Central

Bazan, Justyna; Całkosiński, Ireneusz; Gamian, Andrzej

2012-01-01

One of the most effective molecular diversity techniques is phage display. This technology is based on a direct linkage between phage phenotype and its encapsulated genotype, which leads to presentation of molecule libraries on the phage surface. Phage display is utilized in studying protein-ligand interactions, receptor binding sites and in improving or modifying the affinity of proteins for their binding partners. Generating monoclonal antibodies and improving their affinity, cloning antibodies from unstable hybridoma cells and identifying epitopes, mimotopes and functional or accessible sites from antigens are also important advantages of this technology. Techniques originating from phage display have been applied to transfusion medicine, neurological disorders, mapping vascular addresses and tissue homing of peptides. Phages have been applicable to immunization therapies, which may lead to development of new tools used for treating autoimmune and cancer diseases. This review describes the phage display technology and presents the recent advancements in therapeutic applications of phage display. PMID:22906939

Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

PubMed

Sultana, Azmiri; Lee, Jeffrey E

2015-02-02

Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample. Copyright © 2015 John Wiley & Sons, Inc.

Isothermal titration calorimetry for measuring macromolecule-ligand affinity.

PubMed

Duff, Michael R; Grubbs, Jordan; Howell, Elizabeth E

2011-09-07

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given.

Isothermal Titration Calorimetry for Measuring Macromolecule-Ligand Affinity

PubMed Central

Duff,, Michael R.; Grubbs, Jordan; Howell, Elizabeth E.

2011-01-01

Isothermal titration calorimetry (ITC) is a useful tool for understanding the complete thermodynamic picture of a binding reaction. In biological sciences, macromolecular interactions are essential in understanding the machinery of the cell. Experimental conditions, such as buffer and temperature, can be tailored to the particular binding system being studied. However, careful planning is needed since certain ligand and macromolecule concentration ranges are necessary to obtain useful data. Concentrations of the macromolecule and ligand need to be accurately determined for reliable results. Care also needs to be taken when preparing the samples as impurities can significantly affect the experiment. When ITC experiments, along with controls, are performed properly, useful binding information, such as the stoichiometry, affinity and enthalpy, are obtained. By running additional experiments under different buffer or temperature conditions, more detailed information can be obtained about the system. A protocol for the basic setup of an ITC experiment is given. PMID:21931288

Quantifying Intrinsic Specificity: A Potential Complement to Affinity in Drug Screening

NASA Astrophysics Data System (ADS)

Wang, Jin; Zheng, Xiliang; Yang, Yongliang; Drueckhammer, Dale; Yang, Wei; Verkhivker, Gennardy; Wang, Erkang

2007-11-01

We report here the investigation of a novel description of specificity in protein-ligand binding based on energy landscape theory. We define a new term, intrinsic specificity ratio (ISR), which describes the level of discrimination in binding free energies of the native basin for a protein-ligand complex from the weaker binding states of the same ligand. We discuss the relationship between the intrinsic specificity we defined here and the conventional definition of specificity. In a docking study of molecules with the enzyme COX-2, we demonstrate a statistical correspondence between ISR value and geometrical shapes of the small molecules binding to COX-2. We further observe that the known selective (nonselective) inhibitors of COX-2 have higher (lower) ISR values. We suggest that intrinsic specificity ratio may be a useful new criterion and a complement to affinity in drug screening and in searching for potential drug lead compounds.

X-ray structural studies of quinone reductase 2 nanomolar range inhibitors

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

Pegan, Scott D.; Sturdy, Megan; Ferry, Gilles

Quinone reductase 2 (QR2) is one of two members comprising the mammalian quinone reductase family of enzymes responsible for performing FAD mediated reductions of quinone substrates. In contrast to quinone reductase 1 (QR1) which uses NAD(P)H as its co-substrate, QR2 utilizes a rare group of hydride donors, N-methyl or N-ribosyl nicotinamide. Several studies have linked QR2 to the generation of quinone free radicals, several neuronal degenerative diseases, and cancer. QR2 has been also identified as the third melatonin receptor (MT3) through in cellulo and in vitro inhibition of QR2 by traditional MT3 ligands, and through recent X-ray structures of humanmore » QR2 (hQR2) in complex with melatonin and 2-iodomelatonin. Several MT3 specific ligands have been developed that exhibit both potent in cellulo inhibition of hQR2 nanomolar, affinity for MT3. The potency of these ligands suggest their use as molecular probes for hQR2. However, no definitive correlation between traditionally obtained MT3 ligand affinity and hQR2 inhibition exists limiting our understanding of how these ligands are accommodated in the hQR2 active site. To obtain a clearer relationship between the structures of developed MT3 ligands and their inhibitory properties, in cellulo and in vitro IC{sub 50} values were determined for a representative set of MT3 ligands (MCA-NAT, 2-I-MCANAT, prazosin, S26695, S32797, and S29434). Furthermore, X-ray structures for each of these ligands in complex with hQR2 were determined allowing for a structural evaluation of the binding modes of these ligands in relation to the potency of MT3 ligands.« less

Modulation of PPAR activity via phosphorylation

PubMed Central

Burns, Katherine A.; Vanden Heuvel, John P.

2009-01-01

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear receptor superfamily of transcription factors that respond to specific ligands by altering gene expression in a cell-, developmental- and sex-specific manner. Three subtypes of this receptor have been discovered (PPARα, β and γ), each apparently evolving to fulfill different biological niches. PPARs control a variety of target genes involved in lipid homeostasis, diabetes and cancer. Similar to other nuclear receptors, the PPARs are phosphoproteins and their transcriptional activity is affected by cross-talk with kinases and phosphatases. Phosphorylation by the mitogen-activated protein kinases (ERK- and p38-MAPK), Protein Kinase A and C (PKA, PKC), AMP Kinase (AMPK) and glycogen synthase kinase-3 (GSK3) affect their activity in a ligand-dependent or -independent manner. The effects of phosphorylation depend on the cellular context, receptor subtype and residue metabolized which can be manifested at several steps in the PPAR activation sequence including ligand affinity, DNA binding, coactivator recruitment and proteasomal degradation. The review will summarize the known PPAR kinases that directly act on these receptors, the sites affected and the result of this modification on receptor activity. PMID:17560826

Identification of new ligands for the methionine biosynthesis transcriptional regulator (MetJ) by FAC-MS.

PubMed

Martí-Arbona, Ricardo; Teshima, Munehiro; Anderson, Penelope S; Nowak-Lovato, Kristy L; Hong-Geller, Elizabeth; Unkefer, Clifford J; Unkefer, Pat J

2012-01-01

We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding. Copyright © 2012 S. Karger AG, Basel.

Magneto-nanosensor platform for probing low-affinity protein–protein interactions and identification of a low-affinity PD-L1/PD-L2 interaction

PubMed Central

Lee, Jung-Rok; Bechstein, Daniel J. B.; Ooi, Chin Chun; Patel, Ashka; Gaster, Richard S.; Ng, Elaine; Gonzalez, Lino C.; Wang, Shan X.

2016-01-01

Substantial efforts have been made to understand the interactions between immune checkpoint receptors and their ligands targeted in immunotherapies against cancer. To carefully characterize the complete network of interactions involved and the binding affinities between their extracellular domains, an improved kinetic assay is needed to overcome limitations with surface plasmon resonance (SPR). Here, we present a magneto-nanosensor platform integrated with a microfluidic chip that allows measurement of dissociation constants in the micromolar-range. High-density conjugation of magnetic nanoparticles with prey proteins allows multivalent receptor interactions with sensor-immobilized bait proteins, more closely mimicking natural-receptor clustering on cells. The platform has advantages over traditional SPR in terms of insensitivity of signal responses to pH and salinity, less consumption of proteins and better sensitivities. Using this platform, we characterized the binding affinities of the PD-1—PD-L1/PD-L2 co-inhibitory receptor system, and discovered an unexpected interaction between the two known PD-1 ligands, PD-L1 and PD-L2. PMID:27447090

Synthesis of branched metal nanostructures with controlled architecture and composition

NASA Astrophysics Data System (ADS)

Ortiz, Nancy

On account of their small size, metal nanoparticles are proven to be outstanding catalysts for numerous chemical transformations and represent promising platforms for applications in the fields of electronics, chemical sensing, medicine, and beyond. Many properties of metal nanoparticles are size-dependent and can be further manipulated through their shape and architecture (e.g., spherical vs. branched). Achieving morphology control of nanoparticles through solution-based techniques has proven challenging due to limited knowledge of morphology development in nanosyntheses. To overcome these complications, a systematic examination of the local ligand environment of metal precursors on nanostructure formation was undertaken to evaluate its contribution to nanoparticle nucleation rate and subsequent growth processes. Specifically, this thesis will provide evidence from ex situ studies---Transmission Electron Microscopy (TEM) and UV-visible spectroscopy (UV-Vis)---that support the hypothesis that strongly coordinated ligands delay burst-like nucleation to generate spherical metal nanoparticles and ligands with intermediate binding affinity regulate the gradual reduction of metal precursors to promote aggregated assembly of nanodendrites. These ex situ studies were coupled with a new in situ perspective, providing detailed understanding of metal precursor transformation, its direct relation to nanoparticle morphology development, and the ligand influence towards the formation of structurally complex metal nanostructures, using in situ synchrotron X-ray Diffraction (XRD) and Ultra Small-Angle X-ray Scattering (USAXS). The principles extracted from the study of monometallic nanostructure formation were also found to be generally applicable to the synthesis of bimetallic nanostructures, e.g., Pd-Pt architectures, with either core-shell or alloyed structures that were readily achieved by ligand selection. These outcomes provide a direct connection between fundamental principles of coordination chemistry and nanoparticle formation, with a stronger foundation for the predictive synthesis of future nanomaterials with controllable structural features.

The protein-protein interface evolution acts in a similar way to antibody affinity maturation.

PubMed

Li, Bohua; Zhao, Lei; Wang, Chong; Guo, Huaizu; Wu, Lan; Zhang, Xunming; Qian, Weizhu; Wang, Hao; Guo, Yajun

2010-02-05

Understanding the evolutionary mechanism that acts at the interfaces of protein-protein complexes is a fundamental issue with high interest for delineating the macromolecular complexes and networks responsible for regulation and complexity in biological systems. To investigate whether the evolution of protein-protein interface acts in a similar way as antibody affinity maturation, we incorporated evolutionary information derived from antibody affinity maturation with common simulation techniques to evaluate prediction success rates of the computational method in affinity improvement in four different systems: antibody-receptor, antibody-peptide, receptor-membrane ligand, and receptor-soluble ligand. It was interesting to find that the same evolutionary information could improve the prediction success rates in all the four protein-protein complexes with an exceptional high accuracy (>57%). One of the most striking findings in our present study is that not only in the antibody-combining site but in other protein-protein interfaces almost all of the affinity-enhancing mutations are located at the germline hotspot sequences (RGYW or WA), indicating that DNA hot spot mechanisms may be widely used in the evolution of protein-protein interfaces. Our data suggest that the evolution of distinct protein-protein interfaces may use the same basic strategy under selection pressure to maintain interactions. Additionally, our data indicate that classical simulation techniques incorporating the evolutionary information derived from in vivo antibody affinity maturation can be utilized as a powerful tool to improve the binding affinity of protein-protein complex with a high accuracy.

Docking model of the nicotinic acetylcholine receptor and nitromethylene neonicotinoid derivatives with a longer chiral substituent and their biological activities.

PubMed

Nagaoka, Hikaru; Nishiwaki, Hisashi; Kubo, Takuya; Akamatsu, Miki; Yamauchi, Satoshi; Shuto, Yoshihiro

2015-02-15

In the present study, nitromethylene neonicotinoid derivatives possessing substituents that contain a sulfur atom, oxygen atom or aromatic ring at position 5 on the imidazolidine ring were synthesized to evaluate their affinity for the nicotinic acetylcholine receptor (nAChR) and their insecticidal activity against adult female houseflies. Comparing the receptor affinity of the alkylated derivative with the receptor affinity of compounds possessing either ether or thioether groups revealed that conversion of the carbon atom to a sulfur atom did not influence the receptor affinity, whereas conversion to an oxygen atom was disadvantageous for the receptor affinity. The receptor affinity of compounds possessing a benzyl or phenyl group was lower than that of the unsubstituted compound. Analysis of the three-dimensional quantitative structure-activity relationship using comparative molecular field analysis demonstrated that steric hindrance of the receptor should exist around the C3 of an n-butyl group attached at position 5 on the imidazolidine ring. A docking study of the nAChR-ligand model suggested that the ligand-binding region expands as the length of the substituent increases by brushing against the amino acids that form the binding region. The insecticidal activity of the compounds was positively correlated with the receptor affinity by considering logP and the number of heteroatoms, including sulfur and oxygen atoms, in the substituents, suggesting that the insecticidal activity is influenced by the receptor affinity, hydrophobicity, and metabolic stability of the compounds. Copyright © 2015 Elsevier Ltd. All rights reserved.

Sulfated Metabolites of Polychlorinated Biphenyls Are High-Affinity Ligands for the Thyroid Hormone Transport Protein Transthyretin

PubMed Central

Grimm, Fabian A.; Lehmler, Hans-Joachim; He, Xianran; Robertson, Larry W.

2013-01-01

Background: The displacement of l-thyroxine (T4) from binding sites on transthyretin (TTR) is considered a significant contributing mechanism in polychlorinated biphenyl (PCB)-induced thyroid disruption. Previous research has discovered hydroxylated PCB metabolites (OH-PCBs) as high-affinity ligands for TTR, but the binding potential of conjugated PCB metabolites such as PCB sulfates has not been explored. Objectives: We evaluated the binding of five lower-chlorinated PCB sulfates to human TTR and compared their binding characteristics to those determined for their OH-PCB precursors and for T4. Methods: We used fluorescence probe displacement studies and molecular docking simulations to characterize the binding of PCB sulfates to TTR. The stability of PCB sulfates and the reversibility of these interactions were characterized by HPLC analysis of PCB sulfates after their binding to TTR. The ability of OH-PCBs to serve as substrates for human cytosolic sulfotransferase 1A1 (hSULT1A1) was assessed by OH-PCB–dependent formation of adenosine-3´,5´-diphosphate, an end product of the sulfation reaction. Results: All five PCB sulfates were able to bind to the high-affinity binding site of TTR with equilibrium dissociation constants (Kd values) in the low nanomolar range (4.8–16.8 nM), similar to that observed for T4 (4.7 nM). Docking simulations provided corroborating evidence for these binding interactions and indicated multiple high-affinity modes of binding. All OH-PCB precursors for these sulfates were found to be substrates for hSULT1A1. Conclusions: Our findings show that PCB sulfates are high-affinity ligands for human TTR and therefore indicate, for the first time, a potential relevance for these metabolites in PCB-induced thyroid disruption. PMID:23584369

3D QSAR studies on binding affinities of coumarin natural products for glycosomal GAPDH of Trypanosoma cruzi

NASA Astrophysics Data System (ADS)

Menezes, Irwin R. A.; Lopes, Julio C. D.; Montanari, Carlos A.; Oliva, Glaucius; Pavão, Fernando; Castilho, Marcelo S.; Vieira, Paulo C.; Pupo, M.^onica T.

2003-05-01

Drug design strategies based on Comparative Molecular Field Analysis (CoMFA) have been used to predict the activity of new compounds. The major advantage of this approach is that it permits the analysis of a large number of quantitative descriptors and uses chemometric methods such as partial least squares (PLS) to correlate changes in bioactivity with changes in chemical structure. Because it is often difficult to rationalize all variables affecting the binding affinity of compounds using CoMFA solely, the program GRID was used to describe ligands in terms of their molecular interaction fields, MIFs. The program VolSurf that is able to compress the relevant information present in 3D maps into a few descriptors can treat these GRID fields. The binding affinities of a new set of compounds consisting of 13 coumarins, for one of which the three-dimensional ligand-enzyme bound structure is known, were studied. A final model based on the mentioned programs was independently validated by synthesizing and testing new coumarin derivatives. By relying on our knowledge of the real physical data (i.e., combining crystallographic and binding affinity results), it is also shown that ligand-based design agrees with structure-based design. The compound with the highest binding affinity was the coumarin chalepin, isolated from Rutaceae species, with an IC50 value of 55.5 μM towards the enzyme glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) from glycosomes of the parasite Trypanosoma cruzi, the causative agent of Chagas' disease. The proposed models from GRID MIFs have revealed the importance of lipophilic interactions in modulating the inhibition, but without excluding the dependence on stereo-electronic properties as found from CoMFA fields.

Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations

PubMed Central

Jones, RL; Woodward, DF; Wang, JW; Clark, RL

2011-01-01

BACKGROUND AND PURPOSE The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed. PMID:20973775

Ligand Selectivity Mechanism and Conformational Changes in Guanine Riboswitch by Molecular Dynamics Simulations and Free Energy Calculations.

PubMed

Hu, Guodong; Ma, Aijing; Wang, Jihua

2017-04-24

Riboswitches regulate gene expression through direct and specific interactions with small metabolite molecules. Binding of a ligand to its RNA target is high selectivity and affinity and induces conformational changes of the RNA's secondary and tertiary structure. The structural difference of two purine riboswitches aptamers is caused by only one single mutation, where cytosine 74 in the guanine riboswitch is corresponding to a uracil 74 in adenine riboswitch. Here we employed molecular dynamics (MD) simulation, molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) and thermodynamic integration computational methodologies to evaluate the energetic and conformational changes of ligands binding to purine riboswitches. The snapshots used in MM-PBSA calculation were extracted from ten 50 ns MD simulation trajectories for each complex. These free energy results are in consistent with the experimental data and rationalize the selectivity of the riboswitches for different ligands. In particular, it is found that the loss in binding free energy upon mutation is mainly electrostatic in guanine (GUA) and riboswitch complex. Furthermore, new hydrogen bonds are found in mutated complexes. To reveal the conformational properties of guanine riboswitch, we performed a total of 6 μs MD simulations in both the presence and the absence of the ligand GUA. The MD simulations suggest that the conformation of guanine riboswitch depends on the distance of two groups in the binding pocket of ligand. The conformation is in a close conformation when U51-A52 is close to C74-U75.

Selective affinity labeling of a 27-kDa integral membrane protein in rat liver and kidney with N-bromoacetyl derivatives of L-thyroxine and 3,5,3'-triiodo-L-thyronine.

PubMed

Köhrle, J; Rasmussen, U B; Rokos, H; Leonard, J L; Hesch, R D

1990-04-15

125I-Labeled N-bromoacetyl derivatives of L-thyroxine and L-triiodothyronine were used as alkylating affinity labels to identify rat liver and kidney microsomal membrane proteins which specifically bind thyroid hormones. Affinity label incorporation was analyzed by ethanol precipitation and individual affinity labeled proteins were identified by autoradiography after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Six to eight membrane proteins ranging in size from 17 to 84 kDa were affinity labeled by both bromoacetyl-L-thyroxine (BrAcT4) and bromoacetyl-L-triiodothyronine (BrAcT3). Affinity labeling was time- and temperature-dependent, and both reduced dithiols and detergents increased affinity labeling, predominantly in a 27-kDa protein(s). Up to 80% of the affinity label was associated with a 27-kDa protein (p27) under optimal conditions. Affinity labeling of p27 by 0.4 nM BrAc[125I]L-T4 was blocked by 0.1 microM of the alkylating ligands BrAcT4, BrAcT3, or 100 microM iodoacetate, by 10 microM concentrations of the non-alkylating, reversible ligands N-acetyl-L-thyroxine, 3,3',5'-triiodothyronine, 3,5-diiodosalicylate, and EMD 21388, a T4-antagonistic flavonoid. Neither 10 microM L-T4, nor 10 microM N-acetyltriiodothyronine or 10 microM L-triiodothyronine blocked affinity labeling of p27 or other affinity labeled bands. Affinity labeling of a 17-kDa band was partially inhibited by excess of the alkylating ligands BrAcT4, BrAcT3, and iodoacetate, but labeling of other minor bands was not blocked by excess of the competitors. BrAc[125I]T4 yielded higher affinity label incorporation than BrAc[125I]T3, although similar banding patterns were observed, except that BrAcT3 affinity labeled more intensely a 58,000-Da band in liver and a 53,000-55,000-Da band in kidney. The pattern of other affinity labeled proteins with p27 as the predominant band was similar in liver and kidney. Peptide mapping of affinity labeled p27 and p55 bands by chemical cleavage and protease fragmentation revealed no common bands excluding that p27 is a degradation product of p55. These data indicate that N-bromoacetyl derivatives of T4 and T3 affinity label a limited but similar constellation of membrane proteins with BrAcT4 incorporation greater than that of BrAcT3. One membrane protein (p27) of low abundance (2-5 pmol/mg microsomal protein) with a reactive sulfhydryl group is selectively labeled under conditions identical to those used to measure thyroid hormone 5'-deiodination. Only p27 showed differential affinity labeling in the presence of noncovalently bound inhibitors or substrates on 5'-deiodinase suggesting that p27 is likely to be a component of type I 5'-deiodinase in rat liver and kidney.

Urodilatin: binding properties and stimulation of cGMP generation in rat kidney cells.

PubMed

Saxenhofer, H; Fitzgibbon, W R; Paul, R V

1993-02-01

Urodilatin (URO) [ANP-(95-126)] is an analogue of atrial natriuretic peptide (alpha-ANP) [ANP-(99-126)] that was first isolated from human urine. In rat mesangial cells, URO competed with high affinity for non-guanylate cyclase-coupled ANPR-C receptors [concentration at which 50% labeled ligand is displaced (IC50) approximately 70 pM], but with lesser affinity to the guanylate cyclase-linked ANPR-A receptors (IC50 approximately 800 pM). alpha-ANP bound to both receptors with similar affinity [dissociation constant (Kd) approximately 150 pM]. In papillary collecting duct homogenates, which possess only ANPR-A receptors, the apparent Kd value averaged 229 pM for alpha-ANP and 2.7 nM for URO. Intravenous URO was at least as potent and effective as alpha-ANP in inducing diuresis and natriuresis in anesthetized rats, but URO was approximately 10-fold less potent in stimulating guanosine 3',5'-cyclic monophosphate generation in mesangial and inner medullary collecting duct cells. We conclude that URO has a lesser affinity than alpha-ANP for guanylate cyclase-coupled ANP receptors in the kidney and that the relative natriuretic potency of URO in vivo cannot be directly attributed to its binding characteristics with ANPR-A receptors.

Tripartite ATP-independent Periplasmic (TRAP) Transporters Use an Arginine-mediated Selectivity Filter for High Affinity Substrate Binding*

PubMed Central

Fischer, Marcus; Hopkins, Adam P.; Severi, Emmanuele; Hawkhead, Judith; Bawdon, Daniel; Watts, Andrew G.; Hubbard, Roderick E.; Thomas, Gavin H.

2015-01-01

Tripartite ATP-independent periplasmic (TRAP) transporters are secondary transporters that have evolved an obligate dependence on a substrate-binding protein (SBP) to confer unidirectional transport. Different members of the DctP family of TRAP SBPs have binding sites that recognize a diverse range of organic acid ligands but appear to only share a common electrostatic interaction between a conserved arginine and a carboxylate group in the ligand. We investigated the significance of this interaction using the sialic acid-specific SBP, SiaP, from the Haemophilus influenzae virulence-related SiaPQM TRAP transporter. Using in vitro, in vivo, and structural methods applied to SiaP, we demonstrate that the coordination of the acidic ligand moiety of sialic acid by the conserved arginine (Arg-147) is essential for the function of the transporter as a high affinity scavenging system. However, at high substrate concentrations, the transporter can function in the absence of Arg-147 suggesting that this bi-molecular interaction is not involved in further stages of the transport cycle. As well as being required for high affinity binding, we also demonstrate that the Arg-147 is a strong selectivity filter for carboxylate-containing substrates in TRAP transporters by engineering the SBP to recognize a non-carboxylate-containing substrate, sialylamide, through water-mediated interactions. Together, these data provide biochemical and structural support that TRAP transporters function predominantly as high affinity transporters for carboxylate-containing substrates. PMID:26342690

Non-natural amino acid peptide microarrays to discover Ebola virus glycoprotein ligands.

PubMed

Rabinowitz, Joshua A; Lainson, John C; Johnston, Stephen Albert; Diehnelt, Chris W

2018-02-06

We demonstrate a platform to screen a virus pseudotyped with Ebola virus glycoprotein (GP) against a library of peptides that contain non-natural amino acids to develop GP affinity ligands. This system could be used for rapid development of peptide-based antivirals for other emerging or neglected tropical infectious diseases.

Dissecting the Dynamic Pathways of Stereoselective DNA Threading Intercalation

PubMed Central

Almaqwashi, Ali A.; Andersson, Johanna; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

2016-01-01

DNA intercalators that have high affinity and slow kinetics are developed for potential DNA-targeted therapeutics. Although many natural intercalators contain multiple chiral subunits, only intercalators with a single chiral unit have been quantitatively probed. Dumbbell-shaped DNA threading intercalators represent the next order of structural complexity relative to simple intercalators, and can provide significant insights into the stereoselectivity of DNA-ligand intercalation. We investigated DNA threading intercalation by binuclear ruthenium complex [μ-dppzip(phen)4Ru2]4+ (Piz). Four Piz stereoisomers are defined by the chirality of the intercalating subunit (Ru(phen)2dppz) and the distal subunit (Ru(phen)2ip), respectively, each of which can be either right-handed (Δ) or left-handed (Λ). We used optical tweezers to measure single DNA molecule elongation due to threading intercalation, revealing force-dependent DNA intercalation rates and equilibrium dissociation constants. The force spectroscopy analysis provided the zero-force DNA binding affinity, the equilibrium DNA-ligand elongation Δxeq, and the dynamic DNA structural deformations during ligand association xon and dissociation xoff. We found that Piz stereoisomers exhibit over 20-fold differences in DNA binding affinity, from a Kd of 27 ± 3 nM for (Δ,Λ)-Piz to a Kd of 622 ± 55 nM for (Λ,Δ)-Piz. The striking affinity decrease is correlated with increasing Δxeq from 0.30 ± 0.02 to 0.48 ± 0.02 nm and xon from 0.25 ± 0.01 to 0.46 ± 0.02 nm, but limited xoff changes. Notably, the affinity and threading kinetics is 10-fold enhanced for right-handed intercalating subunits, and 2- to 5-fold enhanced for left-handed distal subunits. These findings demonstrate sterically dispersed transition pathways and robust DNA structural recognition of chiral intercalators, which are critical for optimizing DNA binding affinity and kinetics. PMID:27028636

Ecto-Fc MS identifies ligand-receptor interactions through extracellular domain Fc fusion protein baits and shotgun proteomic analysis

PubMed Central

Savas, Jeffrey N.; De Wit, Joris; Comoletti, Davide; Zemla, Roland; Ghosh, Anirvan

2015-01-01

Ligand-receptor interactions represent essential biological triggers which regulate many diverse and important cellular processes. We have developed a discovery-based proteomic biochemical protocol which couples affinity purification with multidimensional liquid chromatographic tandem mass spectrometry (LCLC-MS/MS) and bioinformatic analysis. Compared to previous approaches, our analysis increases sensitivity, shortens analysis duration, and boosts comprehensiveness. In this protocol, receptor extracellular domains are fused with the Fc region of IgG to generate fusion proteins that are purified from transfected HEK293T cells. These “ecto-Fcs” are coupled to protein A beads and serve as baits for binding assays with prey proteins extracted from rodent brain. After capture, the affinity purified proteins are digested into peptides and comprehensively analyzed by LCLC-MS/MS with ion trap mass spectrometers. In four working days, this protocol can generate shortlists of candidate ligand-receptor protein-protein interactions. Our “Ecto-Fc MS” approach outperforms antibody-based approaches and provides a reproducible and robust framework to identify extracellular ligand – receptor interactions. PMID:25101821

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System.

PubMed

Pliotas, Christos; Grayer, Samuel C; Ekkerman, Silvia; Chan, Anthony K N; Healy, Jess; Marius, Phedra; Bartlett, Wendy; Khan, Amjad; Cortopassi, Wilian A; Chandler, Shane A; Rasmussen, Tim; Benesch, Justin L P; Paton, Robert S; Claridge, Timothy D W; Miller, Samantha; Booth, Ian R; Naismith, James H; Conway, Stuart J

2017-08-15

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme-substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding.

Adenosine Monophosphate Binding Stabilizes the KTN Domain of the Shewanella denitrificans Kef Potassium Efflux System

PubMed Central

2017-01-01

Ligand binding is one of the most fundamental properties of proteins. Ligand functions fall into three basic types: substrates, regulatory molecules, and cofactors essential to protein stability, reactivity, or enzyme–substrate complex formation. The regulation of potassium ion movement in bacteria is predominantly under the control of regulatory ligands that gate the relevant channels and transporters, which possess subunits or domains that contain Rossmann folds (RFs). Here we demonstrate that adenosine monophosphate (AMP) is bound to both RFs of the dimeric bacterial Kef potassium efflux system (Kef), where it plays a structural role. We conclude that AMP binds with high affinity, ensuring that the site is fully occupied at all times in the cell. Loss of the ability to bind AMP, we demonstrate, causes protein, and likely dimer, instability and consequent loss of function. Kef system function is regulated via the reversible binding of comparatively low-affinity glutathione-based ligands at the interface between the dimer subunits. We propose this interfacial binding site is itself stabilized, at least in part, by AMP binding. PMID:28656748

Solution NMR Spectroscopy in Target-Based Drug Discovery.

PubMed

Li, Yan; Kang, Congbao

2017-08-23

Solution NMR spectroscopy is a powerful tool to study protein structures and dynamics under physiological conditions. This technique is particularly useful in target-based drug discovery projects as it provides protein-ligand binding information in solution. Accumulated studies have shown that NMR will play more and more important roles in multiple steps of the drug discovery process. In a fragment-based drug discovery process, ligand-observed and protein-observed NMR spectroscopy can be applied to screen fragments with low binding affinities. The screened fragments can be further optimized into drug-like molecules. In combination with other biophysical techniques, NMR will guide structure-based drug discovery. In this review, we describe the possible roles of NMR spectroscopy in drug discovery. We also illustrate the challenges encountered in the drug discovery process. We include several examples demonstrating the roles of NMR in target-based drug discoveries such as hit identification, ranking ligand binding affinities, and mapping the ligand binding site. We also speculate the possible roles of NMR in target engagement based on recent processes in in-cell NMR spectroscopy.

Design, synthesis, and biological characterization of metabolically stable selective androgen receptor modulators.

PubMed

Marhefka, Craig A; Gao, Wenqing; Chung, Kiwon; Kim, Juhyun; He, Yali; Yin, Donghua; Bohl, Casey; Dalton, James T; Miller, Duane D

2004-02-12

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with K(i) values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo.

Design, Synthesis, and Biological Characterization of Metabolically Stable Selective Androgen Receptor Modulators

PubMed Central

Marhefka, Craig A.; Gao, Wenqing; Chung, Kiwon; Kim, Juhyun; He, Yali; Yin, Donghua; Bohl, Casey; Dalton, James T.; Miller, Duane D.

2007-01-01

A series of nonsteroidal ligands were synthesized as second-generation agonists for the androgen receptor (AR). These ligands were designed to eliminate metabolic sites identified in one of our first-generation AR agonists, which was inactive in vivo due to its rapid metabolism to inactive constituents. The binding affinity of these compounds was evaluated using AR isolated from rat ventral prostate. These second-generation compounds bound the AR in a high affinity and stereoselective manner, with Ki values ranging from about 4 to 130 nM. The ability of these ligands to stimulate AR-mediated transcriptional activation was examined in cells transfected with the human AR and a hormone-dependent luciferase reporter gene. Although some compounds were unable to stimulate AR-mediated transcription, several demonstrated activity similar to that of dihydrotestosterone (DHT, an endogenous steroidal ligand for the AR). We also evaluated the in vivo pharmacologic activity of selected compounds in castrated male rats. Three compounds were identified as selective androgen receptor modulators (SARMs), exhibiting significant anabolic activity while having only moderate to minimal androgenic activity in vivo. PMID:14761201

Thermodynamics and docking of agonists to the β(2)-adrenoceptor determined using [(3)H](R,R')-4-methoxyfenoterol as the marker ligand.

PubMed

Toll, Lawrence; Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A; Tanga, Mary J; Bupp, James E; Wainer, Irving W

2012-06-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β(2)-adrenergic receptor, we developed a new agonist radioligand for binding assays. [(3)H](R,R')-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β(2)-adrenoceptor (AR) population as determined with the antagonist [(3)H]CGP-12177. The β(2)-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and K(i) values determined for fenoterol analogs model much better the cAMP activity of the β(2)-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [(3)H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β(2)-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β(2)-adrenoceptor.

Identification of stepped changes of binding affinity during interactions between the disintegrin rhodostomin and integrin αIIbβ3 in living cells using optical tweezers

NASA Astrophysics Data System (ADS)

Hsieh, Chia-Fen; Chang, Bo-Jui; Pai, Chyi-Huey; Chen, Hsuan-Yi; Chi, Sien; Hsu, Long; Tsai, Jin-Wu; Lin, Chi-Hung

2004-10-01

Integrin receptors serve as both mechanical links and signal transduction mediators between the cell and its environment. Experimental evidence demonstrates that conformational changes and lateral clustering of the integrin proteins may affect their binding to ligands and regulate downstream cellular responses; however, experimental links between the structural and functional correlations of the ligand-receptor interactions are not yet elucidated. In the present report, we utilized optical tweezers to measure the dynamic binding between the snake venom rhodostomin, coated on a microparticle and functioned as a ligand, and the membrane receptor integrin alpha(IIb)beta(3) expressed on a Chinese Hamster Ovary (CHO) cell. A progressive increase of total binding affinity was found between the bead and CHO cell in the first 300 sec following optical tweezers-guided contact. Further analysis of the cumulative data revealed the presence of "unit binding force" presumably exerted by a single rhodostomin-integrin pair. Interestingly, two such units were found. Among the measurements of less total binding forces, presumably taken at the early stage of ligand-receptor interactions, a unit of 4.15 pN per molecule pair was derived. This unit force dropped to 2.54 pN per molecule pair toward the later stage of interactions when the total binding forces were relatively large. This stepped change of single molecule pair binding affinity was not found when mutant rhodostomin proteins were used as ligands (a single unit of 1.81 pN per pair was found). These results were interpreted along with the current knowledge about the conformational changes of integrins during the "molecule activation" process.

Thermodynamics and Docking of Agonists to the β2-Adrenoceptor Determined Using [3H](R,R′)-4-Methoxyfenoterol as the Marker Ligand

PubMed Central

Pajak, Karolina; Plazinska, Anita; Jozwiak, Krzysztof; Jimenez, Lucita; Kozocas, Joseph A.; Tanga, Mary J.; Bupp, James E.; Wainer, Irving W.

2012-01-01

G protein-coupled receptors (GPCRs) are integral membrane proteins that change conformation after ligand binding so that they can transduce signals from an extracellular ligand to a variety of intracellular components. The detailed interaction of a molecule with a G protein-coupled receptor is a complicated process that is influenced by the receptor conformation, thermodynamics, and ligand conformation and stereoisomeric configuration. To better understand the molecular interactions of fenoterol analogs with the β2-adrenergic receptor, we developed a new agonist radioligand for binding assays. [3H](R,R′)-methoxyfenoterol was used to probe the binding affinity for a series of fenoterol stereoisomers and derivatives. The results suggest that the radioligand binds with high affinity to an agonist conformation of the receptor, which represents approximately 25% of the total β2-adrenoceptor (AR) population as determined with the antagonist [3H]CGP-12177. The β2-AR agonists tested in this study have considerably higher affinity for the agonist conformation of the receptor, and Ki values determined for fenoterol analogs model much better the cAMP activity of the β2-AR elicited by these ligands. The thermodynamics of binding are also different when interacting with an agonist conformation, being purely entropy-driven for each fenoterol isomer, rather than a mixture of entropy and enthalpy when the fenoterol isomers binding was determined using [3H]CGP-12177. Finally, computational modeling identified the molecular interactions involved in agonist binding and allow for the prediction of additional novel β2-AR agonists. The study underlines the possibility of using defined radioligand structure to probe a specific conformation of such shape-shifting system as the β2-adrenoceptor. PMID:22434858

NMR studies of DNA oligomers and their interactions with minor groove binding ligands

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

Fagan, Patricia A.

1996-05-01

The cationic peptide ligands distamycin and netropsin bind noncovalently to the minor groove of DNA. The binding site, orientation, stoichiometry, and qualitative affinity of distamycin binding to several short DNA oligomers were investigated by NMR spectroscopy. The oligomers studied contain A,T-rich or I,C-rich binding sites, where I = 2-desaminodeoxyguanosine. I•C base pairs are functional analogs of A•T base pairs in the minor groove. The different behaviors exhibited by distamycin and netropsin binding to various DNA sequences suggested that these ligands are sensitive probes of DNA structure. For sites of five or more base pairs, distamycin can form 1:1 or 2:1more » ligand:DNA complexes. Cooperativity in distamycin binding is low in sites such as AAAAA which has narrow minor grooves, and is higher in sites with wider minor grooves such as ATATAT. The distamycin binding and base pair opening lifetimes of I,C-containing DNA oligomers suggest that the I,C minor groove is structurally different from the A,T minor groove. Molecules which direct chemistry to a specific DNA sequence could be used as antiviral compounds, diagnostic probes, or molecular biology tools. The author studied two ligands in which reactive groups were tethered to a distamycin to increase the sequence specificity of the reactive agent.« less

Effects of a detergent micelle environment on P-glycoprotein (ABCB1)-ligand interactions

PubMed Central

Shukla, Suneet; Abel, Biebele; Chufan, Eduardo E.; Ambudkar, Suresh V.

2017-01-01

P-glycoprotein (P-gp) is a multidrug transporter that uses energy from ATP hydrolysis to export many structurally dissimilar hydrophobic and amphipathic compounds, including anticancer drugs from cells. Several structural studies on purified P-gp have been reported, but only limited and sometimes conflicting information is available on ligand interactions with the isolated transporter in a dodecyl-maltoside detergent environment. In this report we compared the biochemical properties of P-gp in native membranes, detergent micelles, and when reconstituted in artificial membranes. We found that the modulators zosuquidar, tariquidar, and elacridar stimulated the ATPase activity of purified human or mouse P-gp in a detergent micelle environment. In contrast, these drugs inhibited ATPase activity in native membranes or in proteoliposomes, with IC50 values in the 10–40 nm range. Similarly, a 30–150-fold decrease in the apparent affinity for verapamil and cyclic peptide inhibitor QZ59-SSS was observed in detergent micelles compared with native or artificial membranes. Together, these findings demonstrate that the high-affinity site is inaccessible because of either a conformational change or binding of detergent at the binding site in a detergent micelle environment. The ligands bind to a low-affinity site, resulting in altered modulation of P-gp ATPase activity. We, therefore, recommend studying structural and functional aspects of ligand interactions with purified P-gp and other ATP-binding cassette transporters that transport amphipathic or hydrophobic substrates in a detergent-free native or artificial membrane environment. PMID:28283574

Probing Substrate Interactions in the Active Tunnel of a Catalytically Deficient Cellobiohydrolase (Cel7)*

PubMed Central

Colussi, Francieli; Sørensen, Trine H.; Alasepp, Kadri; Kari, Jeppe; Cruys-Bagger, Nicolaj; Windahl, Michael S.; Olsen, Johan P.; Borch, Kim; Westh, Peter

2015-01-01

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 105 m−1, and for longer ligands (DP 5–8) this value was ∼107 m−1. Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔCp) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel. PMID:25477511

Studies on the use of sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucopyranose for the large-scale purification of hepatic glucokinase.

PubMed Central

Holroyde, M J; Chesher, J M; Trayer, I P; Walker, D G

1976-01-01

The synthesis of N-(6-aminohexanoyl)-2-amino-2-deoxy-D-glucose is described and it was shown to be a competitive inhibitor (Ki, 0.75 mM) with respect to glucose of rat hepatic glucokinase (EC 2.7.1.2). After attachment to CNBr-activated Sepharose 4B, this derivative was able to remove glucokinase quantitatively from crude liver extracts and release it when the columns were developed with glucose, glucosamine, N-acetyl-glucosamine or KC1. Repeated exposure of the columns to liver extracts led to rapid loss in their effectiveness as affinity matrices because proteins other than glucokinase are bound to the columns. The nature of such protein binding and methods for the rejuvenation of "used" columns are discussed along with the effect of the mode of preparation of the Sepharose-ligand conjugate and the concentration of bound ligand on the purification of glucokinase. Glucose 6-phosphate dehydrogenase is cited as an example of both non-specific protein binding to the affinity column and of the importance of the control of ligand concentration in removing such non-specifically bound proteins. Some guidelines emerged that should be generally applicable to other systems, particularly those which involve affinity chromatography of enzymes that are present in tissue extracts in very low amounts and possess only a relatively low association constant for the immobilized ligand. PMID:1275893

Identifying Carbohydrate Ligands of a Norovirus P Particle using a Catch and Release Electrospray Ionization Mass Spectrometry Assay

NASA Astrophysics Data System (ADS)

Han, Ling; Kitova, Elena N.; Tan, Ming; Jiang, Xi; Klassen, John S.

2014-01-01

Noroviruses (NoVs), the major cause of epidemic acute gastroenteritis, recognize human histo-blood group antigens (HBGAs), which are present as free oligosaccharides in bodily fluid or glycolipids and glycoproteins on the surfaces of cells. The subviral P particle formed by the protruding (P) domain of the NoV capsid protein serves as a useful model for the study NoV-HBGA interactions. Here, we demonstrate the application of a catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS) assay for screening carbohydrate libraries against the P particle to rapidly identify NoV ligands and potential inhibitors. Carbohydrate libraries of 50 and 146 compounds, which included 18 and 24 analogs of HBGA receptors, respectively, were screened against the P particle of VA387, a member of the predominant GII.4 NoVs. Deprotonated ions corresponding to the P particle bound to carbohydrates were isolated and subjected to collision-induced dissociation to release the ligands in their deprotonated forms. The released ligands were identified by ion mobility separation followed by mass analysis. All 13 and 16 HBGA ligands with intrinsic affinities >500 M-1 were identified in the 50 and the 146 compound libraries, respectively. Furthermore, screening revealed interactions with a series of oligosaccharides with structures found in the cell wall of mycobacteria and human milk. The affinities of these newly discovered ligands are comparable to those of the HBGA receptors, as estimated from the relative abundance of released ligand ions.

In silico design of fragment-based drug targeting host processing α-glucosidase i for dengue fever

NASA Astrophysics Data System (ADS)

Toepak, E. P.; Tambunan, U. S. F.

2017-02-01

Dengue is a major health problem in the tropical and sub-tropical regions. The development of antiviral that targeting dengue’s host enzyme can be more effective and efficient treatment than the viral enzyme. Host enzyme processing α-glucosidase I has an important role in the maturation process of dengue virus envelope glycoprotein. The inhibition of processing α-glucosidase I can become a promising target for dengue fever treatment. The antiviral approach using in silico fragment-based drug design can generate drug candidates with high binding affinity. In this research, 198.621 compounds were obtained from ZINC15 Biogenic Database. These compounds were screened to find the favorable fragments according to Rules of Three and pharmacological properties. The screening fragments were docked into the active site of processing α-glucosidase I. The potential fragment candidates from the molecular docking simulation were linked with castanospermine (CAST) to generate ligands with a better binding affinity. The Analysis of ligand - enzyme interaction showed ligands with code LRS 22, 28, and 47 have the better binding free energy than the standard ligand. Ligand LRS 28 (N-2-4-methyl-5-((1S,3S,6S,7R,8R,8aR)-1,6,7,8-tetrahydroxyoctahydroindolizin-3-yl) pentyl) indolin-1-yl) propionamide) itself among the other ligands has the lowest binding free energy. Pharmacological properties prediction also showed the ligands LRS 22, 28, and 47 can be promising as the dengue fever drug candidates.

Different Epidermal Growth Factor (EGF) Receptor Ligands Show Distinct Kinetics and Biased or Partial Agonism for Homodimer and Heterodimer Formation*

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2014-01-01

The EGF receptor has seven different cognate ligands. Previous work has shown that these different ligands are capable of inducing different biological effects, even in the same cell. To begin to understand the molecular basis for this variation, we used luciferase fragment complementation to measure ligand-induced dimer formation and radioligand binding to study the effect of the ligands on subunit-subunit interactions in EGF receptor (EGFR) homodimers and EGFR/ErbB2 heterodimers. In luciferase fragment complementation imaging studies, amphiregulin (AREG) functioned as a partial agonist, inducing only about half as much total dimerization as the other three ligands. However, unlike the other ligands, AREG showed biphasic kinetics for dimer formation, suggesting that its path for EGF receptor activation involves binding to both monomers and preformed dimers. EGF, TGFα, and betacellulin (BTC) appear to mainly stimulate receptor activation through binding to and dimerization of receptor monomers. In radioligand binding assays, EGF and TGFα exhibited increased affinity for EGFR/ErbB2 heterodimers compared with EGFR homodimers. By contrast, BTC and AREG showed a similar affinity for both dimers. Thus, EGF and TGFα are biased agonists, whereas BTC and AREG are balanced agonists with respect to selectivity of dimer formation. These data suggest that the differences in biological response to different EGF receptor ligands may result from partial agonism for dimer formation, differences in the kinetic pathway utilized to generate activated receptor dimers, and biases in the formation of heterodimers versus homodimers. PMID:25086039

Applications of alginate in bioseparation of proteins.

PubMed

Jain, Sulakshana; Mondal, Kalyani; Gupta, Munishwar N

2006-01-01

Alginate is a polysaccharide that is a block polymer consisting of block units of guluronic acid and mannuronic acid. It shows inherent biological affinity for a variety of enzymes such as pectinase, lipase, phospholipase D, a and ss amylases and glucoamylase. Taking advantage of its precipitation with Ca2+ and the above-mentioned property, alginate has been used for purification of these enzymes by affinity precipitation, aqueous two phase separation, macroaffinity ligand facilitated three phase partitioning, immobilized metal affinity chromatography and expanded bed affinity chromatography. Thus, this versatile marine resource has tremendous potential in bioseparation of proteins.

Protein Surface Structural Recognition in Inactive Areas: A New Immobilization Strategy for Acetylcholinesterase.

PubMed

Diao, Jianxiong; Yu, Xiaolu; Ma, Lin; Li, Yuanqing; Sun, Ying

2018-05-16

This work reported a new method of design for the immobilization of acetylcholinesterase (AChE) based on its molecular structure to improve its sensitivity and stability. The immobilization binding site on the surface of AChE was determined using MOLCAD's multi-channel functionality. Then, 11 molecules ((+)-catechin, (-)-epicatechin, (-)-gallocatechin, hesperetin, naringenin, quercetin, taxifolin, (-)-epicatechin gallate, flupirtine, atropine, and hyoscyamine) were selected from the ZINC database (about 50 000 molecules) as candidate affinity ligands for AChE. The fluorescence results showed that the binding constant K b between AChE and the ligands ranged from 0.01344 × 10 4 to 4.689 × 10 4 M -1 and there was one independent class of binding site for the ligands on AChE. The AChE-ligand binding free energy ranged from -12.14 to -26.65 kJ mol -1 . Naringenin, hesperetin, and quercetin were the three most potent immobilized affinity ligands. In addition, it was confirmed that the binding between the immobilized ligands only occurred at a single site, located in an inactive area on the surface of AChE, and did not affect the enzymatic activity as shown through a competition experiment and enzyme assay. This method based on protein surface structural recognition with high sensitivity and stability can be used as a generic approach for design of the enzyme immobilization and biosensor development.

Prediction of binding affinity and efficacy of thyroid hormone receptor ligands using QSAR and structure-based modeling methods

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

Politi, Regina; Department of Environmental Sciences and Engineering, University of North Carolina, Chapel Hill, NC 27599; Rusyn, Ivan, E-mail: iir@unc.edu

2014-10-01

The thyroid hormone receptor (THR) is an important member of the nuclear receptor family that can be activated by endocrine disrupting chemicals (EDC). Quantitative Structure–Activity Relationship (QSAR) models have been developed to facilitate the prioritization of THR-mediated EDC for the experimental validation. The largest database of binding affinities available at the time of the study for ligand binding domain (LBD) of THRβ was assembled to generate both continuous and classification QSAR models with an external accuracy of R{sup 2} = 0.55 and CCR = 0.76, respectively. In addition, for the first time a QSAR model was developed to predict bindingmore » affinities of antagonists inhibiting the interaction of coactivators with the AF-2 domain of THRβ (R{sup 2} = 0.70). Furthermore, molecular docking studies were performed for a set of THRβ ligands (57 agonists and 15 antagonists of LBD, 210 antagonists of the AF-2 domain, supplemented by putative decoys/non-binders) using several THRβ structures retrieved from the Protein Data Bank. We found that two agonist-bound THRβ conformations could effectively discriminate their corresponding ligands from presumed non-binders. Moreover, one of the agonist conformations could discriminate agonists from antagonists. Finally, we have conducted virtual screening of a chemical library compiled by the EPA as part of the Tox21 program to identify potential THRβ-mediated EDCs using both QSAR models and docking. We concluded that the library is unlikely to have any EDC that would bind to the THRβ. Models developed in this study can be employed either to identify environmental chemicals interacting with the THR or, conversely, to eliminate the THR-mediated mechanism of action for chemicals of concern. - Highlights: • This is the largest curated dataset for ligand binding domain (LBD) of the THRβ. • We report the first QSAR model for antagonists of AF-2 domain of THRβ. • A combination of QSAR and docking enables prediction of both affinity and efficacy. • Models can be used to identify environmental chemicals interacting with THRβ. • Models can be used to eliminate the THRβ-mediated mechanism of action.« less

L-Asp is a useful tool in the purification of the ionotropic glutamate receptor A2 ligand-binding domain.

PubMed

Krintel, Christian; Frydenvang, Karla; Ceravalls de Rabassa, Anna; Kaern, Anne M; Gajhede, Michael; Pickering, Darryl S; Kastrup, Jette S

2014-05-01

In purification of the ionotropic glutamate receptor A2 (GluA2) ligand-binding domain (LBD), L-Glu-supplemented buffers have previously been used for protein stabilization during the procedure. This sometimes hampers structural studies of low-affinity ligands, because L-Glu is difficult to displace, despite extensive dialysis. Here, we show that L-Asp binds to full-length GluA2 with low affinity (Ki = 0.63 mM) and to the GluA2 LBD with even lower affinity (Ki = 2.6 mM), and we use differential scanning fluorimetry to show that L-Asp is able to stabilize the isolated GluA2 LBD. We also show that L-Asp can replace L-Glu during purification, providing both equal yields and purity of the resulting protein sample. Furthermore, we solved three structures of the GluA2 LBD in the presence of 7.5, 50 and 250 mM L-Asp. Surprisingly, with 7.5 mM L-Asp, the GluA2 LBD crystallized as a mixed dimer, with L-Glu being present in one subunit, and neither L-Asp nor L-Glu being present in the other subunit. Thus, residual L-Glu is retained from the expression medium. On the other hand, only L-Asp was found at the binding site when 50 or 250 mM L-Asp was used for crystallization. The binding mode observed for L-Asp at the GluA2 LBD is very similar to that described for L-Glu. Taking our findings together, we have shown that L-Asp can be used instead of L-Glu for ligand-dependent stabilization of the GluA2 LBD during purification. This will enable structural studies of low-affinity ligands for lead optimization in structure-based drug design. Structural data are available in the Protein Data Bank under accession numbers 4O3B (7.5 mM L-Asp), 4O3C (50 mM L-Asp), and 4O3A (250 mM L-Asp). © 2014 FEBS.

The Human Orphan Nuclear Receptor Tailless (TLX, NR2E1) Is Druggable

PubMed Central

Benod, Cindy; Villagomez, Rosa; Filgueira, Carly S.; Hwang, Peter K.; Leonard, Paul G.; Poncet-Montange, Guillaume; Rajagopalan, Senapathy; Fletterick, Robert J.; Gustafsson, Jan-Åke; Webb, Paul

2014-01-01

Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open. PMID:24936658

The human orphan nuclear receptor tailless (TLX, NR2E1) is druggable.

PubMed

Benod, Cindy; Villagomez, Rosa; Filgueira, Carly S; Hwang, Peter K; Leonard, Paul G; Poncet-Montange, Guillaume; Rajagopalan, Senapathy; Fletterick, Robert J; Gustafsson, Jan-Åke; Webb, Paul

2014-01-01

Nuclear receptors (NRs) are an important group of ligand-dependent transcriptional factors. Presently, no natural or synthetic ligand has been identified for a large group of orphan NRs. Small molecules to target these orphan NRs will provide unique resources for uncovering regulatory systems that impact human health and to modulate these pathways with drugs. The orphan NR tailless (TLX, NR2E1), a transcriptional repressor, is a major player in neurogenesis and Neural Stem Cell (NSC) derived brain tumors. No chemical probes that modulate TLX activity are available, and it is not clear whether TLX is druggable. To assess TLX ligand binding capacity, we created homology models of the TLX ligand binding domain (LBD). Results suggest that TLX belongs to an emerging class of NRs that lack LBD helices α1 and α2 and that it has potential to form a large open ligand binding pocket (LBP). Using a medium throughput screening strategy, we investigated direct binding of 20,000 compounds to purified human TLX protein and verified interactions with a secondary (orthogonal) assay. We then assessed effects of verified binders on TLX activity using luciferase assays. As a result, we report identification of three compounds (ccrp1, ccrp2 and ccrp3) that bind to recombinant TLX protein with affinities in the high nanomolar to low micromolar range and enhance TLX transcriptional repressive activity. We conclude that TLX is druggable and propose that our lead compounds could serve as scaffolds to derive more potent ligands. While our ligands potentiate TLX repressive activity, the question of whether it is possible to develop ligands to de-repress TLX activity remains open.

Structure-kinetic relationships--an overlooked parameter in hit-to-lead optimization: a case of cyclopentylamines as chemokine receptor 2 antagonists.

PubMed

Vilums, Maris; Zweemer, Annelien J M; Yu, Zhiyi; de Vries, Henk; Hillger, Julia M; Wapenaar, Hannah; Bollen, Ilse A E; Barmare, Farhana; Gross, Raymond; Clemens, Jeremy; Krenitsky, Paul; Brussee, Johannes; Stamos, Dean; Saunders, John; Heitman, Laura H; Ijzerman, Adriaan P

2013-10-10

Preclinical models of inflammatory diseases (e.g., neuropathic pain, rheumatoid arthritis, and multiple sclerosis) have pointed to a critical role of the chemokine receptor 2 (CCR2) and chemokine ligand 2 (CCL2). However, one of the biggest problems of high-affinity inhibitors of CCR2 is their lack of efficacy in clinical trials. We report a new approach for the design of high-affinity and long-residence-time CCR2 antagonists. We developed a new competition association assay for CCR2, which allows us to investigate the relation of the structure of the ligand and its receptor residence time [i.e., structure-kinetic relationship (SKR)] next to a traditional structure-affinity relationship (SAR). By applying combined knowledge of SAR and SKR, we were able to re-evaluate the hit-to-lead process of cyclopentylamines as CCR2 antagonists. Affinity-based optimization yielded compound 1 with good binding (Ki = 6.8 nM) but very short residence time (2.4 min). However, when the optimization was also based on residence time, the hit-to-lead process yielded compound 22a, a new high-affinity CCR2 antagonist (3.6 nM), with a residence time of 135 min.

Design and synthesis of N-(3,3-diphenylpropenyl)alkanamides as a novel class of high-affinity MT2-selective melatonin receptor ligands.

PubMed

Bedini, Annalida; Spadoni, Gilberto; Gatti, Giuseppe; Lucarini, Simone; Tarzia, Giorgio; Rivara, Silvia; Lorenzi, Simone; Lodola, Alessio; Mor, Marco; Lucini, Valeria; Pannacci, Marilou; Scaglione, Francesco

2006-12-14

A novel series of melatonin receptor ligands was discovered by opening the cyclic scaffolds of known classes of high affinity melatonin receptor antagonists, while retaining the pharmacophore elements postulated by previously described 3D-QSAR and receptor models. Compounds belonging to the classes of 2,3- and [3,3-diphenylprop(en)yl]alkanamides and of o- or [(m-benzyl)phenyl]ethyl-alkanamides were synthesized and tested on MT(1) and MT(2) receptors. The class of 3,3-diphenyl-propenyl-alkanamides was the most interesting one, with compounds having MT(2) receptor affinity similar to that of MLT, remarkable MT(2) selectivity, and partial agonist or antagonist behavior. In particular, the (E)-m-methoxy cyclobutanecarboxamido derivative 18f and the di-(m-methoxy) acetamido one, 18g, have sub-nM affinity for the MT(2) subtype, with more than 100-fold selectivity over MT(1), 18f being an antagonist and 18g a partial agonist on GTPgammaS test. Docking of 18g into a previously developed MT(2) receptor model showed a binding scheme consistent with that of other antagonists. The MT(2) expected binding affinities of the new compounds were calculated by a previously developed 3D-QSAR CoMFA model, giving satisfactory predictions.

SAR by Oxime-Containing Peptide Libraries: Application to Tsg101 Ligand Optimization

PubMed Central

Liu, Fa; Stephen, Andrew G.; Waheed, Abdul A.; Aman, M. Javad; Freed, Eric O.; Fisher, Robert J.; Burke, Terrence R.

2008-01-01

HIV-1 viral assembly requires a direct interaction between a Pro-Thr-Ala-Pro (“PTAP”) motif in the viral protein Gag-p6 and the cellular endosomal sorting factor Tsg101. In an effort to develop competitive inhibitors of this interaction, an SAR study was conducted based on the application of post solid-phase oxime formation involving the sequential insertion of aminooxy-containing residues within a nonamer parent peptide followed by reaction with libraries of aldehydes. Approximately 15–20-fold enhancement in binding affinity was achieved by this approach. PMID:18655064

Receptor-based 3D QSAR analysis of estrogen receptor ligands - merging the accuracy of receptor-based alignments with the computational efficiency of ligand-based methods

NASA Astrophysics Data System (ADS)

Sippl, Wolfgang

2000-08-01

One of the major challenges in computational approaches to drug design is the accurate prediction of binding affinity of biomolecules. In the present study several prediction methods for a published set of estrogen receptor ligands are investigated and compared. The binding modes of 30 ligands were determined using the docking program AutoDock and were compared with available X-ray structures of estrogen receptor-ligand complexes. On the basis of the docking results an interaction energy-based model, which uses the information of the whole ligand-receptor complex, was generated. Several parameters were modified in order to analyze their influence onto the correlation between binding affinities and calculated ligand-receptor interaction energies. The highest correlation coefficient ( r 2 = 0.617, q 2 LOO = 0.570) was obtained considering protein flexibility during the interaction energy evaluation. The second prediction method uses a combination of receptor-based and 3D quantitative structure-activity relationships (3D QSAR) methods. The ligand alignment obtained from the docking simulations was taken as basis for a comparative field analysis applying the GRID/GOLPE program. Using the interaction field derived with a water probe and applying the smart region definition (SRD) variable selection, a significant and robust model was obtained ( r 2 = 0.991, q 2 LOO = 0.921). The predictive ability of the established model was further evaluated by using a test set of six additional compounds. The comparison with the generated interaction energy-based model and with a traditional CoMFA model obtained using a ligand-based alignment ( r 2 = 0.951, q 2 LOO = 0.796) indicates that the combination of receptor-based and 3D QSAR methods is able to improve the quality of the underlying model.

Perturbation-response scanning reveals ligand entry-exit mechanisms of ferric binding protein.

PubMed

Atilgan, Canan; Atilgan, Ali Rana

2009-10-01

We study apo and holo forms of the bacterial ferric binding protein (FBP) which exhibits the so-called ferric transport dilemma: it uptakes iron from the host with remarkable affinity, yet releases it with ease in the cytoplasm for subsequent use. The observations fit the "conformational selection" model whereby the existence of a weakly populated, higher energy conformation that is stabilized in the presence of the ligand is proposed. We introduce a new tool that we term perturbation-response scanning (PRS) for the analysis of remote control strategies utilized. The approach relies on the systematic use of computational perturbation/response techniques based on linear response theory, by sequentially applying directed forces on single-residues along the chain and recording the resulting relative changes in the residue coordinates. We further obtain closed-form expressions for the magnitude and the directionality of the response. Using PRS, we study the ligand release mechanisms of FBP and support the findings by molecular dynamics simulations. We find that the residue-by-residue displacements between the apo and the holo forms, as determined from the X-ray structures, are faithfully reproduced by perturbations applied on the majority of the residues of the apo form. However, once the stabilizing ligand (Fe) is integrated to the system in holo FBP, perturbing only a few select residues successfully reproduces the experimental displacements. Thus, iron uptake by FBP is a favored process in the fluctuating environment of the protein, whereas iron release is controlled by mechanisms including chelation and allostery. The directional analysis that we implement in the PRS methodology implicates the latter mechanism by leading to a few distant, charged, and exposed loop residues. Upon perturbing these, irrespective of the direction of the operating forces, we find that the cap residues involved in iron release are made to operate coherently, facilitating release of the ion.

Identification of 2-arylbenzimidazoles as potent human histamine H4 receptor ligands.

PubMed

Lee-Dutra, Alice; Arienti, Kristen L; Buzard, Daniel J; Hack, Michael D; Khatuya, Haripada; Desai, Pragnya J; Nguyen, Steven; Thurmond, Robin L; Karlsson, Lars; Edwards, James P; Breitenbucher, J Guy

2006-12-01

A series of 2-arylbenzimidazoles was synthesized and found to bind with high affinity to the human histamine H(4) receptor. Structure-activity relationships were investigated through library preparation and evaluation as well as traditional medicinal chemistry approaches, leading to the discovery of compounds with single-digit nanomolar affinity for the H(4) receptor.

SPR Biosensors in Direct Molecular Fishing: Implications for Protein Interactomics.

PubMed

Florinskaya, Anna; Ershov, Pavel; Mezentsev, Yuri; Kaluzhskiy, Leonid; Yablokov, Evgeniy; Medvedev, Alexei; Ivanov, Alexis

2018-05-18

We have developed an original experimental approach based on the use of surface plasmon resonance (SPR) biosensors, applicable for investigation of potential partners involved in protein⁻protein interactions (PPI) as well as protein⁻peptide or protein⁻small molecule interactions. It is based on combining a SPR biosensor, size exclusion chromatography (SEC), mass spectrometric identification of proteins (LC-MS/MS) and direct molecular fishing employing principles of affinity chromatography for isolation of potential partner proteins from the total lysate of biological samples using immobilized target proteins (or small non-peptide compounds) as ligands. Applicability of this approach has been demonstrated within the frame of the Human Proteome Project (HPP) and PPI regulation by a small non-peptide biologically active compound, isatin.

Solution NMR characterization of chemokine CXCL8/IL-8 monomer and dimer binding to glycosaminoglycans: structural plasticity mediates differential binding interactions

PubMed Central

Joseph, Prem Raj B.; Mosier, Philip D.; Desai, Umesh R.; Rajarathnam, Krishna

2015-01-01

Chemokine CXCL8/interleukin-8 (IL-8) plays a crucial role in directing neutrophils and oligodendrocytes to combat infection/injury and tumour cells in metastasis development. CXCL8 exists as monomers and dimers and interaction of both forms with glycosaminoglycans (GAGs) mediate these diverse cellular processes. However, very little is known regarding the structural basis underlying CXCL8–GAG interactions. There are conflicting reports on the affinities, geometry and whether the monomer or dimer is the high-affinity GAG ligand. To resolve these issues, we characterized the binding of a series of heparin-derived oligosaccharides [heparin disaccharide (dp2), heparin tetrasaccharide (dp4), heparin octasaccharide (dp8) and heparin 14-mer (dp14)] to the wild-type (WT) dimer and a designed monomer using solution NMR spectroscopy. The pattern and extent of binding-induced chemical shift perturbation (CSP) varied between dimer and monomer and between longer and shorter oligosaccharides. NMR-based structural models show that different interaction modes coexist and that the nature of interactions varied between monomer and dimer and oligosaccharide length. MD simulations indicate that the binding interface is structurally plastic and provided residue-specific details of the dynamic nature of the binding interface. Binding studies carried out under conditions at which WT CXCL8 exists as monomers and dimers provide unambiguous evidence that the dimer is the high-affinity GAG ligand. Together, our data indicate that a set of core residues function as the major recognition/binding site, a set of peripheral residues define the various binding geometries and that the structural plasticity of the binding interface allows multiplicity of binding interactions. We conclude that structural plasticity most probably regulates in vivo CXCL8 monomer/dimer–GAG interactions and function. PMID:26371375

Are cannabidiol and Δ(9) -tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review.

PubMed

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-02-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ(9) -tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ(9) -tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. © 2014 The British Pharmacological Society.

Are cannabidiol and Δ9-tetrahydrocannabivarin negative modulators of the endocannabinoid system? A systematic review

PubMed Central

McPartland, John M; Duncan, Marnie; Di Marzo, Vincenzo; Pertwee, Roger G

2015-01-01

Based upon evidence that the therapeutic properties of Cannabis preparations are not solely dependent upon the presence of Δ9-tetrahydrocannabinol (THC), pharmacological studies have been recently carried out with other plant cannabinoids (phytocannabinoids), particularly cannabidiol (CBD) and Δ9-tetrahydrocannabivarin (THCV). Results from some of these studies have fostered the view that CBD and THCV modulate the effects of THC via direct blockade of cannabinoid CB1 receptors, thus behaving like first-generation CB1 receptor inverse agonists, such as rimonabant. Here, we review in vitro and ex vivo mechanistic studies of CBD and THCV, and synthesize data from these studies in a meta-analysis. Synthesized data regarding mechanisms are then used to interpret results from recent pre-clinical animal studies and clinical trials. The evidence indicates that CBD and THCV are not rimonabant-like in their action and thus appear very unlikely to produce unwanted CNS effects. They exhibit markedly disparate pharmacological profiles particularly at CB1 receptors: CBD is a very low-affinity CB1 ligand that can nevertheless affect CB1 receptor activity in vivo in an indirect manner, while THCV is a high-affinity CB1 receptor ligand and potent antagonist in vitro and yet only occasionally produces effects in vivo resulting from CB1 receptor antagonism. THCV has also high affinity for CB2 receptors and signals as a partial agonist, differing from both CBD and rimonabant. These cannabinoids illustrate how in vitro mechanistic studies do not always predict in vivo pharmacology and underlie the necessity of testing compounds in vivo before drawing any conclusion on their functional activity at a given target. PMID:25257544

Binding of an antibody mimetic of the human low density lipoprotein receptor to apolipoprotein E is governed through electrostatic forces. Studies using site-directed mutagenesis and molecular modeling.

PubMed

Raffaï, R; Weisgraber, K H; MacKenzie, R; Rupp, B; Rassart, E; Hirama, T; Innerarity, T L; Milne, R

2000-03-10

Monoclonal antibody 2E8 is specific for an epitope that coincides with the binding site of the low density lipoprotein receptor (LDLR) on human apoE. Its reactivity with apoE variants resembles that of the LDLR: it binds well with apoE3 and poorly with apoE2. The heavy chain complementarity-determining region (CDRH) 2 of 2E8 shows homology to the ligand-binding domain of the LDLR. To define better the structural basis of the 2E8/apoE interaction and particularly the role of electrostatic interactions, we generated and characterized a panel of 2E8 variants. Replacement of acidic residues in the 2E8 CDRHs showed that Asp(52), Glu(53), and Asp(56) are essential for high-affinity binding. Although Asp(31) (CDRH1), Glu(58) (CDRH2), and Asp(97) (CDRH3) did not appear to be critical, the Asp(97) --> Ala variant acquired reactivity with apoE2. A Thr(57) --> Glu substitution increased affinity for both apoE3 and apoE2. The affinities of wild-type 2E8 and variants for apoE varied inversely with ionic strength, suggesting that electrostatic forces contribute to both antigen binding and isoform specificity. We propose a model of the 2E8.apoE immune complex that is based on the 2E8 and apoE crystal structures and that is consistent with the apoE-binding properties of wild-type 2E8 and its variants. Given the similarity between the LDLR and 2E8 in terms of specificity, the LDLR/ligand interaction may also have an important electrostatic component.

Cross-activating c-Met/β1 integrin complex drives metastasis and invasive resistance in cancer

PubMed Central

Jahangiri, Arman; Nguyen, Alan; Sidorov, Maxim K.; Yagnik, Garima; Rick, Jonathan; Han, Sung Won; Chen, William; Flanigan, Patrick M.; Schneidman-Duhovny, Dina; Mascharak, Smita; De Lay, Michael; Imber, Brandon; Park, Catherine C.; Matsumoto, Kunio; Lu, Kan; Bergers, Gabriele; Sali, Andrej; Weiss, William A.

2017-01-01

The molecular underpinnings of invasion, a hallmark of cancer, have been defined in terms of individual mediators but crucial interactions between these mediators remain undefined. In xenograft models and patient specimens, we identified a c-Met/β1 integrin complex that formed during significant invasive oncologic processes: breast cancer metastases and glioblastoma invasive resistance to antiangiogenic VEGF neutralizing antibody, bevacizumab. Inducing c-Met/β1 complex formation through an engineered inducible heterodimerization system promoted features crucial to overcoming stressors during metastases or antiangiogenic therapy: migration in the primary site, survival under hypoxia, and extravasation out of circulation. c-Met/β1 complex formation was up-regulated by hypoxia, while VEGF binding VEGFR2 sequestered c-Met and β1 integrin, preventing their binding. Complex formation promoted ligand-independent receptor activation, with integrin-linked kinase phosphorylating c-Met and crystallography revealing the c-Met/β1 complex to maintain the high-affinity β1 integrin conformation. Site-directed mutagenesis verified the necessity for c-Met/β1 binding of amino acids predicted by crystallography to mediate their extracellular interaction. Far-Western blotting and sequential immunoprecipitation revealed that c-Met displaced α5 integrin from β1 integrin, creating a complex with much greater affinity for fibronectin (FN) than α5β1. Thus, tumor cells adapt to microenvironmental stressors induced by metastases or bevacizumab by coopting receptors, which normally promote both cell migration modes: chemotaxis, movement toward concentrations of environmental chemoattractants, and haptotaxis, movement controlled by the relative strengths of peripheral adhesions. Tumor cells then redirect these receptors away from their conventional binding partners, forming a powerful structural c-Met/β1 complex whose ligand-independent cross-activation and robust affinity for FN drive invasive oncologic processes. PMID:28973887

Structural and Functional Characterization of the Kindlin-1 Pleckstrin Homology Domain*

PubMed Central

Yates, Luke A.; Lumb, Craig N.; Brahme, Nina N.; Zalyte, Ruta; Bird, Louise E.; De Colibus, Luigi; Owens, Raymond J.; Calderwood, David A.; Sansom, Mark S. P.; Gilbert, Robert J. C.

2012-01-01

Inside-out activation of integrins is mediated via the binding of talin and kindlin to integrin β-subunit cytoplasmic tails. The kindlin FERM domain is interrupted by a pleckstrin homology (PH) domain within its F2 subdomain. Here, we present data confirming the importance of the kindlin-1 PH domain for integrin activation and its x-ray crystal structure at a resolution of 2.1 Å revealing a C-terminal second α-helix integral to the domain but found only in the kindlin protein family. An isoform-specific salt bridge occludes the canonical phosphoinositide binding site, but molecular dynamics simulations display transient switching to an alternative open conformer. Molecular docking reveals that the opening of the pocket would enable potential ligands to bind within it. Although lipid overlay assays suggested the PH domain binds inositol monophosphates, surface plasmon resonance demonstrated weak affinities for inositol 3,4,5-triphosphate (Ins(3,4,5)P3; KD ∼100 μm) and no monophosphate binding. Removing the salt bridge by site-directed mutagenesis increases the PH domain affinity for Ins(3,4,5)P3 as measured by surface plasmon resonance and enables it to bind PtdIns(3,5)P2 on a dot-blot. Structural comparison with other PH domains suggests that the phosphate binding pocket in the kindlin-1 PH domain is more occluded than in kindlins-2 and -3 due to its salt bridge. In addition, the apparent affinity for Ins(3,4,5)P3 is affected by the presence of PO4 ions in the buffer. We suggest the physiological ligand of the kindlin-1 PH domain is most likely not an inositol phosphate but another phosphorylated species. PMID:23132860

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

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

Bhagavat, Raghu; Kim, Heung -Bok; Kim, Chang -Yub

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived frommore » a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. In conclusion, as the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.« less

Identification of a novel selective PPARγ ligand with a unique binding mode and improved therapeutic profile in vitro

PubMed Central

Yi, Wei; Shi, Jingjing; Zhao, Guanguan; Zhou, X. Edward; Suino-Powell, Kelly; Melcher, Karsten; Xu, H. Eric

2017-01-01

Thiazolidinediones (TZD) function as potent anti-diabetic drugs through their direct action on the nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ), but their therapeutic benefits are compromised by severe side effects. To address this concern, here we developed a potent “hit” compound, VSP-51, which is a novel selective PPARγ-modulating ligand with improved therapeutic profiles in vitro compared to the multi-billion dollar TZD drug rosiglitazone (Rosi). Unlike Rosi, VSP-51 is a partial agonist of PPARγ with improved insulin sensitivity due to its ability to bind PPARγ with high affinity without stimulating adipocyte differentiation and the expression of adipogenesis-related genes. We have determined the crystal structure of the PPARγ ligand-binding domain (LBD) in complex with VSP-51, which revealed a unique mode of binding for VSP-51 and provides the molecular basis for the discrimination between VSP-51 from TZDs and other ligands such as telmisartan, SR1663 and SR1664. Taken together, our findings demonstrate that: a) VSP-51 can serve as a promising candidate for anti-diabetic drug discovery; and b) provide a rational basis for the development of future pharmacological agents targeting PPARγ with advantages over current TZD drugs. PMID:28128331

A genome-wide structure-based survey of nucleotide binding proteins in M. tuberculosis

DOE PAGES

Bhagavat, Raghu; Kim, Heung -Bok; Kim, Chang -Yub; ...

2017-10-02

Nucleoside tri-phosphates (NTP) form an important class of small molecule ligands that participate in, and are essential to a large number of biological processes. Here, we seek to identify the NTP binding proteome (NTPome) in M. tuberculosis (M.tb), a deadly pathogen. Identifying the NTPome is useful not only for gaining functional insights of the individual proteins but also for identifying useful drug targets. From an earlier study, we had structural models of M.tb at a proteome scale from which a set of 13,858 small molecule binding pockets were identified. We use a set of NTP binding sub-structural motifs derived frommore » a previous study and scan the M.tb pocketome, and find that 1,768 proteins or 43% of the proteome can theoretically bind NTP ligands. Using an experimental proteomics approach involving dye-ligand affinity chromatography, we confirm NTP binding to 47 different proteins, of which 4 are hypothetical proteins. Our analysis also provides the precise list of binding site residues in each case, and the probable ligand binding pose. In conclusion, as the list includes a number of known and potential drug targets, the identification of NTP binding can directly facilitate structure-based drug design of these targets.« less

A Direct in Vivo Comparison of the Melanocortin Monovalent Agonist Ac-His-DPhe-Arg-Trp-NH2 versus the Bivalent Agonist Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH2: A Bivalent Advantage.

PubMed

Lensing, Cody J; Adank, Danielle N; Wilber, Stacey L; Freeman, Katie T; Schnell, Sathya M; Speth, Robert C; Zarth, Adam T; Haskell-Luevano, Carrie

2017-06-21

Bivalent ligands targeting putative melanocortin receptor dimers have been developed and characterized in vitro; however, studies of their functional in vivo effects have been limited. The current report compares the effects of homobivalent ligand CJL-1-87, Ac-His-DPhe-Arg-Trp-PEDG20-His-DPhe-Arg-Trp-NH 2 , to monovalent ligand CJL-1-14, Ac-His-DPhe-Arg-Trp-NH 2 , on energy homeostasis in mice after central intracerebroventricular (ICV) administration into the lateral ventricle of the brain. Bivalent ligand CJL-1-87 had noteworthy advantages as an antiobesity probe over CJL-1-14 in a fasting-refeeding in vivo paradigm. Treatment with CJL-1-87 significantly decreased food intake compared to CJL-1-14 or saline (50% less intake 2-8 h after treatment). Furthermore, CJL-1-87 treatment decreased the respiratory exchange ratio (RER) without changing the energy expenditure indicating that fats were being burned as the primary fuel source. Additionally, CJL-1-87 treatment significantly lowered body fat mass percentage 6 h after administration (p < 0.05) without changing the lean mass percentage. The bivalent ligand significantly decreased insulin, C-peptide, leptin, GIP, and resistin plasma levels compared to levels after CJL-1-14 or saline treatments. Alternatively, ghrelin plasma levels were significantly increased. Serum stability of CJL-1-87 and CJL-1-14 (T 1/2 = 6.0 and 16.8 h, respectively) was sufficient to permit physiological effects. The differences in binding affinity of CJL-1-14 compared to CJL-1-87 are speculated as a possible mechanism for the bivalent ligand's unique effects. We also provide in vitro evidence for the formation of a MC3R-MC4R heterodimer complex, for the first time to our knowledge, that may be an unexploited neuronal molecular target. Regardless of the exact mechanism, the advantageous ability of CJL-1-87 compared to CJL-1-14 to increase in vitro binding affinity, increase the duration of action in spite of decreased serum stability, decrease in vivo food intake, decrease mice's body fat percent, and differentially affect mouse hormone levels demonstrates the distinct characteristics achieved from the current melanocortin agonist bivalent design strategy.

Biochemical quality of the pharmaceutically licensed plasma OctaplasLG after implementation of a novel prion protein (PrPSc) removal technology and reduction of the solvent/detergent (S/D) process time.

PubMed

Heger, A; Svae, T-E; Neisser-Svae, A; Jordan, S; Behizad, M; Römisch, J

2009-10-01

A new chromatographic step for the selective binding of pathological prion proteins (PrP(Sc)) to an affinity ligand, developed and optimized for PrP(Sc) capture and attached to synthetic resin particles (PRDT, USA; ProMetic BioSciences Ltd, Isle of Man, UK) was implemented into the manufacturing process of the solvent/detergent (S/D) treated biopharmaceutical quality plasma Octaplas. Pilot batches of Octaplas with the implemented chromatographic step [labelled as OctaplasLG (ligand gel)] were manufactured by Octapharma PPGmbH, Vienna, Austria. The biochemical quality was compared directly after manufacturing as well as after 18 months storage. All samples were tested on global coagulation parameters, fibrinogen levels, activities of coagulation factors and protease inhibitors, ADAMTS13 levels, as well as markers of activated coagulation and fibrinolysis. In addition, von Willebrand factor multimeric analysis was performed. The incorporation of this novel chromatography into the large-scale routine manufacturing process was shown to be technically feasible and the performance of the column was assessed to be excellent. The biochemical studies showed that Octaplas and OctaplasLG produced without and with the new column, respectively, demonstrate an identical biochemical quality. OctaplasLG remained stable over a period of 18 months stored frozen. A parallel reduction of the S/D virus inactivation step from 4-4.5 to 1-1.5 h led to significantly higher activities of plasmin inhibitor. The studies confirmed that the affinity ligand chromatography under the developed conditions can be introduced into the Octaplas manufacturing process, as a mean to reduce potentially present PrP(Sc), without hampering the proven quality of this product.

Identification and Functional Characterization of a Novel Acetylcholine-binding Protein from the Marine Annelid Capitella teleta

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

McCormack, T.; Petrovich,; Mercier, K

2010-01-01

We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) {alpha} subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has beenmore » implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and {alpha}-bungarotoxin bind to ct-AChBP with high affinity, with KD values of 28.7 {micro}M, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K{sub D} = 163 {micro}M). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.« less

Structural and Functional Analyses of a Sterol Carrier Protein in Spodoptera litura

PubMed Central

Xu, Rui; Zheng, Sichun; He, Hongwu; Wan, Jian; Feng, Qili

2014-01-01

Backgrounds In insects, cholesterol is one of the membrane components in cells and a precursor of ecdysteroid biosynthesis. Because insects lack two key enzymes, squalene synthase and lanosterol synthase, in the cholesterol biosynthesis pathway, they cannot autonomously synthesize cholesterol de novo from simple compounds and therefore have to obtain sterols from their diet. Sterol carrier protein (SCP) is a cholesterol-binding protein responsible for cholesterol absorption and transport. Results In this study, a model of the three-dimensional structure of SlSCPx-2 in Spodoptera litura, a destructive polyphagous agricultural pest insect in tropical and subtropical areas, was constructed. Docking of sterol and fatty acid ligands to SlSCPx-2 and ANS fluorescent replacement assay showed that SlSCPx-2 was able to bind with relatively high affinities to cholesterol, stearic acid, linoleic acid, stigmasterol, oleic acid, palmitic acid and arachidonate, implying that SlSCPx may play an important role in absorption and transport of these cholesterol and fatty acids from host plants. Site-directed mutation assay of SlSCPx-2 suggests that amino acid residues F53, W66, F89, F110, I115, T128 and Q131 are critical for the ligand-binding activity of the SlSCPx-2 protein. Virtual ligand screening resulted in identification of several lead compounds which are potential inhibitors of SlSCPx-2. Bioassay for inhibitory effect of five selected compounds showed that AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 inhibited the growth of S. litura larvae. Conclusions Compounds AH-487/41731687, AG-664/14117324, AG-205/36813059 and AG-205/07775053 selected based on structural modeling showed binding affinity to SlSCPx-2 protein and inhibitory effect on the growth of S. litura larvae. PMID:24454688

Crystal structure of glucagon-like peptide-1 in complex with the extracellular domain of the glucagon-like peptide-1 receptor.

PubMed

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic beta-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9-39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Aresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous alpha-helix from Thr(13) to Val(33) when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor.

Crystal Structure of Glucagon-like Peptide-1 in Complex with the Extracellular Domain of the Glucagon-like Peptide-1 Receptor*

PubMed Central

Underwood, Christina Rye; Garibay, Patrick; Knudsen, Lotte Bjerre; Hastrup, Sven; Peters, Günther H.; Rudolph, Rainer; Reedtz-Runge, Steffen

2010-01-01

GLP-1 (glucagon-like peptide-1) is an incretin released from intestinal L-cells in response to food intake. Activation of the GLP-1 receptor potentiates the synthesis and release of insulin from pancreatic β-cells in a glucose-dependent manner. The GLP-1 receptor belongs to class B of the G-protein-coupled receptors, a subfamily characterized by a large N-terminal extracellular ligand binding domain. Exendin-4 and GLP-1 are 50% identical, and exendin-4 is a full agonist with similar affinity and potency for the GLP-1 receptor. We recently solved the crystal structure of the GLP-1 receptor extracellular domain in complex with the competitive antagonist exendin-4(9–39). Interestingly, the isolated extracellular domain binds exendin-4 with much higher affinity than the endogenous agonist GLP-1. Here, we have solved the crystal structure of the extracellular domain in complex with GLP-1 to 2.1 Åresolution. The structure shows that important hydrophobic ligand-receptor interactions are conserved in agonist- and antagonist-bound forms of the extracellular domain, but certain residues in the ligand-binding site adopt a GLP-1-specific conformation. GLP-1 is a kinked but continuous α-helix from Thr13 to Val33 when bound to the extracellular domain. We supplemented the crystal structure with site-directed mutagenesis to link the structural information of the isolated extracellular domain with the binding properties of the full-length receptor. The data support the existence of differences in the binding modes of GLP-1 and exendin-4 on the full-length GLP-1 receptor. PMID:19861722

Molecular basis for the Kallmann syndrome-linked fibroblast growth factor receptor mutation

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

Thurman, Ryan D.; Kathir, Karuppanan Muthusamy; Rajalingam, Dakshinamurthy

Highlights: Black-Right-Pointing-Pointer The structural basis of the Kallmann syndrome is elucidated. Black-Right-Pointing-Pointer Kallmann syndrome mutation (A168S) induces a subtle conformational change(s). Black-Right-Pointing-Pointer Structural interactions mediated by beta-sheet G are most perturbed. Black-Right-Pointing-Pointer Ligand (FGF)-receptor interaction(s) is completely abolished by Kallmann mutation. Black-Right-Pointing-Pointer Kallmann mutation directly affects the FGF signaling process. -- Abstract: Kallmann syndrome (KS) is a developmental disease that expresses in patients as hypogonadotropic hypogonadism and anosmia. KS is commonly associated with mutations in the extracellular D2 domain of the fibroblast growth factor receptor (FGFR). In this study, for the first time, the molecular basis for the FGFR associatedmore » KS mutation (A168S) is elucidated using a variety of biophysical experiments, including multidimensional NMR spectroscopy. Secondary and tertiary structural analysis using far UV circular dichroism, fluorescence and limited trypsin digestion assays suggest that the KS mutation induces subtle tertiary structure change in the D2 domain of FGFR. Results of isothermal titration calorimetry experiments show the KS mutation causes a 10-fold decrease in heparin binding affinity and also a complete loss in ligand (FGF-1) binding. {sup 1}H-{sup 15}N chemical perturbation data suggest that complete loss in the ligand (FGF) binding affinity is triggered by a subtle conformational change that disrupts crucial structural interactions in both the heparin and the FGF binding sites in the D2 domain of FGFR. The novel findings reported in this study are expected to provide valuable clues toward a complete understanding of the other genetic diseases linked to mutations in the FGFR.« less

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

Calvo, Eric; Mans, Ben J.; Ribeiro, José M.C.

The mosquito D7 salivary proteins are encoded by a multigene family related to the arthropod odorant-binding protein (OBP) superfamily. Forms having either one or two OBP domains are found in mosquito saliva. Four single-domain and one two-domain D7 proteins from Anopheles gambiae and Aedes aegypti (AeD7), respectively, were shown to bind biogenic amines with high affinity and with a stoichiometry of one ligand per protein molecule. Sequence comparisons indicated that only the C-terminal domain of AeD7 is homologous to the single-domain proteins from A. gambiae, suggesting that the N-terminal domain may bind a different class of ligands. Here, we describemore » the 3D structure of AeD7 and examine the ligand-binding characteristics of the N- and C-terminal domains. Isothermal titration calorimetry and ligand complex crystal structures show that the N-terminal domain binds cysteinyl leukotrienes (cysLTs) with high affinities (50-60 nM) whereas the C-terminal domain binds biogenic amines. The lipid chain of the cysLT binds in a hydrophobic pocket of the N-terminal domain, whereas binding of norepinephrine leads to an ordering of the C-terminal portion of the C-terminal domain into an alpha-helix that, along with rotations of Arg-176 and Glu-268 side chains, acts to bury the bound ligand.« less

Entropy in molecular recognition by proteins

PubMed Central

Caro, José A.; Harpole, Kyle W.; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G.; Sharp, Kim A.

2017-01-01

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein–ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein–ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein–ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or “entropy meter” also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water–protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins. PMID:28584100

Entropy in molecular recognition by proteins.

PubMed

Caro, José A; Harpole, Kyle W; Kasinath, Vignesh; Lim, Jackwee; Granja, Jeffrey; Valentine, Kathleen G; Sharp, Kim A; Wand, A Joshua

2017-06-20

Molecular recognition by proteins is fundamental to molecular biology. Dissection of the thermodynamic energy terms governing protein-ligand interactions has proven difficult, with determination of entropic contributions being particularly elusive. NMR relaxation measurements have suggested that changes in protein conformational entropy can be quantitatively obtained through a dynamical proxy, but the generality of this relationship has not been shown. Twenty-eight protein-ligand complexes are used to show a quantitative relationship between measures of fast side-chain motion and the underlying conformational entropy. We find that the contribution of conformational entropy can range from favorable to unfavorable, which demonstrates the potential of this thermodynamic variable to modulate protein-ligand interactions. For about one-quarter of these complexes, the absence of conformational entropy would render the resulting affinity biologically meaningless. The dynamical proxy for conformational entropy or "entropy meter" also allows for refinement of the contributions of solvent entropy and the loss in rotational-translational entropy accompanying formation of high-affinity complexes. Furthermore, structure-based application of the approach can also provide insight into long-lived specific water-protein interactions that escape the generic treatments of solvent entropy based simply on changes in accessible surface area. These results provide a comprehensive and unified view of the general role of entropy in high-affinity molecular recognition by proteins.

General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

PubMed

Fei, Xiang; Zavorka, Megan E; Malik, Guillaume; Connelly, Christopher M; MacDonald, Richard G; Berkowitz, David B

2017-08-18

A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

Identifying the receptor subtype selectivity of retinoid X and retinoic acid receptors via quantum mechanics.

PubMed

Tsuji, Motonori; Shudo, Koichi; Kagechika, Hiroyuki

2017-03-01

Understanding and identifying the receptor subtype selectivity of a ligand is an important issue in the field of drug discovery. Using a combination of classical molecular mechanics and quantum mechanical calculations, this report assesses the receptor subtype selectivity for the human retinoid X receptor (hRXR) and retinoic acid receptor (hRAR) ligand-binding domains (LBDs) complexed with retinoid ligands. The calculated energies show good correlation with the experimentally reported binding affinities. The technique proposed here is a promising method as it reveals the origin of the receptor subtype selectivity of selective ligands.

Cloud computing approaches for prediction of ligand binding poses and pathways.

PubMed

Lawrenz, Morgan; Shukla, Diwakar; Pande, Vijay S

2015-01-22

We describe an innovative protocol for ab initio prediction of ligand crystallographic binding poses and highly effective analysis of large datasets generated for protein-ligand dynamics. We include a procedure for setup and performance of distributed molecular dynamics simulations on cloud computing architectures, a model for efficient analysis of simulation data, and a metric for evaluation of model convergence. We give accurate binding pose predictions for five ligands ranging in affinity from 7 nM to > 200 μM for the immunophilin protein FKBP12, for expedited results in cases where experimental structures are difficult to produce. Our approach goes beyond single, low energy ligand poses to give quantitative kinetic information that can inform protein engineering and ligand design.

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

PubMed

Clayton, R N; Shakespear, R A; Duncan, J A; Marshall, J C; Munson, P J; Rodbard, D

1979-12-01

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.

The effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand directed chiral recognition

PubMed Central

Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W.

2011-01-01

The β2 adrenergic receptor (β2-AR) is a model system for studying the ligand recognition process in G-protein coupled receptors. Fenoterol (FEN) is a β2-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van’t Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x’)-isomers is almost entirely enthalpy controlled whereas binding of (R,x’)-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R’)-FEN was shown to selectively activate Gs protein signaling while the (S,R’)- isomer activated both Gi and Gs protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site and the global mechanism of β2-AR activation. Differences in thermodynamic parameters and non-uniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x’)-FEN stereoisomers with a different receptor conformation than the one with which the (S,x’)-isomer interacts. PMID:21618615

Effect of fenoterol stereochemistry on the β2 adrenergic receptor system: ligand-directed chiral recognition.

PubMed

Jozwiak, Krzysztof; Plazinska, Anita; Toll, Lawrence; Jimenez, Lucita; Woo, Anthony Yiu-Ho; Xiao, Rui-Ping; Wainer, Irving W

2011-01-01

The β(2) adrenergic receptor (β(2)-AR) is a model system for studying the ligand recognition process in G protein-coupled receptors. Fenoterol (FEN) is a β(2)-AR selective agonist that has two centers of chirality and exists as four stereoisomers. Radioligand binding studies determined that stereochemistry greatly influences the binding affinity. Subsequent Van't Hoff analysis shows very different thermodynamics of binding depending on the stereoconfiguration of the molecule. The binding of (S,x')-isomers is almost entirely enthalpy controlled whereas binding of (R,x')-isomers is purely entropy driven. Stereochemistry of FEN molecule also affects the coupling of the receptor to different G proteins. In a rat cardiomyocyte contractility model, (R,R')-FEN was shown to selectively activate G(s) protein signaling while the (S,R')-isomer activated both G(i) and G(s) protein. The overall data demonstrate that the chirality at the two chiral centers of the FEN molecule influences the magnitude of binding affinity, thermodynamics of local interactions within the binding site, and the global mechanism of β(2)-AR activation. Differences in thermodynamic parameters and nonuniform G-protein coupling suggest a mechanism of chiral recognition in which observed enantioselectivities arise from the interaction of the (R,x')-FEN stereoisomers with a different receptor conformation than the one with which the (S,x')-isomer interacts. Copyright © 2011 Wiley-Liss, Inc.

Strong DNA deformation required for extremely slow DNA threading intercalation by a binuclear ruthenium complex

PubMed Central

Almaqwashi, Ali A.; Paramanathan, Thayaparan; Lincoln, Per; Rouzina, Ioulia; Westerlund, Fredrik; Williams, Mark C.

2014-01-01

DNA intercalation by threading is expected to yield high affinity and slow dissociation, properties desirable for DNA-targeted therapeutics. To measure these properties, we utilize single molecule DNA stretching to quantify both the binding affinity and the force-dependent threading intercalation kinetics of the binuclear ruthenium complex Δ,Δ-[μ‐bidppz‐(phen)4Ru2]4+ (Δ,Δ-P). We measure the DNA elongation at a range of constant stretching forces using optical tweezers, allowing direct characterization of the intercalation kinetics as well as the amount intercalated at equilibrium. Higher forces exponentially facilitate the intercalative binding, leading to a profound decrease in the binding site size that results in one ligand intercalated at almost every DNA base stack. The zero force Δ,Δ-P intercalation Kd is 44 nM, 25-fold stronger than the analogous mono-nuclear ligand (Δ-P). The force-dependent kinetics analysis reveals a mechanism that requires DNA elongation of 0.33 nm for association, relaxation to an equilibrium elongation of 0.19 nm, and an additional elongation of 0.14 nm from the equilibrium state for dissociation. In cells, a molecule with binding properties similar to Δ,Δ-P may rapidly bind DNA destabilized by enzymes during replication or transcription, but upon enzyme dissociation it is predicted to remain intercalated for several hours, thereby interfering with essential biological processes. PMID:25245944

Multiple Length Peptide-Pheromone Variants Produced by Streptococcus pyogenes Directly Bind Rgg Proteins to Confer Transcriptional Regulation*

PubMed Central

Aggarwal, Chaitanya; Jimenez, Juan Cristobal; Nanavati, Dhaval; Federle, Michael J.

2014-01-01

Streptococcus pyogenes, a human-restricted pathogen, accounts for substantial mortality related to infections worldwide. Recent studies indicate that streptococci produce and respond to several secreted peptide signaling molecules (pheromones), including those known as short hydrophobic peptides (SHPs), to regulate gene expression by a quorum-sensing mechanism. Upon transport into the bacterial cell, pheromones bind to and modulate activity of receptor proteins belonging to the Rgg family of transcription factors. Previously, we reported biofilm regulation by the Rgg2/3 quorum-sensing circuit in S. pyogenes. The aim of this study was to identify the composition of mature pheromones from cell-free culture supernatants that facilitate biofilm formation. Bioluminescent reporters were employed to detect active pheromones in culture supernatants fractionated by reverse-phase chromatography, and mass spectrometry was used to characterize their properties. Surprisingly, multiple SHPs that varied by length were detected. Synthetic peptides of each variant were tested individually using bioluminescence reporters and biofilm growth assays, and although activities differed widely among the group, peptides comprising the C-terminal eight amino acids of the full-length native peptide were most active. Direct Rgg/SHP interactions were determined using a fluorescence polarization assay that utilized FITC-labeled peptide ligands. Peptide receptor affinities were seen to be as low as 500 nm and their binding affinities directly correlated with observed bioactivity. Revelation of naturally produced pheromones along with determination of their affinity for cognate receptors are important steps forward in designing compounds whose purpose is positioned for future therapeutics aimed at treating infections through the interference of bacterial communication. PMID:24958729

Modular scanning FCS quantifies receptor-ligand interactions in living multicellular organisms.

PubMed

Ries, Jonas; Yu, Shuizi Rachel; Burkhardt, Markus; Brand, Michael; Schwille, Petra

2009-09-01

Analysis of receptor-ligand interactions in vivo is key to biology but poses a considerable challenge to quantitative microscopy. Here we combine static-volume, two-focus and dual-color scanning fluorescence correlation spectroscopy to solve this task at cellular resolution in complex biological environments. We quantified the mobility of fibroblast growth factor receptors Fgfr1 and Fgfr4 in cell membranes of living zebrafish embryos and determined their in vivo binding affinities to their ligand Fgf8.

Miniature protein ligands for EVH1 domains: Interplay between affinity, specificity, and cell motility⊥

PubMed Central

Holtzman, Jennifer H.; Woronowicz, Kamil; Golemi-Kotra, Dasantila; Schepartz, Alanna

2008-01-01

Dynamic rearrangements of the actin cytoskeleton power cell motility in contexts ranging from intracellular microbial pathogenesis to axon guidance. The Ena/VASP family proteins--Mena, VASP, and Evl--are believed to control cell motility by serving as a direct link between signaling events and the actin cytoskeleton. Our lab has previously reported a novel miniature protein, pGolemi, which binds with high affinity to the EVH1 domain of Mena (Mena1-112) but not to those of VASP (VASP1-115) or Evl (Evl1-115) and also causes an unusual defect in actin-driven L. monocytogenes motility. Here, we use scanning mutagenesis to examine the effects of single amino acid changes within pGolemi on EVH1 domain affinity and specificity, miniature protein secondary structure, and L. monocytogenes motility. The data suggest that pGolemi contains the expected aPP-like fold and binds Mena1-112 in a manner highly analogous to the proline-rich repeat region of L. monocytogenes ActA protein. Residues throughout pGolemi contribute to both EVH1 domain affinity and paralog specificity. Moreover, the affinities of pGolemi variants for Mena1-112 correlate with selectivity against the EVH1 domains of VASP and Evl. In L. monocytogenes motility assays, speed and speed variability correlate strongly with EVH1 paralog specificity, suggesting that the Ena/VASP paralogs do not play equivalent roles in the process of L. monocytogenes actin tail maturation. PMID:17973491

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition

PubMed Central

Junicke, Henrik; Hart, Jonathan R.; Kisko, Jennifer; Glebov, Oleg; Kirsch, Ilan R.; Barton, Jacqueline K.

2003-01-01

A rhodium(III) complex, rac-[Rh(bpy)2phzi]3+ (bpy, 2,2′-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)2(chrysi)]3+ [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 × 107 M−1, respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design. PMID:12610209

The use of small-molecule structures to complement protein–ligand crystal structures in drug discovery

PubMed Central

Cole, Jason C.

2017-01-01

Many ligand-discovery stories tell of the use of structures of protein–ligand complexes, but the contribution of structural chemistry is such a core part of finding and improving ligands that it is often overlooked. More than 800 000 crystal structures are available to the community through the Cambridge Structural Database (CSD). Individually, these structures can be of tremendous value and the collection of crystal structures is even more helpful. This article provides examples of how small-molecule crystal structures have been used to complement those of protein–ligand complexes to address challenges ranging from affinity, selectivity and bioavailability though to solubility. PMID:28291759

A viral, transporter associated with antigen processing (TAP)-independent, high affinity ligand with alternative interactions endogenously presented by the nonclassical human leukocyte antigen E class I molecule.

PubMed

Lorente, Elena; Infantes, Susana; Abia, David; Barnea, Eilon; Beer, Ilan; García, Ruth; Lasala, Fátima; Jiménez, Mercedes; Mir, Carmen; Morreale, Antonio; Admon, Arie; López, Daniel

2012-10-12

The transporter associated with antigen processing (TAP) enables the flow of viral peptides generated in the cytosol by the proteasome and other proteases to the endoplasmic reticulum, where they complex with nascent human leukocyte antigen (HLA) class I. Later, these peptide-HLA class I complexes can be recognized by CD8(+) lymphocytes. Cancerous cells and infected cells in which TAP is blocked, as well as individuals with unusable TAP complexes, are able to present peptides on HLA class I by generating them through TAP-independent processing pathways. Here, we identify a physiologically processed HLA-E ligand derived from the D8L protein in TAP-deficient vaccinia virus-infected cells. This natural high affinity HLA-E class I ligand uses alternative interactions to the anchor motifs previously described to be presented on nonclassical HLA class I molecules. This octameric peptide was also presented on HLA-Cw1 with similar binding affinity on both classical and nonclassical class I molecules. In addition, this viral peptide inhibits HLA-E-mediated cytolysis by natural killer cells. Comparison between the amino acid sequences of the presenting HLA-E and HLA-Cw1 alleles revealed a shared structural motif in both HLA class molecules, which could be related to their observed similar cross-reactivity affinities. This motif consists of several residues located on the floor of the peptide-binding site. These data expand the role of HLA-E as an antigen-presenting molecule.

Optimization of reverse chemical ecology method: false positive binding of Aenasius bambawalei odorant binding protein 1 caused by uncertain binding mechanism.

PubMed

Li, Q L; Yi, S C; Li, D Z; Nie, X P; Li, S Q; Wang, M-Q; Zhou, A M

2018-06-01

Odorant binding proteins (OBPs) are considered as the core molecular targets in reverse chemical ecology, which is a convenient and efficient method by which to screen potential semiochemicals. Herein, we identified a classic OBP, AbamOBP1 from Aenasius bambawalei, which showed high mRNA expression in male antennae. Fluorescence competitive binding assay (FCBA) results demonstrated that AbamOBP1 has higher binding affinity with ligands at acid pH, suggesting the physiologically inconsistent binding affinity of this protein. Amongst the four compounds with the highest binding affinities at acid pH, 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one were shown to have attractant activity for male adults, whereas (-)-limonene and an analogue of 1-octen-3-ol exhibited nonbehavioural activity. Further homology modelling and fluorescence quenching experiments demonstrated that the stoichiometry of the binding of this protein to these ligands was not 1: 1, suggesting that the results of FCBA were false. In contrast, the apparent association constants (Ka) of fluorescence quenching experiments seemed to be more reliable, because 2, 4, 4-trimethyl-2-pentene and 1-octen-3-one had observably higher Ka than (-)-limonene and 1-octen-3-ol at neutral pH. Based on the characteristics of different OBPs, various approaches should be applied to study their binding affinities with ligands, which could modify and complement the results of FCBA and contribute to the application of reverse chemical ecology. © 2018 The Royal Entomological Society.

Measuring Positive Cooperativity Using the Direct ESI-MS Assay. Cholera Toxin B Subunit Homopentamer Binding to GM1 Pentasaccharide

NASA Astrophysics Data System (ADS)

Lin, Hong; Kitova, Elena N.; Klassen, John S.

2014-01-01

Direct electrospray ionization mass spectrometry (ESI-MS) assay was used to investigate the stepwise binding of the GM1 pentasaccharide β- D-Gal p-(1→3)-β-D-Gal pNAc-(1→4)[α-D-Neu5Ac-(2→3)]-β- D-Gal p-(1→4)-β-D-Glc p (GM1os) to the cholera toxin B subunit homopentamer (CTB5) and to establish conclusively whether GM1os binding is cooperative. Apparent association constants were measured for the stepwise addition of one to five GM1os to CTB5 at pH 6.9 and 22 °C. The intrinsic association constant, which was established from the apparent association constant for the addition of a single GM1os to CTB5, was found to be (3.2 ± 0.2) × 106 M-1. This is in reasonable agreement with the reported value of (6.4 ± 0.3) × 106 M-1, which was measured at pH 7.4 and 25 °C using isothermal titration calorimetry (ITC). Analysis of the apparent association constants provides direct and unambiguous evidence that GM1os binding exhibits small positive cooperativity. Binding was found to be sensitive to the number of ligand-bound nearest neighbor subunits, with the affinities enhanced by a factor of 1.7 and 2.9 when binding occurs next to one or two ligand-bound subunits, respectively. These findings, which provide quantitative support for the binding model proposed by Homans and coworkers [14], highlight the unique strengths of the direct ESI-MS assay for measuring cooperative ligand binding.

A large-scale test of free-energy simulation estimates of protein-ligand binding affinities.

PubMed

Mikulskis, Paulius; Genheden, Samuel; Ryde, Ulf

2014-10-27

We have performed a large-scale test of alchemical perturbation calculations with the Bennett acceptance-ratio (BAR) approach to estimate relative affinities for the binding of 107 ligands to 10 different proteins. Employing 20-Å truncated spherical systems and only one intermediate state in the perturbations, we obtain an error of less than 4 kJ/mol for 54% of the studied relative affinities and a precision of 0.5 kJ/mol on average. However, only four of the proteins gave acceptable errors, correlations, and rankings. The results could be improved by using nine intermediate states in the simulations or including the entire protein in the simulations using periodic boundary conditions. However, 27 of the calculated affinities still gave errors of more than 4 kJ/mol, and for three of the proteins the results were not satisfactory. This shows that the performance of BAR calculations depends on the target protein and that several transformations gave poor results owing to limitations in the molecular-mechanics force field or the restricted sampling possible within a reasonable simulation time. Still, the BAR results are better than docking calculations for most of the proteins.

Hollow fiber based affinity selection combined with high performance liquid chromatography-mass spectroscopy for rapid screening lipase inhibitors from lotus leaf.

PubMed

Tao, Yi; Zhang, Yufeng; Wang, Yi; Cheng, Yiyu

2013-06-27

A novel kind of immobilized enzyme affinity selection strategy based on hollow fibers has been developed for screening inhibitors from extracts of medicinal plants. Lipases from porcine pancreas were adsorbed onto the surface of polypropylene hollow fibers to form a stable matrix for ligand fishing, which was called hollow fibers based affinity selection (HF-AS). A variety of factors related to binding capability, including enzyme concentration, incubation time, temperature, buffer pH and ion strength, were optimized using a known lipase inhibitor hesperidin. The proposed approach was applied in screening potential lipase bound ligands from extracts of lotus leaf, followed by rapid characterization of active compounds using high performance liquid chromatography-mass spectrometry. Three flavonoids including quercetin-3-O-β-D-arabinopyranosyl-(1→2)-β-D-galactopyranoside, quercetin-3-O-β-D-glucuronide and kaempferol-3-O-β-d-glucuronide were identified as lipase inhibitors by the proposed HF-AS approach. Our findings suggested that the hollow fiber-based affinity selection could be a rapid and convenient approach for drug discovery from natural products resources. Copyright © 2013 Elsevier B.V. All rights reserved.

Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats.

PubMed

Bohin, Maxime C; Vincken, Jean-Paul; Westphal, Adrie H; Tripp, Annelise M; Dekker, Peter; van der Hijden, Harry T W M; Gruppen, Harry

2014-09-01

Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with α-casein and β-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to α-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to α-casein, β-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic-casein interactions. Copyright © 2014 Elsevier Ltd. All rights reserved.

Synthesis and evaluation of novel opioid ligands with a C-homomorphinan skeleton.

PubMed

Ishikawa, Kyoko; Mochizuki, Yusuke; Hirayama, Shigeto; Nemoto, Toru; Nagai, Kenichiro; Itoh, Kennosuke; Fujii, Hideaki

2016-05-15

As the reports about C-homomorphinans with the seven-membered C-ring are much fewer than those of morphinan derivatives with a six-membered C-ring, we attempted to synthesize C-homomorphinan derivatives and to evaluate their opioid activities. C-Homomorphinan 5 showed sufficient binding affinities to the opioid receptors. C-Homomorphinan derivatives possessing the δ address moiety such as indole (NTI-type), quinoline, or benzylidene (BNTX-type) functionalities showed the strongest binding affinities for the δ receptor among the three types of opioid receptors, which indicated that the C-homomorphinan skeleton sufficiently functions as a message-part in the ligand. Although NTI-type compound 8 and quinoline compound 9 with C-homomorphinan scaffold exhibited lower affinities and selectivities for the δ receptor than the corresponding morphinan derivatives did, both the binding affinity and selectivity for the δ receptor of BNTX-type compound 12 with a seven-membered C-ring were improved compared with the corresponding compounds with a six-membered C-ring including BNTX itself. BNTX-Type compound 12 was the most selective δ receptor antagonist among the tested compounds. Copyright © 2016 Elsevier Ltd. All rights reserved.

Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands.

PubMed Central

Kinnally, K W; Zorov, D B; Antonenko, Y N; Snyder, S H; McEnery, M W; Tedeschi, H

1993-01-01

The mitochrondrial benzodiazepine receptor (mBzR) binds a subset of benzodiazepines and isoquinoline carboxamides with nanomolar affinity and consists of the voltage-dependent anion channel, the adenine nucleotide translocator, and an 18-kDa protein. The effect of ligands of the mBzR on two inner mitochondrial membrane channel activities was determined with patch-clamp techniques. The relative inhibitory potencies of the drugs resemble their binding affinities for the mBzR. Ro5-4864 and protoporphyrin IX inhibit activity of the multiple conductance channel (MCC) and the mitochondrial centum-picosiemen (mCtS) channel activities at nanomolar concentrations. PK11195 inhibits mCtS activity at similar levels. Higher concentrations of protoporphyrin IX induce MCC but possibly not mCtS activity. Clonazepam, which has low affinity for mBzR, is at least 500 times less potent at both channel activities. Ro15-1788, which also has a low mBzR affinity, inhibits MCC at very high concentrations (16 microM). The findings indicate an association of these two channel activities with the proteins forming the mBzR complex and are consistent with an interaction of inner and outer membrane channels. PMID:7679505

Surface Plasmon Resonance kinetic analysis of the interaction between G-quadruplex nucleic acids and an anti-G-quadruplex monoclonal antibody.

PubMed

Lago, Sara; Nadai, Matteo; Rossetto, Monica; Richter, Sara N

2018-06-01

G-quadruplexes (G4s) are nucleic acids secondary structures formed in guanine-rich sequences. Anti-G4 antibodies represent a tool for the direct investigation of G4s in cells. Surface Plasmon Resonance (SPR) is a highly sensitive technology, suitable for assessing the affinity between biomolecules. We here aimed at improving the orientation of an anti-G4 antibody on the SPR sensor chip to optimize detection of binding antigens. SPR was employed to characterize the anti-G4 antibody interaction with G4 and non-G4 oligonucleotides. Dextran-functionalized sensor chips were used both in covalent coupling and capturing procedures. The use of two leading molecule for orienting the antibody of interest allowed to improve its activity from completely non-functional to 65% active. The specificity of the anti-G4 antobody for G4 structures could thus be assessed with high sensitivity and reliability. Optimization of the immobilization protocol for SPR biosensing, allowed us to determine the anti-G4 antibody affinity and specificity for G4 antigens with higher sensitivity with respect to other in vitro assays such as ELISA. Anti-G4 antibody specificity is a fundamental assumption for the future utilization of this kind of antibodies for monitoring G4s directly in cells. The heterogeneous orientation of amine-coupling immobilized ligands is a general problem that often leads to partial or complete inactivation of the molecules. Here we describe a new strategy for improving ligand orientation: driving it from two sides. This principle can be virtually applied to every molecule that loses its activity or is poorly immobilized after standard coupling to the SPR chip surface. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

NASA Astrophysics Data System (ADS)

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-04-01

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region.

PubMed

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-04-07

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine.

The low binding affinity of D-serine at the ionotropic glutamate receptor GluD2 can be attributed to the hinge region

PubMed Central

Tapken, Daniel; Steffensen, Thomas Bielefeldt; Leth, Rasmus; Kristensen, Lise Baadsgaard; Gerbola, Alexander; Gajhede, Michael; Jørgensen, Flemming Steen; Olsen, Lars; Kastrup, Jette Sandholm

2017-01-01

Ionotropic glutamate receptors (iGluRs) are responsible for most of the fast excitatory communication between neurons in our brain. The GluD2 receptor is a puzzling member of the iGluR family: It is involved in synaptic plasticity, plays a role in human diseases, e.g. ataxia, binds glycine and D-serine with low affinity, yet no ligand has been discovered so far that can activate its ion channel. In this study, we show that the hinge region connecting the two subdomains of the GluD2 ligand-binding domain is responsible for the low affinity of D-serine, by analysing GluD2 mutants with electrophysiology, isothermal titration calorimetry and molecular dynamics calculations. The hinge region is highly variable among iGluRs and fine-tunes gating activity, suggesting that in GluD2 this region has evolved to only respond to micromolar concentrations of D-serine. PMID:28387240

Utilizing combinatorial engineering to develop Tie2 targeting antagonistic angiopoetin-2 ligands as candidates for anti-angiogenesis therapy.

PubMed

Shlamkovich, Tomer; Aharon, Lidan; Barton, William A; Papo, Niv

2017-05-16

In many human cancers, the receptor tyrosine kinase (RTK) Tie2 plays important roles in mediating proliferation, survival, migration and angiogenesis. Thus, molecules that could potently inhibit activation of the Tie2 receptor would have a significant impact on cancer therapy. Nevertheless, attempts to develop Tie2-targeted inhibitors have met with little success, and there is currently no FDA-approved therapeutic selectively targeting Tie2. We used a combinatorial protein engineering approach to develop a new generation of angiopoietin (Ang)2-derived Tie2 antagonists as potential cancer therapeutics and as tools to study angiogenesis. The construct for designing a yeast surface display (YSD) library of potential antagonists was an Ang2 binding domain (Ang2-BD) that retains Tie2 binding ability but prevents ligand multimerization and receptor dimerization and activation. This mutant library was then screened by quantitative high-throughput flow cytometric sorting to identify Ang2-BD variants with increased expression, stability and affinity to Tie2. The selected variants were recombinantly expressed and showed high affinity to soluble and cellular Tie2 and strongly inhibited both Tie2 phosphorylation and endothelial capillary tube formation and cell invasion compared to the parental Ang2-BD. The significance of the study lies in the insight it provides into the sequence-structure-function relationships and mechanism of action of the antagonistic Ang mutants. The approach of using a natural protein ligand as a molecular scaffold for engineering high-affinity agents can be applied to other ligands to create functional protein antagonists against additional biomedical targets.

Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme

PubMed Central

Leong, Max K.; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

2017-01-01

The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r2 = 0.928–0.988,  = 0.894–0.954, RMSE = 0.002–0.412, s = 0.001–0.214), and the predicted pKi values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r2 = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q2 = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery. PMID:28059133

Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme.

PubMed

Leong, Max K; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

2017-01-06

The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r 2  = 0.928-0.988,  = 0.894-0.954, RMSE = 0.002-0.412, s = 0.001-0.214), and the predicted pK i values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r 2  = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q 2  = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery.

Structure-based design of competitive ligands to target Spon2 in gastric cancer: An integration of molecular modeling and in vitro assay.

PubMed

Xu, Zhenglei; Yu, Zhichao; Nai, Shumei; Shi, Ruiyue; Tang, Qinhong; Zhang, Haiyang; Ye, Lijuan; Wang, Lisheng; Hong, Yincai

2017-10-01

Spon2 is a proto-oncogene matrix protein that plays an essential role in the tumorigenesis and metastasis of gastric cancer. The protein has recently been found to function as a guanine nucleotide exchange factor through the activation of RhoGTPase. Here, computational modeling and bioinformatics analysis were employed to investigate the molecular mechanism and biological implication underlying Spon2 autoinhibition. It is revealed that the binding of PxxP motif to SH domain can stabilize the intramolecular interaction between the N-terminal helix and DH domain of Spon2, thus shifting the protein into an autoinhibitory state. Here, we proposed releasing Spon2 autoinhibition by targeting SH domain with competitive peptide ligands. To verify this notion, the PxxP sequence was adopted as the start to derive an array of efficient SH binders by using a structure-based rational design strategy, which were then substantiated with fluorescence spectroscopy analysis and guanine nucleotide exchange test. Consequently, the obtained peptide ligands were determined to have a moderate or high affinity for SH domain; they can also enhance Spon2 exchange activity by 1.2-6.1 folds, exhibiting a significant correlation with their SH-binding affinity (Pearson's coefficient=0.92). In addition, neutral substitution of conserved residues in a high-affinity peptide ligand can largely reduce its Spon2-activating potency, confirming that the designed peptide activates Spon2 by competitively disrupting SH-PxxP interaction. Copyright © 2017 Elsevier Inc. All rights reserved.

Prediction of N-Methyl-D-Aspartate Receptor GluN1-Ligand Binding Affinity by a Novel SVM-Pose/SVM-Score Combinatorial Ensemble Docking Scheme

NASA Astrophysics Data System (ADS)

Leong, Max K.; Syu, Ren-Guei; Ding, Yi-Lung; Weng, Ching-Feng

2017-01-01

The glycine-binding site of the N-methyl-D-aspartate receptor (NMDAR) subunit GluN1 is a potential pharmacological target for neurodegenerative disorders. A novel combinatorial ensemble docking scheme using ligand and protein conformation ensembles and customized support vector machine (SVM)-based models to select the docked pose and to predict the docking score was generated for predicting the NMDAR GluN1-ligand binding affinity. The predicted root mean square deviation (RMSD) values in pose by SVM-Pose models were found to be in good agreement with the observed values (n = 30, r2 = 0.928-0.988,  = 0.894-0.954, RMSE = 0.002-0.412, s = 0.001-0.214), and the predicted pKi values by SVM-Score were found to be in good agreement with the observed values for the training samples (n = 24, r2 = 0.967,  = 0.899, RMSE = 0.295, s = 0.170) and test samples (n = 13, q2 = 0.894, RMSE = 0.437, s = 0.202). When subjected to various statistical validations, the developed SVM-Pose and SVM-Score models consistently met the most stringent criteria. A mock test asserted the predictivity of this novel docking scheme. Collectively, this accurate novel combinatorial ensemble docking scheme can be used to predict the NMDAR GluN1-ligand binding affinity for facilitating drug discovery.

Competition effects in cation binding to humic acid: Conditional affinity spectra for fixed total metal concentration conditions

NASA Astrophysics Data System (ADS)

David, Calin; Mongin, Sandrine; Rey-Castro, Carlos; Galceran, Josep; Companys, Encarnació; Garcés, José Luis; Salvador, José; Puy, Jaume; Cecilia, Joan; Lodeiro, Pablo; Mas, Francesc

2010-09-01

Information on the Pb and Cd binding to a purified Aldrich humic acid (HA) is obtained from the influence of different fixed total metal concentrations on the acid-base titrations of this ligand. NICA (Non-Ideal Competitive Adsorption) isotherm has been used for a global quantitative description of the binding, which has then been interpreted by plotting the Conditional Affinity Spectra of the H + binding at fixed total metal concentrations (CAScTM). This new physicochemical tool, here introduced, allows the interpretation of binding results in terms of distributions of proton binding energies. A large increase in the acidity of the phenolic sites as the total metal concentration increases, especially in presence of Pb, is revealed from the shift of the CAScTM towards lower affinities. The variance of the CAScTM distribution, which can be used as a direct measure of the heterogeneity, also shows a significant dependence on the total metal concentration. A discussion of the factors that influence the heterogeneity of the HA under the conditions of each experiment is provided, so that the smoothed pattern exhibited by the titration curves can be justified.

Estimation of relative free energies of binding using pre-computed ensembles based on the single-step free energy perturbation and the site-identification by Ligand competitive saturation approaches.

PubMed

Raman, E Prabhu; Lakkaraju, Sirish Kaushik; Denny, Rajiah Aldrin; MacKerell, Alexander D

2017-06-05

Accurate and rapid estimation of relative binding affinities of ligand-protein complexes is a requirement of computational methods for their effective use in rational ligand design. Of the approaches commonly used, free energy perturbation (FEP) methods are considered one of the most accurate, although they require significant computational resources. Accordingly, it is desirable to have alternative methods of similar accuracy but greater computational efficiency to facilitate ligand design. In the present study relative free energies of binding are estimated for one or two non-hydrogen atom changes in compounds targeting the proteins ACK1 and p38 MAP kinase using three methods. The methods include standard FEP, single-step free energy perturbation (SSFEP) and the site-identification by ligand competitive saturation (SILCS) ligand grid free energy (LGFE) approach. Results show the SSFEP and SILCS LGFE methods to be competitive with or better than the FEP results for the studied systems, with SILCS LGFE giving the best agreement with experimental results. This is supported by additional comparisons with published FEP data on p38 MAP kinase inhibitors. While both the SSFEP and SILCS LGFE approaches require a significant upfront computational investment, they offer a 1000-fold computational savings over FEP for calculating the relative affinities of ligand modifications once those pre-computations are complete. An illustrative example of the potential application of these methods in the context of screening large numbers of transformations is presented. Thus, the SSFEP and SILCS LGFE approaches represent viable alternatives for actively driving ligand design during drug discovery and development. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Monitoring β-arrestin recruitment via β-lactamase enzyme fragment complementation: purification of peptide E as a low-affinity ligand for mammalian bombesin receptors.

PubMed

Ikeda, Yuichi; Kumagai, Hidetoshi; Okazaki, Hiroaki; Fujishiro, Mitsuhiro; Motozawa, Yoshihiro; Nomura, Seitaro; Takeda, Norifumi; Toko, Haruhiro; Takimoto, Eiki; Akazawa, Hiroshi; Morita, Hiroyuki; Suzuki, Jun-ichi; Yamazaki, Tsutomu; Komuro, Issei; Yanagisawa, Masashi

2015-01-01

Identification of cognate ligands for G protein-coupled receptors (GPCRs) provides a starting point for understanding novel regulatory mechanisms. Although GPCR ligands have typically been evaluated through the activation of heterotrimeric G proteins, recent studies have shown that GPCRs signal not only through G proteins but also through β-arrestins. As such, monitoring β-arrestin signaling instead of G protein signaling will increase the likelihood of identifying currently unknown ligands, including β-arrestin-biased agonists. Here, we developed a cell-based assay for monitoring ligand-dependent GPCR-β-arrestin interaction via β-lactamase enzyme fragment complementation. Inter alia, β-lactamase is a superior reporter enzyme because of its cell-permeable fluorescent substrate. This substrate makes the assay non-destructive and compatible with fluorescence-activated cell sorting (FACS). In a reporter cell, complementary fragments of β-lactamase (α and ω) were fused to β-arrestin 2 and GPCR, respectively. Ligand stimulation initiated the interaction of these chimeric proteins (β-arrestin-α and GPCR-ω), and this inducible interaction was measured through reconstituted β-lactamase activity. Utilizing this system, we screened various mammalian tissue extracts for agonistic activities on human bombesin receptor subtype 3 (hBRS3). We purified peptide E as a low-affinity ligand for hBRS3, which was also found to be an agonist for the other two mammalian bombesin receptors such as gastrin-releasing peptide receptor (GRPR) and neuromedin B receptor (NMBR). Successful purification of peptide E has validated the robustness of this assay. We conclude that our newly developed system will facilitate the discovery of GPCR ligands.

Unravelling intrinsic efficacy and ligand bias at G protein coupled receptors: A practical guide to assessing functional data.

PubMed

Stott, Lisa A; Hall, David A; Holliday, Nicholas D

2016-02-01

Stephenson's empirical definition of an agonist, as a ligand with binding affinity and intrinsic efficacy (the ability to activate the receptor once bound), underpins classical receptor pharmacology. Quantifying intrinsic efficacy using functional concentration response relationships has always presented an experimental challenge. The requirement for realistic determination of efficacy is emphasised by recent developments in our understanding of G protein coupled receptor (GPCR) agonists, with recognition that some ligands stabilise different active conformations of the receptor, leading to pathway-selective, or biased agonism. Biased ligands have potential as therapeutics with improved selectivity and clinical efficacy, but there are also pitfalls to the identification of pathway selective effects. Here we explore the basics of concentration response curve analysis, beginning with the need to distinguish ligand bias from other influences of the functional system under study. We consider the different approaches that have been used to quantify and compare biased ligands, many of which are based on the Black and Leff operational model of agonism. Some of the practical issues that accompany these analyses are highlighted, with opportunities to improve estimates in future, particularly in the separation of true agonist intrinsic efficacy from the contributions of system dependent coupling efficiency. Such methods are by their nature practical approaches, and all rely on Stephenson's separation of affinity and efficacy parameters, which are interdependent at the mechanistic level. Nevertheless, operational analysis methods can be justified by mechanistic models of GPCR activation, and if used wisely are key elements to biased ligand identification. Copyright © 2015 Elsevier Inc. All rights reserved.

High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor

PubMed Central

Pichlo, Magdalena; Bungert-Plümke, Stefanie; Weyand, Ingo; Seifert, Reinhard; Bönigk, Wolfgang; Strünker, Timo; Kashikar, Nachiket Dilip; Goodwin, Normann; Müller, Astrid; Körschen, Heinz G.; Collienne, Ursel; Pelzer, Patric; Van, Qui; Enderlein, Jörg; Klemm, Clementine; Krause, Eberhard; Trötschel, Christian; Poetsch, Ansgar; Kremmer, Elisabeth

2014-01-01

Guanylyl cyclases (GCs), which synthesize the messenger cyclic guanosine 3′,5′-monophosphate, control several sensory functions, such as phototransduction, chemosensation, and thermosensation, in many species from worms to mammals. The GC chemoreceptor in sea urchin sperm can decode chemoattractant concentrations with single-molecule sensitivity. The molecular and cellular underpinnings of such ultrasensitivity are not known for any eukaryotic chemoreceptor. In this paper, we show that an exquisitely high density of 3 × 105 GC chemoreceptors and subnanomolar ligand affinity provide a high ligand-capture efficacy and render sperm perfect absorbers. The GC activity is terminated within 150 ms by dephosphorylation steps of the receptor, which provides a means for precise control of the GC lifetime and which reduces “molecule noise.” Compared with other ultrasensitive sensory systems, the 10-fold signal amplification by the GC receptor is surprisingly low. The hallmarks of this signaling mechanism provide a blueprint for chemical sensing in small compartments, such as olfactory cilia, insect antennae, or even synaptic boutons. PMID:25135936

Structural basis of ligand recognition in 5-HT3 receptors

PubMed Central

Kesters, Divya; Thompson, Andrew J; Brams, Marijke; van Elk, René; Spurny, Radovan; Geitmann, Matthis; Villalgordo, Jose M; Guskov, Albert; Helena Danielson, U; Lummis, Sarah C R; Smit, August B; Ulens, Chris

2013-01-01

The 5-HT3 receptor is a pentameric serotonin-gated ion channel, which mediates rapid excitatory neurotransmission and is the target of a therapeutically important class of anti-emetic drugs, such as granisetron. We report crystal structures of a binding protein engineered to recognize the agonist serotonin and the antagonist granisetron with affinities comparable to the 5-HT3 receptor. In the serotonin-bound structure, we observe hydrophilic interactions with loop E-binding site residues, which might enable transitions to channel opening. In the granisetron-bound structure, we observe a critical cation–π interaction between the indazole moiety of the ligand and a cationic centre in loop D, which is uniquely present in the 5-HT3 receptor. We use a series of chemically tuned granisetron analogues to demonstrate the energetic contribution of this electrostatic interaction to high-affinity ligand binding in the human 5-HT3 receptor. Our study offers the first structural perspective on recognition of serotonin and antagonism by anti-emetics in the 5-HT3 receptor. PMID:23196367

Synthesis and characterization of a Eu-DTPA-PEGO-MSH(4) derivative for evaluation of binding of multivalent molecules to melanocortin receptors.

PubMed

Xu, Liping; Vagner, Josef; Alleti, Ramesh; Rao, Venkataramanarao; Jagadish, Bhumasamudram; Morse, David L; Hruby, Victor J; Gillies, Robert J; Mash, Eugene A

2010-04-15

A labeled variant of MSH(4), a tetrapeptide that binds to the human melanocortin 4 receptor (hMC4R) with low microM affinity, was prepared by solid-phase synthesis methods, purified, and characterized. The labeled ligand, Eu-DTPA-PEGO-His-dPhe-Arg-Trp-NH(2), exhibited a K(d) for hMC4R of 9.1+/-1.4 microM, approximately 10-fold lower affinity than the parental ligand. The labeled MSH(4) derivative was employed in a competitive binding assay to characterize the interactions of hMC4R with monovalent and divalent MSH(4) constructs derived from squalene. The results were compared with results from a similar assay that employed a more potent labeled ligand, Eu-DTPA-NDP-alpha-MSH. While results from the latter assay reflected only statistical effects, results from the former assay reflected a mixture of statistical, proximity, and/or cooperative binding effects. Copyright 2010 Elsevier Ltd. All rights reserved.

Molecular characterization and functional analysis of pheromone binding protein 1 from Cydia pomonella (L.).

PubMed

Tian, Z; Zhang, Y

2016-12-01

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. © 2016 The Royal Entomological Society.

Predicting the affinity of Farnesoid X Receptor ligands through a hierarchical ranking protocol: a D3R Grand Challenge 2 case study

NASA Astrophysics Data System (ADS)

Réau, Manon; Langenfeld, Florent; Zagury, Jean-François; Montes, Matthieu

2018-01-01

The Drug Design Data Resource (D3R) Grand Challenges are blind contests organized to assess the state-of-the-art methods accuracy in predicting binding modes and relative binding free energies of experimentally validated ligands for a given target. The second stage of the D3R Grand Challenge 2 (GC2) was focused on ranking 102 compounds according to their predicted affinity for Farnesoid X Receptor. In this task, our workflow was ranked 5th out of the 77 submissions in the structure-based category. Our strategy consisted in (1) a combination of molecular docking using AutoDock 4.2 and manual edition of available structures for binding poses generation using SeeSAR, (2) the use of HYDE scoring for pose selection, and (3) a hierarchical ranking using HYDE and MM/GBSA. In this report, we detail our pose generation and ligands ranking protocols and provide guidelines to be used in a prospective computer aided drug design program.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

PubMed

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Vascular development in the retina and inner ear: control by Norrin and Frizzled-4, a high-affinity ligand-receptor pair.

PubMed

Xu, Qiang; Wang, Yanshu; Dabdoub, Alain; Smallwood, Philip M; Williams, John; Woods, Chad; Kelley, Matthew W; Jiang, Li; Tasman, William; Zhang, Kang; Nathans, Jeremy

2004-03-19

Incomplete retinal vascularization occurs in both Norrie disease and familial exudative vitreoretinopathy (FEVR). Norrin, the protein product of the Norrie disease gene, is a secreted protein of unknown biochemical function. One form of FEVR is caused by defects in Frizzled-4 (Fz4), a presumptive Wnt receptor. We show here that Norrin and Fz4 function as a ligand-receptor pair based on (1) the similarity in vascular phenotypes caused by Norrin and Fz4 mutations in humans and mice, (2) the specificity and high affinity of Norrin-Fz4 binding, (3) the high efficiency with which Norrin induces Fz4- and Lrp-dependent activation of the classical Wnt pathway, and (4) the signaling defects displayed by disease-associated variants of Norrin and Fz4. These data define a Norrin-Fz4 signaling system that plays a central role in vascular development in the eye and ear, and they indicate that ligands unrelated to Wnts can act through Fz receptors.

Ligand-directed tosyl chemistry for in situ native protein labeling and engineering in living systems: from basic properties to applications.

PubMed

Tsukiji, Shinya; Hamachi, Itaru

2014-08-01

The ability to introduce any chemical probe to any endogenous target protein in its native environment, that is in cells and in vivo, is anticipated to provide various new exciting tools for biological and biomedical research. Although still at the prototype stage, the ligand-directed tosyl (LDT) chemistry is a novel type of affinity labeling technique that we developed for such a dream. This chemistry allows for modifying native proteins by various chemical probes with high specificity in various biological settings ranging from in vitro (in test tubes) to in living cells and in vivo. Since the first report, the list of proteins that are successfully labeled by the LDT chemistry has been increasing. A growing number of studies have demonstrated its utility to create semisynthetic proteins directly in cellular contexts. The in situ generated semisynthetic proteins are applicable for various types of analysis and imaging of intracellular biological processes. In this review, we summarize the basic properties of the LDT chemistry and its applications toward in situ engineering and analysis of native proteins in living systems. Current limitations and future challenges of this area are also described. Copyright © 2014 Elsevier Ltd. All rights reserved.

Nucleic Acid Ligands With Protein-like Side Chains: Modified Aptamers and Their Use as Diagnostic and Therapeutic Agents

PubMed Central

Rohloff, John C; Gelinas, Amy D; Jarvis, Thale C; Ochsner, Urs A; Schneider, Daniel J; Gold, Larry; Janjic, Nebojsa

2014-01-01

Limited chemical diversity of nucleic acid libraries has long been suspected to be a major constraining factor in the overall success of SELEX (Systematic Evolution of Ligands by EXponential enrichment). Despite this constraint, SELEX has enjoyed considerable success over the past quarter of a century as a result of the enormous size of starting libraries and conformational richness of nucleic acids. With judicious introduction of functional groups absent in natural nucleic acids, the “diversity gap” between nucleic acid–based ligands and protein-based ligands can be substantially bridged, to generate a new class of ligands that represent the best of both worlds. We have explored the effect of various functional groups at the 5-position of uracil and found that hydrophobic aromatic side chains have the most profound influence on the success rate of SELEX and allow the identification of ligands with very low dissociation rate constants (named Slow Off-rate Modified Aptamers or SOMAmers). Such modified nucleotides create unique intramolecular motifs and make direct contacts with proteins. Importantly, SOMAmers engage their protein targets with surfaces that have significantly more hydrophobic character compared with conventional aptamers, thereby increasing the range of epitopes that are available for binding. These improvements have enabled us to build a collection of SOMAmers to over 3,000 human proteins encompassing major families such as growth factors, cytokines, enzymes, hormones, and receptors, with additional SOMAmers aimed at pathogen and rodent proteins. Such a large and growing collection of exquisite affinity reagents expands the scope of possible applications in diagnostics and therapeutics. PMID:25291143

Discovering ligands for a microRNA precursor with peptoid microarrays

PubMed Central

Chirayil, Sara; Chirayil, Rachel; Luebke, Kevin J.

2009-01-01

We have screened peptoid microarrays to identify specific ligands for the RNA hairpin precursor of miR-21, a microRNA involved in cancer and heart disease. Microarrays were printed by spotting a library of 7680 N-substituted oligoglycines (peptoids) onto glass slides. Two compounds on the array specifically bind RNA having the sequence and predicted secondary structure of the miR-21 precursor hairpin and have specific affinity for the target in solution. Their binding induces a conformational change around the hairpin loop, and the most specific compound recognizes the loop sequence and a bulged uridine in the proximal duplex. Functional groups contributing affinity and specificity were identified, and by varying a critical methylpyridine group, a compound with a dissociation constant of 1.9 μM for the miR-21 precursor hairpin and a 20-fold discrimination against a closely-related hairpin was created. This work describes a systematic approach to discovery of ligands for specific pre-defined novel RNA structures. It demonstrates discovery of new ligands for an RNA for which no specific lead compounds were previously known by screening a microarray of small molecules. PMID:19561197

Binding cooperativity between a ligand carbonyl group and a hydrophobic side chain can be enhanced by additional H-bonds in a distance dependent manner: A case study with thrombin inhibitors.

PubMed

Said, Ahmed M; Hangauer, David G

2015-01-01

One of the underappreciated non-covalent binding factors, which can significantly affect ligand-protein binding affinity, is the cooperativity between ligand functional groups. Using four different series of thrombin inhibitors, we reveal a strong positive cooperativity between an H-bond accepting carbonyl functionality and the adjacent P3 hydrophobic side chain. Adding an H-bond donating amine adjacent to the P3 hydrophobic side chain further increases this positive cooperativity thereby improving the Ki by as much as 546-fold. In contrast, adding an amidine multiple H-bond/salt bridge group in the distal S1 pocket does not affect this cooperativity. An analysis of the crystallographic B-factors of the ligand groups inside the binding site indicates that the strong cooperativity is mainly due to a significant mutual reduction in the residual mobility of the hydrophobic side chain and the H-bonding functionalities that is absent when the separation distance is large. This type of cooperativity is important to encode in binding affinity prediction software, and to consider in SAR studies. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

Characterization of 5-HT₁A receptors and their complexes with G-proteins in budded baculovirus particles using fluorescence anisotropy of Bodipy-FL-NAN-190.

PubMed

Tõntson, Lauri; Kopanchuk, Sergei; Rinken, Ago

2014-02-01

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass<1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+). Copyright © 2014 Elsevier Ltd. All rights reserved.