Combinatorial techniques to efficiently investigate and optimize organic thin film processing and properties.

PubMed

Wieberger, Florian; Kolb, Tristan; Neuber, Christian; Ober, Christopher K; Schmidt, Hans-Werner

2013-04-08

In this article we present several developed and improved combinatorial techniques to optimize processing conditions and material properties of organic thin films. The combinatorial approach allows investigations of multi-variable dependencies and is the perfect tool to investigate organic thin films regarding their high performance purposes. In this context we develop and establish the reliable preparation of gradients of material composition, temperature, exposure, and immersion time. Furthermore we demonstrate the smart application of combinations of composition and processing gradients to create combinatorial libraries. First a binary combinatorial library is created by applying two gradients perpendicular to each other. A third gradient is carried out in very small areas and arranged matrix-like over the entire binary combinatorial library resulting in a ternary combinatorial library. Ternary combinatorial libraries allow identifying precise trends for the optimization of multi-variable dependent processes which is demonstrated on the lithographic patterning process. Here we verify conclusively the strong interaction and thus the interdependency of variables in the preparation and properties of complex organic thin film systems. The established gradient preparation techniques are not limited to lithographic patterning. It is possible to utilize and transfer the reported combinatorial techniques to other multi-variable dependent processes and to investigate and optimize thin film layers and devices for optical, electro-optical, and electronic applications.

Structure-based design of combinatorial mutagenesis libraries

PubMed Central

Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

2015-01-01

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called “Structure-based Optimization of Combinatorial Mutagenesis” (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. PMID:25611189

Structure-based design of combinatorial mutagenesis libraries.

PubMed

Verma, Deeptak; Grigoryan, Gevorg; Bailey-Kellogg, Chris

2015-05-01

The development of protein variants with improved properties (thermostability, binding affinity, catalytic activity, etc.) has greatly benefited from the application of high-throughput screens evaluating large, diverse combinatorial libraries. At the same time, since only a very limited portion of sequence space can be experimentally constructed and tested, an attractive possibility is to use computational protein design to focus libraries on a productive portion of the space. We present a general-purpose method, called "Structure-based Optimization of Combinatorial Mutagenesis" (SOCoM), which can optimize arbitrarily large combinatorial mutagenesis libraries directly based on structural energies of their constituents. SOCoM chooses both positions and substitutions, employing a combinatorial optimization framework based on library-averaged energy potentials in order to avoid explicitly modeling every variant in every possible library. In case study applications to green fluorescent protein, β-lactamase, and lipase A, SOCoM optimizes relatively small, focused libraries whose variants achieve energies comparable to or better than previous library design efforts, as well as larger libraries (previously not designable by structure-based methods) whose variants cover greater diversity while still maintaining substantially better energies than would be achieved by representative random library approaches. By allowing the creation of large-scale combinatorial libraries based on structural calculations, SOCoM promises to increase the scope of applicability of computational protein design and improve the hit rate of discovering beneficial variants. While designs presented here focus on variant stability (predicted by total energy), SOCoM can readily incorporate other structure-based assessments, such as the energy gap between alternative conformational or bound states. © 2015 The Protein Society.

Application of computer assisted combinatorial chemistry in antivirial, antimalarial and anticancer agents design

NASA Astrophysics Data System (ADS)

Burello, E.; Bologa, C.; Frecer, V.; Miertus, S.

Combinatorial chemistry and technologies have been developed to a stage where synthetic schemes are available for generation of a large variety of organic molecules. The innovative concept of combinatorial design assumes that screening of a large and diverse library of compounds will increase the probability of finding an active analogue among the compounds tested. Since the rate at which libraries are screened for activity currently constitutes a limitation to the use of combinatorial technologies, it is important to be selective about the number of compounds to be synthesized. Early experience with combinatorial chemistry indicated that chemical diversity alone did not result in a significant increase in the number of generated lead compounds. Emphasis has therefore been increasingly put on the use of computer assisted combinatorial chemical techniques. Computational methods are valuable in the design of virtual libraries of molecular models. Selection strategies based on computed physicochemical properties of the models or of a target compound are introduced to reduce the time and costs of library synthesis and screening. In addition, computational structure-based library focusing methods can be used to perform in silico screening of the activity of compounds against a target receptor by docking the ligands into the receptor model. Three case studies are discussed dealing with the design of targeted combinatorial libraries of inhibitors of HIV-1 protease, P. falciparum plasmepsin and human urokinase as potential antivirial, antimalarial and anticancer drugs. These illustrate library focusing strategies.

Library fingerprints: a novel approach to the screening of virtual libraries.

PubMed

Klon, Anthony E; Diller, David J

2007-01-01

We propose a novel method to prioritize libraries for combinatorial synthesis and high-throughput screening that assesses the viability of a particular library on the basis of the aggregate physical-chemical properties of the compounds using a naïve Bayesian classifier. This approach prioritizes collections of related compounds according to the aggregate values of their physical-chemical parameters in contrast to single-compound screening. The method is also shown to be useful in screening existing noncombinatorial libraries when the compounds in these libraries have been previously clustered according to their molecular graphs. We show that the method used here is comparable or superior to the single-compound virtual screening of combinatorial libraries and noncombinatorial libraries and is superior to the pairwise Tanimoto similarity searching of a collection of combinatorial libraries.

An Indexed Combinatorial Library: The Synthesis and Testing of Insect Repellents

NASA Astrophysics Data System (ADS)

Miles, William H.; Gelato, Kathy A.; Pompizzi, Kristen M.; Scarbinsky, Aislinn M.; Albrecht, Brian K.; Reynolds, Elaine R.

2001-04-01

An indexed combinatorial library of amides was prepared by the reaction of amines and acid chlorides. A simple test for insect repellency using fruit flies (Drosophila melanogaster) allowed the determination of the most repellent sublibraries. The student-generated data were collected and analyzed to determine the most active amide(s) in the library. This experiment illustrates the fundamentals of combinatorial chemistry, a field that has undergone explosive growth in the last decade.

In vitro and direct in vivo testing of mixture-based combinatorial libraries for the identification of highly active and specific opiate ligands.

PubMed

Houghten, Richard A; Dooley, Colette T; Appel, Jon R

2006-05-26

The use of combinatorial libraries for the identification of novel opiate and related ligands in opioid receptor assays is reviewed. Case studies involving opioid assays used to demonstrate the viability of combinatorial libraries are described. The identification of new opioid peptides composed of L-amino acids, D-amino acids, or L-, D-, and unnatural amino acids is reviewed. New opioid compounds have also been identified from peptidomimetic libraries, such as peptoids and alkylated dipeptides, and those identified from acyclic (eg, polyamine, urea) and heterocyclic (eg, bicyclic guanidine) libraries are reviewed.

Increased Diversity of Libraries from Libraries: Chemoinformatic Analysis of Bis-Diazacyclic Libraries

PubMed Central

López-Vallejo, Fabian; Nefzi, Adel; Bender, Andreas; Owen, John R.; Nabney, Ian T.; Houghten, Richard A.; Medina-Franco, Jose L.

2011-01-01

Combinatorial libraries continue to play a key role in drug discovery. To increase structural diversity, several experimental methods have been developed. However, limited efforts have been performed so far to quantify the diversity of the broadly used diversity-oriented synthetic (DOS) libraries. Herein we report a comprehensive characterization of 15 bis-diazacyclic combinatorial libraries obtained through libraries from libraries, which is a DOS approach. Using MACCS keys, radial and different pharmacophoric fingerprints as well as six molecular properties, it was demonstrated the increased structural and property diversity of the libraries from libraries over the individual libraries. Comparison of the libraries to existing drugs, NCI Diversity and the Molecular Libraries Small Molecule Repository revealed the structural uniqueness of the combinatorial libraries (mean similarity < 0.5 for any fingerprint representation). In particular, bis-cyclic thiourea libraries were the most structurally dissimilar to drugs retaining drug-like character in property space. This study represents the first comprehensive quantification of the diversity of libraries from libraries providing a solid quantitative approach to compare and contrast the diversity of DOS libraries with existing drugs or any other compound collection. PMID:21294850

Construction of a scFv Library with Synthetic, Non-combinatorial CDR Diversity.

PubMed

Bai, Xuelian; Shim, Hyunbo

2017-01-01

Many large synthetic antibody libraries have been designed, constructed, and successfully generated high-quality antibodies suitable for various demanding applications. While synthetic antibody libraries have many advantages such as optimized framework sequences and a broader sequence landscape than natural antibodies, their sequence diversities typically are generated by random combinatorial synthetic processes which cause the incorporation of many undesired CDR sequences. Here, we describe the construction of a synthetic scFv library using oligonucleotide mixtures that contain predefined, non-combinatorially synthesized CDR sequences. Each CDR is first inserted to a master scFv framework sequence and the resulting single-CDR libraries are subjected to a round of proofread panning. The proofread CDR sequences are assembled to produce the final scFv library with six diversified CDRs.

Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry

PubMed Central

Drożdż, Wojciech; Kołodziejski, Michał; Markiewicz, Grzegorz; Jenczak, Anna; Stefankiewicz, Artur R.

2015-01-01

We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines. PMID:26193265

Generation of a Multicomponent Library of Disulfide Donor-Acceptor Architectures Using Dynamic Combinatorial Chemistry.

PubMed

Drożdż, Wojciech; Kołodziejski, Michał; Markiewicz, Grzegorz; Jenczak, Anna; Stefankiewicz, Artur R

2015-07-17

We describe here the generation of new donor-acceptor disulfide architectures obtained in aqueous solution at physiological pH. The application of a dynamic combinatorial chemistry approach allowed us to generate a large number of new disulfide macrocyclic architectures together with a new type of [2]catenanes consisting of four distinct components. Up to fifteen types of structurally-distinct dynamic architectures have been generated through one-pot disulfide exchange reactions between four thiol-functionalized aqueous components. The distribution of disulfide products formed was found to be strongly dependent on the structural features of the thiol components employed. This work not only constitutes a success in the synthesis of topologically- and morphologically-complex targets, but it may also open new horizons for the use of this methodology in the construction of molecular machines.

Exploring the formation pathways of donor-acceptor catenanes in aqueous dynamic combinatorial libraries.

PubMed

Cougnon, Fabien B L; Au-Yeung, Ho Yu; Pantoş, G Dan; Sanders, Jeremy K M

2011-03-09

The discovery through dynamic combinatorial chemistry (DCC) of a new generation of donor-acceptor [2]catenanes highlights the power of DCC to access unprecedented structures. While conventional thinking has limited the scope of donor-acceptor catenanes to strictly alternating stacks of donor (D) and acceptor (A) aromatic units, DCC is demonstrated in this paper to give access to unusual DAAD, DADD, and ADAA stacks. Each of these catenanes has specific structural requirements, allowing control of their formation. On the basis of these results, and on the observation that the catenanes represent kinetic bottlenecks in the reaction pathway, we propose a mechanism that explains and predicts the structures formed. Furthermore, the spontaneous assembly of catenanes in aqueous dynamic systems gives a fundamental insight into the role played by hydrophobic effect and donor-acceptor interactions when building such complex architectures.

Combinatorial chemistry on solid support in the search for central nervous system agents.

PubMed

Zajdel, Paweł; Pawłowski, Maciej; Martinez, Jean; Subra, Gilles

2009-08-01

The advent of combinatorial chemistry was one of the most important developments, that has significantly contributed to the drug discovery process. Within just a few years, its initial concept aimed at production of libraries containing huge number of compounds (thousands to millions), so called screening libraries, has shifted towards preparation of small and medium-sized rationally designed libraries. When applicable, the use of solid supports for the generation of libraries has been a real breakthrough in enhancing productivity. With a limited amount of resin and simple manual workups, the split/mix procedure may generate thousands of bead-tethered compounds. Beads can be chemically or physically encoded to facilitate the identification of a hit after the biological assay. Compartmentalization of solid supports using small reactors like teabags, kans or pellicular discrete supports like Lanterns resulted in powerful sort and combine technologies, relying on codes 'written' on the reactor, and thus reducing the need for automation and improving the number of compounds synthesized. These methods of solid-phase combinatorial chemistry have been recently supported by introduction of solid-supported reagents and scavenger resins. The first part of this review discusses the general premises of combinatorial chemistry and some methods used in the design of primary and focused combinatorial libraries. The aim of the second part is to present combinatorial chemistry methodologies aimed at discovering bioactive compounds acting on diverse GPCR involved in central nervous system disorders.

Optimized Reaction Conditions for Amide Bond Formation in DNA-Encoded Combinatorial Libraries.

PubMed

Li, Yizhou; Gabriele, Elena; Samain, Florent; Favalli, Nicholas; Sladojevich, Filippo; Scheuermann, Jörg; Neri, Dario

2016-08-08

DNA-encoded combinatorial libraries are increasingly being used as tools for the discovery of small organic binding molecules to proteins of biological or pharmaceutical interest. In the majority of cases, synthetic procedures for the formation of DNA-encoded combinatorial libraries incorporate at least one step of amide bond formation between amino-modified DNA and a carboxylic acid. We investigated reaction conditions and established a methodology by using 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide, 1-hydroxy-7-azabenzotriazole and N,N'-diisopropylethylamine (EDC/HOAt/DIPEA) in combination, which provided conversions greater than 75% for 423/543 (78%) of the carboxylic acids tested. These reaction conditions were efficient with a variety of primary and secondary amines, as well as with various types of amino-modified oligonucleotides. The reaction conditions, which also worked efficiently over a broad range of DNA concentrations and reaction scales, should facilitate the synthesis of novel DNA-encoded combinatorial libraries.

Successful application of the dual-vector system II in creating a reliable phage-displayed combinatorial Fab library.

PubMed

Song, Suk-yoon; Hur, Byung-ung; Lee, Kyung-woo; Choi, Hyo-jung; Kim, Sung-soo; Kang, Goo; Cha, Sang-hoon

2009-03-31

The dual-vector system-II (DVS-II), which allows efficient display of Fab antibodies on phage, has been reported previously, but its practical applicability in a phage-displayed antibody library has not been verified. To resolve this issue, we created two small combinatorial human Fab antibody libraries using the DVS-II, and isolation of target-specific antibodies was attempted. Biopanning of one antibody library, termed DVFAB-1L library, which has a 1.3 x 10(7) combinatorial antibody complexity, against fluorescein-BSA resulted in successful isolation of human Fab clones specific for the antigen despite the presence of only a single light chain in the library. By using the unique feature of the DVS-II, an antibody library of a larger size, named DVFAB-131L, which has a 1.5 x 10(9) combinatorial antibody complexity, was also generated in a rapid manner by combining 1.3 x 10(7) heavy chains and 131 light chains and more diverse anti-fluorescein-BSA Fab antibody clones were successfully obtained. Our results demonstrate that the DVS-II can be applied readily in creating phage-displayed antibody libraries with much less effort, and target-specific antibody clones can be isolated reliably via light chain promiscuity of antibody molecule.

Evaluating the Effect of Peptoid Lipophilicity on Antimicrobial Potency, Cytotoxicity, and Combinatorial Library Design.

PubMed

Turkett, Jeremy A; Bicker, Kevin L

2017-04-10

Growing prevalence of antibiotic resistant bacterial infections necessitates novel antimicrobials, which could be rapidly identified from combinatorial libraries. We report the use of the peptoid library agar diffusion (PLAD) assay to screen peptoid libraries against the ESKAPE pathogens, including the optimization of assay conditions for each pathogen. Work presented here focuses on the tailoring of combinatorial peptoid library design through a detailed study of how peptoid lipophilicity relates to antibacterial potency and mammalian cell toxicity. The information gleaned from this optimization was then applied using the aforementioned screening method to examine the relative potency of peptoid libraries against Staphylococcus aureus, Acinetobacter baumannii, and Enterococcus faecalis prior to and following functionalization with long alkyl tails. The data indicate that overall peptoid hydrophobicity and not simply alkyl tail length is strongly correlated with mammalian cell toxicity. Furthermore, this work demonstrates the utility of the PLAD assay in rapidly evaluating the effect of molecular property changes in similar libraries.

Preparation of 24 ternary thin film materials libraries on a single substrate in one experiment for irreversible high-throughput studies.

PubMed

Buenconsejo, Pio John S; Siegel, Alexander; Savan, Alan; Thienhaus, Sigurd; Ludwig, Alfred

2012-01-09

For different areas of combinatorial materials science, it is desirable to have multiple materials libraries: especially for irreversible high-throughput studies, like, for example, corrosion resistance testing in different media or annealing of complete materials libraries at different temperatures. Therefore a new combinatorial sputter-deposition process was developed which yields 24 materials libraries in one experiment on a single substrate. It is discussed with the example of 24 Ti-Ni-Ag materials libraries. They are divided based on the composition coverage and orientation of composition gradient into two sets of 12 nearly identical materials libraries. Each materials library covers at least 30-40% of the complete ternary composition range. An acid etch test in buffered-HF solution was performed, illustrating the feasibility of our approach for destructive materials characterization. The results revealed that within the composition range of Ni < 30 at.%, the films were severely etched. The composition range which shows reversible martensitic transformations was confirmed to be outside this region. The high output of the present method makes it attractive for combinatorial studies requiring multiple materials libraries.

Fabrication and characterization of thin-film phosphor combinatorial libraries

NASA Astrophysics Data System (ADS)

Mordkovich, V. Z.; Jin, Zhengwu; Yamada, Y.; Fukumura, T.; Kawasaki, M.; Koinuma, H.

2002-05-01

The laser molecular beam epitaxy method was employed to fabricate thin-film combinatorial libraries of ZnO-based phosphors on different substrates. Fabrication of both pixel libraries, on the example of Fe-doped ZnO, and spread libraries, on the example of Eu-doped ZnO, has been demonstrated. Screening of the Fe-doped ZnO libraries led to the discovery of weak green cathodoluminescence with the maximum efficiency at the Fe content of 0.58 mol %. Screening of the Eu-doped ZnO libraries led to the discovery of unusual reddish-violet cathodoluminescence which is observed in a broad range of Eu concentration. No photoluminescence was registered in either system.

Building synthetic gene circuits from combinatorial libraries: screening and selection strategies.

PubMed

Schaerli, Yolanda; Isalan, Mark

2013-07-01

The promise of wide-ranging biotechnology applications inspires synthetic biologists to design novel genetic circuits. However, building such circuits rationally is still not straightforward and often involves painstaking trial-and-error. Mimicking the process of natural selection can help us to bridge the gap between our incomplete understanding of nature's design rules and our desire to build functional networks. By adopting the powerful method of directed evolution, which is usually applied to protein engineering, functional networks can be obtained through screening or selecting from randomised combinatorial libraries. This review first highlights the practical options to introduce combinatorial diversity into gene circuits and then examines strategies for identifying the potentially rare library members with desired functions, either by screening or selection.

Similarity searching and scaffold hopping in synthetically accessible combinatorial chemistry spaces.

PubMed

Boehm, Markus; Wu, Tong-Ying; Claussen, Holger; Lemmen, Christian

2008-04-24

Large collections of combinatorial libraries are an integral element in today's pharmaceutical industry. It is of great interest to perform similarity searches against all virtual compounds that are synthetically accessible by any such library. Here we describe the successful application of a new software tool CoLibri on 358 combinatorial libraries based on validated reaction protocols to create a single chemistry space containing over 10 (12) possible products. Similarity searching with FTrees-FS allows the systematic exploration of this space without the need to enumerate all product structures. The search result is a set of virtual hits which are synthetically accessible by one or more of the existing reaction protocols. Grouping these virtual hits by their synthetic protocols allows the rapid design and synthesis of multiple follow-up libraries. Such library ideas support hit-to-lead design efforts for tasks like follow-up from high-throughput screening hits or scaffold hopping from one hit to another attractive series.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry.

PubMed

Gurevich-Messina, Juan M; Giudicessi, Silvana L; Martínez-Ceron, María C; Acosta, Gerardo; Erra-Balsells, Rosa; Cascone, Osvaldo; Albericio, Fernando; Camperi, Silvia A

2015-01-01

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.

Combinatorial Investigation of ZrO2-Based Dielectric Materials for Dynamic Random-Access Memory Capacitors

NASA Astrophysics Data System (ADS)

Kiyota, Yuji; Itaka, Kenji; Iwashita, Yuta; Adachi, Tetsuya; Chikyow, Toyohiro; Ogura, Atsushi

2011-06-01

We investigated zirconia (ZrO2)-based material libraries in search of new dielectric materials for dynamic random-access memory (DRAM) by combinatorial-pulsed laser deposition (combi-PLD). We found that the substitution of yttrium (Y) to Zr sites in the ZrO2 system suppressed the leakage current effectively. The metal-insulator-metal (MIM) capacitor property of this system showed a leakage current density of less than 5×10-7 A/cm2 and the dielectric constant was 20. Moreover, the addition of titanium (Ti) or tantalum (Ta) to this system caused the dielectric constant to increase to ˜25 within the allowed leakage level of 5×10-7 A/cm2. Therefore, Zr-Y-Ti-O and Zr-Y-Ta-O systems have good potentials for use as new materials with high dielectric constants of DRAM capacitors instead of silicon dioxides (SiO2).

Selective host molecules obtained by dynamic adaptive chemistry.

PubMed

Matache, Mihaela; Bogdan, Elena; Hădade, Niculina D

2014-02-17

Up till 20 years ago, in order to endow molecules with function there were two mainstream lines of thought. One was to rationally design the positioning of chemical functionalities within candidate molecules, followed by an iterative synthesis-optimization process. The second was the use of a "brutal force" approach of combinatorial chemistry coupled with advanced screening for function. Although both methods provided important results, "rational design" often resulted in time-consuming efforts of modeling and synthesis only to find that the candidate molecule was not performing the designed job. "Combinatorial chemistry" suffered from a fundamental limitation related to the focusing of the libraries employed, often using lead compounds that limit its scope. Dynamic constitutional chemistry has developed as a combination of the two approaches above. Through the rational use of reversible chemical bonds together with a large plethora of precursor libraries, one is now able to build functional structures, ranging from quite simple molecules up to large polymeric structures. Thus, by introduction of the dynamic component within the molecular recognition processes, a new perspective of deciphering the world of the molecular events has aroused together with a new field of chemistry. Since its birth dynamic constitutional chemistry has continuously gained attention, in particular due to its ability to easily create from scratch outstanding molecular structures as well as the addition of adaptive features. The fundamental concepts defining the dynamic constitutional chemistry have been continuously extended to currently place it at the intersection between the supramolecular chemistry and newly defined adaptive chemistry, a pivotal feature towards evolutive chemistry. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

High-throughput screening for combinatorial thin-film library of thermoelectric materials.

PubMed

Watanabe, Masaki; Kita, Takuji; Fukumura, Tomoteru; Ohtomo, Akira; Ueno, Kazunori; Kawasaki, Masashi

2008-01-01

A high-throughput method has been developed to evaluate the Seebeck coefficient and electrical resistivity of combinatorial thin-film libraries of thermoelectric materials from room temperature to 673 K. Thin-film samples several millimeters in size were deposited on an integrated Al2O3 substrate with embedded lead wires and local heaters for measurement of the thermopower under a controlled temperature gradient. An infrared camera was used for real-time observation of the temperature difference Delta T between two electrical contacts on the sample to obtain the Seebeck coefficient. The Seebeck coefficient and electrical resistivity of constantan thin films were shown to be almost identical to standard data for bulk constantan. High-throughput screening was demonstrated for a thermoelectric Mg-Si-Ge combinatorial library.

Sequence-independent construction of ordered combinatorial libraries with predefined crossover points.

PubMed

Jézéquel, Laetitia; Loeper, Jacqueline; Pompon, Denis

2008-11-01

Combinatorial libraries coding for mosaic enzymes with predefined crossover points constitute useful tools to address and model structure-function relationships and for functional optimization of enzymes based on multivariate statistics. The presented method, called sequence-independent generation of a chimera-ordered library (SIGNAL), allows easy shuffling of any predefined amino acid segment between two or more proteins. This method is particularly well adapted to the exchange of protein structural modules. The procedure could also be well suited to generate ordered combinatorial libraries independent of sequence similarities in a robotized manner. Sequence segments to be recombined are first extracted by PCR from a single-stranded template coding for an enzyme of interest using a biotin-avidin-based method. This technique allows the reduction of parental template contamination in the final library. Specific PCR primers allow amplification of two complementary mosaic DNA fragments, overlapping in the region to be exchanged. Fragments are finally reassembled using a fusion PCR. The process is illustrated via the construction of a set of mosaic CYP2B enzymes using this highly modular approach.

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules

PubMed Central

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis. PMID:26355961

A Robust and Versatile Method of Combinatorial Chemical Synthesis of Gene Libraries via Hierarchical Assembly of Partially Randomized Modules.

PubMed

Popova, Blagovesta; Schubert, Steffen; Bulla, Ingo; Buchwald, Daniela; Kramer, Wilfried

2015-01-01

A major challenge in gene library generation is to guarantee a large functional size and diversity that significantly increases the chances of selecting different functional protein variants. The use of trinucleotides mixtures for controlled randomization results in superior library diversity and offers the ability to specify the type and distribution of the amino acids at each position. Here we describe the generation of a high diversity gene library using tHisF of the hyperthermophile Thermotoga maritima as a scaffold. Combining various rational criteria with contingency, we targeted 26 selected codons of the thisF gene sequence for randomization at a controlled level. We have developed a novel method of creating full-length gene libraries by combinatorial assembly of smaller sub-libraries. Full-length libraries of high diversity can easily be assembled on demand from smaller and much less diverse sub-libraries, which circumvent the notoriously troublesome long-term archivation and repeated proliferation of high diversity ensembles of phages or plasmids. We developed a generally applicable software tool for sequence analysis of mutated gene sequences that provides efficient assistance for analysis of library diversity. Finally, practical utility of the library was demonstrated in principle by assessment of the conformational stability of library members and isolating protein variants with HisF activity from it. Our approach integrates a number of features of nucleic acids synthetic chemistry, biochemistry and molecular genetics to a coherent, flexible and robust method of combinatorial gene synthesis.

Design of combinatorial libraries for the exploration of virtual hits from fragment space searches with LoFT.

PubMed

Lessel, Uta; Wellenzohn, Bernd; Fischer, J Robert; Rarey, Matthias

2012-02-27

A case study is presented illustrating the design of a focused CDK2 library. The scaffold of the library was detected by a feature trees search in a fragment space based on reactions from combinatorial chemistry. For the design the software LoFT (Library optimizer using Feature Trees) was used. The special feature called FTMatch was applied to restrict the parts of the queries where the reagents are permitted to match. This way a 3D scoring function could be simulated. Results were compared with alternative designs by GOLD docking and ROCS 3D alignments.

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

Design of diversity and focused combinatorial libraries in drug discovery.

PubMed

Young, S Stanley; Ge, Nanxiang

2004-05-01

Using well-characterized chemical reactions and readily available monomers, chemists are able to create sets of compounds, termed libraries, which are useful in drug discovery processes. The design of combinatorial chemical libraries can be complex and there has been much information recently published offering suggestions on how the design process can be carried out. This review focuses on literature with the goal of organizing current thinking. At this point in time, it is clear that benchmarking of current suggested methods is required as opposed to further new methods.

The mathematics of a successful deconvolution: a quantitative assessment of mixture-based combinatorial libraries screened against two formylpeptide receptors.

PubMed

Santos, Radleigh G; Appel, Jon R; Giulianotti, Marc A; Edwards, Bruce S; Sklar, Larry A; Houghten, Richard A; Pinilla, Clemencia

2013-05-30

In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays.

Combinatorially-generated library of 6-fluoroquinolone analogs as potential novel antitubercular agents: a chemometric and molecular modeling assessment.

PubMed

Minovski, Nikola; Perdih, Andrej; Solmajer, Tom

2012-05-01

The virtual combinatorial chemistry approach as a methodology for generating chemical libraries of structurally-similar analogs in a virtual environment was employed for building a general mixed virtual combinatorial library with a total of 53.871 6-FQ structural analogs, introducing the real synthetic pathways of three well known 6-FQ inhibitors. The druggability properties of the generated combinatorial 6-FQs were assessed using an in-house developed drug-likeness filter integrating the Lipinski/Veber rule-sets. The compounds recognized as drug-like were used as an external set for prediction of the biological activity values using a neural-networks (NN) model based on an experimentally-determined set of active 6-FQs. Furthermore, a subset of compounds was extracted from the pool of drug-like 6-FQs, with predicted biological activity, and subsequently used in virtual screening (VS) campaign combining pharmacophore modeling and molecular docking studies. This complex scheme, a powerful combination of chemometric and molecular modeling approaches provided novel QSAR guidelines that could aid in the further lead development of 6-FQs agents.

Combinatorial screening of halide perovskite thin films and solar cells by mask-defined IR laser molecular beam epitaxy

NASA Astrophysics Data System (ADS)

Kawashima, Kazuhiro; Okamoto, Yuji; Annayev, Orazmuhammet; Toyokura, Nobuo; Takahashi, Ryota; Lippmaa, Mikk; Itaka, Kenji; Suzuki, Yoshikazu; Matsuki, Nobuyuki; Koinuma, Hideomi

2017-12-01

As an extension of combinatorial molecular layer epitaxy via ablation of perovskite oxides by a pulsed excimer laser, we have developed a laser molecular beam epitaxy (MBE) system for parallel integration of nano-scaled thin films of organic-inorganic hybrid materials. A pulsed infrared (IR) semiconductor laser was adopted for thermal evaporation of organic halide (A-site: CH3NH3I) and inorganic halide (B-site: PbI2) powder targets to deposit repeated A/B bilayer films where the thickness of each layer was controlled on molecular layer scale by programming the evaporation IR laser pulse number, length, or power. The layer thickness was monitored with an in situ quartz crystal microbalance and calibrated against ex situ stylus profilometer measurements. A computer-controlled movable mask system enabled the deposition of combinatorial thin film libraries, where each library contains a vertically homogeneous film with spatially programmable A- and B-layer thicknesses. On the composition gradient film, a hole transport Spiro-OMeTAD layer was spin-coated and dried followed by the vacuum evaporation of Ag electrodes to form the solar cell. The preliminary cell performance was evaluated by measuring I-V characteristics at seven different positions on the 12.5 mm × 12.5 mm combinatorial library sample with seven 2 mm × 4 mm slits under a solar simulator irradiation. The combinatorial solar cell library clearly demonstrated that the energy conversion efficiency sharply changes from nearly zero to 10.2% as a function of the illumination area in the library. The exploration of deposition parameters for obtaining optimum performance could thus be greatly accelerated. Since the thickness ratio of PbI2 and CH3NH3I can be freely chosen along the shadow mask movement, these experiments show the potential of this system for high-throughput screening of optimum chemical composition in the binary film library and application to halide perovskite solar cell.

Combinatorial screening of halide perovskite thin films and solar cells by mask-defined IR laser molecular beam epitaxy.

PubMed

Kawashima, Kazuhiro; Okamoto, Yuji; Annayev, Orazmuhammet; Toyokura, Nobuo; Takahashi, Ryota; Lippmaa, Mikk; Itaka, Kenji; Suzuki, Yoshikazu; Matsuki, Nobuyuki; Koinuma, Hideomi

2017-01-01

As an extension of combinatorial molecular layer epitaxy via ablation of perovskite oxides by a pulsed excimer laser, we have developed a laser molecular beam epitaxy (MBE) system for parallel integration of nano-scaled thin films of organic-inorganic hybrid materials. A pulsed infrared (IR) semiconductor laser was adopted for thermal evaporation of organic halide (A-site: CH 3 NH 3 I) and inorganic halide (B-site: PbI 2 ) powder targets to deposit repeated A/B bilayer films where the thickness of each layer was controlled on molecular layer scale by programming the evaporation IR laser pulse number, length, or power. The layer thickness was monitored with an in situ quartz crystal microbalance and calibrated against ex situ stylus profilometer measurements. A computer-controlled movable mask system enabled the deposition of combinatorial thin film libraries, where each library contains a vertically homogeneous film with spatially programmable A- and B-layer thicknesses. On the composition gradient film, a hole transport Spiro-OMeTAD layer was spin-coated and dried followed by the vacuum evaporation of Ag electrodes to form the solar cell. The preliminary cell performance was evaluated by measuring I - V characteristics at seven different positions on the 12.5 mm × 12.5 mm combinatorial library sample with seven 2 mm × 4 mm slits under a solar simulator irradiation. The combinatorial solar cell library clearly demonstrated that the energy conversion efficiency sharply changes from nearly zero to 10.2% as a function of the illumination area in the library. The exploration of deposition parameters for obtaining optimum performance could thus be greatly accelerated. Since the thickness ratio of PbI 2 and CH 3 NH 3 I can be freely chosen along the shadow mask movement, these experiments show the potential of this system for high-throughput screening of optimum chemical composition in the binary film library and application to halide perovskite solar cell.

A Rapid Python-Based Methodology for Target-Focused Combinatorial Library Design.

PubMed

Li, Shiliang; Song, Yuwei; Liu, Xiaofeng; Li, Honglin

2016-01-01

The chemical space is so vast that only a small portion of it has been examined. As a complementary approach to systematically probe the chemical space, virtual combinatorial library design has extended enormous impacts on generating novel and diverse structures for drug discovery. Despite the favorable contributions, high attrition rates in drug development that mainly resulted from lack of efficacy and side effects make it increasingly challenging to discover good chemical starting points. In most cases, focused libraries, which are restricted to particular regions of the chemical space, are deftly exploited to maximize hit rate and improve efficiency at the beginning of the drug discovery and drug development pipeline. This paper presented a valid methodology for fast target-focused combinatorial library design in both reaction-based and production-based ways with the library creating rates of approximately 70,000 molecules per second. Simple, quick and convenient operating procedures are the specific features of the method. SHAFTS, a hybrid 3D similarity calculation software, was embedded to help refine the size of the libraries and improve hit rates. Two target-focused (p38-focused and COX2-focused) libraries were constructed efficiently in this study. This rapid library enumeration method is portable and applicable to any other targets for good chemical starting points identification collaborated with either structure-based or ligand-based virtual screening.

Generation of Synthetic Copolymer Libraries by Combinatorial Assembly on Nucleic Acid Templates.

PubMed

Kong, Dehui; Yeung, Wayland; Hili, Ryan

2016-07-11

Recent advances in nucleic acid-templated copolymerization have expanded the scope of sequence-controlled synthetic copolymers beyond the molecular architectures witnessed in nature. This has enabled the power of molecular evolution to be applied to synthetic copolymer libraries to evolve molecular function ranging from molecular recognition to catalysis. This Review seeks to summarize different approaches available to generate sequence-defined monodispersed synthetic copolymer libraries using nucleic acid-templated polymerization. Key concepts and principles governing nucleic acid-templated polymerization, as well as the fidelity of various copolymerization technologies, will be described. The Review will focus on methods that enable the combinatorial generation of copolymer libraries and their molecular evolution for desired function.

A Simple Combinatorial Codon Mutagenesis Method for Targeted Protein Engineering.

PubMed

Belsare, Ketaki D; Andorfer, Mary C; Cardenas, Frida S; Chael, Julia R; Park, Hyun June; Lewis, Jared C

2017-03-17

Directed evolution is a powerful tool for optimizing enzymes, and mutagenesis methods that improve enzyme library quality can significantly expedite the evolution process. Here, we report a simple method for targeted combinatorial codon mutagenesis (CCM). To demonstrate the utility of this method for protein engineering, CCM libraries were constructed for cytochrome P450 BM3 , pfu prolyl oligopeptidase, and the flavin-dependent halogenase RebH; 10-26 sites were targeted for codon mutagenesis in each of these enzymes, and libraries with a tunable average of 1-7 codon mutations per gene were generated. Each of these libraries provided improved enzymes for their respective transformations, which highlights the generality, simplicity, and tunability of CCM for targeted protein engineering.

The Mathematics of a Successful Deconvolution: A Quantitative Assessment of Mixture-Based Combinatorial Libraries Screened Against Two Formylpeptide Receptors

PubMed Central

Santos, Radleigh G.; Appel, Jon R.; Giulianotti, Marc A.; Edwards, Bruce S.; Sklar, Larry A.; Houghten, Richard A.; Pinilla, Clemencia

2014-01-01

In the past 20 years, synthetic combinatorial methods have fundamentally advanced the ability to synthesize and screen large numbers of compounds for drug discovery and basic research. Mixture-based libraries and positional scanning deconvolution combine two approaches for the rapid identification of specific scaffolds and active ligands. Here we present a quantitative assessment of the screening of 32 positional scanning libraries in the identification of highly specific and selective ligands for two formylpeptide receptors. We also compare and contrast two mixture-based library approaches using a mathematical model to facilitate the selection of active scaffolds and libraries to be pursued for further evaluation. The flexibility demonstrated in the differently formatted mixture-based libraries allows for their screening in a wide range of assays. PMID:23722730

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4

NASA Astrophysics Data System (ADS)

Evensen, Erik; Joseph-McCarthy, Diane; Weiss, Gregory A.; Schreiber, Stuart L.; Karplus, Martin

2007-07-01

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.

Review of high-throughput techniques for detecting solid phase Transformation from material libraries produced by combinatorial methods

NASA Technical Reports Server (NTRS)

Lee, Jonathan A.

2005-01-01

High-throughput measurement techniques are reviewed for solid phase transformation from materials produced by combinatorial methods, which are highly efficient concepts to fabricate large variety of material libraries with different compositional gradients on a single wafer. Combinatorial methods hold high potential for reducing the time and costs associated with the development of new materials, as compared to time-consuming and labor-intensive conventional methods that test large batches of material, one- composition at a time. These high-throughput techniques can be automated to rapidly capture and analyze data, using the entire material library on a single wafer, thereby accelerating the pace of materials discovery and knowledge generation for solid phase transformations. The review covers experimental techniques that are applicable to inorganic materials such as shape memory alloys, graded materials, metal hydrides, ferric materials, semiconductors and industrial alloys.

How to synthesize macrocycles efficiently by using virtual combinatorial libraries.

PubMed

Storm, Ole; Lüning, Ulrich

2002-02-15

The selection of different diimines 4 a-c by alkaline earth ions from a virtual combinatorial library (VCL) is described. The products were stabilized by reduction to the diamines 6 a-c; this allowed easy analysis. The library can be directed toward different target molecules 6 a-c upon addition of alkaline earth ions with different radii. Competition experiments show the possibility of synthesizing the macrocycles 6 a, 6 b, and 6 c simultaneously when using Mg(2+), Ca(2+), and Ba(2+) as template ions. The scope of this thermodynamically controlled, reversible approach for macrocycle syntheses is illustrated.

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

PubMed Central

Mondal, Milon; Radeva, Nedyalka; Fanlo‐Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard

2016-01-01

Abstract Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization. PMID:27400756

Identification of Osteoconductive and Biodegradable Polymers from a Combinatorial Polymer Library

PubMed Central

Brey, Darren M.; Chung, Cindy; Hankenson, Kurt D.; Garino, Jonathon P.; Burdick, Jason A.

2012-01-01

Combinatorial polymer syntheses are now being utilized to create libraries of materials with potential utility for a wide variety of biomedical applications. We recently developed a library of photopolymerizable and biodegradable poly(β-amino ester)s (PBAEs) that possessed a range of tunable properties. In this work, the PBAE library was assessed for candidate materials that met design criteria (e.g., physical properties such as degradation and mechanical strength and in vitro cell viability and osteoconductive behavior) for scaffolding in mineralized tissue repair. The most promising candidate, A6, was then processed into 3-dimensional porous scaffolds and implanted subcutaneously and only presented a mild inflammatory response. The scaffolds were then implanted intramuscularly and into a critically-sized cranial defect either alone or loaded with bone morphogenetic protein-2 (BMP-2). The samples in both locations displayed mineralized tissue formation in the presence of BMP-2, as evident through radiographs, micro-computed tomography, and histology, while samples without BMP-2 showed minimal or no mineralized tissue. These results illustrate a process to identify a candidate scaffolding material from a combinatorial polymer library, and specifically for the identification of an osteoconductive scaffold with osteoinductive properties via the inclusion of a growth factor. PMID:20198696

Template-based combinatorial enumeration of virtual compound libraries for lipids

PubMed Central

2012-01-01

A variety of software packages are available for the combinatorial enumeration of virtual libraries for small molecules, starting from specifications of core scaffolds with attachments points and lists of R-groups as SMILES or SD files. Although SD files include atomic coordinates for core scaffolds and R-groups, it is not possible to control 2-dimensional (2D) layout of the enumerated structures generated for virtual compound libraries because different packages generate different 2D representations for the same structure. We have developed a software package called LipidMapsTools for the template-based combinatorial enumeration of virtual compound libraries for lipids. Virtual libraries are enumerated for the specified lipid abbreviations using matching lists of pre-defined templates and chain abbreviations, instead of core scaffolds and lists of R-groups provided by the user. 2D structures of the enumerated lipids are drawn in a specific and consistent fashion adhering to the framework for representing lipid structures proposed by the LIPID MAPS consortium. LipidMapsTools is lightweight, relatively fast and contains no external dependencies. It is an open source package and freely available under the terms of the modified BSD license. PMID:23006594

Template-based combinatorial enumeration of virtual compound libraries for lipids.

PubMed

Sud, Manish; Fahy, Eoin; Subramaniam, Shankar

2012-09-25

A variety of software packages are available for the combinatorial enumeration of virtual libraries for small molecules, starting from specifications of core scaffolds with attachments points and lists of R-groups as SMILES or SD files. Although SD files include atomic coordinates for core scaffolds and R-groups, it is not possible to control 2-dimensional (2D) layout of the enumerated structures generated for virtual compound libraries because different packages generate different 2D representations for the same structure. We have developed a software package called LipidMapsTools for the template-based combinatorial enumeration of virtual compound libraries for lipids. Virtual libraries are enumerated for the specified lipid abbreviations using matching lists of pre-defined templates and chain abbreviations, instead of core scaffolds and lists of R-groups provided by the user. 2D structures of the enumerated lipids are drawn in a specific and consistent fashion adhering to the framework for representing lipid structures proposed by the LIPID MAPS consortium. LipidMapsTools is lightweight, relatively fast and contains no external dependencies. It is an open source package and freely available under the terms of the modified BSD license.

Combining on-chip synthesis of a focused combinatorial library with computational target prediction reveals imidazopyridine GPCR ligands.

PubMed

Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Schneider, Gisbert

2014-01-07

Using the example of the Ugi three-component reaction we report a fast and efficient microfluidic-assisted entry into the imidazopyridine scaffold, where building block prioritization was coupled to a new computational method for predicting ligand-target associations. We identified an innovative GPCR-modulating combinatorial chemotype featuring ligand-efficient adenosine A1/2B and adrenergic α1A/B receptor antagonists. Our results suggest the tight integration of microfluidics-assisted synthesis with computer-based target prediction as a viable approach to rapidly generate bioactivity-focused combinatorial compound libraries with high success rates. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Balancing focused combinatorial libraries based on multiple GPCR ligands

NASA Astrophysics Data System (ADS)

Soltanshahi, Farhad; Mansley, Tamsin E.; Choi, Sun; Clark, Robert D.

2006-08-01

G-Protein coupled receptors (GPCRs) are important targets for drug discovery, and combinatorial chemistry is an important tool for pharmaceutical development. The absence of detailed structural information, however, limits the kinds of combinatorial design techniques that can be applied to GPCR targets. This is particularly problematic given the current emphasis on focused combinatorial libraries. By linking an incremental construction method (OptDesign) to the very fast shape-matching capability of ChemSpace, we have created an efficient method for designing targeted sublibraries that are topomerically similar to known actives. Multi-objective scoring allows consideration of multiple queries (actives) simultaneously. This can lead to a distribution of products skewed towards one particular query structure, however, particularly when the ligands of interest are quite dissimilar to one another. A novel pivoting technique is described which makes it possible to generate promising designs even under those circumstances. The approach is illustrated by application to some serotonergic agonists and chemokine antagonists.

Secretory Overexpression of Bacillus thermocatenulatus Lipase in Saccharomyces cerevisiae Using Combinatorial Library Strategy.

PubMed

Kajiwara, Shota; Yamada, Ryosuke; Ogino, Hiroyasu

2018-04-10

Simple and cost-effective lipase expression host microorganisms are highly desirable. A combinatorial library strategy is used to improve the secretory expression of lipase from Bacillus thermocatenulatus (BTL2) in the culture supernatant of Saccharomyces cerevisiae. A plasmid library including expression cassettes composed of sequences encoding one of each 15 promoters, 15 secretion signals, and 15 terminators derived from yeast species, S. cerevisiae, Pichia pastoris, and Hansenula polymorpha, is constructed. The S. cerevisiae transformant YPH499/D4, comprising H. polymorpha GAP promoter, S. cerevisiae SAG1 secretion signal, and P. pastoris AOX1 terminator, is selected by high-throughput screening. This transformant expresses BTL2 extra-cellularly with a 130-fold higher than the control strain, comprising S. cerevisiae PGK1 promoter, S. cerevisiae α-factor secretion signal, and S. cerevisiae PGK1 terminator, after cultivation for 72 h. This combinatorial library strategy holds promising potential for application in the optimization of the secretory expression of proteins in yeast. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Scaffold diversification enhances effectiveness of a superlibrary of hyperthermophilic proteins.

PubMed

Hussain, Mahmud; Gera, Nimish; Hill, Andrew B; Rao, Balaji M

2013-01-18

The use of binding proteins from non-immunoglobulin scaffolds has become increasingly common in biotechnology and medicine. Typically, binders are isolated from a combinatorial library generated by mutating a single scaffold protein. In contrast, here we generated a "superlibrary" or "library-of-libraries" of 4 × 10(8) protein variants by mutagenesis of seven different hyperthermophilic proteins; six of the seven proteins have not been used as scaffolds prior to this study. Binding proteins for five different model targets were successfully isolated from this library. Binders obtained were derived from five out of the seven scaffolds. Strikingly, binders from this modestly sized superlibrary have affinities comparable or higher than those obtained from a library with 1000-fold higher sequence diversity but derived from a single stable scaffold. Thus scaffold diversification, i.e., randomization of multiple different scaffolds, is a powerful alternate strategy for combinatorial library construction.

Design of self-coded combinatorial libraries to facilitate direct analysis of ligands by mass spectrometry.

PubMed

Hughes, I

1998-09-24

The direct analysis of selected components from combinatorial libraries by sensitive methods such as mass spectrometry is potentially more efficient than deconvolution and tagging strategies since additional steps of resynthesis or introduction of molecular tags are avoided. A substituent selection procedure is described that eliminates the mass degeneracy commonly observed in libraries prepared by "split-and-mix" methods, without recourse to high-resolution mass measurements. A set of simple rules guides the choice of substituents such that all components of the library have unique nominal masses. Additional rules extend the scope by ensuring that characteristic isotopic mass patterns distinguish isobaric components. The method is applicable to libraries having from two to four varying substituent groups and can encode from a few hundred to several thousand components. No restrictions are imposed on the manner in which the "self-coded" library is synthesized or screened.

A Combinatorial Platform for the Optimization of Peptidomimetic Methyl-Lysine Reader Antagonists

NASA Astrophysics Data System (ADS)

Barnash, Kimberly D.

Post-translational modification of histone N-terminal tails mediates chromatin compaction and, consequently, DNA replication, transcription, and repair. While numerous post-translational modifications decorate histone tails, lysine methylation is an abundant mark important for both gene activation and repression. Methyl-lysine (Kme) readers function through binding mono-, di-, or trimethyl-lysine. Chemical intervention of Kme readers faces numerous challenges due to the broad surface-groove interactions between readers and their cognate histone peptides; yet, the increasing interest in understanding chromatin-modifying complexes suggests tractable lead compounds for Kme readers are critical for elucidating the mechanisms of chromatin dysregulation in disease states and validating the druggability of these domains and complexes. The successful discovery of a peptide-derived chemical probe, UNC3866, for the Polycomb repressive complex 1 (PRC1) chromodomain Kme readers has proven the potential for selective peptidomimetic inhibition of reader function. Unfortunately, the systematic modification of peptides-to-peptidomimetics is a costly and inefficient strategy for target-class hit discovery against Kme readers. Through the exploration of biased chemical space via combinatorial on-bead libraries, we have developed two concurrent methodologies for Kme reader chemical probe discovery. We employ biased peptide combinatorial libraries as a hit discovery strategy with subsequent optimization via iterative targeted libraries. Peptide-to-peptidomimetic optimization through targeted library design was applied based on structure-guided library design around the interaction of the endogenous peptide ligand with three target Kme readers. Efforts targeting the WD40 reader EED led to the discovery of the 3-mer peptidomimetic ligand UNC5115 while combinatorial repurposing of UNC3866 for off-target chromodomains resulted in the discovery of UNC4991, a CDYL/2-selective ligand, and UNC4848, a MPP8 and CDYL/2 ligand. Ultimately, our efforts demonstrate the generalizability of a peptidomimetic combinatorial platform for the optimization of Kme reader ligands in a target class manner.

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.

Linkage of Recognition and Replication Functions by Assembling Combinatorial Antibody Fab Libraries Along Phage Surfaces

NASA Astrophysics Data System (ADS)

Kang, Angray S.; Barbas, Carlos F.; Janda, Kim D.; Benkovic, Stephen J.; Lerner, Richard A.

1991-05-01

We describe a method based on a phagemid vector with helper phage rescue for the construction and rapid analysis of combinatorial antibody Fab libraries. This approach should allow the generation and selection of many monoclonal antibodies. Antibody genes are expressed in concert with phage morphogenesis, thereby allowing incorporation of functional Fab molecules along the surface of filamentous phage. The power of the method depends upon the linkage of recognition and replication functions and is not limited to antibody molecules.

Combinatorial screening of halide perovskite thin films and solar cells by mask-defined IR laser molecular beam epitaxy

PubMed Central

Kawashima, Kazuhiro; Okamoto, Yuji; Annayev, Orazmuhammet; Toyokura, Nobuo; Takahashi, Ryota; Lippmaa, Mikk; Itaka, Kenji; Suzuki, Yoshikazu; Matsuki, Nobuyuki; Koinuma, Hideomi

2017-01-01

Abstract As an extension of combinatorial molecular layer epitaxy via ablation of perovskite oxides by a pulsed excimer laser, we have developed a laser molecular beam epitaxy (MBE) system for parallel integration of nano-scaled thin films of organic–inorganic hybrid materials. A pulsed infrared (IR) semiconductor laser was adopted for thermal evaporation of organic halide (A-site: CH3NH3I) and inorganic halide (B-site: PbI2) powder targets to deposit repeated A/B bilayer films where the thickness of each layer was controlled on molecular layer scale by programming the evaporation IR laser pulse number, length, or power. The layer thickness was monitored with an in situ quartz crystal microbalance and calibrated against ex situ stylus profilometer measurements. A computer-controlled movable mask system enabled the deposition of combinatorial thin film libraries, where each library contains a vertically homogeneous film with spatially programmable A- and B-layer thicknesses. On the composition gradient film, a hole transport Spiro-OMeTAD layer was spin-coated and dried followed by the vacuum evaporation of Ag electrodes to form the solar cell. The preliminary cell performance was evaluated by measuring I-V characteristics at seven different positions on the 12.5 mm × 12.5 mm combinatorial library sample with seven 2 mm × 4 mm slits under a solar simulator irradiation. The combinatorial solar cell library clearly demonstrated that the energy conversion efficiency sharply changes from nearly zero to 10.2% as a function of the illumination area in the library. The exploration of deposition parameters for obtaining optimum performance could thus be greatly accelerated. Since the thickness ratio of PbI2 and CH3NH3I can be freely chosen along the shadow mask movement, these experiments show the potential of this system for high-throughput screening of optimum chemical composition in the binary film library and application to halide perovskite solar cell. PMID:28567176

Identification of avocado (Persea americana) pulp proteins by nano-LC-MS/MS via combinatorial peptide ligand libraries.

PubMed

Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

2012-09-01

Avocado (Persea americana) proteins have been scarcely studied despite their importance, especially in food related allergies. The proteome of avocado pulp was explored in depth by extracting proteins with capture by combinatorial peptide ligand libraries at pH 7.4 and under conditions mimicking reverse-phase capture at pH 2.2. The total number of unique gene products identified amounts to 1012 proteins, of which 174 are in common with the control, untreated sample, 190 are present only in the control and 648 represent the new species detected via combinatorial peptide ligand libraries of all combined eluates and likely represent low-abundance proteins. Among the 1012 proteins, it was possible to identify the already known avocado allergen Pers a 1 and different proteins susceptible to be allergens such as a profilin, a polygalacturonase, a thaumatin-like protein, a glucanase, and an isoflavone reductase like protein. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Iterative optimization of xylose catabolism in Saccharomyces cerevisiae using combinatorial expression tuning.

PubMed

Latimer, Luke N; Dueber, John E

2017-06-01

A common challenge in metabolic engineering is rapidly identifying rate-controlling enzymes in heterologous pathways for subsequent production improvement. We demonstrate a workflow to address this challenge and apply it to improving xylose utilization in Saccharomyces cerevisiae. For eight reactions required for conversion of xylose to ethanol, we screened enzymes for functional expression in S. cerevisiae, followed by a combinatorial expression analysis to achieve pathway flux balancing and identification of limiting enzymatic activities. In the next round of strain engineering, we increased the copy number of these limiting enzymes and again tested the eight-enzyme combinatorial expression library in this new background. This workflow yielded a strain that has a ∼70% increase in biomass yield and ∼240% increase in xylose utilization. Finally, we chromosomally integrated the expression library. This library enriched for strains with multiple integrations of the pathway, which likely were the result of tandem integrations mediated by promoter homology. Biotechnol. Bioeng. 2017;114: 1301-1309. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Reducing codon redundancy and screening effort of combinatorial protein libraries created by saturation mutagenesis.

PubMed

Kille, Sabrina; Acevedo-Rocha, Carlos G; Parra, Loreto P; Zhang, Zhi-Gang; Opperman, Diederik J; Reetz, Manfred T; Acevedo, Juan Pablo

2013-02-15

Saturation mutagenesis probes define sections of the vast protein sequence space. However, even if randomization is limited this way, the combinatorial numbers problem is severe. Because diversity is created at the codon level, codon redundancy is a crucial factor determining the necessary effort for library screening. Additionally, due to the probabilistic nature of the sampling process, oversampling is required to ensure library completeness as well as a high probability to encounter all unique variants. Our trick employs a special mixture of three primers, creating a degeneracy of 22 unique codons coding for the 20 canonical amino acids. Therefore, codon redundancy and subsequent screening effort is significantly reduced, and a balanced distribution of codon per amino acid is achieved, as demonstrated exemplarily for a library of cyclohexanone monooxygenase. We show that this strategy is suitable for any saturation mutagenesis methodology to generate less-redundant libraries.

Human Monoclonal Antibodies Against a Plethora of Viral Pathogens From Single Combinatorial Libraries

NASA Astrophysics Data System (ADS)

Williamson, R. Anthony; Burioni, Roberto; Sanna, Pietro P.; Partridge, Lynda J.; Barbas, Carlos F., III; Burton, Dennis R.

1993-05-01

Conventional antibody generation usually requires active immunization with antigen immediately prior to the preparation procedure. Combinatorial antibody library technology offers the possibility of cloning a range of antibody specificities at a single point in time and then accessing these specificities at will. Here we show that human monoclonal antibody Fab fragments against a plethora of infectious agents can be readily derived from a single library. Further examination of a number of libraries shows that whenever antibody against a pathogen can be detected in the serum of the donor, then specific antibodies can be derived from the corresponding library. We describe the generation of human Fab fragments against herpes simplex virus types 1 and 2, human cytomegalovirus, varicella zoster virus, rubella, human immunodeficiency virus type 1, and respiratory syncytial virus. The antibodies are shown to be highly specific and a number are effective in neutralizing virus in vitro.

Rationally reduced libraries for combinatorial pathway optimization minimizing experimental effort.

PubMed

Jeschek, Markus; Gerngross, Daniel; Panke, Sven

2016-03-31

Rational flux design in metabolic engineering approaches remains difficult since important pathway information is frequently not available. Therefore empirical methods are applied that randomly change absolute and relative pathway enzyme levels and subsequently screen for variants with improved performance. However, screening is often limited on the analytical side, generating a strong incentive to construct small but smart libraries. Here we introduce RedLibs (Reduced Libraries), an algorithm that allows for the rational design of smart combinatorial libraries for pathway optimization thereby minimizing the use of experimental resources. We demonstrate the utility of RedLibs for the design of ribosome-binding site libraries by in silico and in vivo screening with fluorescent proteins and perform a simple two-step optimization of the product selectivity in the branched multistep pathway for violacein biosynthesis, indicating a general applicability for the algorithm and the proposed heuristics. We expect that RedLibs will substantially simplify the refactoring of synthetic metabolic pathways.

Discovery of potent inhibitors of soluble epoxide hydrolase by combinatorial library design and structure-based virtual screening.

PubMed

Xing, Li; McDonald, Joseph J; Kolodziej, Steve A; Kurumbail, Ravi G; Williams, Jennifer M; Warren, Chad J; O'Neal, Janet M; Skepner, Jill E; Roberds, Steven L

2011-03-10

Structure-based virtual screening was applied to design combinatorial libraries to discover novel and potent soluble epoxide hydrolase (sEH) inhibitors. X-ray crystal structures revealed unique interactions for a benzoxazole template in addition to the conserved hydrogen bonds with the catalytic machinery of sEH. By exploitation of the favorable binding elements, two iterations of library design based on amide coupling were employed, guided principally by the docking results of the enumerated virtual products. Biological screening of the libraries demonstrated as high as 90% hit rate, of which over two dozen compounds were single digit nanomolar sEH inhibitors by IC(50) determination. In total the library design and synthesis produced more than 300 submicromolar sEH inhibitors. In cellular systems consistent activities were demonstrated with biochemical measurements. The SAR understanding of the benzoxazole template provides valuable insights into discovery of novel sEH inhibitors as therapeutic agents.

Bulk combinatorial synthesis and high throughput characterization for rapid assessment of magnetic materials: Application of laser engineered net shaping (LENS)

DOE PAGES

Geng, J.; Nlebedim, I. C.; Besser, M. F.; ...

2016-04-15

A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. Furthermore, the Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

Predictive Array Design. A method for sampling combinatorial chemistry library space.

PubMed

Lipkin, M J; Rose, V S; Wood, J

2002-01-01

A method, Predictive Array Design, is presented for sampling combinatorial chemistry space and selecting a subarray for synthesis based on the experimental design method of Latin Squares. The method is appropriate for libraries with three sites of variation. Libraries with four sites of variation can be designed using the Graeco-Latin Square. Simulated annealing is used to optimise the physicochemical property profile of the sub-array. The sub-array can be used to make predictions of the activity of compounds in the all combinations array if we assume each monomer has a relatively constant contribution to activity and that the activity of a compound is composed of the sum of the activities of its constitutive monomers.

Sequencing thousands of single-cell genomes with combinatorial indexing.

PubMed

Vitak, Sarah A; Torkenczy, Kristof A; Rosenkrantz, Jimi L; Fields, Andrew J; Christiansen, Lena; Wong, Melissa H; Carbone, Lucia; Steemers, Frank J; Adey, Andrew

2017-03-01

Single-cell genome sequencing has proven valuable for the detection of somatic variation, particularly in the context of tumor evolution. Current technologies suffer from high library construction costs, which restrict the number of cells that can be assessed and thus impose limitations on the ability to measure heterogeneity within a tissue. Here, we present single-cell combinatorial indexed sequencing (SCI-seq) as a means of simultaneously generating thousands of low-pass single-cell libraries for detection of somatic copy-number variants. We constructed libraries for 16,698 single cells from a combination of cultured cell lines, primate frontal cortex tissue and two human adenocarcinomas, and obtained a detailed assessment of subclonal variation within a pancreatic tumor.

Design of focused and restrained subsets from extremely large virtual libraries.

PubMed

Jamois, Eric A; Lin, Chien T; Waldman, Marvin

2003-11-01

With the current and ever-growing offering of reagents along with the vast palette of organic reactions, virtual libraries accessible to combinatorial chemists can reach sizes of billions of compounds or more. Extracting practical size subsets for experimentation has remained an essential step in the design of combinatorial libraries. A typical approach to computational library design involves enumeration of structures and properties for the entire virtual library, which may be unpractical for such large libraries. This study describes a new approach termed as on the fly optimization (OTFO) where descriptors are computed as needed within the subset optimization cycle and without intermediate enumeration of structures. Results reported herein highlight the advantages of coupling an ultra-fast descriptor calculation engine to subset optimization capabilities. We also show that enumeration of properties for the entire virtual library may not only be unpractical but also wasteful. Successful design of focused and restrained subsets can be achieved while sampling only a small fraction of the virtual library. We also investigate the stability of the method and compare results obtained from simulated annealing (SA) and genetic algorithms (GA).

On the suitability of different representations of solid catalysts for combinatorial library design by genetic algorithms.

PubMed

Gobin, Oliver C; Schüth, Ferdi

2008-01-01

Genetic algorithms are widely used to solve and optimize combinatorial problems and are more often applied for library design in combinatorial chemistry. Because of their flexibility, however, their implementation can be challenging. In this study, the influence of the representation of solid catalysts on the performance of genetic algorithms was systematically investigated on the basis of a new, constrained, multiobjective, combinatorial test problem with properties common to problems in combinatorial materials science. Constraints were satisfied by penalty functions, repair algorithms, or special representations. The tests were performed using three state-of-the-art evolutionary multiobjective algorithms by performing 100 optimization runs for each algorithm and test case. Experimental data obtained during the optimization of a noble metal-free solid catalyst system active in the selective catalytic reduction of nitric oxide with propene was used to build up a predictive model to validate the results of the theoretical test problem. A significant influence of the representation on the optimization performance was observed. Binary encodings were found to be the preferred encoding in most of the cases, and depending on the experimental test unit, repair algorithms or penalty functions performed best.

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

PubMed

Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

2016-08-01

Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

Combinatorial synthesis of bimetallic complexes with three halogeno bridges.

PubMed

Gauthier, Sébastien; Quebatte, Laurent; Scopelliti, Rosario; Severin, Kay

2004-06-07

Methods for the synthesis of bimetallic complexes in which two different metal fragments are connected by three chloro or bromo bridges are reported. The reactions are general, fast, and give rise to structurally defined products in quantitative yields. Therefore, they are ideally suited for generating a library of homo- and heterobimetallic complexes in a combinatorial fashion. This is of special interest for applications in homogeneous catalysis. Selected members of this library were synthesized and comprehensively characterized; single-crystal X-ray analyses were performed for 15 new bimetallic compounds.

Evaluation of the Optimum Composition of Low-Temperature Fuel Cell Electrocatalysts for Methanol Oxidation by Combinatorial Screening.

PubMed

Antolini, Ermete

2017-02-13

Combinatorial chemistry and high-throughput screening represent an innovative and rapid tool to prepare and evaluate a large number of new materials, saving time and expense for research and development. Considering that the activity and selectivity of catalysts depend on complex kinetic phenomena, making their development largely empirical in practice, they are prime candidates for combinatorial discovery and optimization. This review presents an overview of recent results of combinatorial screening of low-temperature fuel cell electrocatalysts for methanol oxidation. Optimum catalyst compositions obtained by combinatorial screening were compared with those of bulk catalysts, and the effect of the library geometry on the screening of catalyst composition is highlighted.

Combinatorial Nano-Bio Interfaces.

PubMed

Cai, Pingqiang; Zhang, Xiaoqian; Wang, Ming; Wu, Yun-Long; Chen, Xiaodong

2018-06-08

Nano-bio interfaces are emerging from the convergence of engineered nanomaterials and biological entities. Despite rapid growth, clinical translation of biomedical nanomaterials is heavily compromised by the lack of comprehensive understanding of biophysicochemical interactions at nano-bio interfaces. In the past decade, a few investigations have adopted a combinatorial approach toward decoding nano-bio interfaces. Combinatorial nano-bio interfaces comprise the design of nanocombinatorial libraries and high-throughput bioevaluation. In this Perspective, we address challenges in combinatorial nano-bio interfaces and call for multiparametric nanocombinatorics (composition, morphology, mechanics, surface chemistry), multiscale bioevaluation (biomolecules, organelles, cells, tissues/organs), and the recruitment of computational modeling and artificial intelligence. Leveraging combinatorial nano-bio interfaces will shed light on precision nanomedicine and its potential applications.

Development of hydrogel TentaGel shell-core beads for ultrahigh throughput solution-phase screening of encoded OBOC combinatorial small molecule libraries.

PubMed

Baek, Hyoung Gee; Liu, Ruiwu; Lam, Kit S

2009-01-01

The one-bead one-compound (OBOC) combinatorial library method enables the rapid generation and screening of millions of discrete chemical compounds on beads. Most of the OBOC screening methods require the library compounds to remain tethered to the bead during screening process. Methods have also been developed to release library compounds from immobilized beads for in situ solution phase or "lawn" assays. However, this latter approach, while extremely powerful, is severely limited by the lack of suitable solid supports for such assays. Here, we report on the development of a novel hydrogel TentaGel shell-core (HTSC) bead in which hydrogel is grafted onto the polystyrene-based TentaGel (TG) bead as an outer shell (5-80 mum thick) via free radical surface-initiated polymerization. This novel shell-core bilayer resin enables the preparation of encoded OBOC combinatorial small molecule libraries, such that the library compounds reside on the highly hydrophilic outer layer and the coding tags reside in the polystyrene-based TG core. Using fluorescein as a model small molecule compound, we have demonstrated that fluorescein molecules that have been linked covalently to the hydrogel shell via a disulfide bond could readily diffuse out of the hydrogel layer into the bead surrounding after reduction with dithiothreitol. In contrast, under identical condition, the released fluorescein molecules remained bound to unmodified TG bead. We have prepared an encoded OBOC small molecule library on the novel shell-core beads and demonstrated that the beads can be readily decoded.

Combinatorial Libraries As a Tool for the Discovery of Novel, Broad-Spectrum Antibacterial Agents Targeting the ESKAPE Pathogens.

PubMed

Fleeman, Renee; LaVoi, Travis M; Santos, Radleigh G; Morales, Angela; Nefzi, Adel; Welmaker, Gregory S; Medina-Franco, José L; Giulianotti, Marc A; Houghten, Richard A; Shaw, Lindsey N

2015-04-23

Mixture based synthetic combinatorial libraries offer a tremendous enhancement for the rate of drug discovery, allowing the activity of millions of compounds to be assessed through the testing of exponentially fewer samples. In this study, we used a scaffold-ranking library to screen 37 different libraries for antibacterial activity against the ESKAPE pathogens. Each library contained between 10000 and 750000 structural analogues for a total of >6 million compounds. From this, we identified a bis-cyclic guanidine library that displayed strong antibacterial activity. A positional scanning library for these compounds was developed and used to identify the most effective functional groups at each variant position. Individual compounds were synthesized that were broadly active against all ESKAPE organisms at concentrations <2 μM. In addition, these compounds were bactericidal, had antibiofilm effects, showed limited potential for the development of resistance, and displayed almost no toxicity when tested against human lung cells and erythrocytes. Using a murine model of peritonitis, we also demonstrate that these agents are highly efficacious in vivo.

Combinatorial fabrication and screening of organic light-emitting device arrays

NASA Astrophysics Data System (ADS)

Shinar, Joseph; Shinar, Ruth; Zhou, Zhaoqun

2007-11-01

The combinatorial fabrication and screening of 2-dimensional (2-d) small molecular UV-violet organic light-emitting device (OLED) arrays, 1-d blue-to-red arrays, 1-d intense white OLED libraries, 1-d arrays to study Förster energy transfer in guest-host OLEDs, and 2-d arrays to study exciplex emission from OLEDs is described. The results demonstrate the power of combinatorial approaches for screening OLED materials and configurations, and for studying their basic properties.

London University Search Instrument: a combinatorial robot for high-throughput methods in ceramic science.

PubMed

Wang, Jian; Evans, Julian R G

2005-01-01

This paper describes the design, construction, and operation of the London University Search Instrument (LUSI) which was recently commissioned to create and test combinatorial libraries of ceramic compositions. The instrument uses commercially available powders, milled as necessary to create thick-film libraries by ink-jet printing. Multicomponent mixtures are prepared by well plate reformatting of ceramic inks. The library tiles are robotically loaded into a flatbed furnace and, when fired, transferred to a 2-axis high-resolution measurement table fitted with a hot plate where measurements of, for example, optical or electrical properties can be made. Data are transferred to a dedicated high-performance computer. The possibilities for remote interrogation and search steering are discussed.

Mapping protease substrates using a biotinylated phage substrate library.

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

Scholle, M. D.; Kriplani, U.; Pabon, A.

We describe a bacteriophage M13 substrate library encoding the AviTag (BirA substrate) and combinatorial heptamer peptides displayed at the N terminus of the mature form of capsid protein III. Phages are biotinylated efficiently (> or = 50%) when grown in E. coli cells coexpressing BirA, and such viral particles can be immobilized on a streptavidin-coated support and released by protease cleavage within the combinatorial peptide. We have used this library to map the specificity of human Factor Xa and a neuropeptidase, neurolysin (EC3.4.24.16). Validation by analysis of isolated peptide substrates has revealed that neurolysin recognizes the motif hydrophobic-X-Pro-Arg-hydrophobic, where Arg-hydrophobicmore » is the scissile bond.« less

Parallel and automated library synthesis of 2-long alkyl chain benzoazoles and azole[4,5-b]pyridines under microwave irradiation.

PubMed

Martínez-Palou, Rafael; Zepeda, L Gerardo; Höpfl, Herbert; Montoya, Ascensión; Guzmán-Lucero, Diego J; Guzmán, Javier

2005-01-01

A versatile route to 40-membered library of 2-long alkyl chain substituted benzoazoles (1 and 2) and azole[4,5-b]pyridines (3 and 4) via microwave-assisted combinatorial synthesis was developed. The reactions were carried out in both monomode and multimode microwave oven. With the latter, all reactions were performed in high-throughput experimental settings consisting of an 8 x 5 combinatorial library designed to synthesize 40 compounds. Each step, from the addition of reagents to the recovery of final products, was automated. The microwave-assisted N-long chain alkylation reactions of 2-alkyl-1H-benzimidazole (1) and 2-alkyl-1H-benzimidazole[4,5-b] pyridines (3) were also studied.

Programming gene expression with combinatorial promoters

PubMed Central

Cox, Robert Sidney; Surette, Michael G; Elowitz, Michael B

2007-01-01

Promoters control the expression of genes in response to one or more transcription factors (TFs). The architecture of a promoter is the arrangement and type of binding sites within it. To understand natural genetic circuits and to design promoters for synthetic biology, it is essential to understand the relationship between promoter function and architecture. We constructed a combinatorial library of random promoter architectures. We characterized 288 promoters in Escherichia coli, each containing up to three inputs from four different TFs. The library design allowed for multiple −10 and −35 boxes, and we observed varied promoter strength over five decades. To further analyze the functional repertoire, we defined a representation of promoter function in terms of regulatory range, logic type, and symmetry. Using these results, we identified heuristic rules for programming gene expression with combinatorial promoters. PMID:18004278

On-bead combinatorial synthesis and imaging of chemical exchange saturation transfer magnetic resonance imaging agents to identify factors that influence water exchange.

PubMed

Napolitano, Roberta; Soesbe, Todd C; De León-Rodríguez, Luis M; Sherry, A Dean; Udugamasooriya, D Gomika

2011-08-24

The sensitivity of magnetic resonance imaging (MRI) contrast agents is highly dependent on the rate of water exchange between the inner sphere of a paramagnetic ion and bulk water. Normally, identifying a paramagnetic complex that has optimal water exchange kinetics is done by synthesizing and testing one compound at a time. We report here a rapid, economical on-bead combinatorial synthesis of a library of imaging agents. Eighty different 1,4,7,10-tetraazacyclododecan-1,4,7,10-tetraacetic acid (DOTA)-tetraamide peptoid derivatives were prepared on beads using a variety of charged, uncharged but polar, hydrophobic, and variably sized primary amines. A single chemical exchange saturation transfer image of the on-bead library easily distinguished those compounds having the most favorable water exchange kinetics. This combinatorial approach will allow rapid screening of libraries of imaging agents to identify the chemical characteristics of a ligand that yield the most sensitive imaging agents. This technique could be automated and readily adapted to other types of MRI or magnetic resonance/positron emission tomography agents as well.

Massively multiplex single-cell Hi-C

PubMed Central

Ramani, Vijay; Deng, Xinxian; Qiu, Ruolan; Gunderson, Kevin L; Steemers, Frank J; Disteche, Christine M; Noble, William S; Duan, Zhijun; Shendure, Jay

2016-01-01

We present single-cell combinatorial indexed Hi-C (sciHi-C), which applies the concept of combinatorial cellular indexing to chromosome conformation capture. In this proof-of-concept, we generate and sequence six sciHi-C libraries comprising a total of 10,696 single cells. We use sciHi-C data to separate cells by karytoypic and cell-cycle state differences and identify cell-to-cell heterogeneity in mammalian chromosomal conformation. Our results demonstrate that combinatorial indexing is a generalizable strategy for single-cell genomics. PMID:28135255

Combinatorial Optimization of Heterogeneous Catalysts Used in the Growth of Carbon Nanotubes

NASA Technical Reports Server (NTRS)

Cassell, Alan M.; Verma, Sunita; Delzeit, Lance; Meyyappan, M.; Han, Jie

2000-01-01

Libraries of liquid-phase catalyst precursor solutions were printed onto iridium-coated silicon substrates and evaluated for their effectiveness in catalyzing the growth of multi-walled carbon nanotubes (MWNTs) by chemical vapor deposition (CVD). The catalyst precursor solutions were composed of inorganic salts and a removable tri-block copolymer (EO)20(PO)70(EO)20 (EO = ethylene oxide, PO = propylene oxide) structure-directing agent (SDA), dissolved in ethanol/methanol mixtures. Sample libraries were quickly assayed using scanning electron microscopy after CVD growth to identify active catalysts and CVD conditions. Composition libraries and focus libraries were then constructed around the active spots identified in the discovery libraries to understand how catalyst precursor composition affects the yield, density, and quality of the nanotubes. Successful implementation of combinatorial optimization methods in the development of highly active, carbon nanotube catalysts is demonstrated, as well as the identification of catalyst formulations that lead to varying densities and shapes of aligned nanotube towers.

Automated Combinatorial Chemistry in the Organic Chemistry Majors Laboratory

ERIC Educational Resources Information Center

Nichols, Christopher J.; Hanne, Larry F.

2010-01-01

A multidisciplinary experiment has been developed in which students each synthesize a combinatorial library of 48 hydrazones with the aid of a liquid-handling robot. Each product is then subjected to a Kirby-Bauer disk diffusion assay to assess its antibacterial activity. Students gain experience working with automation and at the…

Synthesis of small combinatorial libraries of natural products: identification and quantification of new long-chain 3-methyl-2-alkanones from the root essential oil of Inula helenium L. (Asteraceae).

PubMed

Radulović, Niko S; Denić, Marija S; Stojanović-Radić, Zorica Z

2014-01-01

Recently, a potent anti-staphylococcal activity of Inula helenium L. (Asteraceae) root essential oil was reported. Also, bioassay guided fractionation of the oil pointed to eudesmane sesquiterpene lactones and a series of unidentified constituents as the main carriers of the observed activity. To identify nine new constituents (long-chain 3-methyl-2-alkanones) from a fraction of this root essential oil with a low minimum inhibitory concentration value (0.8 µg/mL) by employing a synthetic methodology that leads to the formation of a small combinatorial library of these compounds. The identity of these constituents was inferred from mass spectral fragmentation patterns and GC retention data. A library of 3-methyl-2-alkanones (C11 -C19 homologous series) was synthesised in three steps starting from methyl acetoacetate and the corresponding alkyl halides. The synthetic library was also screened for in vitro anti-microbial activity. Gas chromatographic analyses of I. helenium essential oil samples with spiked compounds from the synthesised library corroborated the tentative identifications of the long-chain 3-methyl-2-alkanones. The availability of these anti-microbial compounds from this library made it possible to construct GC/FID calibration curves and determine their content in the plant material: 0.08 - 24.2 mg/100 g of dry roots. The small combinatorial library approach enabled the first unequivocal identification of long-chain 3-methyl-2-alkanones as plant secondary metabolites, and, also, allowed determination of not only a single compound and biological properties, but those of a group of structurally related compounds. Copyright © 2013 John Wiley & Sons, Ltd.

Self-optimizing charge-transfer energy phenomena in metallosupramolecular complexes by dynamic constitutional self-sorting.

PubMed

Legrand, Yves-Marie; van der Lee, Arie; Barboiu, Mihail

2007-11-12

In this paper we report an extended series of 2,6-(iminoarene)pyridine-type ZnII complexes [(Lii)2Zn]II, which were surveyed for their ability to self-exchange both their ligands and their aromatic arms and to form different homoduplex and heteroduplex complexes in solution. The self-sorting of heteroduplex complexes is likely to be the result of geometric constraints. Whereas the imine-exchange process occurs quantitatively in 1:1 mixtures of [(Lii)2Zn]II complexes, the octahedral coordination process around the metal ion defines spatial-frustrated exchanges that involve the selective formation of heterocomplexes of two, by two different substituents; the bulkiest ones (pyrene in principle) specifically interact with the pseudoterpyridine core, sterically hindering the least bulky ones, which are intermolecularly stacked with similar ligands of neighboring molecules. Such a self-sorting process defined by the specific self-constitution of the ligands exchanging their aromatic substituents is self-optimized by a specific control over their spatial orientation around a metal center within the complex. They ultimately show an improved charge-transfer energy function by virtue of the dynamic amplification of self-optimized heteroduplex architectures. These systems therefore illustrate the convergence of the combinatorial self-sorting of the dynamic combinatorial libraries (DCLs) strategy and the constitutional self-optimized function.

Quantum mechanical energy-based screening of combinatorially generated library of tautomers. TauTGen: a tautomer generator program.

PubMed

Harańczyk, Maciej; Gutowski, Maciej

2007-01-01

We describe a procedure of finding low-energy tautomers of a molecule. The procedure consists of (i) combinatorial generation of a library of tautomers, (ii) screening based on the results of geometry optimization of initial structures performed at the density functional level of theory, and (iii) final refinement of geometry for the top hits at the second-order Möller-Plesset level of theory followed by single-point energy calculations at the coupled cluster level of theory with single, double, and perturbative triple excitations. The library of initial structures of various tautomers is generated with TauTGen, a tautomer generator program. The procedure proved to be successful for these molecular systems for which common chemical knowledge had not been sufficient to predict the most stable structures.

Combinatorial synthesis of ceramic materials

DOEpatents

Lauf, Robert J [Oak Ridge, TN; Walls, Claudia A [Oak Ridge, TN; Boatner, Lynn A [Oak Ridge, TN

2010-02-23

A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

Combinatorial synthesis of ceramic materials

DOEpatents

Lauf, Robert J.; Walls, Claudia A.; Boatner, Lynn A.

2006-11-14

A combinatorial library includes a gelcast substrate defining a plurality of cavities in at least one surface thereof; and a plurality of gelcast test materials in the cavities, at least two of the test materials differing from the substrate in at least one compositional characteristic, the two test materials differing from each other in at least one compositional characteristic.

Orthogonal combinatorial mutagenesis: a codon-level combinatorial mutagenesis method useful for low multiplicity and amino acid-scanning protocols

PubMed Central

Gaytán, Paul; Yáñez, Jorge; Sánchez, Filiberto; Soberón, Xavier

2001-01-01

We describe here a method to generate combinatorial libraries of oligonucleotides mutated at the codon-level, with control of the mutagenesis rate so as to create predictable binomial distributions of mutants. The method allows enrichment of the libraries with single, double or larger multiplicity of amino acid replacements by appropriate choice of the mutagenesis rate, depending on the concentration of synthetic precursors. The method makes use of two sets of deoxynucleoside-phosphoramidites bearing orthogonal protecting groups [4,4′-dimethoxytrityl (DMT) and 9-fluorenylmethoxycarbonyl (Fmoc)] in the 5′ hydroxyl. These phosphoramidites are divergently combined during automated synthesis in such a way that wild-type codons are assembled with commercial DMT-deoxynucleoside-methyl-phosphoramidites while mutant codons are assembled with Fmoc-deoxynucleoside-methyl-phosphoramidites in an NNG/C fashion in a single synthesis column. This method is easily automated and suitable for low mutagenesis rates and large windows, such as those required for directed evolution and alanine scanning. Through the assembly of three oligonucleotide libraries at different mutagenesis rates, followed by cloning at the polylinker region of plasmid pUC18 and sequencing of 129 clones, we concluded that the method performs essentially as intended. PMID:11160911

Multiobjective optimization of combinatorial libraries.

PubMed

Agrafiotis, D K

2002-01-01

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

Comparison of dissimilarity measures for cluster analysis of X-ray diffraction data from combinatorial libraries

NASA Astrophysics Data System (ADS)

Iwasaki, Yuma; Kusne, A. Gilad; Takeuchi, Ichiro

2017-12-01

Machine learning techniques have proven invaluable to manage the ever growing volume of materials research data produced as developments continue in high-throughput materials simulation, fabrication, and characterization. In particular, machine learning techniques have been demonstrated for their utility in rapidly and automatically identifying potential composition-phase maps from structural data characterization of composition spread libraries, enabling rapid materials fabrication-structure-property analysis and functional materials discovery. A key issue in development of an automated phase-diagram determination method is the choice of dissimilarity measure, or kernel function. The desired measure reduces the impact of confounding structural data issues on analysis performance. The issues include peak height changes and peak shifting due to lattice constant change as a function of composition. In this work, we investigate the choice of dissimilarity measure in X-ray diffraction-based structure analysis and the choice of measure's performance impact on automatic composition-phase map determination. Nine dissimilarity measures are investigated for their impact in analyzing X-ray diffraction patterns for a Fe-Co-Ni ternary alloy composition spread. The cosine, Pearson correlation coefficient, and Jensen-Shannon divergence measures are shown to provide the best performance in the presence of peak height change and peak shifting (due to lattice constant change) when the magnitude of peak shifting is unknown. With prior knowledge of the maximum peak shifting, dynamic time warping in a normalized constrained mode provides the best performance. This work also serves to demonstrate a strategy for rapid analysis of a large number of X-ray diffraction patterns in general beyond data from combinatorial libraries.

Quantum Efficiency and Bandgap Analysis for Combinatorial Photovoltaics: Sorting Activity of Cu–O Compounds in All-Oxide Device Libraries

PubMed Central

2014-01-01

All-oxide-based photovoltaics (PVs) encompass the potential for extremely low cost solar cells, provided they can obtain an order of magnitude improvement in their power conversion efficiencies. To achieve this goal, we perform a combinatorial materials study of metal oxide based light absorbers, charge transporters, junctions between them, and PV devices. Here we report the development of a combinatorial internal quantum efficiency (IQE) method. IQE measures the efficiency associated with the charge separation and collection processes, and thus is a proxy for PV activity of materials once placed into devices, discarding optical properties that cause uncontrolled light harvesting. The IQE is supported by high-throughput techniques for bandgap fitting, composition analysis, and thickness mapping, which are also crucial parameters for the combinatorial investigation cycle of photovoltaics. As a model system we use a library of 169 solar cells with a varying thickness of sprayed titanium dioxide (TiO2) as the window layer, and covarying thickness and composition of binary compounds of copper oxides (Cu–O) as the light absorber, fabricated by Pulsed Laser Deposition (PLD). The analysis on the combinatorial devices shows the correlation between compositions and bandgap, and their effect on PV activity within several device configurations. The analysis suggests that the presence of Cu4O3 plays a significant role in the PV activity of binary Cu–O compounds. PMID:24410367

Rapid identification of areas of interest in thin film materials libraries by combining electrical, optical, X-ray diffraction, and mechanical high-throughput measurements: a case study for the system Ni-Al.

PubMed

Thienhaus, S; Naujoks, D; Pfetzing-Micklich, J; König, D; Ludwig, A

2014-12-08

The efficient identification of compositional areas of interest in thin film materials systems fabricated by combinatorial deposition methods is essential in combinatorial materials science. We use a combination of compositional screening by EDX together with high-throughput measurements of electrical and optical properties of thin film libraries to determine efficiently the areas of interest in a materials system. Areas of interest are compositions which show distinctive properties. The crystallinity of the thus determined areas is identified by X-ray diffraction. Additionally, by using automated nanoindentation across the materials library, mechanical data of the thin films can be obtained which complements the identification of areas of interest. The feasibility of this approach is demonstrated by using a Ni-Al thin film library as a reference system. The obtained results promise that this approach can be used for the case of ternary and higher order systems.

Solid-phase synthesis and screening of N-acylated polyamine (NAPA) combinatorial libraries for protein binding.

PubMed

Iera, Jaclyn A; Jenkins, Lisa M Miller; Kajiyama, Hiroshi; Kopp, Jeffrey B; Appella, Daniel H

2010-11-15

Inhibitors for protein-protein interactions are challenging to design, in part due to the unique and complex architectures of each protein's interaction domain. Most approaches to develop inhibitors for these interactions rely on rational design, which requires prior structural knowledge of the target and its ligands. In the absence of structural information, a combinatorial approach may be the best alternative to finding inhibitors of a protein-protein interaction. Current chemical libraries, however, consist mostly of molecules designed to inhibit enzymes. In this manuscript, we report the synthesis and screening of a library based on an N-acylated polyamine (NAPA) scaffold that we designed to have specific molecular features necessary to inhibit protein-protein interactions. Screens of the library identified a member with favorable binding properties to the HIV viral protein R (Vpr), a regulatory protein from HIV, that is involved in numerous interactions with other proteins critical for viral replication. Published by Elsevier Ltd.

Viral morphogenesis is the dominant source of sequence censorship in M13 combinatorial peptide phage display.

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

Rodi, D. J.; Soares, A. S.; Makowski, L.

Novel statistical methods have been developed and used to quantitate and annotate the sequence diversity within combinatorial peptide libraries on the basis of small numbers (1-200) of sequences selected at random from commercially available M13 p3-based phage display libraries. These libraries behave statistically as though they correspond to populations containing roughly 4.0{+-}1.6% of the random dodecapeptides and 7.9{+-}2.6% of the random constrained heptapeptides that are theoretically possible within the phage populations. Analysis of amino acid residue occurrence patterns shows no demonstrable influence on sequence censorship by Escherichia coli tRNA isoacceptor profiles or either overall codon or Class II codon usagemore » patterns, suggesting no metabolic constraints on recombinant p3 synthesis. There is an overall depression in the occurrence of cysteine, arginine and glycine residues and an overabundance of proline, threonine and histidine residues. The majority of position-dependent amino acid sequence bias is clustered at three positions within the inserted peptides of the dodecapeptide library, +1, +3 and +12 downstream from the signal peptidase cleavage site. Conformational tendency measures of the peptides indicate a significant preference for inserts favoring a {beta}-turn conformation. The observed protein sequence limitations can primarily be attributed to genetic codon degeneracy and signal peptidase cleavage preferences. These data suggest that for applications in which maximal sequence diversity is essential, such as epitope mapping or novel receptor identification, combinatorial peptide libraries should be constructed using codon-corrected trinucleotide cassettes within vector-host systems designed to minimize morphogenesis-related censorship.« less

cDREM: inferring dynamic combinatorial gene regulation.

PubMed

Wise, Aaron; Bar-Joseph, Ziv

2015-04-01

Genes are often combinatorially regulated by multiple transcription factors (TFs). Such combinatorial regulation plays an important role in development and facilitates the ability of cells to respond to different stresses. While a number of approaches have utilized sequence and ChIP-based datasets to study combinational regulation, these have often ignored the combinational logic and the dynamics associated with such regulation. Here we present cDREM, a new method for reconstructing dynamic models of combinatorial regulation. cDREM integrates time series gene expression data with (static) protein interaction data. The method is based on a hidden Markov model and utilizes the sparse group Lasso to identify small subsets of combinatorially active TFs, their time of activation, and the logical function they implement. We tested cDREM on yeast and human data sets. Using yeast we show that the predicted combinatorial sets agree with other high throughput genomic datasets and improve upon prior methods developed to infer combinatorial regulation. Applying cDREM to study human response to flu, we were able to identify several combinatorial TF sets, some of which were known to regulate immune response while others represent novel combinations of important TFs.

Strategies for the construction and use of peptide and antibody libraries displayed on phages.

PubMed

Pini, Alessandro; Giuliani, Andrea; Ricci, Claudia; Runci, Ylenia; Bracci, Luisa

2004-12-01

Combinatorial chemistry and biology have become popular methods for the identification of bio-active molecules in drug discovery. A widely used technique in combinatorial biology is "phage display", by which peptides, antibody fragments and enzymes are displayed on the surface of bacteriophages, and can be selected by simple procedures of biopanning. The construction of phage libraries of peptides or antibody fragments provides a huge source of ligands and bio-active molecules that can be isolated from the library without laborious studies on antigen characteristics and prediction of ligand structure. This "irrational" approach for the construction of new drugs is extremely rapid and is now used by thousands of laboratories world-wide. The bottleneck in this procedure is the availability of large reliable libraries that can be used repeatedly over the years without loss of ligand expression and diversity. Construction of personalized libraries is therefore important for public and private laboratories engaged in the isolation of specific molecules for therapeutic or diagnostic use. Here we report the general strategies for constructing large phage peptide and antibody libraries, based on the experience of researchers who built the world's most widely used libraries. Particular attention is paid to advanced strategies for the construction, preservation and panning.

Device for preparing combinatorial libraries in powder metallurgy.

PubMed

Yang, Shoufeng; Evans, Julian R G

2004-01-01

This paper describes a powder-metering, -mixing, and -dispensing mechanism that can be used as a method for producing large numbers of samples for metallurgical evaluation or electrical or mechanical testing from multicomponent metal and cermet powder systems. It is designed to make use of the same commercial powders that are used in powder metallurgy and, therefore, to produce samples that are faithful to the microstructure of finished products. The particle assemblies produced by the device could be consolidated by die pressing, isostatic pressing, laser sintering, or direct melting. The powder metering valve provides both on/off and flow rate control of dry powders in open capillaries using acoustic vibration. The valve is simple and involves no relative movement, avoiding seizure with fine powders. An orchestra of such valves can be arranged on a building platform to prepare multicomponent combinatorial libraries. As with many combinatorial devices, identification and evaluation of sources of mixing error as a function of sample size is mandatory. Such an analysis is presented.

Combinatorial Screening for Transgenic Yeasts with High Cellulase Activities in Combination with a Tunable Expression System

PubMed Central

Ito, Yoichiro; Yamanishi, Mamoru; Ikeuchi, Akinori; Imamura, Chie; Matsuyama, Takashi

2015-01-01

Combinatorial screening used together with a broad library of gene expression cassettes is expected to produce a powerful tool for the optimization of the simultaneous expression of multiple enzymes. Recently, we proposed a highly tunable protein expression system that utilized multiple genome-integrated target genes to fine-tune enzyme expression in yeast cells. This tunable system included a library of expression cassettes each composed of three gene-expression control elements that in different combinations produced a wide range of protein expression levels. In this study, four gene expression cassettes with graded protein expression levels were applied to the expression of three cellulases: cellobiohydrolase 1, cellobiohydrolase 2, and endoglucanase 2. After combinatorial screening for transgenic yeasts simultaneously secreting these three cellulases, we obtained strains with higher cellulase expressions than a strain harboring three cellulase-expression constructs within one high-performance gene expression cassette. These results show that our method will be of broad use throughout the field of metabolic engineering. PMID:26692026

Combinatorial Strategies for the Development of Bulk Metallic Glasses

NASA Astrophysics Data System (ADS)

Ding, Shiyan

The systematic identification of multi-component alloys out of the vast composition space is still a daunting task, especially in the development of bulk metallic glasses that are typically based on three or more elements. In order to address this challenge, combinatorial approaches have been proposed. However, previous attempts have not successfully coupled the synthesis of combinatorial libraries with high-throughput characterization methods. The goal of my dissertation is to develop efficient high-throughput characterization methods, optimized to identify glass formers systematically. Here, two innovative approaches have been invented. One is to measure the nucleation temperature in parallel for up-to 800 compositions. The composition with the lowest nucleation temperature has a reasonable agreement with the best-known glass forming composition. In addition, the thermoplastic formability of a metallic glass forming system is determined through blow molding a compositional library. Our results reveal that the composition with the largest thermoplastic deformation correlates well with the best-known formability composition. I have demonstrated both methods as powerful tools to develop new bulk metallic glasses.

De Novo Proteins with Life-Sustaining Functions Are Structurally Dynamic.

PubMed

Murphy, Grant S; Greisman, Jack B; Hecht, Michael H

2016-01-29

Designing and producing novel proteins that fold into stable structures and provide essential biological functions are key goals in synthetic biology. In initial steps toward achieving these goals, we constructed a combinatorial library of de novo proteins designed to fold into 4-helix bundles. As described previously, screening this library for sequences that function in vivo to rescue conditionally lethal mutants of Escherichia coli (auxotrophs) yielded several de novo sequences, termed SynRescue proteins, which rescued four different E. coli auxotrophs. In an effort to understand the structural requirements necessary for auxotroph rescue, we investigated the biophysical properties of the SynRescue proteins, using both computational and experimental approaches. Results from circular dichroism, size-exclusion chromatography, and NMR demonstrate that the SynRescue proteins are α-helical and relatively stable. Surprisingly, however, they do not form well-ordered structures. Instead, they form dynamic structures that fluctuate between monomeric and dimeric states. These findings show that a well-ordered structure is not a prerequisite for life-sustaining functions, and suggests that dynamic structures may have been important in the early evolution of protein function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Selection of stably folded proteins by phage-display with proteolysis.

PubMed

Bai, Yawen; Feng, Hanqiao

2004-05-01

To facilitate the process of protein design and learn the basic rules that control the structure and stability of proteins, combinatorial methods have been developed to select or screen proteins with desired properties from libraries of mutants. One such method uses phage-display and proteolysis to select stably folded proteins. This method does not rely on specific properties of proteins for selection. Therefore, in principle it can be applied to any protein. Since its first demonstration in 1998, the method has been used to create hyperthermophilic proteins, to evolve novel folded domains from a library generated by combinatorial shuffling of polypeptide segments and to convert a partially unfolded structure to a fully folded protein.

Automating gene library synthesis by structure-based combinatorial protein engineering: examples from plant sesquiterpene synthases.

PubMed

Dokarry, Melissa; Laurendon, Caroline; O'Maille, Paul E

2012-01-01

Structure-based combinatorial protein engineering (SCOPE) is a homology-independent recombination method to create multiple crossover gene libraries by assembling defined combinations of structural elements ranging from single mutations to domains of protein structure. SCOPE was originally inspired by DNA shuffling, which mimics recombination during meiosis, where mutations from parental genes are "shuffled" to create novel combinations in the resulting progeny. DNA shuffling utilizes sequence identity between parental genes to mediate template-switching events (the annealing and extension of one parental gene fragment on another) in PCR reassembly reactions to generate crossovers and hence recombination between parental genes. In light of the conservation of protein structure and degeneracy of sequence, SCOPE was developed to enable the "shuffling" of distantly related genes with no requirement for sequence identity. The central principle involves the use of oligonucleotides to encode for crossover regions to choreograph template-switching events during PCR assembly of gene fragments to create chimeric genes. This approach was initially developed to create libraries of hybrid DNA polymerases from distantly related parents, and later developed to create a combinatorial mutant library of sesquiterpene synthases to explore the catalytic landscapes underlying the functional divergence of related enzymes. This chapter presents a simplified protocol of SCOPE that can be integrated with different mutagenesis techniques and is suitable for automation by liquid-handling robots. Two examples are presented to illustrate the application of SCOPE to create gene libraries using plant sesquiterpene synthases as the model system. In the first example, we outline how to create an active-site library as a series of complex mixtures of diverse mutants. In the second example, we outline how to create a focused library as an array of individual clones to distil minimal combinations of functionally important mutations. Through these examples, the principles of the technique are illustrated and the suitability of automating various aspects of the procedure for given applications are discussed. Copyright © 2012 Elsevier Inc. All rights reserved.

Synthesis and cell-free cloning of DNA libraries using programmable microfluidics

PubMed Central

Yehezkel, Tuval Ben; Rival, Arnaud; Raz, Ofir; Cohen, Rafael; Marx, Zipora; Camara, Miguel; Dubern, Jean-Frédéric; Koch, Birgit; Heeb, Stephan; Krasnogor, Natalio; Delattre, Cyril; Shapiro, Ehud

2016-01-01

Microfluidics may revolutionize our ability to write synthetic DNA by addressing several fundamental limitations associated with generating novel genetic constructs. Here we report the first de novo synthesis and cell-free cloning of custom DNA libraries in sub-microliter reaction droplets using programmable digital microfluidics. Specifically, we developed Programmable Order Polymerization (POP), Microfluidic Combinatorial Assembly of DNA (M-CAD) and Microfluidic In-vitro Cloning (MIC) and applied them to de novo synthesis, combinatorial assembly and cell-free cloning of genes, respectively. Proof-of-concept for these methods was demonstrated by programming an autonomous microfluidic system to construct and clone libraries of yeast ribosome binding sites and bacterial Azurine, which were then retrieved in individual droplets and validated. The ability to rapidly and robustly generate designer DNA molecules in an autonomous manner should have wide application in biological research and development. PMID:26481354

Combinatorial genetic perturbation to refine metabolic circuits for producing biofuels and biochemicals.

PubMed

Kim, Hyo Jin; Turner, Timothy Lee; Jin, Yong-Su

2013-11-01

Recent advances in metabolic engineering have enabled microbial factories to compete with conventional processes for producing fuels and chemicals. Both rational and combinatorial approaches coupled with synthetic and systematic tools play central roles in metabolic engineering to create and improve a selected microbial phenotype. Compared to knowledge-based rational approaches, combinatorial approaches exploiting biological diversity and high-throughput screening have been demonstrated as more effective tools for improving various phenotypes of interest. In particular, identification of unprecedented targets to rewire metabolic circuits for maximizing yield and productivity of a target chemical has been made possible. This review highlights general principles and the features of the combinatorial approaches using various libraries to implement desired phenotypes for strain improvement. In addition, recent applications that harnessed the combinatorial approaches to produce biofuels and biochemicals will be discussed. Copyright © 2013 Elsevier Inc. All rights reserved.

Creating the New from the Old: Combinatorial Libraries Generation with Machine-Learning-Based Compound Structure Optimization.

PubMed

Podlewska, Sabina; Czarnecki, Wojciech M; Kafel, Rafał; Bojarski, Andrzej J

2017-02-27

The growing computational abilities of various tools that are applied in the broadly understood field of computer-aided drug design have led to the extreme popularity of virtual screening in the search for new biologically active compounds. Most often, the source of such molecules consists of commercially available compound databases, but they can also be searched for within the libraries of structures generated in silico from existing ligands. Various computational combinatorial approaches are based solely on the chemical structure of compounds, using different types of substitutions for new molecules formation. In this study, the starting point for combinatorial library generation was the fingerprint referring to the optimal substructural composition in terms of the activity toward a considered target, which was obtained using a machine learning-based optimization procedure. The systematic enumeration of all possible connections between preferred substructures resulted in the formation of target-focused libraries of new potential ligands. The compounds were initially assessed by machine learning methods using a hashed fingerprint to represent molecules; the distribution of their physicochemical properties was also investigated, as well as their synthetic accessibility. The examination of various fingerprints and machine learning algorithms indicated that the Klekota-Roth fingerprint and support vector machine were an optimal combination for such experiments. This study was performed for 8 protein targets, and the obtained compound sets and their characterization are publically available at http://skandal.if-pan.krakow.pl/comb_lib/ .

High-throughput combinatorial chemical bath deposition: The case of doping Cu (In, Ga) Se film with antimony

NASA Astrophysics Data System (ADS)

Yan, Zongkai; Zhang, Xiaokun; Li, Guang; Cui, Yuxing; Jiang, Zhaolian; Liu, Wen; Peng, Zhi; Xiang, Yong

2018-01-01

The conventional methods for designing and preparing thin film based on wet process remain a challenge due to disadvantages such as time-consuming and ineffective, which hinders the development of novel materials. Herein, we present a high-throughput combinatorial technique for continuous thin film preparation relied on chemical bath deposition (CBD). The method is ideally used to prepare high-throughput combinatorial material library with low decomposition temperatures and high water- or oxygen-sensitivity at relatively high-temperature. To check this system, a Cu(In, Ga)Se (CIGS) thin films library doped with 0-19.04 at.% of antimony (Sb) was taken as an example to evaluate the regulation of varying Sb doping concentration on the grain growth, structure, morphology and electrical properties of CIGS thin film systemically. Combined with the Energy Dispersive Spectrometer (EDS), X-ray Photoelectron Spectroscopy (XPS), automated X-ray Diffraction (XRD) for rapid screening and Localized Electrochemical Impedance Spectroscopy (LEIS), it was confirmed that this combinatorial high-throughput system could be used to identify the composition with the optimal grain orientation growth, microstructure and electrical properties systematically, through accurately monitoring the doping content and material composition. According to the characterization results, a Sb2Se3 quasi-liquid phase promoted CIGS film-growth model has been put forward. In addition to CIGS thin film reported here, the combinatorial CBD also could be applied to the high-throughput screening of other sulfide thin film material systems.

Phenotype-information-phenotype cycle for deconvolution of combinatorial antibody libraries selected against complex systems.

PubMed

Zhang, Hongkai; Torkamani, Ali; Jones, Teresa M; Ruiz, Diana I; Pons, Jaume; Lerner, Richard A

2011-08-16

Use of large combinatorial antibody libraries and next-generation sequencing of nucleic acids are two of the most powerful methods in modern molecular biology. The libraries are screened using the principles of evolutionary selection, albeit in real time, to enrich for members with a particular phenotype. This selective process necessarily results in the loss of information about less-fit molecules. On the other hand, sequencing of the library, by itself, gives information that is mostly unrelated to phenotype. If the two methods could be combined, the full potential of very large molecular libraries could be realized. Here we report the implementation of a phenotype-information-phenotype cycle that integrates information and gene recovery. After selection for phage-encoded antibodies that bind to targets expressed on the surface of Escherichia coli, the information content of the selected pool is obtained by pyrosequencing. Sequences that encode specific antibodies are identified by a bioinformatic analysis and recovered by a stringent affinity method that is uniquely suited for gene isolation from a highly degenerate collection of nucleic acids. This approach can be generalized for selection of antibodies against targets that are present as minor components of complex systems.

System dynamics of subcellular transport.

PubMed

Chen, Vivien Y; Khersonsky, Sonya M; Shedden, Kerby; Chang, Young Tae; Rosania, Gus R

2004-01-01

In pharmacokinetic experiments, interpretations often hinge on treating cells as a "black box": a single, lumped compartment or boundary. Here, a combinatorial library of fluorescent small molecules was used to visualize subcellular transport pathways in living cells, using a kinetic, high content imaging system to monitor spatiotemporal variations of intracellular probe distribution. Most probes accumulate in cytoplasmic vesicles and probe kinetics conform to a nested, two-compartment dynamical system. At steady state, probes preferentially partition from the extracellular medium to the cytosol, and from the cytosol to cytoplasmic vesicles, with hydrophobic molecules favoring sequestration. Altogether, these results point to a general organizing principle underlying the system dynamics of subcellular, small molecule transport. In addition to plasma membrane permeability, subcellular transport phenomena can determine the active concentration of small molecules in the cytosol and the efflux of small molecules from cells. Fundamentally, direct observation of intracellular probe distribution challenges the simple boundary model of classical pharmacokinetics, which considers cells as static permeability barriers.

Application of visual basic in high-throughput mass spectrometry-directed purification of combinatorial libraries.

PubMed

Li, B; Chan, E C Y

2003-01-01

We present an approach to customize the sample submission process for high-throughput purification (HTP) of combinatorial parallel libraries using preparative liquid chromatography electrospray ionization mass spectrometry. In this study, Visual Basic and Visual Basic for Applications programs were developed using Microsoft Visual Basic 6 and Microsoft Excel 2000, respectively. These programs are subsequently applied for the seamless electronic submission and handling of data for HTP. Functions were incorporated into these programs where medicinal chemists can perform on-line verification of the purification status and on-line retrieval of postpurification data. The application of these user friendly and cost effective programs in our HTP technology has greatly increased our work efficiency by reducing paper work and manual manipulation of data.

Going nuts for nuts? The trace proteome of a Cola drink, as detected via combinatorial peptide ligand libraries.

PubMed

D'Amato, Alfonsina; Fasoli, Elisa; Kravchuk, Alexander V; Righetti, Pier Giorgio

2011-05-06

The "invisible" proteome of a Cola drink, stated to be produced with a kola nut extract, has been investigated via capture with combinatorial peptide ligand libraries (CPLL). Indeed, a few proteins in the M(r) 15-20 kDa range could be identified by treating large beverage volumes (1 L) and performing the capture with CPLLs at very acidic pH values (pH 2.2) under conditions mimicking reverse-phase adsorption. Ascertaining the presence of proteins deriving from plant extracts has confirmed the genuineness of such beverage and suggests the possibility of certifying whether soft drinks present on the market are indeed made with vegetable extracts or only with artificial chemical flavoring.

Poisson Statistics of Combinatorial Library Sampling Predict False Discovery Rates of Screening

PubMed Central

2017-01-01

Microfluidic droplet-based screening of DNA-encoded one-bead-one-compound combinatorial libraries is a miniaturized, potentially widely distributable approach to small molecule discovery. In these screens, a microfluidic circuit distributes library beads into droplets of activity assay reagent, photochemically cleaves the compound from the bead, then incubates and sorts the droplets based on assay result for subsequent DNA sequencing-based hit compound structure elucidation. Pilot experimental studies revealed that Poisson statistics describe nearly all aspects of such screens, prompting the development of simulations to understand system behavior. Monte Carlo screening simulation data showed that increasing mean library sampling (ε), mean droplet occupancy, or library hit rate all increase the false discovery rate (FDR). Compounds identified as hits on k > 1 beads (the replicate k class) were much more likely to be authentic hits than singletons (k = 1), in agreement with previous findings. Here, we explain this observation by deriving an equation for authenticity, which reduces to the product of a library sampling bias term (exponential in k) and a sampling saturation term (exponential in ε) setting a threshold that the k-dependent bias must overcome. The equation thus quantitatively describes why each hit structure’s FDR is based on its k class, and further predicts the feasibility of intentionally populating droplets with multiple library beads, assaying the micromixtures for function, and identifying the active members by statistical deconvolution. PMID:28682059

Combinatorial Methods for Exploring Complex Materials

NASA Astrophysics Data System (ADS)

Amis, Eric J.

2004-03-01

Combinatorial and high-throughput methods have changed the paradigm of pharmaceutical synthesis and have begun to have a similar impact on materials science research. Already there are examples of combinatorial methods used for inorganic materials, catalysts, and polymer synthesis. For many investigations the primary goal has been discovery of new material compositions that optimize properties such as phosphorescence or catalytic activity. In the midst of the excitement generated to "make things", another opportunity arises for materials science to "understand things" by using the efficiency of combinatorial methods. We have shown that combinatorial methods hold potential for rapid and systematic generation of experimental data over the multi-parameter space typical of investigations in polymer physics. We have applied the combinatorial approach to studies of polymer thin films, biomaterials, polymer blends, filled polymers, and semicrystalline polymers. By combining library fabrication, high-throughput measurements, informatics, and modeling we can demonstrate validation of the methodology, new observations, and developments toward predictive models. This talk will present some of our latest work with applications to coating stability, multi-component formulations, and nanostructure assembly.

Combinatorial games with a pass: a dynamical systems approach.

PubMed

Morrison, Rebecca E; Friedman, Eric J; Landsberg, Adam S

2011-12-01

By treating combinatorial games as dynamical systems, we are able to address a longstanding open question in combinatorial game theory, namely, how the introduction of a "pass" move into a game affects its behavior. We consider two well known combinatorial games, 3-pile Nim and 3-row Chomp. In the case of Nim, we observe that the introduction of the pass dramatically alters the game's underlying structure, rendering it considerably more complex, while for Chomp, the pass move is found to have relatively minimal impact. We show how these results can be understood by recasting these games as dynamical systems describable by dynamical recursion relations. From these recursion relations, we are able to identify underlying structural connections between these "games with passes" and a recently introduced class of "generic (perturbed) games." This connection, together with a (non-rigorous) numerical stability analysis, allows one to understand and predict the effect of a pass on a game.

Double Dutch: A Tool for Designing Combinatorial Libraries of Biological Systems.

PubMed

Roehner, Nicholas; Young, Eric M; Voigt, Christopher A; Gordon, D Benjamin; Densmore, Douglas

2016-06-17

Recently, semirational approaches that rely on combinatorial assembly of characterized DNA components have been used to engineer biosynthetic pathways. In practice, however, it is not practical to assemble and test millions of pathway variants in order to elucidate how different DNA components affect the behavior of a pathway. To address this challenge, we apply a rigorous mathematical approach known as design of experiments (DOE) that can be used to construct empirical models of system behavior without testing all variants. To support this approach, we have developed a tool named Double Dutch, which uses a formal grammar and heuristic algorithms to automate the process of DOE library design. Compared to designing by hand, Double Dutch enables users to more efficiently and scalably design libraries of pathway variants that can be used in a DOE framework and uniquely provides a means to flexibly balance design considerations of statistical analysis, construction cost, and risk of homologous recombination, thereby demonstrating the utility of automating decision making when faced with complex design trade-offs.

Combinatorial investigation of Fe–B thin-film nanocomposites

PubMed Central

Brunken, Hayo; Grochla, Dario; Savan, Alan; Kieschnick, Michael; Meijer, Jan D; Ludwig, Alfred

2011-01-01

Combinatorial magnetron sputter deposition from elemental targets was used to create Fe–B composition spread type thin film materials libraries on thermally oxidized 4-in. Si wafers. The materials libraries consisting of wedge-type multilayer thin films were annealed at 500 or 700 °C to transform the multilayers into multiphase alloys. The libraries were characterized by nuclear reaction analysis, Rutherford backscattering, nanoindentation, vibrating sample magnetometry, x-ray diffraction (XRD) and transmission electron microscopy (TEM). Young's modulus and hardness values were related to the annealing parameters, structure and composition of the films. The magnetic properties of the films were improved by annealing in a H2 atmosphere, showing a more than tenfold decrease in the coercive field values in comparison to those of the vacuum-annealed films. The hardness values increased from 8 to 18 GPa when the annealing temperature was increased from 500 to 700 °C. The appearance of Fe2B phases, as revealed by XRD and TEM, had a significant effect on the mechanical properties of the films. PMID:27877435

Vector Design Tour de Force: Integrating Combinatorial and Rational Approaches to Derive Novel Adeno-associated Virus Variants

PubMed Central

Marsic, Damien; Govindasamy, Lakshmanan; Currlin, Seth; Markusic, David M; Tseng, Yu-Shan; Herzog, Roland W; Agbandje-McKenna, Mavis; Zolotukhin, Sergei

2014-01-01

Methodologies to improve existing adeno-associated virus (AAV) vectors for gene therapy include either rational approaches or directed evolution to derive capsid variants characterized by superior transduction efficiencies in targeted tissues. Here, we integrated both approaches in one unified design strategy of “virtual family shuffling” to derive a combinatorial capsid library whereby only variable regions on the surface of the capsid are modified. Individual sublibraries were first assembled in order to preselect compatible amino acid residues within restricted surface-exposed regions to minimize the generation of dead-end variants. Subsequently, the successful families were interbred to derive a combined library of ~8 × 105 complexity. Next-generation sequencing of the packaged viral DNA revealed capsid surface areas susceptible to directed evolution, thus providing guidance for future designs. We demonstrated the utility of the library by deriving an AAV2-based vector characterized by a 20-fold higher transduction efficiency in murine liver, now equivalent to that of AAV8. PMID:25048217

Combinatorial antibody libraries from survivors of the Turkish H5N1 avian influenza outbreak reveal virus neutralization strategies.

PubMed

Kashyap, Arun K; Steel, John; Oner, Ahmet F; Dillon, Michael A; Swale, Ryann E; Wall, Katherine M; Perry, Kimberly J; Faynboym, Aleksandr; Ilhan, Mahmut; Horowitz, Michael; Horowitz, Lawrence; Palese, Peter; Bhatt, Ramesh R; Lerner, Richard A

2008-04-22

The widespread incidence of H5N1 influenza viruses in bird populations poses risks to human health. Although the virus has not yet adapted for facile transmission between humans, it can cause severe disease and often death. Here we report the generation of combinatorial antibody libraries from the bone marrow of five survivors of the recent H5N1 avian influenza outbreak in Turkey. To date, these libraries have yielded >300 unique antibodies against H5N1 viral antigens. Among these antibodies, we have identified several broadly reactive neutralizing antibodies that could be used for passive immunization against H5N1 virus or as guides for vaccine design. The large number of antibodies obtained from these survivors provide a detailed immunochemical analysis of individual human solutions to virus neutralization in the setting of an actual virulent influenza outbreak. Remarkably, three of these antibodies neutralized both H1 and H5 subtype influenza viruses.

Unnatural amino acids increase sensitivity and provide for the design of highly selective caspase substrates

PubMed Central

Poreba, M; Kasperkiewicz, P; Snipas, S J; Fasci, D; Salvesen, G S; Drag, M

2014-01-01

Traditional combinatorial peptidyl substrate library approaches generally utilize natural amino acids, limiting the usefulness of this tool in generating selective substrates for proteases that share similar substrate specificity profiles. To address this limitation, we synthesized a Hybrid Combinatorial Substrate Library (HyCoSuL) with the general formula of Ac-P4-P3-P2-Asp-ACC, testing the approach on a family of closely related proteases – the human caspases. The power of this library for caspase discrimination extends far beyond traditional PS-SCL approach, as in addition to 19 natural amino acids we also used 110 diverse unnatural amino acids that can more extensively explore the chemical space represented by caspase-active sites. Using this approach we identified and employed peptide-based substrates that provided excellent discrimination between individual caspases, allowing us to simultaneously resolve the individual contribution of the apical caspase-9 and the executioner caspase-3 and caspase-7 in the development of cytochrome-c-dependent apoptosis for the first time. PMID:24832467

Design of ultrasensitive probes for human neutrophil elastase through hybrid combinatorial substrate library profiling

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Parker, Heather; Winterbourn, Christine C.; Salvesen, Guy S.; Drag, Marcin

2014-01-01

The exploration of protease substrate specificity is generally restricted to naturally occurring amino acids, limiting the degree of conformational space that can be surveyed. We substantially enhanced this by incorporating 102 unnatural amino acids to explore the S1–S4 pockets of human neutrophil elastase. This approach provides hybrid natural and unnatural amino acid sequences, and thus we termed it the Hybrid Combinatorial Substrate Library. Library results were validated by the synthesis of individual tetrapeptide substrates, with the optimal substrate demonstrating more than three orders of magnitude higher catalytic efficiency than commonly used substrates of elastase. This optimal substrate was converted to an activity-based probe that demonstrated high selectivity and revealed the specific presence of active elastase during the process of neutrophil extracellular trap formation. We propose that this approach can be successfully used for any type of endopeptidase to deliver high activity and selectivity in substrates and probes. PMID:24550277

Dynamic covalent chemistry of bisimines at the solid/liquid interface monitored by scanning tunnelling microscopy.

PubMed

Ciesielski, Artur; El Garah, Mohamed; Haar, Sébastien; Kovaříček, Petr; Lehn, Jean-Marie; Samorì, Paolo

2014-11-01

Dynamic covalent chemistry relies on the formation of reversible covalent bonds under thermodynamic control to generate dynamic combinatorial libraries. It provides access to numerous types of complex functional architectures, and thereby targets several technologically relevant applications, such as in drug discovery, (bio)sensing and dynamic materials. In liquid media it was proved that by taking advantage of the reversible nature of the bond formation it is possible to combine the error-correction capacity of supramolecular chemistry with the robustness of covalent bonding to generate adaptive systems. Here we show that double imine formation between 4-(hexadecyloxy)benzaldehyde and different α,ω-diamines as well as reversible bistransimination reactions can be achieved at the solid/liquid interface, as monitored on the submolecular scale by in situ scanning tunnelling microscopy imaging. Our modular approach enables the structurally controlled reversible incorporation of various molecular components to form sophisticated covalent architectures, which opens up perspectives towards responsive multicomponent two-dimensional materials and devices.

The Use of Electrospray Mass Spectrometry to Determine Speciation in a Dynamic Combinatorial Library for Anion Recognition

PubMed Central

Phillips, Hazel I A; Chernikov, Aleksey V; Fletcher, Nicholas C; Ashcroft, Alison E; Ault, James R; Filby, Maria H; Wilson, Andrew J

2012-01-01

The composition of a dynamic mixture of similar 2,2′-bipyridine complexes of iron(II) bearing either an amide (5-benzylamido-2,2′-bipyridine and 5-(2-methoxyethane)amido-2,2′-bipyridine) or an ester (2,2′-bipyridine-5-carboxylic acid benzylester and 2,2′-bipyridine-5-carboxylic acid 2-methoxyethane ester) side chain have been evaluated by electrospray mass spectroscopy in acetonitrile. The time taken for the complexes to come to equilibrium appears to be dependent on the counteranion, with chloride causing a rapid redistribution of two preformed heteroleptic complexes (of the order of 1 hour), whereas the time it takes in the presence of tetrafluoroborate salts is in excess of 24 h. Similarly the final distribution of products is dependent on the anion present, with the presence of chloride, and to a lesser extent bromide, preferring three amide-functionalized ligands, and a slight preference for an appended benzyl over a methoxyethyl group. Furthermore, for the first time, this study shows that the distribution of a dynamic library of metal complexes monitored by ESI-MS can adapt following the introduction of a different anion, in this case tetrabutylammonium chloride to give the most favoured heteroleptic complex despite the increasing ionic strength of the solution. PMID:22996943

High-throughput characterization of film thickness in thin film materials libraries by digital holographic microscopy.

PubMed

Lai, Yiu Wai; Krause, Michael; Savan, Alan; Thienhaus, Sigurd; Koukourakis, Nektarios; Hofmann, Martin R; Ludwig, Alfred

2011-10-01

A high-throughput characterization technique based on digital holography for mapping film thickness in thin-film materials libraries was developed. Digital holographic microscopy is used for fully automatic measurements of the thickness of patterned films with nanometer resolution. The method has several significant advantages over conventional stylus profilometry: it is contactless and fast, substrate bending is compensated, and the experimental setup is simple. Patterned films prepared by different combinatorial thin-film approaches were characterized to investigate and demonstrate this method. The results show that this technique is valuable for the quick, reliable and high-throughput determination of the film thickness distribution in combinatorial materials research. Importantly, it can also be applied to thin films that have been structured by shadow masking.

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

PubMed

Makeyev, E V; Kolb, V A; Spirin, A S

1999-02-12

A novel cloning-independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single-chain antibody repertoire and express it directly in a cell-free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Applications of high throughput (combinatorial) methodologies to electronic, magnetic, optical, and energy-related materials

NASA Astrophysics Data System (ADS)

Green, Martin L.; Takeuchi, Ichiro; Hattrick-Simpers, Jason R.

2013-06-01

High throughput (combinatorial) materials science methodology is a relatively new research paradigm that offers the promise of rapid and efficient materials screening, optimization, and discovery. The paradigm started in the pharmaceutical industry but was rapidly adopted to accelerate materials research in a wide variety of areas. High throughput experiments are characterized by synthesis of a "library" sample that contains the materials variation of interest (typically composition), and rapid and localized measurement schemes that result in massive data sets. Because the data are collected at the same time on the same "library" sample, they can be highly uniform with respect to fixed processing parameters. This article critically reviews the literature pertaining to applications of combinatorial materials science for electronic, magnetic, optical, and energy-related materials. It is expected that high throughput methodologies will facilitate commercialization of novel materials for these critically important applications. Despite the overwhelming evidence presented in this paper that high throughput studies can effectively inform commercial practice, in our perception, it remains an underutilized research and development tool. Part of this perception may be due to the inaccessibility of proprietary industrial research and development practices, but clearly the initial cost and availability of high throughput laboratory equipment plays a role. Combinatorial materials science has traditionally been focused on materials discovery, screening, and optimization to combat the extremely high cost and long development times for new materials and their introduction into commerce. Going forward, combinatorial materials science will also be driven by other needs such as materials substitution and experimental verification of materials properties predicted by modeling and simulation, which have recently received much attention with the advent of the Materials Genome Initiative. Thus, the challenge for combinatorial methodology will be the effective coupling of synthesis, characterization and theory, and the ability to rapidly manage large amounts of data in a variety of formats.

[Status of libraries and databases for natural products at abroad].

PubMed

Zhao, Li-Mei; Tan, Ning-Hua

2015-01-01

For natural products are one of the important sources for drug discovery, libraries and databases of natural products are significant for the development and research of natural products. At present, most of compound libraries at abroad are synthetic or combinatorial synthetic molecules, resulting to access natural products difficult; for information of natural products are scattered with different standards, it is difficult to construct convenient, comprehensive and large-scale databases for natural products. This paper reviewed the status of current accessing libraries and databases for natural products at abroad and provided some important information for the development of libraries and database for natural products.

Designing a multiroute synthesis scheme in combinatorial chemistry.

PubMed

Akavia, Adi; Senderowitz, Hanoch; Lerner, Alon; Shamir, Ron

2004-01-01

Solid-phase mix-and-split combinatorial synthesis is often used to produce large arrays of compounds to be tested during the various stages of the drug development process. This method can be represented by a synthesis graph in which nodes correspond to grow operations and arcs to beads transferred among the different reaction vessels. In this work, we address the problem of designing such a graph which maximizes the number of produced target compounds (namely, compounds out of an input library of desired molecules), given constraints on the number of beads used for library synthesis and on the number of reaction vessels available for concurrent grow steps. We present a heuristic based on a discrete search for solving this problem, test our solution on several data sets, explore its behavior, and show that it achieves good performance.

Development of a large peptoid-DOTA combinatorial library.

PubMed

Singh, Jaspal; Lopes, Daniel; Gomika Udugamasooriya, D

2016-09-01

Conventional one-bead one-compound (OBOC) library synthesis is typically used to identify molecules with therapeutic value. The design and synthesis of OBOC libraries that contain molecules with imaging or even potentially therapeutic and diagnostic capacities (e.g. theranostic agents) has been overlooked. The development of a therapeutically active molecule with a built-in imaging component for a certain target is a daunting task, and structure-based rational design might not be the best approach. We hypothesize to develop a combinatorial library with potentially therapeutic and imaging components fused together in each molecule. Such molecules in the library can be used to screen, identify, and validate as direct theranostic candidates against targets of interest. As the first step in achieving that aim, we developed an on-bead library of 153,600 Peptoid-DOTA compounds in which the peptoids are the target-recognizing and potentially therapeutic components and the DOTA is the imaging component. We attached the DOTA scaffold to TentaGel beads using one of the four arms of DOTA, and we built a diversified 6-mer peptoid library on the remaining three arms. We evaluated both the synthesis and the mass spectrometric sequencing capacities of the test compounds and of the final library. The compounds displayed unique ionization patterns including direct breakages of the DOTA scaffold into two units, allowing clear decoding of the sequences. Our approach provides a facile synthesis method for the complete on-bead development of large peptidomimetic-DOTA libraries for screening against biological targets for the identification of potential theranostic agents in the future. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 673-684, 2016. © 2016 The Authors. Biopolymers Published by Wiley Periodicals, Inc.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-11-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis.

A binary plasmid system for shuffling combinatorial antibody libraries.

PubMed Central

Collet, T A; Roben, P; O'Kennedy, R; Barbas, C F; Burton, D R; Lerner, R A

1992-01-01

We have used a binary system of replicon-compatible plasmids to test the potential for promiscuous recombination of heavy and light chains within sets of human Fab fragments isolated from combinatorial antibody libraries. Antibody molecules showed a surprising amount of promiscuity in that a particular heavy chain could recombine with multiple light chains with retention of binding to a protein antigen. The degree to which a given heavy chain productively paired with any light chain to bind antigen varied from 43% to 100% and depended strongly on the heavy-chain sequence. Such productive crosses resulted in a set of Fab fragments of similar apparent binding constants, which seemed to differ mainly in the amount of active Fab fragment produced in the bacterial cell. The dominance of the heavy chain in the antibody-antigen interaction was further explored in a set of directed crosses, in which heavy and light chains derived from antigen-specific clones were crossed with nonrelated heavy and light chains. In these crosses, an Fab fragment retained antigen binding only if it contained a heavy chain from an antigen-specific clone. In no case did the light chain confer detectable affinity when paired with indifferent heavy chains. The surprising promiscuity of heavy chains has ramifications for the evaluation of the diversity of combinatorial libraries made against protein antigens and should allow the combination of one such promiscuous heavy chain with an engineered light chain to form an Fab fragment carrying synthetic cofactors to assist in antibody catalysis. Images PMID:1438192

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.

Protocols for the Design of Kinase-focused Compound Libraries.

PubMed

Jacoby, Edgar; Wroblowski, Berthold; Buyck, Christophe; Neefs, Jean-Marc; Meyer, Christophe; Cummings, Maxwell D; van Vlijmen, Herman

2018-05-01

Protocols for the design of kinase-focused compound libraries are presented. Kinase-focused compound libraries can be differentiated based on the design goal. Depending on whether the library should be a discovery library specific for one particular kinase, a general discovery library for multiple distinct kinase projects, or even phenotypic screening, there exists today a variety of in silico methods to design candidate compound libraries. We address the following scenarios: 1) Datamining of SAR databases and kinase focused vendor catalogues; 2) Predictions and virtual screening; 3) Structure-based design of combinatorial kinase inhibitors; 4) Design of covalent kinase inhibitors; 5) Design of macrocyclic kinase inhibitors; and 6) Design of allosteric kinase inhibitors and activators. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemical Space of DNA-Encoded Libraries.

PubMed

Franzini, Raphael M; Randolph, Cassie

2016-07-28

In recent years, DNA-encoded chemical libraries (DECLs) have attracted considerable attention as a potential discovery tool in drug development. Screening encoded libraries may offer advantages over conventional hit discovery approaches and has the potential to complement such methods in pharmaceutical research. As a result of the increased application of encoded libraries in drug discovery, a growing number of hit compounds are emerging in scientific literature. In this review we evaluate reported encoded library-derived structures and identify general trends of these compounds in relation to library design parameters. We in particular emphasize the combinatorial nature of these libraries. Generally, the reported molecules demonstrate the ability of this technology to afford hits suitable for further lead development, and on the basis of them, we derive guidelines for DECL design.

Genetic Selection for Improved Abzymes in E. Coli

DTIC Science & Technology

1994-08-03

immunoassay to scemen antibody phage libraries directly for catalysis of a bimolecular Diels - Alder reaction and have identified several active clones...sensitive growth selection assay in F coli for catalysts with chorismate mutase activity; and (3) we identified new abzymes for a Diels - Alder ...generated combinatorial antibody libraries from mRNA isolated from the spleens of mice hyperimmunized with transition state analogs for Diels - Alder and

Parallel solution-phase synthesis of a 2-aminothiazole library including fully automated work-up.

PubMed

Buchstaller, Hans-Peter; Anlauf, Uwe

2011-02-01

A straightforward and effective procedure for the solution phase preparation of a 2-aminothiazole combinatorial library is described. Reaction, work-up and isolation of the title compounds as free bases was accomplished in a fully automated fashion using the Chemspeed ASW 2000 automated synthesizer. The compounds were obtained in good yields and excellent purities without any further purification procedure.

Neural Meta-Memes Framework for Combinatorial Optimization

NASA Astrophysics Data System (ADS)

Song, Li Qin; Lim, Meng Hiot; Ong, Yew Soon

In this paper, we present a Neural Meta-Memes Framework (NMMF) for combinatorial optimization. NMMF is a framework which models basic optimization algorithms as memes and manages them dynamically when solving combinatorial problems. NMMF encompasses neural networks which serve as the overall planner/coordinator to balance the workload between memes. We show the efficacy of the proposed NMMF through empirical study on a class of combinatorial problem, the quadratic assignment problem (QAP).

Inhibition of multidrug resistant Listeria monocytogenes by peptides isolated from combinatorial phage display libraries.

PubMed

Flachbartova, Z; Pulzova, L; Bencurova, E; Potocnakova, L; Comor, L; Bednarikova, Z; Bhide, M

2016-01-01

The aim of the study was to isolate and characterize novel antimicrobial peptides from peptide phage library with antimicrobial activity against multidrug resistant Listeria monocytogenes. Combinatorial phage-display library was used to affinity select peptides binding to the cell surface of multidrug resistant L. monocytogenes. After several rounds of affinity selection followed by sequencing, three peptides were revealed as the most promising candidates. Peptide L2 exhibited features common to antimicrobial peptides (AMPs), and was rich in Asp, His and Lys residues. Peptide L3 (NSWIQAPDTKSI), like peptide L2, inhibited bacterial growth in vitro, without any hemolytic or cytotoxic effects on eukaryotic cells. L1 peptide showed no inhibitory effect on Listeria. Structurally, peptides L2 and L3 formed random coils composed of α-helix and β-sheet units. Peptides L2 and L3 exhibited antimicrobial activity against multidrug resistant isolates of L. monocytogenes with no haemolytic or toxic effects. Both peptides identified in this study have the potential to be beneficial in human and veterinary medicine. Copyright © 2016 Elsevier GmbH. All rights reserved.

Combinatorial peptide libraries and biometric score matrices permit the quantitative analysis of specific and degenerate interactions between clonotypic TCR and MHC peptide ligands.

PubMed

Zhao, Y; Gran, B; Pinilla, C; Markovic-Plese, S; Hemmer, B; Tzou, A; Whitney, L W; Biddison, W E; Martin, R; Simon, R

2001-08-15

The interaction of TCRs with MHC peptide ligands can be highly flexible, so that many different peptides are recognized by the same TCR in the context of a single restriction element. We provide a quantitative description of such interactions, which allows the identification of T cell epitopes and molecular mimics. The response of T cell clones to positional scanning synthetic combinatorial libraries is analyzed with a mathematical approach that is based on a model of independent contribution of individual amino acids to peptide Ag recognition. This biometric analysis compares the information derived from these libraries composed of trillions of decapeptides with all the millions of decapeptides contained in a protein database to rank and predict the most stimulatory peptides for a given T cell clone. We demonstrate the predictive power of the novel strategy and show that, together with gene expression profiling by cDNA microarrays, it leads to the identification of novel candidate autoantigens in the inflammatory autoimmune disease, multiple sclerosis.

A phage display vector optimized for the generation of human antibody combinatorial libraries and the molecular cloning of monoclonal antibody fragments.

PubMed

Solforosi, Laura; Mancini, Nicasio; Canducci, Filippo; Clementi, Nicola; Sautto, Giuseppe Andrea; Diotti, Roberta Antonia; Clementi, Massimo; Burioni, Roberto

2012-07-01

A novel phagemid vector, named pCM, was optimized for the cloning and display of antibody fragment (Fab) libraries on the surface of filamentous phage. This vector contains two long DNA "stuffer" fragments for easier differentiation of the correctly cut forms of the vector. Moreover, in pCM the fragment at the heavy-chain cloning site contains an acid phosphatase-encoding gene allowing an easy distinction of the Escherichia coli cells containing the unmodified form of the phagemid versus the heavy-chain fragment coding cDNA. In pCM transcription of heavy-chain Fd/gene III and light chain is driven by a single lacZ promoter. The light chain is directed to the periplasm by the ompA signal peptide, whereas the heavy-chain Fd/coat protein III is trafficked by the pelB signal peptide. The phagemid pCM was used to generate a human combinatorial phage display antibody library that allowed the selection of a monoclonal Fab fragment antibody directed against the nucleoprotein (NP) of Influenza A virus.

Sensitive electrospray mass spectrometry analysis of one-bead-one-compound peptide libraries labeled by quaternary ammonium salts.

PubMed

Bąchor, Remigiusz; Cydzik, Marzena; Rudowska, Magdalena; Kluczyk, Alicja; Stefanowicz, Piotr; Szewczuk, Zbigniew

2012-08-01

A rapid and straightforward method for high-throughput analysis of single resin beads from one-bead-one-compound combinatorial libraries with high resolution electrospray ionization tandem mass spectrometry (HR ESI-MS/MS) is presented. The application of an efficient method of peptide derivatization by quaternary ammonium salts (QAS) formation increases ionization efficiency and reduces the detection limit, allowing analysis of trace amounts of compounds by ESI-MS. Peptides, synthesized on solid support, contain a new cleavable linker composed of a Peg spacer (9-aza-3,6,12,15-tetraoxa-10-on-heptadecanoic acid), lysine with ɛ-amino group marked by the N,N,N-triethylglycine salt, and methionine, which makes possible the selective cleavage by cyanogen bromide. Even a small portion of peptides derivatized by QAS cleaved from a single resin bead is sufficient for sequencing by HR ESI-MS/MS experiments. The developed strategy was applied to a small training library of α chymotrypsin substrates. The obtained results confirm the applicability of the proposed method in combinatorial chemistry.

A Combinatorial H4 Tail Library to Explore the Histone Code

PubMed Central

Garske, Adam L.; Craciun, Gheorghe; Denu, John M.

2008-01-01

Histone modifications modulate chromatin structure and function. A posttranslational modification-randomized, combinatorial library based on the first twenty-one residues of histone H4 was designed for systematic examination of proteins that interpret a histone code. The 800-member library represented all permutations of most known modifications within the N-terminal tail of histone H4. To determine its utility in a protein-binding assay, the on-bead library was screened with an antibody directed against phosphoserine 1 of H4. Among the hits, 59/60 sequences were phosphorylated at S1, while 30/30 of those selected from the non-hits were unphosphorylated. A 512-member version of the library was then used to determine the binding specificity of the double tudor domain of hJMJD2A, a histone demethylase involved in transcriptional repression. Global linear least squares fitting of modifications from the identified peptides (40 hits and 34 non-hits) indicated that methylation of K20 was the primary determinant for binding, but that phosphorylation/acetylation on neighboring sites attenuated the interaction. To validate the on-bead screen, isothermal titration calorimetry was performed with thirteen H4 peptides. Dissociation constants ranged from 1 mM - 1μM and corroborated the screening results. The general approach should be useful for probing the specificity of any histone-binding protein. PMID:18616348

Molecular insights of protein contour recognition with ligand pharmacophoric sites through combinatorial library design and MD simulation in validating HTLV-1 PR inhibitors.

PubMed

Selvaraj, Chandrabose; Omer, Ankur; Singh, Poonam; Singh, Sanjeev Kumar

2015-01-01

Retroviruses HIV-1 and HTLV-1 are chiefly considered to be the most dangerous pathogens in Homo sapiens. These two viruses have structurally unique protease (PR) enzymes, which are having common function of its replication mechanism. Though HIV PR drugs failed to inhibit HTLV-1 infections, they emphatically emphasise the need for designing new lead compounds against HTLV-1 PR. Therefore, we tried to understand the binding level interactions through the charge environment present in both ligand and protein active sites. The domino effect illustrates that libraries of purvalanol-A are attuned to fill allosteric binding site of HTLV-1 PR through molecular recognition and shows proper binding of ligand pharmacophoric features in receptor contours. Our screening evaluates seven compounds from purvalanol-A libraries, and these compounds' pharmacophore searches for an appropriate place in the binding site and it places well according to respective receptor contour surfaces. Thus our result provides a platform for the progress of more effective compounds, which are better in free energy calculation, molecular docking, ADME and molecular dynamics studies. Finally, this research provided novel chemical scaffolds for HTLV-1 drug discovery.

Extremal problems for topological indices in combinatorial chemistry.

PubMed

Tichy, Robert F; Wagner, Stephan

2005-09-01

Topological indices of molecular graphs are related to several physicochemical characteristics; recently, the inverse problem for some of these indices has been studied, and it has some applications in the design of combinatorial libraries for drug discovery. It is thus very natural to study also extremal problems for these indices, i.e., finding graphs having minimal or maximal index. In this paper, these questions will be discussed for three different indices, namely the sigma-index, the c-index and the Z-index, with emphasis on the sigma-index.

Laser direct writing of combinatorial libraries of idealized cellular constructs: Biomedical applications

NASA Astrophysics Data System (ADS)

Schiele, Nathan R.; Koppes, Ryan A.; Corr, David T.; Ellison, Karen S.; Thompson, Deanna M.; Ligon, Lee A.; Lippert, Thomas K. M.; Chrisey, Douglas B.

2009-03-01

The ability to control cell placement and to produce idealized cellular constructs is essential for understanding and controlling intercellular processes and ultimately for producing engineered tissue replacements. We have utilized a novel intra-cavity variable aperture excimer laser operated at 193 nm to reproducibly direct write mammalian cells with micrometer resolution to form a combinatorial array of idealized cellular constructs. We deposited patterns of human dermal fibroblasts, mouse myoblasts, rat neural stem cells, human breast cancer cells, and bovine pulmonary artery endothelial cells to study aspects of collagen network formation, breast cancer progression, and neural stem cell proliferation, respectively. Mammalian cells were deposited by matrix assisted pulsed laser evaporation direct write from ribbons comprised of a UV transparent quartz coated with either a thin layer of extracellular matrix or triazene as a dynamic release layer using CAD/CAM control. We demonstrate that through optical imaging and incorporation of a machine vision algorithm, specific cells on the ribbon can be laser deposited in spatial coherence with respect to geometrical arrays and existing cells on the receiving substrate. Having the ability to direct write cells into idealized cellular constructs can help to answer many biomedical questions and advance tissue engineering and cancer research.

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

PubMed

Martínez-Ceron, María C; Giudicessi, Silvana L; Marani, Mariela M; Albericio, Fernando; Cascone, Osvaldo; Erra-Balsells, Rosa; Camperi, Silvia A

2010-05-15

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix. Copyright 2010 Elsevier Inc. All rights reserved.

Phage display for the discovery of hydroxyapatite-associated peptides.

PubMed

Jin, Hyo-Eon; Chung, Woo-Jae; Lee, Seung-Wuk

2013-01-01

In nature, proteins play a critical role in the biomineralization process. Understanding how different peptide or protein sequences selectively interact with the target crystal is of great importance. Identifying such protein structures is one of the critical steps in verifying the molecular mechanisms of biomineralization. One of the promising ways to obtain such information for a particular crystal surface is to screen combinatorial peptide libraries in a high-throughput manner. Among the many combinatorial library screening procedures, phage display is a powerful method to isolate such proteins and peptides. In this chapter, we will describe our established methods to perform phage display with inorganic crystal surfaces. Specifically, we will use hydroxyapatite as a model system for discovery of apatite-associated proteins in bone or tooth biomineralization studies. This model approach can be generalized to other desired crystal surfaces using the same experimental design principles with a little modification of the procedures. © 2013 Elsevier Inc. All rights reserved.

Heuristic Implementation of Dynamic Programming for Matrix Permutation Problems in Combinatorial Data Analysis

ERIC Educational Resources Information Center

Brusco, Michael J.; Kohn, Hans-Friedrich; Stahl, Stephanie

2008-01-01

Dynamic programming methods for matrix permutation problems in combinatorial data analysis can produce globally-optimal solutions for matrices up to size 30x30, but are computationally infeasible for larger matrices because of enormous computer memory requirements. Branch-and-bound methods also guarantee globally-optimal solutions, but computation…

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery.

PubMed

O'Reilly, Rachel K; Turberfield, Andrew J; Wilks, Thomas R

2017-10-17

Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify "successful" structures. The largest libraries made by conventional synthesis are currently of the order of 10 8 distinct molecules. To put this in context, there are 10 13 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure-a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery-imagine being able to evolve molecules for light harvesting, or catalysts for CO 2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous "DNA robots" have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution.

The Evolution of DNA-Templated Synthesis as a Tool for Materials Discovery

PubMed Central

2017-01-01

Conspectus Precise control over reactivity and molecular structure is a fundamental goal of the chemical sciences. Billions of years of evolution by natural selection have resulted in chemical systems capable of information storage, self-replication, catalysis, capture and production of light, and even cognition. In all these cases, control over molecular structure is required to achieve a particular function: without structural control, function may be impaired, unpredictable, or impossible. The search for molecules with a desired function is often achieved by synthesizing a combinatorial library, which contains many or all possible combinations of a set of chemical building blocks (BBs), and then screening this library to identify “successful” structures. The largest libraries made by conventional synthesis are currently of the order of 108 distinct molecules. To put this in context, there are 1013 ways of arranging the 21 proteinogenic amino acids in chains up to 10 units long. Given that we know that a number of these compounds have potent biological activity, it would be highly desirable to be able to search them all to identify leads for new drug molecules. Large libraries of oligonucleotides can be synthesized combinatorially and translated into peptides using systems based on biological replication such as mRNA display, with selected molecules identified by DNA sequencing; but these methods are limited to BBs that are compatible with cellular machinery. In order to search the vast tracts of chemical space beyond nucleic acids and natural peptides, an alternative approach is required. DNA-templated synthesis (DTS) could enable us to meet this challenge. DTS controls chemical product formation by using the specificity of DNA hybridization to bring selected reactants into close proximity, and is capable of the programmed synthesis of many distinct products in the same reaction vessel. By making use of dynamic, programmable DNA processes, it is possible to engineer a system that can translate instructions coded as a sequence of DNA bases into a chemical structure—a process analogous to the action of the ribosome in living organisms but with the potential to create a much more chemically diverse set of products. It is also possible to ensure that each product molecule is tagged with its identifying DNA sequence. Compound libraries synthesized in this way can be exposed to selection against suitable targets, enriching successful molecules. The encoding DNA can then be amplified using the polymerase chain reaction and decoded by DNA sequencing. More importantly, the DNA instruction sequences can be mutated and reused during multiple rounds of amplification, translation, and selection. In other words, DTS could be used as the foundation for a system of synthetic molecular evolution, which could allow us to efficiently search a vast chemical space. This has huge potential to revolutionize materials discovery—imagine being able to evolve molecules for light harvesting, or catalysts for CO2 fixation. The field of DTS has developed to the point where a wide variety of reactions can be performed on a DNA template. Complex architectures and autonomous “DNA robots” have been implemented for the controlled assembly of BBs, and these mechanisms have in turn enabled the one-pot synthesis of large combinatorial libraries. Indeed, DTS libraries are being exploited by pharmaceutical companies and have already found their way into drug lead discovery programs. This Account explores the processes involved in DTS and highlights the challenges that remain in creating a general system for molecular discovery by evolution. PMID:28915003

Natural products and combinatorial chemistry: back to the future.

PubMed

Ortholand, Jean-Yves; Ganesan, A

2004-06-01

The introduction of high-throughput synthesis and combinatorial chemistry has precipitated a global decline in the screening of natural products by the pharmaceutical industry. Some companies terminated their natural products program, despite the unproven success of the new technologies. This was a premature decision, as natural products have a long history of providing important medicinal agents. Furthermore, they occupy a complementary region of chemical space compared with the typical synthetic compound library. For these reasons, the interest in natural products has been rekindled. Various approaches have evolved that combine the power of natural products and organic chemistry, ranging from the combinatorial total synthesis of analogues to the exploration of natural product scaffolds and the design of completely unnatural molecules that resemble natural products in their molecular characteristics.

SO2 and NH3 gas adsorption on a ternary ZnO/CuO/CuCl2 impregnated activated carbon evaluated using combinatorial methods.

PubMed

Romero, Jennifer V; Smith, Jock W H; Sullivan, Braden M; Croll, Lisa M; Dahn, J R

2012-01-09

Ternary libraries of 64 ZnO/CuO/CuCl(2) impregnated activated carbon samples were prepared on untreated or HNO(3)-treated carbon and evaluated for their SO(2) and NH(3) gas adsorption properties gravimetrically using a combinatorial method. CuCl(2) is shown to be a viable substitute for HNO(3) and some compositions of ternary ZnO/CuO/CuCl(2) impregnated carbon samples prepared on untreated carbon provided comparable SO(2) and NH(3) gas removal capacities to the materials prepared on HNO(3)-treated carbon. Through combinatorial methods, it was determined that the use of HNO(3) in this multigas adsorbent formulation can be avoided.

DNA Assembly Techniques for Next Generation Combinatorial Biosynthesis of Natural Products

PubMed Central

Cobb, Ryan E.; Ning, Jonathan C.; Zhao, Huimin

2013-01-01

Natural product scaffolds remain important leads for pharmaceutical development. However, transforming a natural product into a drug entity often requires derivatization to enhance the compound’s therapeutic properties. A powerful method by which to perform this derivatization is combinatorial biosynthesis, the manipulation of the genes in the corresponding pathway to divert synthesis towards novel derivatives. While these manipulations have traditionally been carried out via restriction digestion/ligation-based cloning, the shortcomings of such techniques limit their throughput and thus the scope of corresponding combinatorial biosynthesis experiments. In the burgeoning field of synthetic biology, the demand for facile DNA assembly techniques has promoted the development of a host of novel DNA assembly strategies. Here we describe the advantages of these recently-developed tools for rapid, efficient synthesis of large DNA constructs. We also discuss their potential to facilitate the simultaneous assembly of complete libraries of natural product biosynthetic pathways, ushering in the next generation of combinatorial biosynthesis. PMID:24127070

Solution-phase parallel synthesis of hexahydro-1H-isoindolone libraries via tactical combination of Cu-catalyzed three-component coupling and Diels-Alder reactions.

PubMed

Zhang, Lei; Lushington, Gerald H; Neuenswander, Benjamin; Hershberger, John C; Malinakova, Helena C

2008-01-01

Parallel solution-phase synthesis of combinatorial libraries of hexahydro-1 H-isoindolones exploiting a novel "tactical combination" of Cu-catalyzed three-component coupling and Diels-Alder reactions was accomplished. Three distinct libraries consisting of 24 members (library I), 60 members (library II), and 32 members (library III) were constructed. Variation of three substituents on the isoindolone scaffold in library I was exclusively achieved by the choice of the building blocks. In the syntheses of libraries II and III, sublibraries of isoindolone scaffolds were prepared initially in a one-pot/two-step process and were further diversified via Pd-catalyzed Suzuki cross-coupling reaction with boronic acids at two different diversification points. The Lipinski profiles and calculated ADME properties of the compounds are also reported.

Design, synthesis and selection of DNA-encoded small-molecule libraries.

PubMed

Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

2009-09-01

Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

Optical tools for high-throughput screening of abrasion resistance of combinatorial libraries of organic coatings

NASA Astrophysics Data System (ADS)

Potyrailo, Radislav A.; Chisholm, Bret J.; Olson, Daniel R.; Brennan, Michael J.; Molaison, Chris A.

2002-02-01

Design, validation, and implementation of an optical spectroscopic system for high-throughput analysis of combinatorially developed protective organic coatings are reported. Our approach replaces labor-intensive coating evaluation steps with an automated system that rapidly analyzes 8x6 arrays of coating elements that are deposited on a plastic substrate. Each coating element of the library is 10 mm in diameter and 2 to 5 micrometers thick. Performance of coatings is evaluated with respect to their resistance to wear abrasion because this parameter is one of the primary considerations in end-use applications. Upon testing, the organic coatings undergo changes that are impossible to quantitatively predict using existing knowledge. Coatings are abraded using industry-accepted abrasion test methods at single-or multiple-abrasion conditions, followed by high- throughput analysis of abrasion-induced light scatter. The developed automated system is optimized for the analysis of diffusively scattered light that corresponds to 0 to 30% haze. System precision of 0.1 to 2.5% relative standard deviation provides capability for the reliable ranking of coatings performance. While the system was implemented for high-throughput screening of combinatorially developed organic protective coatings for automotive applications, it can be applied to a variety of other applications where materials ranking can be achieved using optical spectroscopic tools.

Developing recombinant antibodies for biomarker detection

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

Baird, Cheryl L.; Fischer, Christopher J.; Pefaur, Noah B.

2010-10-01

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (eg, yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype-binding conferred by an antibody fragment-with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune librariesmore » provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.« less

Concept of combinatorial de novo design of drug-like molecules by particle swarm optimization.

PubMed

Hartenfeller, Markus; Proschak, Ewgenij; Schüller, Andreas; Schneider, Gisbert

2008-07-01

We present a fast stochastic optimization algorithm for fragment-based molecular de novo design (COLIBREE, Combinatorial Library Breeding). The search strategy is based on a discrete version of particle swarm optimization. Molecules are represented by a scaffold, which remains constant during optimization, and variable linkers and side chains. Different linkers represent virtual chemical reactions. Side-chain building blocks were obtained from pseudo-retrosynthetic dissection of large compound databases. Here, ligand-based design was performed using chemically advanced template search (CATS) topological pharmacophore similarity to reference ligands as fitness function. A weighting scheme was included for particle swarm optimization-based molecular design, which permits the use of many reference ligands and allows for positive and negative design to be performed simultaneously. In a case study, the approach was applied to the de novo design of potential peroxisome proliferator-activated receptor subtype-selective agonists. The results demonstrate the ability of the technique to cope with large combinatorial chemistry spaces and its applicability to focused library design. The technique was able to perform exploitation of a known scheme and at the same time explorative search for novel ligands within the framework of a given molecular core structure. It thereby represents a practical solution for compound screening in the early hit and lead finding phase of a drug discovery project.

One step DNA assembly for combinatorial metabolic engineering.

PubMed

Coussement, Pieter; Maertens, Jo; Beauprez, Joeri; Van Bellegem, Wouter; De Mey, Marjan

2014-05-01

The rapid and efficient assembly of multi-step metabolic pathways for generating microbial strains with desirable phenotypes is a critical procedure for metabolic engineering, and remains a significant challenge in synthetic biology. Although several DNA assembly methods have been developed and applied for metabolic pathway engineering, many of them are limited by their suitability for combinatorial pathway assembly. The introduction of transcriptional (promoters), translational (ribosome binding site (RBS)) and enzyme (mutant genes) variability to modulate pathway expression levels is essential for generating balanced metabolic pathways and maximizing the productivity of a strain. We report a novel, highly reliable and rapid single strand assembly (SSA) method for pathway engineering. The method was successfully optimized and applied to create constructs containing promoter, RBS and/or mutant enzyme libraries. To demonstrate its efficiency and reliability, the method was applied to fine-tune multi-gene pathways. Two promoter libraries were simultaneously introduced in front of two target genes, enabling orthogonal expression as demonstrated by principal component analysis. This shows that SSA will increase our ability to tune multi-gene pathways at all control levels for the biotechnological production of complex metabolites, achievable through the combinatorial modulation of transcription, translation and enzyme activity. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

Parallel solid-phase synthesis and high-throughput 1H NMR evaluation of a 96-member 1,2,4-trisubstituted-pyrimidin-6-one-5-carboxylic acid library.

PubMed

Hamper, Bruce C; Kesselring, Allen S; Chott, Robert C; Yang, Shengtian

2009-01-01

A solid-phase organic synthesis method has been developed for the preparation of trisubstituted pyrimidin-6-one carboxylic acids 12, which allows elaboration to a 3-dimensional combinatorial library. Three substituents are introduced by initial Knoevenagel condensation of an aldehyde and malonate ester resin 7 to give resin bound 1. Cyclization of 1 with an N-substituted amidine 10, oxidation, and cleavage afforded pyrimidinone 12. The initial solid-phase reaction sequence was followed by gel-phase (19)FNMR and direct-cleavage (1)H NMR of intermediate resins to determine the optimal conditions. The scope of the method for library production was determined by investigation of a 3 x 4 pilot library of twelve compounds. Cyclocondensation of N-methylamidines and 7 followed by CAN oxidation gave mixtures of the resin bound pyrimidin-6-one 11 and the regioisomeric pyrimidin-4-one 15, which after cleavage from the resin afforded a nearly 1:1 mixture of pyrimidin-6-one and pyrimidin-4-one carboxylic acids 12 and 16, respectively. The regiochemical assignment was confirmed by ROESY1D and gHMBC NMR experiments. A library was prepared using 8 aldehydes, 3 nitriles, and 4 amines to give a full combinatorial set of 96 pyrimidinones 12. Confirmation of structural identity and purity was carried out by LCMS using coupled ELS detection and by high-throughput flow (1)H NMR.

A diversity-oriented synthesis strategy enabling the combinatorial-type variation of macrocyclic peptidomimetic scaffolds.

PubMed

Isidro-Llobet, Albert; Hadje Georgiou, Kathy; Galloway, Warren R J D; Giacomini, Elisa; Hansen, Mette R; Méndez-Abt, Gabriela; Tan, Yaw Sing; Carro, Laura; Sore, Hannah F; Spring, David R

2015-04-21

Macrocyclic peptidomimetics are associated with a broad range of biological activities. However, despite such potentially valuable properties, the macrocyclic peptidomimetic structural class is generally considered as being poorly explored within drug discovery. This has been attributed to the lack of general methods for producing collections of macrocyclic peptidomimetics with high levels of structural, and thus shape, diversity. In particular, there is a lack of scaffold diversity in current macrocyclic peptidomimetic libraries; indeed, the efficient construction of diverse molecular scaffolds presents a formidable general challenge to the synthetic chemist. Herein we describe a new, advanced strategy for the diversity-oriented synthesis (DOS) of macrocyclic peptidomimetics that enables the combinatorial variation of molecular scaffolds (core macrocyclic ring architectures). The generality and robustness of this DOS strategy is demonstrated by the step-efficient synthesis of a structurally diverse library of over 200 macrocyclic peptidomimetic compounds, each based around a distinct molecular scaffold and isolated in milligram quantities, from readily available building-blocks. To the best of our knowledge this represents an unprecedented level of scaffold diversity in a synthetically derived library of macrocyclic peptidomimetics. Cheminformatic analysis indicated that the library compounds access regions of chemical space that are distinct from those addressed by top-selling brand-name drugs and macrocyclic natural products, illustrating the value of our DOS approach to sample regions of chemical space underexploited in current drug discovery efforts. An analysis of three-dimensional molecular shapes illustrated that the DOS library has a relatively high level of shape diversity.

Combinatorial preparation and characterization of thin-film multilayer electro-optical devices.

PubMed

Neuber, Christian; Bäte, Markus; Thelakkat, Mukundan; Schmidt, Hans-Werner; Hänsel, Helmut; Zettl, Heiko; Krausch, Georg

2007-07-01

In this article we present a setup for the combinatorial vapor deposition of thin-film multilayer devices as well as methods for the fast and efficient analytic screening of the libraries obtained. The preparation setup is based on a commercially available evaporation chamber equipped with various evaporation sources for both organic and metallic materials. The combinatorial approach is realized by the combination of a rotation stage for the substrate, a five-mask sampler, and an additional mask whose position can be deliberately varied along one axis during the evaporation process. The latter is used to evaporate linear as well as step gradients by continuous or stepwise movement of a shutter mask. The mask sampler allows to define the sectors of the library and to evaporate more complex structures, e.g., an electrode layout. Finally, the simultaneous evaporation of two or more materials enables us to produce layers of varying composition ratio in general and doped materials, in particular. For the control of the evaporation process we have developed an automation software, which is particularly helpful for complex library designs and which grants excellent repeatability of experiments. Efficient and fast characterization of the obtained libraries is realized by (i) a purely optical setup and (ii) an electro-optical setup. (i) The UV/vis reader FLASHScan 530 permits to map out the UV/vis absorbance or fluorescence of the whole library. The UV/vis absorbance is primarily used to determine layer thicknesses and to confirm thickness uniformity across larger regions. The fluorescence measurements are used to determine the composition of layers containing fluorescent dyes. (ii) For a detailed short- and long-term electro-optical analysis we have developed an automated measurement system, which allows the characterization of 8x8 optoelectronic devices and to study their degradation behavior. Both solar cells and organic light-emitting diodes can be tested. Finally, we have developed a data analysis software to extract characteristic values from the huge amount of data and with this facilitate the finding of systematic dependencies.

Nanostructured Ti-Ta thin films synthesized by combinatorial glancing angle sputter deposition

NASA Astrophysics Data System (ADS)

Motemani, Yahya; Khare, Chinmay; Savan, Alan; Hans, Michael; Paulsen, Alexander; Frenzel, Jan; Somsen, Christoph; Mücklich, Frank; Eggeler, Gunther; Ludwig, Alfred

2016-12-01

Ti-Ta alloys are attractive materials for applications in actuators as well as biomedical implants. When fabricated as thin films, these alloys can potentially be employed as microactuators, components for micro-implantable devices and coatings on surgical implants. In this study, Ti100-x Ta x (x = 21, 30) nanocolumnar thin films are fabricated by glancing angle deposition (GLAD) at room temperature using Ti73Ta27 and Ta sputter targets. Crystal structure, morphology and microstructure of the nanostructured thin films are systematically investigated by XRD, SEM and TEM, respectively. Nanocolumns of ˜150-160 nm in width are oriented perpendicular to the substrate for both Ti79Ta21 and Ti70Ta30 compositions. The disordered α″ martensite phase with orthorhombic structure is formed in room temperature as-deposited thin films. The columns are found to be elongated small single crystals which are aligned perpendicular to the (20\\bar{4}) and (204) planes of α″ martensite, indicating that the films’ growth orientation is mainly dominated by these crystallographic planes. Laser pre-patterned substrates are utilized to obtain periodic nanocolumnar arrays. The differences in seed pattern, and inter-seed distances lead to growth of multi-level porous nanostructures. Using a unique sputter deposition geometry consisting of Ti73Ta27 and Ta sputter sources, a nanocolumnar Ti-Ta materials library was fabricated on a static substrate by a co-deposition process (combinatorial-GLAD approach). In this library, a composition spread developed between Ti72.8Ta27.2 and Ti64.4Ta35.6, as confirmed by high-throughput EDX analysis. The morphology over the materials library varies from well-isolated nanocolumns to fan-like nanocolumnar structures. The influence of two sputter sources is investigated by studying the resulting column angle on the materials library. The presented nanostructuring methods including the use of the GLAD technique along with pre-patterning and a combinatorial materials library fabrication strategy offer a promising technological approach for investigating Ti-Ta thin films for a range of applications. The proposed approaches can be similarly implemented for other materials systems which can benefit from the formation of a nanocolumnar morphology.

Discovery of Cationic Polymers for Non-viral Gene Delivery using Combinatorial Approaches

PubMed Central

Barua, Sutapa; Ramos, James; Potta, Thrimoorthy; Taylor, David; Huang, Huang-Chiao; Montanez, Gabriela; Rege, Kaushal

2015-01-01

Gene therapy is an attractive treatment option for diseases of genetic origin, including several cancers and cardiovascular diseases. While viruses are effective vectors for delivering exogenous genes to cells, concerns related to insertional mutagenesis, immunogenicity, lack of tropism, decay and high production costs necessitate the discovery of non-viral methods. Significant efforts have been focused on cationic polymers as non-viral alternatives for gene delivery. Recent studies have employed combinatorial syntheses and parallel screening methods for enhancing the efficacy of gene delivery, biocompatibility of the delivery vehicle, and overcoming cellular level barriers as they relate to polymer-mediated transgene uptake, transport, transcription, and expression. This review summarizes and discusses recent advances in combinatorial syntheses and parallel screening of cationic polymer libraries for the discovery of efficient and safe gene delivery systems. PMID:21843141

Application of combinatorial biocatalysis for a unique ring expansion of dihydroxymethylzearalenone.

PubMed

Rich, Joseph O; Budde, Cheryl L; McConeghey, Luke D; Cotterill, Ian C; Mozhaev, Vadim V; Singh, Sheo B; Goetz, Michael A; Zhao, Annie; Michels, Peter C; Khmelnitsky, Yuri L

2009-06-01

Combinatorial biocatalysis was applied to generate a diverse set of dihydroxymethylzearalenone analogs with modified ring structure. In one representative chemoenzymatic reaction sequence, dihydroxymethylzearalenone was first subjected to a unique enzyme-catalyzed oxidative ring opening reaction that creates two new carboxylic groups on the molecule. These groups served as reaction sites for further derivatization involving biocatalytic ring closure reactions with structurally diverse bifunctional reagents, including different diols and diamines. As a result, a library of cyclic bislactones and bislactams was created, with modified ring structures covering chemical space and structure activity relationships unattainable by conventional synthetic means.

Molecular Library Synthesis Using Complex Substrates: Expanding the Framework of Triterpenoids

PubMed Central

Ignatenko, Vasily A.; Han, Yong; Tochtrop, Gregory P.

2013-01-01

The remodelling of a natural product core framework by means of diversity-oriented synthesis (DOS) is a valuable approach to access diverse/biologically relevant chemical space and to overcome the limitations of combinatorial-type compounds. Here we provide proof of principle and a thorough conformational analysis for a general strategy whereby the inherent complexity of a starting material is used to define the regio- and stereochemical outcomes of reactions in chemical library construction. This is in contrast to the traditional DOS logic employing reaction development and catalysis to drive library diversity PMID:23245400

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

Geng, J.; Nlebedim, I. C.; Besser, M. F.

A bulk combinatorial approach for synthesizing alloy libraries using laser engineered net shaping (LENS; i.e., 3D printing) was utilized to rapidly assess material systems for magnetic applications. The LENS system feeds powders in different ratios into a melt pool created by a laser to synthesize samples with bulk (millimeters) dimensions. By analyzing these libraries with autosampler differential scanning calorimeter/thermal gravimetric analysis and vibrating sample magnetometry, we are able to rapidly characterize the thermodynamic and magnetic properties of the libraries. Furthermore, the Fe-Co binary alloy was used as a model system and the results were compared with data in the literature.

Toxin Inhibition - Deconvolution Strategies and Assay Screening of Combinatorial Peptide Libraries

DTIC Science & Technology

2007-08-01

that could serve as lead compounds in the development of drug therapies to toxins. The libraries have typical structures of X I - X2 - hinge - X3 - X4...or passive vaccines have limited efficacies. There are no drug prophylaxes or therapies available. This technical report describes research to provide...broadly applicable drug discovery methods for a wide range of toxins. Future Work: The work conducted in the TIF project raised and renewed interest in

Intracellular directed evolution of proteins from combinatorial libraries based on conditional phage replication.

PubMed

Brödel, Andreas K; Jaramillo, Alfonso; Isalan, Mark

2017-09-01

Directed evolution is a powerful tool to improve the characteristics of biomolecules. Here we present a protocol for the intracellular evolution of proteins with distinct differences and advantages in comparison with established techniques. These include the ability to select for a particular function from a library of protein variants inside cells, minimizing undesired coevolution and propagation of nonfunctional library members, as well as allowing positive and negative selection logics using basally active promoters. A typical evolution experiment comprises the following stages: (i) preparation of a combinatorial M13 phagemid (PM) library expressing variants of the gene of interest (GOI) and preparation of the Escherichia coli host cells; (ii) multiple rounds of an intracellular selection process toward a desired activity; and (iii) the characterization of the evolved target proteins. The system has been developed for the selection of new orthogonal transcription factors (TFs) but is capable of evolving any gene-or gene circuit function-that can be linked to conditional M13 phage replication. Here we demonstrate our approach using as an example the directed evolution of the bacteriophage λ cI TF against two synthetic bidirectional promoters. The evolved TF variants enable simultaneous activation and repression against their engineered promoters and do not cross-react with the wild-type promoter, thus ensuring orthogonality. This protocol requires no special equipment, allowing synthetic biologists and general users to evolve improved biomolecules within ∼7 weeks.

Defining RNA motif-aminoglycoside interactions via two-dimensional combinatorial screening and structure-activity relationships through sequencing.

PubMed

Velagapudi, Sai Pradeep; Disney, Matthew D

2013-10-15

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3×3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure-activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif-aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site. Copyright © 2013 Elsevier Ltd. All rights reserved.

Defining RNA motif–aminoglycoside interactions via two-dimensional combinatorial screening and structure–activity relationships through sequencing

PubMed Central

Velagapudi, Sai Pradeep; Disney, Matthew D.

2013-01-01

RNA is an extremely important target for the development of chemical probes of function or small molecule therapeutics. Aminoglycosides are the most well studied class of small molecules to target RNA. However, the RNA motifs outside of the bacterial rRNA A-site that are likely to be bound by these compounds in biological systems is largely unknown. If such information were known, it could allow for aminoglycosides to be exploited to target other RNAs and, in addition, could provide invaluable insights into potential bystander targets of these clinically used drugs. We utilized two-dimensional combinatorial screening (2DCS), a library-versus-library screening approach, to select the motifs displayed in a 3 × 3 nucleotide internal loop library and in a 6-nucleotide hairpin library that bind with high affinity and selectivity to six aminoglycoside derivatives. The selected RNA motifs were then analyzed using structure–activity relationships through sequencing (StARTS), a statistical approach that defines the privileged RNA motif space that binds a small molecule. StARTS allowed for the facile annotation of the selected RNA motif–aminoglycoside interactions in terms of affinity and selectivity. The interactions selected by 2DCS generally have nanomolar affinities, which is higher affinity than the binding of aminoglycosides to a mimic of their therapeutic target, the bacterial rRNA A-site. PMID:23719281

Identification of a small molecule inhibitor of the aminoglycoside 6'-N-acetyltransferase type Ib [AAC(6')-Ib] using mixture-based combinatorial libraries.

PubMed

Tran, Tung; Chiem, Kevin; Jani, Saumya; Arivett, Brock A; Lin, David L; Lad, Rupali; Jimenez, Verónica; Farone, Mary B; Debevec, Ginamarie; Santos, Radleigh; Giulianotti, Marc; Pinilla, Clemencia; Tolmasky, Marcelo E

2018-05-01

The aminoglycoside, 6'-N-acetyltransferase type Ib [AAC(6')-Ib] is the most widely distributed enzyme among AAC(6')-I-producing Gram-negative pathogens and confers resistance to clinically relevant aminoglycosides, including amikacin. This enzyme is therefore an ideal target for enzymatic inhibitors that could overcome resistance to aminoglycosides. The search for inhibitors was carried out using mixture-based combinatorial libraries, the scaffold ranking approach, and the positional scanning strategy. A library with high inhibitory activity had pyrrolidine pentamine scaffold and was selected for further analysis. This library contained 738,192 compounds with functionalities derived from 26 different amino acids (R1, R2 and R3) and 42 different carboxylic acids (R4) in four R-group functionalities. The most active compounds all contained S-phenyl (R1 and R3) and S-hydromethyl (R2) functionalities at three locations and differed at the R4 position. The compound containing 3-phenylbutyl at R4 (compound 206) was a robust enzymatic inhibitor in vitro, in combination with amikacin it potentiated the inhibition of growth of three resistant bacteria in culture, and it improved survival when used as treatment of Galleria mellonella infected with aac(6')-Ib-harboring Klebsiella pneumoniae and Acinetobacter baumannii strains. Copyright © 2018 Elsevier B.V. and International Society of Chemotherapy. All rights reserved.

High efficiency family shuffling based on multi-step PCR and in vivo DNA recombination in yeast: statistical and functional analysis of a combinatorial library between human cytochrome P450 1A1 and 1A2.

PubMed

Abécassis, V; Pompon, D; Truan, G

2000-10-15

The design of a family shuffling strategy (CLERY: Combinatorial Libraries Enhanced by Recombination in Yeast) associating PCR-based and in vivo recombination and expression in yeast is described. This strategy was tested using human cytochrome P450 CYP1A1 and CYP1A2 as templates, which share 74% nucleotide sequence identity. Construction of highly shuffled libraries of mosaic structures and reduction of parental gene contamination were two major goals. Library characterization involved multiprobe hybridization on DNA macro-arrays. The statistical analysis of randomly selected clones revealed a high proportion of chimeric genes (86%) and a homogeneous representation of the parental contribution among the sequences (55.8 +/- 2.5% for parental sequence 1A2). A microtiter plate screening system was designed to achieve colorimetric detection of polycyclic hydrocarbon hydroxylation by transformed yeast cells. Full sequences of five randomly picked and five functionally selected clones were analyzed. Results confirmed the shuffling efficiency and allowed calculation of the average length of sequence exchange and mutation rates. The efficient and statistically representative generation of mosaic structures by this type of family shuffling in a yeast expression system constitutes a novel and promising tool for structure-function studies and tuning enzymatic activities of multicomponent eucaryote complexes involving non-soluble enzymes.

Ribosome Display of Combinatorial Antibody Libraries Derived from Mice Immunized with Heat-Killed Xylella fastidiosa and the Selection of MopB-Specific Single-Chain Antibodies

PubMed Central

Azizi, Armaghan; Arora, Arinder; Markiv, Anatoliy; Lampe, David J.; Miller, Thomas A.

2012-01-01

Pierce's disease is a devastating lethal disease of Vitus vinifera grapevines caused by the bacterium Xylella fastidiosa. There is no cure for Pierce's disease, and control is achieved predominantly by suppressing transmission of the glassy-winged sharpshooter insect vector. We present a simple robust approach for the generation of panels of recombinant single-chain antibodies against the surface-exposed elements of X. fastidiosa that may have potential use in diagnosis and/or disease transmission blocking studies. In vitro combinatorial antibody ribosome display libraries were assembled from immunoglobulin transcripts rescued from the spleens of mice immunized with heat-killed X. fastidiosa. The libraries were used in a single round of selection against an outer membrane protein, MopB, resulting in the isolation of a panel of recombinant antibodies. The potential use of selected anti-MopB antibodies was demonstrated by the successful application of the 4XfMopB3 antibody in an enzyme-linked immunosorbent assay (ELISA), a Western blot assay, and an immunofluorescence assay (IFA). These immortalized in vitro recombinant single-chain antibody libraries generated against heat-killed X. fastidiosa are a resource for the Pierce's disease research community that may be readily accessed for the isolation of antibodies against a plethora of X. fastidiosa surface-exposed antigenic molecules. PMID:22327580

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening

PubMed Central

2017-01-01

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing. PMID:28199790

An Integrated Microfluidic Processor for DNA-Encoded Combinatorial Library Functional Screening.

PubMed

MacConnell, Andrew B; Price, Alexander K; Paegel, Brian M

2017-03-13

DNA-encoded synthesis is rekindling interest in combinatorial compound libraries for drug discovery and in technology for automated and quantitative library screening. Here, we disclose a microfluidic circuit that enables functional screens of DNA-encoded compound beads. The device carries out library bead distribution into picoliter-scale assay reagent droplets, photochemical cleavage of compound from the bead, assay incubation, laser-induced fluorescence-based assay detection, and fluorescence-activated droplet sorting to isolate hits. DNA-encoded compound beads (10-μm diameter) displaying a photocleavable positive control inhibitor pepstatin A were mixed (1920 beads, 729 encoding sequences) with negative control beads (58 000 beads, 1728 encoding sequences) and screened for cathepsin D inhibition using a biochemical enzyme activity assay. The circuit sorted 1518 hit droplets for collection following 18 min incubation over a 240 min analysis. Visual inspection of a subset of droplets (1188 droplets) yielded a 24% false discovery rate (1166 pepstatin A beads; 366 negative control beads). Using template barcoding strategies, it was possible to count hit collection beads (1863) using next-generation sequencing data. Bead-specific barcodes enabled replicate counting, and the false discovery rate was reduced to 2.6% by only considering hit-encoding sequences that were observed on >2 beads. This work represents a complete distributable small molecule discovery platform, from microfluidic miniaturized automation to ultrahigh-throughput hit deconvolution by sequencing.

Combinatorial discovery of new methanol-tolerant non-noble metal cathode electrocatalysts for direct methanol fuel cells.

PubMed

Yu, Jong-Sung; Kim, Min-Sik; Kim, Jung Ho

2010-12-14

Combinatorial synthesis and screening were used to identify methanol-tolerant non-platinum cathode electrocatalysts for use in direct methanol fuel cells (DMFCs). Oxygen reduction consumes protons at the surface of DMFC cathode catalysts. In combinatorial screening, this pH change allows one to differentiate active catalysts using fluorescent acid-base indicators. Combinatorial libraries of carbon-supported catalyst compositions containing Ru, Mo, W, Sn, and Se were screened. Ternary and quaternary compositions containing Ru, Sn, Mo, Se were more active than the "standard" Alonso-Vante catalyst, Ru(3)Mo(0.08)Se(2), when tested in liquid-feed DMFCs. Physical characterization of the most active catalysts by powder X-ray diffraction, gas adsorption, and X-ray photoelectron spectroscopy revealed that the predominant crystalline phase was hexagonal close-packed (hcp) ruthenium, and showed a surface mostly covered with oxide. The best new catalyst, Ru(7.0)Sn(1.0)Se(1.0), was significantly more active than Ru(3)Se(2)Mo(0.08), even though the latter contained smaller particles.

On the Computer Generation of Adaptive Numerical Libraries

DTIC Science & Technology

2010-05-01

D.; Borowski, P.; Clark, T.; Clerc, D.; Dachsel, H.; Deegan , M.; Dyall, K.; Elwood, D.; Bibliography 123 Glendening, E.; Gutowski, M.; Hess, A...Science, pages 72–83. Springer, 2007. 84 Curry, Haskell B.; Feys, Robert; Craig , William. Combinatory Logic, volume 1. North-Holland Publishing

Widening and diversifying the proteome capture by combinatorial peptide ligand libraries via Alcian Blue dye binding.

PubMed

Candiano, Giovanni; Santucci, Laura; Petretto, Andrea; Lavarello, Chiara; Inglese, Elvira; Bruschi, Maurizio; Ghiggeri, Gian Marco; Boschetti, Egisto; Righetti, Pier Giorgio

2015-01-01

Combinatorial peptide ligand libraries (CPLLs) tend to bind complex molecules such as dyes due to their aromatic, heterocyclic, hydrophobic, and ionic nature that may affect the protein capture specificity. In this experimental work Alcian Blue 8GX, a positively charged phthalocyanine dye well-known to bind to glycoproteins and to glucosaminoglycans, was adsorbed on a chemically modified CPLL solid phase, and the behavior of the resulting conjugate was then investigated. The control and dye-adsorbed beads were used to harvest the human urinary proteome at physiological pH, this resulting in a grand total of 1151 gene products identified after the capture. Although the Alcian Blue-modified CPLL incremented the total protein capture by 115 species, it particularly enriched some families among the harvested proteins, such as glycoproteins and nucleotide-binding proteins. This study teaches that it is possible, via the two combined harvest mechanisms, to drive the CPLL capture toward the enrichment of specific protein categories.

Data Mining and Machine Learning Tools for Combinatorial Material Science of All-Oxide Photovoltaic Cells.

PubMed

Yosipof, Abraham; Nahum, Oren E; Anderson, Assaf Y; Barad, Hannah-Noa; Zaban, Arie; Senderowitz, Hanoch

2015-06-01

Growth in energy demands, coupled with the need for clean energy, are likely to make solar cells an important part of future energy resources. In particular, cells entirely made of metal oxides (MOs) have the potential to provide clean and affordable energy if their power conversion efficiencies are improved. Such improvements require the development of new MOs which could benefit from combining combinatorial material sciences for producing solar cells libraries with data mining tools to direct synthesis efforts. In this work we developed a data mining workflow and applied it to the analysis of two recently reported solar cell libraries based on Titanium and Copper oxides. Our results demonstrate that QSAR models with good prediction statistics for multiple solar cells properties could be developed and that these models highlight important factors affecting these properties in accord with experimental findings. The resulting models are therefore suitable for designing better solar cells. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Combinatorial ligand libraries as a two-dimensional method for proteome analysis.

PubMed

Santucci, Laura; Candiano, Giovanni; Petretto, Andrea; Lavarello, Chiara; Bruschi, Maurizio; Ghiggeri, Gian Marco; Citterio, Attilio; Righetti, Pier Giorgio

2013-07-05

The present report tries to assess the possibility of performing capture of proteomes via combinatorial peptide ligand libraries (CPLL) in a two-dimensional (2D) mode, i.e. via orthogonal complementarity in the capture phase. To that aim, serum proteins are captured at physiological pH either at low ionic strength (25mM NaCl) or at high concentrations of lyotropic salts of the Hofmeister series (1M ammonium sulphate) favouring hydrophobic interaction. Indeed such 2D mechanisms seems to be operative, since 52% of the captured proteins are common to the two capture modes, 20% are specific only of the "ionic" interaction mode and 28% are found only in the "hydrophobically" driven interaction. As an additional bonus, losses of protein species from the initial sample, one of the major drawbacks of CPLLs, are diminished to about 5% and are found only in the ionic capture, whereas the hydrophobically engendered capture is loss-free. Copyright © 2013 Elsevier B.V. All rights reserved.

Statistical theory of combinatorial libraries of folding proteins: energetic discrimination of a target structure.

PubMed

Zou, J; Saven, J G

2000-02-11

A self-consistent theory is presented that can be used to estimate the number and composition of sequences satisfying a predetermined set of constraints. The theory is formulated so as to examine the features of sequences having a particular value of Delta=E(f)-(u), where E(f) is the energy of sequences when in a target structure and (u) is an average energy of non-target structures. The theory yields the probabilities w(i)(alpha) that each position i in the sequence is occupied by a particular monomer type alpha. The theory is applied to a simple lattice model of proteins. Excellent agreement is observed between the theory and the results of exact enumerations. The theory provides a quantitative framework for the design and interpretation of combinatorial experiments involving proteins, where a library of amino acid sequences is searched for sequences that fold to a desired structure. Copyright 2000 Academic Press.

Proteomic analysis of sweet algerian apricot kernels (Prunus armeniaca L.) by combinatorial peptide ligand libraries and LC-MS/MS.

PubMed

Ghorab, Hamida; Lammi, Carmen; Arnoldi, Anna; Kabouche, Zahia; Aiello, Gilda

2018-01-15

An investigation on the proteome of the sweet kernel of apricot, based on equalisation with combinatorial peptide ligand libraries (CPLLs), SDS-PAGE, nLC-ESI-MS/MS, and database search, permitted identifying 175 proteins. Gene ontology analysis indicated that their main molecular functions are in nucleotide binding (20.9%), hydrolase activities (10.6%), kinase activities (7%), and catalytic activity (5.6%). A protein-protein association network analysis using STRING software permitted to build an interactomic map of all detected proteins, characterised by 34 interactions. In order to forecast the potential health benefits deriving from the consumption of these proteins, the two most abundant, i.e. Prunin 1 and 2, were enzymatically digested in silico predicting 10 and 14 peptides, respectively. Searching their sequences in the database BIOPEP, it was possible to suggest a variety of bioactivities, including dipeptidyl peptidase-IV (DPP-IV) and angiotensin converting enzyme I (ACE) inhibition, glucose uptake stimulation and antioxidant properties. Copyright © 2017 Elsevier Ltd. All rights reserved.

Phage selection of peptide "microantibodies".

PubMed

Fujiwara, Daisuke; Fujii, Ikuo

2013-01-01

A bioactive peptide capable of inhibiting protein-protein interactions has the potential to be a molecular tool for biological studies and a therapeutic by disrupting aberrant interactions involved in diseases. We have developed combinatorial libraries of peptides with helix-loop-helix structure, from which the isolated peptides have the constrained structure to reduce entropy costs in binding, resulting in high binding affinities for target molecules. Previously, we designed a de novo peptide of helix-loop-helix structure that we termed a "microantibody." Using the microantibody as a library scaffold, we have constructed a phage-display library to successfully isolate molecular-targeting peptides against a cytokine receptor (granulocyte colony-stimulating factor receptor), a protein kinase (Aurora-A), and a ganglioside (GM1). Protocols in this article describe a general procedure for the library construction and the library screening.

Combinatorial study of Ni-Ti-Pt ternary metal gate electrodes on HfO{sub 2} for the advanced gate stack

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

Chang, K.-S.; Green, M. L.; Suehle, J.

2006-10-02

The authors have fabricated combinatorial Ni-Ti-Pt ternary metal gate thin film libraries on HfO{sub 2} using magnetron co-sputtering to investigate flatband voltage shift ({delta}V{sub fb}), work function ({phi}{sub m}), and leakage current density (J{sub L}) variations. A more negative {delta}V{sub fb} is observed close to the Ti-rich corner than at the Ni- and Pt-rich corners, implying smaller {phi}{sub m} near the Ti-rich corners and higher {phi}{sub m} near the Ni- and Pt-rich corners. In addition, measured J{sub L} values can be explained consistently with the observed {phi}{sub m} variations. Combinatorial methodologies prove to be useful in surveying the large compositionalmore » space of ternary alloy metal gate electrode systems.« less

Combinatorial Algorithms to Enable Computational Science and Engineering: Work from the CSCAPES Institute

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

Boman, Erik G.; Catalyurek, Umit V.; Chevalier, Cedric

2015-01-16

This final progress report summarizes the work accomplished at the Combinatorial Scientific Computing and Petascale Simulations Institute. We developed Zoltan, a parallel mesh partitioning library that made use of accurate hypergraph models to provide load balancing in mesh-based computations. We developed several graph coloring algorithms for computing Jacobian and Hessian matrices and organized them into a software package called ColPack. We developed parallel algorithms for graph coloring and graph matching problems, and also designed multi-scale graph algorithms. Three PhD students graduated, six more are continuing their PhD studies, and four postdoctoral scholars were advised. Six of these students and Fellowsmore » have joined DOE Labs (Sandia, Berkeley), as staff scientists or as postdoctoral scientists. We also organized the SIAM Workshop on Combinatorial Scientific Computing (CSC) in 2007, 2009, and 2011 to continue to foster the CSC community.« less

Fast Modeling of Binding Affinities by Means of Superposing Significant Interaction Rules (SSIR) Method

PubMed Central

Besalú, Emili

2016-01-01

The Superposing Significant Interaction Rules (SSIR) method is described. It is a general combinatorial and symbolic procedure able to rank compounds belonging to combinatorial analogue series. The procedure generates structure-activity relationship (SAR) models and also serves as an inverse SAR tool. The method is fast and can deal with large databases. SSIR operates from statistical significances calculated from the available library of compounds and according to the previously attached molecular labels of interest or non-interest. The required symbolic codification allows dealing with almost any combinatorial data set, even in a confidential manner, if desired. The application example categorizes molecules as binding or non-binding, and consensus ranking SAR models are generated from training and two distinct cross-validation methods: leave-one-out and balanced leave-two-out (BL2O), the latter being suited for the treatment of binary properties. PMID:27240346

Combinatorial studies of (1-x)Na0.5Bi0.5TiO3-xBaTiO3 thin-film chips

NASA Astrophysics Data System (ADS)

Cheng, Hong-Wei; Zhang, Xue-Jin; Zhang, Shan-Tao; Feng, Yan; Chen, Yan-Feng; Liu, Zhi-Guo; Cheng, Guang-Xi

2004-09-01

Applying a combinatorial methodology, (1-x)Na0.5Bi0.5TiO3-xBaTiO3 (NBT-BT) thin-film chips were fabricated on (001)-LaAlO3 substrates by pulsed laser deposition with a few quaternary masks. A series of NBT-BT library with the composition of BT ranged from 0 to 44% was obtained with uniform composition and well crystallinity. The relation between the concentration of NBT-BT and their structural and dielectric properties were investigated by x-ray diffraction (XRD), evanescent microwave probe, atomic force microscopy, and Raman spectroscopy. An obvious morphotropic phase boundary (MPB) was established to be about 9% BT by XRD, Raman frequency shift, and dielectric anomaly, different from the well-known MPB of the materials. The result shows the high efficiency of combinatorial method in searching new relaxor ferroelectrics.

Preparation and infrared/raman classification of 630 spectroscopically encoded styrene copolymers.

PubMed

Fenniri, Hicham; Chun, Sangki; Terreau, Owen; Bravo-Vasquez, Juan-Pablo

2008-01-01

The barcoded resins (BCRs) were introduced recently as a platform for encoded combinatorial chemistry. One of the main challenges yet to be overcome is the demonstration that a large number of BCRs could be generated and classified with high confidence. Here, we describe the synthesis and classification of 630 polystyrene-based copolymers prepared from the combinatorial association of 15 spectroscopically active styrene monomers. Each of the 630 copolymers displayed a unique vibrational fingerprint (infrared and Raman), which was converted into a spectral vector. To each of the 630 copolymers, a vector of the known (reference) composition was assigned. Unknown (prediction) vectors were decoded using multivariate data analysis. From the inner product of the reference and prediction vectors, a correlation map comparing 396 900 copolymer pairs (630 x 630) was generated. In 100% of the cases, the highest correlation was obtained for polymer pairs in which the reference and prediction vectors correspond to copolymers prepared from identical styrene monomers, thus demonstrating the high reliability of this encoding strategy. We have also established that the spectroscopic barcodes generated from the Raman and infrared spectra are independent of the copolymers' morphology (beaded versus bulk polymers). Besides the demonstration of the generality of the polymer barcoding strategy, the analytical methods developed here could in principle be extended to the investigation of the composition and purity of any other synthetic polymer and biopolymer library, or even scaffold-based combinatorial libraries.

DeviceEditor visual biological CAD canvas

PubMed Central

2012-01-01

Background Biological Computer Aided Design (bioCAD) assists the de novo design and selection of existing genetic components to achieve a desired biological activity, as part of an integrated design-build-test cycle. To meet the emerging needs of Synthetic Biology, bioCAD tools must address the increasing prevalence of combinatorial library design, design rule specification, and scar-less multi-part DNA assembly. Results We report the development and deployment of web-based bioCAD software, DeviceEditor, which provides a graphical design environment that mimics the intuitive visual whiteboard design process practiced in biological laboratories. The key innovations of DeviceEditor include visual combinatorial library design, direct integration with scar-less multi-part DNA assembly design automation, and a graphical user interface for the creation and modification of design specification rules. We demonstrate how biological designs are rendered on the DeviceEditor canvas, and we present effective visualizations of genetic component ordering and combinatorial variations within complex designs. Conclusions DeviceEditor liberates researchers from DNA base-pair manipulation, and enables users to create successful prototypes using standardized, functional, and visual abstractions. Open and documented software interfaces support further integration of DeviceEditor with other bioCAD tools and software platforms. DeviceEditor saves researcher time and institutional resources through correct-by-construction design, the automation of tedious tasks, design reuse, and the minimization of DNA assembly costs. PMID:22373390

Chimeric Rhinoviruses Displaying MPER Epitopes Elicit Anti-HIV Neutralizing Responses

PubMed Central

Yi, Guohua; Lapelosa, Mauro; Bradley, Rachel; Mariano, Thomas M.; Dietz, Denise Elsasser; Hughes, Scott; Wrin, Terri; Petropoulos, Chris; Gallicchio, Emilio; Levy, Ronald M.; Arnold, Eddy; Arnold, Gail Ferstandig

2013-01-01

Background The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER) of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV. Methodology/Principle Findings Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV) displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested. Conclusions Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection. PMID:24039745

Immobilized OBOC combinatorial bead array to facilitate multiplicative screening.

PubMed

Xiao, Wenwu; Bononi, Fernanda C; Townsend, Jared; Li, Yuanpei; Liu, Ruiwu; Lam, Kit S

2013-07-01

One-bead-one-compound (OBOC) combinatorial library screening has been broadly utilized for the last two decades to identify small molecules, peptides or peptidomimetics targeting variable screening probes such as cell surface receptors, bacteria, protein kinases, phosphatases, proteases etc. In previous screening methods, library beads were suspended in solution and screened against one single probe. Only the positive beads were tracked and isolated for additional screens and finally selected for chemical decoding. During this process, the remaining negative beads were not tracked and discarded. Here we report a novel bead immobilization method such that a bead library array can be conveniently prepared and screened in its entirety, sequentially many times with a series of distinct probes. This method not only allows us to increase the screening efficiency but also permits us to determine the binding profile of each and every library bead against a large number of target receptors. As proof of concept, we serially screened a random OBOC disulfide containing cyclic heptapeptide library with three water soluble dyes as model probes: malachite green, bromocresol purple and indigo carmine. This multiplicative screening approach resulted in a rapid determination of the binding profile of each and every bead respective to each of the three dyes. Beads that interacted with malachite green only, bromocresol purple only, or both indigo carmine and bromocresol purple were isolated, and their peptide sequences were determined with microsequencer. Ultimately, the novel OBOC multiplicative screening approach could play a key role in the enhancement of existing on-bead assays such as whole cell binding, bacteria binding, protein binding, posttranslational modifications etc. with increased efficiency, capacity, and specificity.

Why is combinatorial communication rare in the natural world, and why is language an exception to this trend?

PubMed

Scott-Phillips, Thomas C; Blythe, Richard A

2013-11-06

In a combinatorial communication system, some signals consist of the combinations of other signals. Such systems are more efficient than equivalent, non-combinatorial systems, yet despite this they are rare in nature. Why? Previous explanations have focused on the adaptive limits of combinatorial communication, or on its purported cognitive difficulties, but neither of these explains the full distribution of combinatorial communication in the natural world. Here, we present a nonlinear dynamical model of the emergence of combinatorial communication that, unlike previous models, considers how initially non-communicative behaviour evolves to take on a communicative function. We derive three basic principles about the emergence of combinatorial communication. We hence show that the interdependence of signals and responses places significant constraints on the historical pathways by which combinatorial signals might emerge, to the extent that anything other than the most simple form of combinatorial communication is extremely unlikely. We also argue that these constraints can be bypassed if individuals have the socio-cognitive capacity to engage in ostensive communication. Humans, but probably no other species, have this ability. This may explain why language, which is massively combinatorial, is such an extreme exception to nature's general trend for non-combinatorial communication.

Combinatorially Screened Peptide as Targeted Covalent Binder: Alteration of Bait-Conjugated Peptide to Reactive Modifier.

PubMed

Uematsu, Shuta; Tabuchi, Yudai; Ito, Yuji; Taki, Masumi

2018-06-01

A peptide-type covalent binder for a target protein was obtained by combinatorial screening of fluoroprobe-conjugated peptide libraries on bacteriophage T7. The solvatochromic fluoroprobe works as a bait during the affinity selection process of phage display. To obtain the targeted covalent binder, the bait in the selected consensus peptide was altered into a reactive warhead possessing a sulfonyl fluoride. The reaction efficiency and site/position specificity of the covalent conjugation between the binder and the target protein were evaluated by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and rationalized by a protein-ligand docking simulation.

A Road Less Traveled By: Exploring a Decade of Ellman Chemistry

PubMed Central

Shelat, Anang A.; Guy, R. Kiplin

2009-01-01

The Ellman group has been one of the most influential in the development and widespread adoption of combinatorial chemistry techniques for biomedical research. Their work has included substantial methodological development for library synthesis with a particular focus on new scaffolds rationally targeted to biomolecules of interest and biologically relevant natural products. Herein we analyze a representative set of libraries from this group with respect to their biological and biomedical relevance in comparison to existing drugs and probe compounds. This analysis reveals that the Ellman group has not only provided new methodologies to the community but also provided libraries with unique potential for further biological study. PMID:18343129

DNA-Encoded Chemical Libraries: A Selection System Based on Endowing Organic Compounds with Amplifiable Information.

PubMed

Neri, Dario; Lerner, Richard A

2018-06-20

The discovery of organic ligands that bind specifically to proteins is a central problem in chemistry, biology, and the biomedical sciences. The encoding of individual organic molecules with distinctive DNA tags, serving as amplifiable identification bar codes, allows the construction and screening of combinatorial libraries of unprecedented size, thus facilitating the discovery of ligands to many different protein targets. Fundamentally, one links powers of genetics and chemical synthesis. After the initial description of DNA-encoded chemical libraries in 1992, several experimental embodiments of the technology have been reduced to practice. This review provides a historical account of important milestones in the development of DNA-encoded chemical libraries, a survey of relevant ongoing research activities, and a glimpse into the future.

Learning to generate combinatorial action sequences utilizing the initial sensitivity of deterministic dynamical systems.

PubMed

Nishimoto, Ryu; Tani, Jun

2004-09-01

This study shows how sensory-action sequences of imitating finite state machines (FSMs) can be learned by utilizing the deterministic dynamics of recurrent neural networks (RNNs). Our experiments indicated that each possible combinatorial sequence can be recalled by specifying its respective initial state value and also that fractal structures appear in this initial state mapping after the learning converges. We also observed that the sequences of mimicking FSMs are encoded utilizing the transient regions rather than the invariant sets of the evolved dynamical systems of the RNNs.

Improved Modeling of Side-Chain–Base Interactions and Plasticity in Protein–DNA Interface Design

PubMed Central

Thyme, Summer B.; Baker, David; Bradley, Philip

2012-01-01

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed “motifs”) was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein–DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. PMID:22426128

EcoFlex: A Multifunctional MoClo Kit for E. coli Synthetic Biology.

PubMed

Moore, Simon J; Lai, Hung-En; Kelwick, Richard J R; Chee, Soo Mei; Bell, David J; Polizzi, Karen Marie; Freemont, Paul S

2016-10-21

Golden Gate cloning is a prominent DNA assembly tool in synthetic biology for the assembly of plasmid constructs often used in combinatorial pathway optimization, with a number of assembly kits developed specifically for yeast and plant-based expression. However, its use for synthetic biology in commonly used bacterial systems such as Escherichia coli has surprisingly been overlooked. Here, we introduce EcoFlex a simplified modular package of DNA parts for a variety of applications in E. coli, cell-free protein synthesis, protein purification and hierarchical assembly of transcription units based on the MoClo assembly standard. The kit features a library of constitutive promoters, T7 expression, RBS strength variants, synthetic terminators, protein purification tags and fluorescence proteins. We validate EcoFlex by assembling a 68-part containing (20 genes) plasmid (31 kb), characterize in vivo and in vitro library parts, and perform combinatorial pathway assembly, using pooled libraries of either fluorescent proteins or the biosynthetic genes for the antimicrobial pigment violacein as a proof-of-concept. To minimize pathway screening, we also introduce a secondary module design site to simplify MoClo pathway optimization. In summary, EcoFlex provides a standardized and multifunctional kit for a variety of applications in E. coli synthetic biology.

Improved modeling of side-chain--base interactions and plasticity in protein--DNA interface design.

PubMed

Thyme, Summer B; Baker, David; Bradley, Philip

2012-06-08

Combinatorial sequence optimization for protein design requires libraries of discrete side-chain conformations. The discreteness of these libraries is problematic, particularly for long, polar side chains, since favorable interactions can be missed. Previously, an approach to loop remodeling where protein backbone movement is directed by side-chain rotamers predicted to form interactions previously observed in native complexes (termed "motifs") was described. Here, we show how such motif libraries can be incorporated into combinatorial sequence optimization protocols and improve native complex recapitulation. Guided by the motif rotamer searches, we made improvements to the underlying energy function, increasing recapitulation of native interactions. To further test the methods, we carried out a comprehensive experimental scan of amino acid preferences in the I-AniI protein-DNA interface and found that many positions tolerated multiple amino acids. This sequence plasticity is not observed in the computational results because of the fixed-backbone approximation of the model. We improved modeling of this diversity by introducing DNA flexibility and reducing the convergence of the simulated annealing algorithm that drives the design process. In addition to serving as a benchmark, this extensive experimental data set provides insight into the types of interactions essential to maintain the function of this potential gene therapy reagent. Published by Elsevier Ltd.

Combinatorial library based engineering of Candida antarctica lipase A for enantioselective transacylation of sec-alcohols in organic solvent.

PubMed

Wikmark, Ylva; Svedendahl Humble, Maria; Bäckvall, Jan-E

2015-03-27

A method for determining lipase enantioselectivity in the transacylation of sec-alcohols in organic solvent was developed. The method was applied to a model library of Candida antarctica lipase A (CalA) variants for improved enantioselectivity (E values) in the kinetic resolution of 1-phenylethanol in isooctane. A focused combinatorial gene library simultaneously targeting seven positions in the enzyme active site was designed. Enzyme variants were immobilized on nickel-coated 96-well microtiter plates through a histidine tag (His6-tag), screened for transacylation of 1-phenylethanol in isooctane, and analyzed by GC. The highest enantioselectivity was shown by the double mutant Y93L/L367I. This enzyme variant gave an E value of 100 (R), which is a dramatic improvement on the wild-type CalA (E=3). This variant also showed high to excellent enantioselectivity for other secondary alcohols tested. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Tailoring gas sensor arrays via the design of short peptides sequences as binding elements.

PubMed

Mascini, Marcello; Pizzoni, Daniel; Perez, German; Chiarappa, Emilio; Di Natale, Corrado; Pittia, Paola; Compagnone, Dario

2017-07-15

A semi-combinatorial virtual approach was used to prepare peptide-based gas sensors with binding properties towards five different chemical classes (alcohols, aldehydes, esters, hydrocarbons and ketones). Molecular docking simulations were conducted for a complete tripeptide library (8000 elements) versus 58 volatile compounds belonging to those five chemical classes. By maximizing the differences between chemical classes, a subset of 120 tripeptides was extracted and used as scaffolds for generating a combinatorial library of 7912 tetrapeptides. This library was processed in an analogous way to the former. Five tetrapeptides (IHRI, KSDS, LGFD, TGKF and WHVS) were chosen depending on their virtual affinity and cross-reactivity for the experimental step. The five peptides were covalently bound to gold nanoparticles by adding a terminal cysteine to each tetrapeptide and deposited onto 20MHz quartz crystal microbalances to construct the gas sensors. The behavior of peptides after this chemical modification was simulated at the pH range used in the immobilization step. ΔF signals analyzed by principal component analysis matched the virtually screened data. The array was able to clearly discriminate the 13 volatile compounds tested based on their hydrophobicity and hydrophilicity molecules as well as the molecular weight. Copyright © 2016 Elsevier B.V. All rights reserved.

Phage display vectors for in vivo recombination of immunoglobulin heavy and light chain genes to make large combinatorial libraries.

PubMed

Tsurushita, N; Fu, H; Warren, C

1996-06-12

New phage display vectors for in vivo recombination of immunoglobulin (Ig) heavy (VH) and light (VL) chain variable genes, to make single-chain Fv fragments (scFv), were constructed. The VH and VL genes of monoclonal antibody (mAb) EP-5C7, which binds to both human E- and P-selectin, were cloned into a pUC19-derived plasmid vector, pCW93, and a pACYC184-derived phagemid vector, pCW99, respectively. Upon induction of Cre recombinase (phage P1 recombinase), the VH and VL genes were efficiently recombined into the same plasmid via the two loxP sites (phage P1 recombination sites), one located downstream from a VH gene in pCW93 and another upstream from a VL gene in pCW99. In the resulting phagemid, the loxP sequence also encodes a polypeptide linker connecting the VH and VL domains to form a scFv of EP-5C7. Whether expressed on the phage surface or as a soluble form, the EP-5C7 scFv showed specific binding to human E- and P-selectin. This phagemid vector system provides a way to recombine VH and VL gene libraries efficiently in vivo to make extremely large Ig combinatorial libraries.

Structure-Directed and Tailored Diversity Synthetic Antibody Libraries Yield Novel Anti-EGFR Antagonists.

PubMed

Miersch, Shane; Maruthachalam, Bharathikumar Vellalore; Geyer, C Ronald; Sidhu, Sachdev S

2017-05-19

We tested whether grafting an interaction domain into the hypervariable loop of a combinatorial antibody library could promote targeting to a specific epitope. Formation of the epidermal growth factor receptor (EGFR) signaling heterodimer involves extensive contacts mediated by a "dimerization loop." We grafted the dimerization loop into the third hypervariable loop of a synthetic antigen-binding fragment (Fab) library and diversified other loops using a tailored diversity strategy. This structure-directed Fab library and a naı̈ve synthetic Fab library were used to select Fabs against EGFR. Both libraries yielded high affinity Fabs that bound to overlapping epitopes on cell-surface EGFR, inhibited receptor activation, and targeted epitopes distinct from those of cetuximab and panitumumab. Epitope mapping experiments revealed complex sites of interaction, comprised of domains I and II but not exclusively localized to the receptor dimerization loop. These results validate the grafting approach for designing Fab libraries and also underscore the versatility of naı̈ve synthetic libraries.

Microwave-assisted one-pot synthesis of ring-fused aminals under catalyst- and solvent-free conditions

EPA Science Inventory

Heterocyclic compounds hold a special place in drug discovery and variety of techniques such as combinatorial synthesis, parallel synthesis, and automated library production to increase the output of these entities has been developed. Although most of these techniques are rapid a...

High-Throughput Ligand Discovery Reveals a Sitewise Gradient of Diversity in Broadly Evolved Hydrophilic Fibronectin Domains

PubMed Central

Woldring, Daniel R.; Holec, Patrick V.; Zhou, Hong; Hackel, Benjamin J.

2015-01-01

Discovering new binding function via a combinatorial library in small protein scaffolds requires balance between appropriate mutations to introduce favorable intermolecular interactions while maintaining intramolecular integrity. Sitewise constraints exist in a non-spatial gradient from diverse to conserved in evolved antibody repertoires; yet non-antibody scaffolds generally do not implement this strategy in combinatorial libraries. Despite the fact that biased amino acid distributions, typically elevated in tyrosine, serine, and glycine, have gained wider use in synthetic scaffolds, these distributions are still predominantly applied uniformly to diversified sites. While select sites in fibronectin domains and DARPins have shown benefit from sitewise designs, they have not been deeply evaluated. Inspired by this disparity between diversity distributions in natural libraries and synthetic scaffold libraries, we hypothesized that binders resulting from discovery and evolution would exhibit a non-spatial, sitewise gradient of amino acid diversity. To identify sitewise diversities consistent with efficient evolution in the context of a hydrophilic fibronectin domain, >105 binders to six targets were evolved and sequenced. Evolutionarily favorable amino acid distributions at 25 sites reveal Shannon entropies (range: 0.3–3.9; median: 2.1; standard deviation: 1.1) supporting the diversity gradient hypothesis. Sitewise constraints in evolved sequences are consistent with complementarity, stability, and consensus biases. Implementation of sitewise constrained diversity enables direct selection of nanomolar affinity binders validating an efficient strategy to balance inter- and intra-molecular interaction demands at each site. PMID:26383268

cGRNB: a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets.

PubMed

Xu, Huayong; Yu, Hui; Tu, Kang; Shi, Qianqian; Wei, Chaochun; Li, Yuan-Yuan; Li, Yi-Xue

2013-01-01

We are witnessing rapid progress in the development of methodologies for building the combinatorial gene regulatory networks involving both TFs (Transcription Factors) and miRNAs (microRNAs). There are a few tools available to do these jobs but most of them are not easy to use and not accessible online. A web server is especially needed in order to allow users to upload experimental expression datasets and build combinatorial regulatory networks corresponding to their particular contexts. In this work, we compiled putative TF-gene, miRNA-gene and TF-miRNA regulatory relationships from forward-engineering pipelines and curated them as built-in data libraries. We streamlined the R codes of our two separate forward-and-reverse engineering algorithms for combinatorial gene regulatory network construction and formalized them as two major functional modules. As a result, we released the cGRNB (combinatorial Gene Regulatory Networks Builder): a web server for constructing combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. The cGRNB enables two major network-building modules, one for MPGE (miRNA-perturbed gene expression) datasets and the other for parallel miRNA/mRNA expression datasets. A miRNA-centered two-layer combinatorial regulatory cascade is the output of the first module and a comprehensive genome-wide network involving all three types of combinatorial regulations (TF-gene, TF-miRNA, and miRNA-gene) are the output of the second module. In this article we propose cGRNB, a web server for building combinatorial gene regulatory networks through integrated engineering of seed-matching sequence information and gene expression datasets. Since parallel miRNA/mRNA expression datasets are rapidly accumulated by the advance of next-generation sequencing techniques, cGRNB will be very useful tool for researchers to build combinatorial gene regulatory networks based on expression datasets. The cGRNB web-server is free and available online at http://www.scbit.org/cgrnb.

Experimental Design for Combinatorial and High Throughput Materials Development

NASA Astrophysics Data System (ADS)

Cawse, James N.

2002-12-01

In the past decade, combinatorial and high throughput experimental methods have revolutionized the pharmaceutical industry, allowing researchers to conduct more experiments in a week than was previously possible in a year. Now high throughput experimentation is rapidly spreading from its origins in the pharmaceutical world to larger industrial research establishments such as GE and DuPont, and even to smaller companies and universities. Consequently, researchers need to know the kinds of problems, desired outcomes, and appropriate patterns for these new strategies. Editor James Cawse's far-reaching study identifies and applies, with specific examples, these important new principles and techniques. Experimental Design for Combinatorial and High Throughput Materials Development progresses from methods that are now standard, such as gradient arrays, to mathematical developments that are breaking new ground. The former will be particularly useful to researchers entering the field, while the latter should inspire and challenge advanced practitioners. The book's contents are contributed by leading researchers in their respective fields. Chapters include: -High Throughput Synthetic Approaches for the Investigation of Inorganic Phase Space -Combinatorial Mapping of Polymer Blends Phase Behavior -Split-Plot Designs -Artificial Neural Networks in Catalyst Development -The Monte Carlo Approach to Library Design and Redesign This book also contains over 200 useful charts and drawings. Industrial chemists, chemical engineers, materials scientists, and physicists working in combinatorial and high throughput chemistry will find James Cawse's study to be an invaluable resource.

DNA-Encoded Solid-Phase Synthesis: Encoding Language Design and Complex Oligomer Library Synthesis.

PubMed

MacConnell, Andrew B; McEnaney, Patrick J; Cavett, Valerie J; Paegel, Brian M

2015-09-14

The promise of exploiting combinatorial synthesis for small molecule discovery remains unfulfilled due primarily to the "structure elucidation problem": the back-end mass spectrometric analysis that significantly restricts one-bead-one-compound (OBOC) library complexity. The very molecular features that confer binding potency and specificity, such as stereochemistry, regiochemistry, and scaffold rigidity, are conspicuously absent from most libraries because isomerism introduces mass redundancy and diverse scaffolds yield uninterpretable MS fragmentation. Here we present DNA-encoded solid-phase synthesis (DESPS), comprising parallel compound synthesis in organic solvent and aqueous enzymatic ligation of unprotected encoding dsDNA oligonucleotides. Computational encoding language design yielded 148 thermodynamically optimized sequences with Hamming string distance ≥ 3 and total read length <100 bases for facile sequencing. Ligation is efficient (70% yield), specific, and directional over 6 encoding positions. A series of isomers served as a testbed for DESPS's utility in split-and-pool diversification. Single-bead quantitative PCR detected 9 × 10(4) molecules/bead and sequencing allowed for elucidation of each compound's synthetic history. We applied DESPS to the combinatorial synthesis of a 75,645-member OBOC library containing scaffold, stereochemical and regiochemical diversity using mixed-scale resin (160-μm quality control beads and 10-μm screening beads). Tandem DNA sequencing/MALDI-TOF MS analysis of 19 quality control beads showed excellent agreement (<1 ppt) between DNA sequence-predicted mass and the observed mass. DESPS synergistically unites the advantages of solid-phase synthesis and DNA encoding, enabling single-bead structural elucidation of complex compounds and synthesis using reactions normally considered incompatible with unprotected DNA. The widespread availability of inexpensive oligonucleotide synthesis, enzymes, DNA sequencing, and PCR make implementation of DESPS straightforward, and may prompt the chemistry community to revisit the synthesis of more complex and diverse libraries.

A diversity-oriented synthesis strategy enabling the combinatorial-type variation of macrocyclic peptidomimetic scaffolds† †Electronic supplementary information (ESI) available: Experimental procedures, characterization data and details of the computational analyses. See DOI: 10.1039/c5ob00371g Click here for additional data file.

PubMed Central

Isidro-Llobet, Albert; Hadje Georgiou, Kathy; Galloway, Warren R. J. D.; Giacomini, Elisa; Hansen, Mette R.; Méndez-Abt, Gabriela; Tan, Yaw Sing; Carro, Laura; Sore, Hannah F.

2015-01-01

Macrocyclic peptidomimetics are associated with a broad range of biological activities. However, despite such potentially valuable properties, the macrocyclic peptidomimetic structural class is generally considered as being poorly explored within drug discovery. This has been attributed to the lack of general methods for producing collections of macrocyclic peptidomimetics with high levels of structural, and thus shape, diversity. In particular, there is a lack of scaffold diversity in current macrocyclic peptidomimetic libraries; indeed, the efficient construction of diverse molecular scaffolds presents a formidable general challenge to the synthetic chemist. Herein we describe a new, advanced strategy for the diversity-oriented synthesis (DOS) of macrocyclic peptidomimetics that enables the combinatorial variation of molecular scaffolds (core macrocyclic ring architectures). The generality and robustness of this DOS strategy is demonstrated by the step-efficient synthesis of a structurally diverse library of over 200 macrocyclic peptidomimetic compounds, each based around a distinct molecular scaffold and isolated in milligram quantities, from readily available building-blocks. To the best of our knowledge this represents an unprecedented level of scaffold diversity in a synthetically derived library of macrocyclic peptidomimetics. Cheminformatic analysis indicated that the library compounds access regions of chemical space that are distinct from those addressed by top-selling brand-name drugs and macrocyclic natural products, illustrating the value of our DOS approach to sample regions of chemical space underexploited in current drug discovery efforts. An analysis of three-dimensional molecular shapes illustrated that the DOS library has a relatively high level of shape diversity. PMID:25778821

Molecular Determinants of Estrogen Receptor Alpha Stability

DTIC Science & Technology

2008-07-01

presence of E2. This question can be addressed by a T7 phage display screen using a breast cancer cell library and DNA-bound ERα in the presence of...conformation of ERα induced by 27HC versus E2. To accomplish this, we performed combinatorial phage display using a modified M13 phage display screen

Finding the Right Candidate for the Right Position: A Fast NMR-Assisted Combinatorial Method for Optimizing Nucleic Acids Binders.

PubMed

Jiménez-Moreno, Ester; Montalvillo-Jiménez, Laura; Santana, Andrés G; Gómez, Ana M; Jiménez-Osés, Gonzalo; Corzana, Francisco; Bastida, Agatha; Jiménez-Barbero, Jesús; Cañada, Francisco Javier; Gómez-Pinto, Irene; González, Carlos; Asensio, Juan Luis

2016-05-25

Development of strong and selective binders from promiscuous lead compounds represents one of the most expensive and time-consuming tasks in drug discovery. We herein present a novel fragment-based combinatorial strategy for the optimization of multivalent polyamine scaffolds as DNA/RNA ligands. Our protocol provides a quick access to a large variety of regioisomer libraries that can be tested for selective recognition by combining microdialysis assays with simple isotope labeling and NMR experiments. To illustrate our approach, 20 small libraries comprising 100 novel kanamycin-B derivatives have been prepared and evaluated for selective binding to the ribosomal decoding A-Site sequence. Contrary to the common view of NMR as a low-throughput technique, we demonstrate that our NMR methodology represents a valuable alternative for the detection and quantification of complex mixtures, even integrated by highly similar or structurally related derivatives, a common situation in the context of a lead optimization process. Furthermore, this study provides valuable clues about the structural requirements for selective A-site recognition.

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

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

Liu, Rongming; Liang, Liya; Garst, Andrew D.

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)Hmore » metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.« less

Directed combinatorial mutagenesis of Escherichia coli for complex phenotype engineering

DOE PAGES

Liu, Rongming; Liang, Liya; Garst, Andrew D.; ...

2018-03-29

Strain engineering for industrial production requires a targeted improvement of multiple complex traits, which range from pathway flux to tolerance to mixed sugar utilization. Here, we report the use of an iterative CRISPR EnAbled Trackable genome Engineering (iCREATE) method to engineer rapid glucose and xylose co-consumption and tolerance to hydrolysate inhibitors in E. coli. Deep mutagenesis libraries were rationally designed, constructed, and screened to target ~40,000 mutations across 30 genes. These libraries included global and high-level regulators that regulate global gene expression, transcription factors that play important roles in genome-level transcription, enzymes that function in the sugar transport system, NAD(P)Hmore » metabolism, and the aldehyde reduction system. Specific mutants that conferred increased growth in mixed sugars and hydrolysate tolerance conditions were isolated, confirmed, and evaluated for changes in genome-wide expression levels. As a result, we tested the strain with positive combinatorial mutations for 3-hydroxypropionic acid (3HP) production under high furfural and high acetate hydrolysate fermentation, which demonstrated a 7- and 8-fold increase in 3HP productivity relative to the parent strain, respectively.« less

Microfluidic Synthesis of Composite Cross-Gradient Materials for Investigating Cell–Biomaterial Interactions

PubMed Central

He, Jiankang; Du, Yanan; Guo, Yuqi; Hancock, Matthew J.; Wang, Ben; Shin, Hyeongho; Wu, Jinhui; Li, Dichen; Khademhosseini, Ali

2010-01-01

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high-throughput screening of cell–material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed “hit”) materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross-gradients of gelatin and chitosan for investigating cell–biomaterial interactions. The microfluidic synthesis of the cross-gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross-gradients were characterized by Fourier transform infrared spectrometry and X-ray photoelectron spectrometry. The three-dimensional (3D) porous gelatin/chitosan cross-gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient-dependent manner. We envision that our microfluidic cross-gradient platform may accelerate the material development processes involved in a wide range of biomedical applications. PMID:20721897

Algorithms for optimizing cross-overs in DNA shuffling.

PubMed

He, Lu; Friedman, Alan M; Bailey-Kellogg, Chris

2012-03-21

DNA shuffling generates combinatorial libraries of chimeric genes by stochastically recombining parent genes. The resulting libraries are subjected to large-scale genetic selection or screening to identify those chimeras with favorable properties (e.g., enhanced stability or enzymatic activity). While DNA shuffling has been applied quite successfully, it is limited by its homology-dependent, stochastic nature. Consequently, it is used only with parents of sufficient overall sequence identity, and provides no control over the resulting chimeric library. This paper presents efficient methods to extend the scope of DNA shuffling to handle significantly more diverse parents and to generate more predictable, optimized libraries. Our CODNS (cross-over optimization for DNA shuffling) approach employs polynomial-time dynamic programming algorithms to select codons for the parental amino acids, allowing for zero or a fixed number of conservative substitutions. We first present efficient algorithms to optimize the local sequence identity or the nearest-neighbor approximation of the change in free energy upon annealing, objectives that were previously optimized by computationally-expensive integer programming methods. We then present efficient algorithms for more powerful objectives that seek to localize and enhance the frequency of recombination by producing "runs" of common nucleotides either overall or according to the sequence diversity of the resulting chimeras. We demonstrate the effectiveness of CODNS in choosing codons and allocating substitutions to promote recombination between parents targeted in earlier studies: two GAR transformylases (41% amino acid sequence identity), two very distantly related DNA polymerases, Pol X and β (15%), and beta-lactamases of varying identity (26-47%). Our methods provide the protein engineer with a new approach to DNA shuffling that supports substantially more diverse parents, is more deterministic, and generates more predictable and more diverse chimeric libraries.

Bead-based screening in chemical biology and drug discovery.

PubMed

Komnatnyy, Vitaly V; Nielsen, Thomas E; Qvortrup, Katrine

2018-06-11

High-throughput screening is an important component of the drug discovery process. The screening of libraries containing hundreds of thousands of compounds requires assays amenable to miniaturisation and automization. Combinatorial chemistry holds a unique promise to deliver structurally diverse libraries for early drug discovery. Among the various library forms, the one-bead-one-compound (OBOC) library, where each bead carries many copies of a single compound, holds the greatest potential for the rapid identification of novel hits against emerging drug targets. However, this potential has not yet been fully realized due to a number of technical obstacles. In this feature article, we review the progress that has been made in bead-based library screening and its application to the discovery of bioactive compounds. We identify the key challenges of this approach and highlight key steps needed for making a greater impact in the field.

Combinatorial selection of molecular conformations and supramolecular synthons in quercetin cocrystal landscapes: a route to ternary solids

PubMed Central

Dubey, Ritesh; Desiraju, Gautam R.

2015-01-01

The crystallization of 28 binary and ternary cocrystals of quercetin with dibasic coformers is analyzed in terms of a combinatorial selection from a solution of preferred molecular conformations and supramolecular synthons. The crystal structures are characterized by distinctive O—H⋯N and O—H⋯O based synthons and are classified as nonporous, porous and helical. Variability in molecular conformation and synthon structure led to an increase in the energetic and structural space around the crystallization event. This space is the crystal structure landscape of the compound and is explored by fine-tuning the experimental conditions of crystallization. In the landscape context, we develop a strategy for the isolation of ternary cocrystals with the use of auxiliary template molecules to reduce the molecular and supramolecular ‘confusion’ that is inherent in a molecule like quercetin. The absence of concomitant polymorphism in this study highlights the selectivity in conformation and synthon choice from the virtual combinatorial library in solution. PMID:26175900

Thermoelectric properties of the LaCoO3-LaCrO3 system using a high-throughput combinatorial approach

NASA Astrophysics Data System (ADS)

Talley, K. R.; Barron, S. C.; Nguyen, N.; Wong-Ng, W.; Martin, J.; Zhang, Y. L.; Song, X.

2017-02-01

A combinatorial film of the LaCo1-xCrxO3 system was fabricated using the LaCoO3 and LaCrO3 targets at the NIST Pulsed Laser Deposition (PLD) facility. As the ionic size of Cr3+ is greater than that of Co3+, the unit cell volume of the series increases with increasing x. Using a custom screening tool, the Seebeck coefficient of LaCo1-xCrxO3 approaches a measured maximum of 286 μV/K, near to the cobalt-rich end of the film library (with x ≈ 0.49). The resistivity value increases continuously with increasing x. The measured power factor, PF, of this series, which is related to the efficiency of energy conversion, also exhibits a maximum at the composition of x ≈ 0.49, which corresponds to the maximum value of the Seebeck coefficient. Our results illustrate the efficiency of applying the high-throughput combinatorial technique to study thermoelectric materials.

Two is better than one; toward a rational design of combinatorial therapy.

PubMed

Chen, Sheng-Hong; Lahav, Galit

2016-12-01

Drug combination is an appealing strategy for combating the heterogeneity of tumors and evolution of drug resistance. However, the rationale underlying combinatorial therapy is often not well established due to lack of understandings of the specific pathways responding to the drugs, and their temporal dynamics following each treatment. Here we present several emerging trends in harnessing properties of biological systems for the optimal design of drug combinations, including the type of drugs, specific concentration, sequence of addition and the temporal schedule of treatments. We highlight recent studies showing different approaches for efficient design of drug combinations including single-cell signaling dynamics, adaption and pathway crosstalk. Finally, we discuss novel and feasible approaches that can facilitate the optimal design of combinatorial therapy. Copyright © 2016 Elsevier Ltd. All rights reserved.

Phage display biopanning and isolation of target-unrelated peptides: in search of nonspecific binders hidden in a combinatorial library.

PubMed

Bakhshinejad, Babak; Zade, Hesam Motaleb; Shekarabi, Hosna Sadat Zahed; Neman, Sara

2016-12-01

Phage display is known as a powerful methodology for the identification of targeting ligands that specifically bind to a variety of targets. The high-throughput screening of phage display combinatorial peptide libraries is performed through the affinity selection method of biopanning. Although phage display selection has proven very successful in the discovery of numerous high-affinity target-binding peptides with potential application in drug discovery and delivery, the enrichment of false-positive target-unrelated peptides (TUPs) without any actual affinity towards the target remains a major problem of library screening. Selection-related TUPs may emerge because of binding to the components of the screening system rather than the target. Propagation-related TUPs may arise as a result of faster growth rate of some phage clones enabling them to outcompete slow-propagating clones. Amplification of the library between rounds of biopanning makes a significant contribution to the selection of phage clones with propagation advantage. Distinguishing nonspecific TUPs from true target binders is of particular importance for the translation of biopanning findings from basic research to clinical applications. Different experimental and in silico approaches are applied to assess the specificity of phage display-derived peptides towards the target. Bioinformatic tools are playing a rapidly growing role in the analysis of biopanning data and identification of target-irrelevant TUPs. Recent progress in the introduction of efficient strategies for TUP detection holds enormous promise for the discovery of clinically relevant cell- and tissue-homing peptides and paves the way for the development of novel targeted diagnostic and therapeutic platforms in pharmaceutical areas.

Variable epitope libraries: new vaccine immunogens capable of inducing broad human immunodeficiency virus type 1-neutralizing antibody response.

PubMed

Charles-Niño, Claudia; Pedroza-Roldan, Cesar; Viveros, Monica; Gevorkian, Goar; Manoutcharian, Karen

2011-07-18

The extreme antigenic variability of human immunodeficiency virus (HIV) leads to immune escape of the virus, representing a major challenge in the design of effective vaccine. We have developed a novel concept for immunogen construction based on introduction of massive mutations within the epitopes targeting antigenically variable pathogens and diseases. Previously, we showed that these immunogens carrying large combinatorial libraries of mutated epitope variants, termed as variable epitope libraries (VELs), induce potent, broad and long lasting CD8+IFN-γ+ T-cell response. Moreover, we demonstrated that these T cells recognize more than 50% of heavily mutated variants (5 out of 10 amino acid positions were mutated in each epitope variant) of HIV-1 gp120 V3 loop-derived cytotoxic T lymphocyte epitope (RGPGRAFVTI) in mice. The constructed VELs had complexities of 10000 and 12500 individual members, generated as plasmid DNA or as M13 phage display combinatorial libraries, respectively, and with structural composition RGPGXAXXXX or XGXGXAXVXI, where X is any of 20 natural amino acids. Here, we demonstrated that sera from mice immunized with these VELs are capable of neutralizing 5 out of 10 viral isolates from Tier 2 reference panel of subtype B envelope clones, including HIV-1 isolates which are known to be resistant to neutralization by several potent monoclonal antibodies, described previously. These data indicate the feasibility of the application of immunogens based on VEL concept as an alternative approach for the development of molecular vaccines against antigenically variable pathogens. Copyright © 2011 Elsevier Ltd. All rights reserved.

A generic approach to engineer antibody pH-switches using combinatorial histidine scanning libraries and yeast display.

PubMed

Schröter, Christian; Günther, Ralf; Rhiel, Laura; Becker, Stefan; Toleikis, Lars; Doerner, Achim; Becker, Janine; Schönemann, Andreas; Nasu, Daichi; Neuteboom, Berend; Kolmar, Harald; Hock, Björn

2015-01-01

There is growing interest in the fast and robust engineering of protein pH-sensitivity that aims to reduce binding at acidic pH, compared to neutral pH. Here, we describe a novel strategy for the incorporation of pH-sensitive antigen binding functions into antibody variable domains using combinatorial histidine scanning libraries and yeast surface display. The strategy allows simultaneous screening for both, high affinity binding at pH 7.4 and pH-sensitivity, and excludes conventional negative selection steps. As proof of concept, we applied this strategy to incorporate pH-dependent antigen binding into the complementary-determining regions of adalimumab. After 3 consecutive rounds of separate heavy and light chain library screening, pH-sensitive variants could be isolated. Heavy and light chain mutations were combined, resulting in 3 full-length antibody variants that revealed sharp, reversible pH-dependent binding profiles. Dissociation rate constants at pH 6.0 increased 230- to 780-fold, while high affinity binding at pH 7.4 in the sub-nanomolar range was retained. Furthermore, binding to huFcRn and thermal stability were not affected by histidine substitutions. Overall, this study emphasizes a generalizable strategy for engineering pH-switch functions potentially applicable to a variety of antibodies and further proteins-based therapeutics.

Utilizing pulsed laser deposition lateral inhomogeneity as a tool in combinatorial material science.

PubMed

Keller, David A; Ginsburg, Adam; Barad, Hannah-Noa; Shimanovich, Klimentiy; Bouhadana, Yaniv; Rosh-Hodesh, Eli; Takeuchi, Ichiro; Aviv, Hagit; Tischler, Yaakov R; Anderson, Assaf Y; Zaban, Arie

2015-04-13

Pulsed laser deposition (PLD) is widely used in combinatorial material science, as it enables rapid fabrication of different composite materials. Nevertheless, this method was usually limited to small substrates, since PLD deposition on large substrate areas results in severe lateral inhomogeneity. A few technical solutions for this problem have been suggested, including the use of different designs of masks, which were meant to prevent inhomogeneity in the thickness, density, and oxidation state of a layer, while only the composition is allowed to be changed. In this study, a possible way to take advantage of the large scale deposition inhomogeneity is demonstrated, choosing an iron oxide PLD-deposited library with continuous compositional spread (CCS) as a model system. An Fe₂O₃-Nb₂O₅ library was fabricated using PLD, without any mask between the targets and the substrate. The library was measured using high-throughput scanners for electrical, structural, and optical properties. A decrease in electrical resistivity that is several orders of magnitude lower than pure α-Fe₂O₃ was achieved at ∼20% Nb-O (measured at 47 and 267 °C) but only at points that are distanced from the center of the PLD plasma plume. Using hierarchical clustering analysis, we show that the PLD inhomogeneity can be used as an additional degree of freedom, helping, in this case, to achieve iron oxide with much lower resistivity.

Screening of a Combinatorial Library of Organic Polymers for the Solid-Phase Extraction of Patulin from Apple Juice

PubMed Central

Giovannoli, Cristina; Spano, Giulia; Di Nardo, Fabio; Anfossi, Laura; Baggiani, Claudio

2017-01-01

Patulin is a water-soluble mycotoxin produced by several species of fungi. Governmental bodies have placed it under scrutiny for its potential negative health effects, and maximum residue limits are fixed in specific food matrices to protect consumers’ health. Confirmatory analysis of patulin in complex food matrices can be a difficult task, and sample clean-up treatments are frequently necessary before instrumental analyses. With the aim of simplifying the clean-up step, we prepared a 256-member combinatorial polymeric library based on 16 functional monomers, four cross-linkers and four different porogenic solvents. The library was screened for the binding towards patulin in different media (acetonitrile and citrate buffer at pH 3.2), with the goal of identifying polymer formulations with good binding properties towards the target compound. As a proof of concept, a methacrylic acid-co-pentaerithrytole tetraacrylate polymer prepared in chloroform was successfully used as a solid-phase extraction material for the clean-up and extraction of patulin from apple juice. Clean chromatographic patterns and acceptable recoveries were obtained for juice spiked with patulin at concentration levels of 25 (64 ± 12%), 50 (83 ± 5.6%) and 100 μg L−1 (76 ± 4.5%). The within-day and between-day reproducibility evaluated at a concentration level of 25 μg L−1 were 5.6 and 7.6%, respectively. PMID:28531103

Scoring of Side-Chain Packings: An Analysis of Weight Factors and Molecular Dynamics Structures.

PubMed

Colbes, Jose; Aguila, Sergio A; Brizuela, Carlos A

2018-02-26

The protein side-chain packing problem (PSCPP) is a central task in computational protein design. The problem is usually modeled as a combinatorial optimization problem, which consists of searching for a set of rotamers, from a given rotamer library, that minimizes a scoring function (SF). The SF is a weighted sum of terms, that can be decomposed in physics-based and knowledge-based terms. Although there are many methods to obtain approximate solutions for this problem, all of them have similar performances and there has not been a significant improvement in recent years. Studies on protein structure prediction and protein design revealed the limitations of current SFs to achieve further improvements for these two problems. In the same line, a recent work reported a similar result for the PSCPP. In this work, we ask whether or not this negative result regarding further improvements in performance is due to (i) an incorrect weighting of the SFs terms or (ii) the constrained conformation resulting from the protein crystallization process. To analyze these questions, we (i) model the PSCPP as a bi-objective combinatorial optimization problem, optimizing, at the same time, the two most important terms of two SFs of state-of-the-art algorithms and (ii) performed a preprocessing relaxation of the crystal structure through molecular dynamics to simulate the protein in the solvent and evaluated the performance of these two state-of-the-art SFs under these conditions. Our results indicate that (i) no matter what combination of weight factors we use the current SFs will not lead to better performances and (ii) the evaluated SFs will not be able to improve performance on relaxed structures. Furthermore, the experiments revealed that the SFs and the methods are biased toward crystallized structures.

A signaling visualization toolkit to support rational design of combination therapies and biomarker discovery: SiViT.

PubMed

Bown, James L; Shovman, Mark; Robertson, Paul; Boiko, Andrei; Goltsov, Alexey; Mullen, Peter; Harrison, David J

2017-05-02

Targeted cancer therapy aims to disrupt aberrant cellular signalling pathways. Biomarkers are surrogates of pathway state, but there is limited success in translating candidate biomarkers to clinical practice due to the intrinsic complexity of pathway networks. Systems biology approaches afford better understanding of complex, dynamical interactions in signalling pathways targeted by anticancer drugs. However, adoption of dynamical modelling by clinicians and biologists is impeded by model inaccessibility. Drawing on computer games technology, we present a novel visualization toolkit, SiViT, that converts systems biology models of cancer cell signalling into interactive simulations that can be used without specialist computational expertise. SiViT allows clinicians and biologists to directly introduce for example loss of function mutations and specific inhibitors. SiViT animates the effects of these introductions on pathway dynamics, suggesting further experiments and assessing candidate biomarker effectiveness. In a systems biology model of Her2 signalling we experimentally validated predictions using SiViT, revealing the dynamics of biomarkers of drug resistance and highlighting the role of pathway crosstalk. No model is ever complete: the iteration of real data and simulation facilitates continued evolution of more accurate, useful models. SiViT will make accessible libraries of models to support preclinical research, combinatorial strategy design and biomarker discovery.

One-Bead-Two-Compound Thioether Bridged Macrocyclic γ-AApeptide Screening Library against EphA2.

PubMed

Shi, Yan; Challa, Sridevi; Sang, Peng; She, Fengyu; Li, Chunpu; Gray, Geoffrey M; Nimmagadda, Alekhya; Teng, Peng; Odom, Timothy; Wang, Yan; van der Vaart, Arjan; Li, Qi; Cai, Jianfeng

2017-11-22

Identification of molecular ligands that recognize peptides or proteins is significant but poses a fundamental challenge in chemical biology and biomedical sciences. Development of cyclic peptidomimetic library is scarce, and thus discovery of cyclic peptidomimetic ligands for protein targets is rare. Herein we report the unprecedented one-bead-two-compound (OBTC) combinatorial library based on a novel class of the macrocyclic peptidomimetics γ-AApeptides. In the library, we utilized the coding peptide tags synthesized with Dde-protected α-amino acids, which were orthogonal to solid phase synthesis of γ-AApeptides. Employing the thioether linkage, the desired macrocyclic γ-AApeptides were found to be effective for ligand identification. Screening the library against the receptor tyrosine kinase EphA2 led to the discovery of one lead compound that tightly bound to EphA2 (K d = 81 nM) and potently antagonized EphA2-mediated signaling. This new approach of macrocyclic peptidomimetic library may lead to a novel platform for biomacromolecular surface recognition and function modulation.

PERMutation Using Transposase Engineering (PERMUTE): A Simple Approach for Constructing Circularly Permuted Protein Libraries.

PubMed

Jones, Alicia M; Atkinson, Joshua T; Silberg, Jonathan J

2017-01-01

Rearrangements that alter the order of a protein's sequence are used in the lab to study protein folding, improve activity, and build molecular switches. One of the simplest ways to rearrange a protein sequence is through random circular permutation, where native protein termini are linked together and new termini are created elsewhere through random backbone fission. Transposase mutagenesis has emerged as a simple way to generate libraries encoding different circularly permuted variants of proteins. With this approach, a synthetic transposon (called a permuteposon) is randomly inserted throughout a circularized gene to generate vectors that express different permuted variants of a protein. In this chapter, we outline the protocol for constructing combinatorial libraries of circularly permuted proteins using transposase mutagenesis, and we describe the different permuteposons that have been developed to facilitate library construction.

Beyond directed evolution - semi-rational protein engineering and design

PubMed Central

Lutz, Stefan

2010-01-01

Over the last two decades, directed evolution has transformed the field of protein engineering. The advances in understanding protein structure and function, in no insignificant part a result of directed evolution studies, are increasingly empowering scientists and engineers to device more effective methods for manipulating and tailoring biocatalysts. Abandoning large combinatorial libraries, the focus has shifted to small, functionally-rich libraries and rational design. A critical component to the success of these emerging engineering strategies are computational tools for the evaluation of protein sequence datasets and the analysis of conformational variations of amino acids in proteins. Highlighting the opportunities and limitations of such approaches, this review focuses on recent engineering and design examples that require screening or selection of small libraries. PMID:20869867

Diversity-oriented combinatorial biosynthesis of benzenediol lactone scaffolds by subunit shuffling of fungal polyketide synthases.

PubMed

Xu, Yuquan; Zhou, Tong; Zhang, Shuwei; Espinosa-Artiles, Patricia; Wang, Luoyi; Zhang, Wei; Lin, Min; Gunatilaka, A A Leslie; Zhan, Jixun; Molnár, István

2014-08-26

Combinatorial biosynthesis aspires to exploit the promiscuity of microbial anabolic pathways to engineer the synthesis of new chemical entities. Fungal benzenediol lactone (BDL) polyketides are important pharmacophores with wide-ranging bioactivities, including heat shock response and immune system modulatory effects. Their biosynthesis on a pair of sequentially acting iterative polyketide synthases (iPKSs) offers a test case for the modularization of secondary metabolic pathways into "build-couple-pair" combinatorial synthetic schemes. Expression of random pairs of iPKS subunits from four BDL model systems in a yeast heterologous host created a diverse library of BDL congeners, including a polyketide with an unnatural skeleton and heat shock response-inducing activity. Pairwise heterocombinations of the iPKS subunits also helped to illuminate the innate, idiosyncratic programming of these enzymes. Even in combinatorial contexts, these biosynthetic programs remained largely unchanged, so that the iPKSs built their cognate biosynthons, coupled these building blocks into chimeric polyketide intermediates, and catalyzed intramolecular pairing to release macrocycles or α-pyrones. However, some heterocombinations also provoked stuttering, i.e., the relaxation of iPKSs chain length control to assemble larger homologous products. The success of such a plug and play approach to biosynthesize novel chemical diversity bodes well for bioprospecting unnatural polyketides for drug discovery.

Solid-Phase Synthesis of Diverse Peptide Tertiary Amides By Reductive Amination

PubMed Central

Pels, Kevin; Kodadek, Thomas

2015-01-01

The synthesis of libraries of conformationally-constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as peptide tertiary amide (PTA). PTAs are strongly biased conformationally due to allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers. PMID:25695359

Solid-phase synthesis of diverse peptide tertiary amides by reductive amination.

PubMed

Pels, Kevin; Kodadek, Thomas

2015-03-09

The synthesis of libraries of conformationally constrained peptide-like oligomers is an important goal in combinatorial chemistry. In this regard an attractive building block is the N-alkylated peptide, also known as a peptide tertiary amide (PTA). PTAs are conformationally constrained because of allylic 1,3 strain interactions. We report here an improved synthesis of these species on solid supports through the use of reductive amination chemistry using amino acid-terminated, bead-displayed oligomers and diverse aldehydes. The utility of this chemistry is demonstrated by the synthesis of a library of 10,000 mixed peptoid-PTA oligomers.

Modulating weak interactions for molecular recognition: a dynamic combinatorial analysis for assessing the contribution of electrostatics to the stability of CH-π bonds in water.

PubMed

Jiménez-Moreno, Ester; Gómez, Ana M; Bastida, Agatha; Corzana, Francisco; Jiménez-Oses, Gonzalo; Jiménez-Barbero, Jesús; Asensio, Juan Luis

2015-03-27

Electrostatic and charge-transfer contributions to CH-π complexes can be modulated by attaching electron-withdrawing substituents to the carbon atom. While clearly stabilizing in the gas phase, the outcome of this chemical modification in water is more difficult to predict. Herein we provide a definitive and quantitative answer to this question employing a simple strategy based on dynamic combinatorial chemistry. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A combinatorial histidine scanning library approach to engineer highly pH-dependent protein switches

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

Murtaugh, Megan L.; Fanning, Sean W.; Sharma, Tressa M.

2012-09-05

There is growing interest in the development of protein switches, which are proteins whose function, such as binding a target molecule, can be modulated through environmental triggers. Efforts to engineer highly pH sensitive protein-protein interactions typically rely on the rational introduction of ionizable groups in the protein interface. Such experiments are typically time intensive and often sacrifice the protein's affinity at the permissive pH. The underlying thermodynamics of proton-linkage dictate that the presence of multiple ionizable groups, which undergo a pK{sub a} change on protein binding, are necessary to result in highly pH-dependent binding. To test this hypothesis, a novelmore » combinatorial histidine library was developed where every possible combination of histidine and wild-type residue is sampled throughout the interface of a model anti-RNase A single domain VHH antibody. Antibodies were coselected for high-affinity binding and pH-sensitivity using an in vitro, dual-function selection strategy. The resulting antibodies retained near wild-type affinity yet became highly sensitive to small decreases in pH, drastically decreasing their binding affinity, due to the incorporation of multiple histidine groups. Several trends were observed, such as histidine 'hot-spots,' which will help enhance the development of pH switch proteins as well as increase our understanding of the role of ionizable residues in protein interfaces. Overall, the combinatorial approach is rapid, general, and robust and should be capable of producing highly pH-sensitive protein affinity reagents for a number of different applications.« less

DC-Analyzer-facilitated combinatorial strategy for rapid directed evolution of functional enzymes with multiple mutagenesis sites.

PubMed

Wang, Xiong; Zheng, Kai; Zheng, Huayu; Nie, Hongli; Yang, Zujun; Tang, Lixia

2014-12-20

Iterative saturation mutagenesis (ISM) has been shown to be a powerful method for directed evolution. In this study, the approach was modified (termed M-ISM) by combining the single-site saturation mutagenesis method with a DC-Analyzer-facilitated combinatorial strategy, aiming to evolve novel biocatalysts efficiently in the case where multiple sites are targeted simultaneously. Initially, all target sites were explored individually by constructing single-site saturation mutagenesis libraries. Next, the top two to four variants in each library were selected and combined using the DC-Analyzer-facilitated combinatorial strategy. In addition to site-saturation mutagenesis, iterative saturation mutagenesis also needed to be performed. The advantages of M-ISM over ISM were that the screening effort is greatly reduced, and the entire M-ISM procedure was less time-consuming. The M-ISM strategy was successfully applied to the randomization of halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) when five interesting sites were targeted simultaneously. After screening 900 clones in total, six positive mutants were obtained. These mutants exhibited 4.0- to 9.3-fold higher k(cat) values than did the wild-type HheC toward 1,3-dichloro-2-propanol. However, with the ISM strategy, the best hit showed a 5.9-fold higher k(cat) value toward 1,3-DCP than the wild-type HheC, which was obtained after screening 4000 clones from four rounds of mutagenesis. Therefore, M-ISM could serve as a simple and efficient version of ISM for the randomization of target genes with multiple positions of interest.

Invention as a combinatorial process: evidence from US patents

PubMed Central

Youn, Hyejin; Strumsky, Deborah; Bettencourt, Luis M. A.; Lobo, José

2015-01-01

Invention has been commonly conceptualized as a search over a space of combinatorial possibilities. Despite the existence of a rich literature, spanning a variety of disciplines, elaborating on the recombinant nature of invention, we lack a formal and quantitative characterization of the combinatorial process underpinning inventive activity. Here, we use US patent records dating from 1790 to 2010 to formally characterize invention as a combinatorial process. To do this, we treat patented inventions as carriers of technologies and avail ourselves of the elaborate system of technology codes used by the United States Patent and Trademark Office to classify the technologies responsible for an invention's novelty. We find that the combinatorial inventive process exhibits an invariant rate of ‘exploitation’ (refinements of existing combinations of technologies) and ‘exploration’ (the development of new technological combinations). This combinatorial dynamic contrasts sharply with the creation of new technological capabilities—the building blocks to be combined—that has significantly slowed down. We also find that, notwithstanding the very reduced rate at which new technologies are introduced, the generation of novel technological combinations engenders a practically infinite space of technological configurations. PMID:25904530

A combinatorial code for pattern formation in Drosophila oogenesis.

PubMed

Yakoby, Nir; Bristow, Christopher A; Gong, Danielle; Schafer, Xenia; Lembong, Jessica; Zartman, Jeremiah J; Halfon, Marc S; Schüpbach, Trudi; Shvartsman, Stanislav Y

2008-11-01

Two-dimensional patterning of the follicular epithelium in Drosophila oogenesis is required for the formation of three-dimensional eggshell structures. Our analysis of a large number of published gene expression patterns in the follicle cells suggests that they follow a simple combinatorial code based on six spatial building blocks and the operations of union, difference, intersection, and addition. The building blocks are related to the distribution of inductive signals, provided by the highly conserved epidermal growth factor receptor and bone morphogenetic protein signaling pathways. We demonstrate the validity of the code by testing it against a set of patterns obtained in a large-scale transcriptional profiling experiment. Using the proposed code, we distinguish 36 distinct patterns for 81 genes expressed in the follicular epithelium and characterize their joint dynamics over four stages of oogenesis. The proposed combinatorial framework allows systematic analysis of the diversity and dynamics of two-dimensional transcriptional patterns and guides future studies of gene regulation.

Protein and Antibody Engineering by Phage Display

PubMed Central

Frei, J.C.; Lai, J.R.

2017-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. PMID:27586328

Identification of Broad-Based HIV-1 Protease Inhibitors From Combinatorial Libraries

PubMed Central

Chang, Max W.; Giffin, Michael J.; Muller, Rolf; Savage, Jeremiah; Lin, Ying C.; Hong, Sukwon; Jin, Wei; Whitby, Landon R.; Elder, John H.; Boger, Dale L.; Torbett, Bruce E.

2011-01-01

Clinically approved inhibitors of HIV-1 protease function via a competitive mechanism. A particular vulnerability of competitive inhibitors is their sensitivity to increases in substrate concentration, as may occur during virion assembly, budding and processing into a mature, infectious viral particle. Advances in chemical synthesis have led to the development of new chemical libraries with high diversity using rapid in-solution syntheses. These libraries have been previously shown to be effective at disrupting protein-protein and protein-nucleic acid interfaces. We have screened 44,000 compounds from such a library to identify inhibitors of HIV-1 protease. One compound was identified that inhibits wild type protease, as well as a drug-resistant protease with 6 mutations. Moreover, analysis of this compound suggests an allosteric, non-competitive mechanism of inhibition and may represent a starting point for an additional strategy for anti-retroviral therapy. PMID:20507280

Advancement and applications of peptide phage display technology in biomedical science.

PubMed

Wu, Chien-Hsun; Liu, I-Ju; Lu, Ruei-Min; Wu, Han-Chung

2016-01-19

Combinatorial phage library is a powerful research tool for high-throughput screening of protein interactions. Of all available molecular display techniques, phage display has proven to be the most popular approach. Screening phage-displayed random peptide libraries is an effective means of identifying peptides that can bind target molecules and regulate their function. Phage-displayed peptide libraries can be used for (i) B-cell and T-cell epitope mapping, (ii) selection of bioactive peptides bound to receptors or proteins, disease-specific antigen mimics, peptides bound to non-protein targets, cell-specific peptides, or organ-specific peptides, and (iii) development of peptide-mediated drug delivery systems and other applications. Targeting peptides identified using phage display technology may be useful for basic research and translational medicine. In this review article, we summarize the latest technological advancements in the application of phage-displayed peptide libraries to applied biomedical sciences.

Patent review.

PubMed

Roy, Anuradha

2011-05-01

The section on patent review will be focused in the areas of interest to the readers of CCHTS. The search was conducted using the following key words: combinatorial chemistry, high throughput screening, drug repurposing, chemical library, high content screening, drug discovery and natural products. All patents highlighted here are identified by the patent number issued either by the World Intellectual Property Organization or by a regional patent office.

Patent review.

PubMed

Roy, Anuradha; McGee, James E

2011-08-01

The section on patent review will be focused in the areas of interest to the readers of CCHTS. The search was conducted using the following key words: combinatorial chemistry, high throughput screening, drug repurposing, chemical library, high content screening, drug discovery and natural products. All patents highlighted here are identified by the patent number issued either by the World Intellectual Property Organization or by a regional patent office.

In-depth proteomic analysis of banana (Musa spp.) fruit with combinatorial peptide ligand libraries.

PubMed

Esteve, Clara; D'Amato, Alfonsina; Marina, María Luisa; García, María Concepción; Righetti, Pier Giorgio

2013-01-01

Musa ssp. is among the world's leading fruit crops. Although a strong interest on banana biochemistry exists in the scientific community, focused on metabolite composition, proteins have been scarcely investigated even if they play an important role in food allergy and stability, are a source of biologically active peptides, and can provide information about nutritional aspects of this fruit. In this work we have employed the combinatorial peptide ligand libraries after different types of protein extractions, for searching the very low-abundance proteins in banana. The use of advanced MS techniques and Musa ssp. mRNAs database in combination with the Uniprot_viridiplantae database allowed us to identify 1131 proteins. Among this huge amount of proteins we found several already known allergens such as Mus a 1, pectinesterase, superoxide dismutase, and potentially new allergens. Additionally several enzymes involved in degradation of starch granules and strictly correlated to ripening stage were identified. This is the first in-depth exploration of the banana fruit proteome and one of the largest descriptions of the proteome of any vegetable system. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Expediting Combinatorial Data Set Analysis by Combining Human and Algorithmic Analysis.

PubMed

Stein, Helge Sören; Jiao, Sally; Ludwig, Alfred

2017-01-09

A challenge in combinatorial materials science remains the efficient analysis of X-ray diffraction (XRD) data and its correlation to functional properties. Rapid identification of phase-regions and proper assignment of corresponding crystal structures is necessary to keep pace with the improved methods for synthesizing and characterizing materials libraries. Therefore, a new modular software called htAx (high-throughput analysis of X-ray and functional properties data) is presented that couples human intelligence tasks used for "ground-truth" phase-region identification with subsequent unbiased verification by an algorithm to efficiently analyze which phases are present in a materials library. Identified phases and phase-regions may then be correlated to functional properties in an expedited manner. For the functionality of htAx to be proven, two previously published XRD benchmark data sets of the materials systems Al-Cr-Fe-O and Ni-Ti-Cu are analyzed by htAx. The analysis of ∼1000 XRD patterns takes less than 1 day with htAx. The proposed method reliably identifies phase-region boundaries and robustly identifies multiphase structures. The method also addresses the problem of identifying regions with previously unpublished crystal structures using a special daisy ternary plot.

Automated Lead Optimization of MMP-12 Inhibitors Using a Genetic Algorithm.

PubMed

Pickett, Stephen D; Green, Darren V S; Hunt, David L; Pardoe, David A; Hughes, Ian

2011-01-13

Traditional lead optimization projects involve long synthesis and testing cycles, favoring extensive structure-activity relationship (SAR) analysis and molecular design steps, in an attempt to limit the number of cycles that a project must run to optimize a development candidate. Microfluidic-based chemistry and biology platforms, with cycle times of minutes rather than weeks, lend themselves to unattended autonomous operation. The bottleneck in the lead optimization process is therefore shifted from synthesis or test to SAR analysis and design. As such, the way is open to an algorithm-directed process, without the need for detailed user data analysis. Here, we present results of two synthesis and screening experiments, undertaken using traditional methodology, to validate a genetic algorithm optimization process for future application to a microfluidic system. The algorithm has several novel features that are important for the intended application. For example, it is robust to missing data and can suggest compounds for retest to ensure reliability of optimization. The algorithm is first validated on a retrospective analysis of an in-house library embedded in a larger virtual array of presumed inactive compounds. In a second, prospective experiment with MMP-12 as the target protein, 140 compounds are submitted for synthesis over 10 cycles of optimization. Comparison is made to the results from the full combinatorial library that was synthesized manually and tested independently. The results show that compounds selected by the algorithm are heavily biased toward the more active regions of the library, while the algorithm is robust to both missing data (compounds where synthesis failed) and inactive compounds. This publication places the full combinatorial library and biological data into the public domain with the intention of advancing research into algorithm-directed lead optimization methods.

Automated Lead Optimization of MMP-12 Inhibitors Using a Genetic Algorithm

PubMed Central

2010-01-01

Traditional lead optimization projects involve long synthesis and testing cycles, favoring extensive structure−activity relationship (SAR) analysis and molecular design steps, in an attempt to limit the number of cycles that a project must run to optimize a development candidate. Microfluidic-based chemistry and biology platforms, with cycle times of minutes rather than weeks, lend themselves to unattended autonomous operation. The bottleneck in the lead optimization process is therefore shifted from synthesis or test to SAR analysis and design. As such, the way is open to an algorithm-directed process, without the need for detailed user data analysis. Here, we present results of two synthesis and screening experiments, undertaken using traditional methodology, to validate a genetic algorithm optimization process for future application to a microfluidic system. The algorithm has several novel features that are important for the intended application. For example, it is robust to missing data and can suggest compounds for retest to ensure reliability of optimization. The algorithm is first validated on a retrospective analysis of an in-house library embedded in a larger virtual array of presumed inactive compounds. In a second, prospective experiment with MMP-12 as the target protein, 140 compounds are submitted for synthesis over 10 cycles of optimization. Comparison is made to the results from the full combinatorial library that was synthesized manually and tested independently. The results show that compounds selected by the algorithm are heavily biased toward the more active regions of the library, while the algorithm is robust to both missing data (compounds where synthesis failed) and inactive compounds. This publication places the full combinatorial library and biological data into the public domain with the intention of advancing research into algorithm-directed lead optimization methods. PMID:24900251

Application of High-Throughput Seebeck Microprobe Measurements on Thermoelectric Half-Heusler Thin Film Combinatorial Material Libraries.

PubMed

Ziolkowski, Pawel; Wambach, Matthias; Ludwig, Alfred; Mueller, Eckhard

2018-01-08

In view of the variety and complexity of thermoelectric (TE) material systems, combinatorial approaches to materials development come to the fore for identifying new promising compounds. The success of this approach is related to the availability and reliability of high-throughput characterization methods for identifying interrelations between materials structures and properties within the composition spread libraries. A meaningful characterization starts with determination of the Seebeck coefficient as a major feature of TE materials. Its measurement, and hence the accuracy and detectability of promising material compositions, may be strongly affected by thermal and electrical measurement conditions. This work illustrates the interrelated effects of the substrate material, the layer thickness, and spatial property distributions of thin film composition spread libraries, which are studied experimentally by local thermopower scans by means of the Potential and Seebeck Microprobe (PSM). The study is complemented by numerical evaluation. Material libraries of the half-Heusler compound system Ti-Ni-Sn were deposited on selected substrates (Si, AlN, Al 2 O 3 ) by magnetron sputtering. Assuming homogeneous properties of a film, significant decrease of the detected thermopower S m can be expected on substrates with higher thermal conductivity, yielding an underestimation of materials thermopower between 15% and 50%, according to FEM (finite element methods) simulations. Thermally poor conducting substrates provide a better accuracy with thermopower underestimates lower than 8%, but suffer from a lower spatial resolution. According to FEM simulations, local scanning of sharp thermopower peaks on lowly conductive substrates is linked to an additional deviation of the measured thermopower of up to 70% compared to homogeneous films, which is 66% higher than for corresponding cases on substrates with higher thermal conductivity of this study.

Human recombinant Fab fragment from combinatorial libraries of a B-cell lymphoma patient recognizes core protein of chondroitin sulphate proteoglycan 4.

PubMed

Egami, Yoko; Narushima, Yuta; Ohshima, Motohiro; Yoshida, Akira; Yoneta, Naruki; Masaki, Yasufumi; Itoh, Kunihiko

2018-01-01

CD antigens are well known as therapeutic targets of B-cell lymphoma. To isolate therapeutic antibodies that recognize novel targets other than CD antigens, we constructed a phage display combinatorial antibody Fab library from bone marrow lymphocytes of B-cell lymphoma patient. To eliminate antibodies reactive with known B-cell lymphoma antigen, non-hematopoietic and patient's sera reactive HeLaS3 cells was selected as a target of whole cell panning. Five rounds of panning against live HeLaS3 cells retrieved single Fab clone, termed AHSA (Antibody to HeLa Surface Antigen). Using phage display random peptide library, LSYLEP was identified as an epitope sequence of AHSA. LC-MS/MS analysis of AHSA-precipitated HeLaS3 cell lysates detected several fragments corresponding to the sequence of chondroitin sulphate proteoglycan 4 (CSPG4) core protein. Since LSYLEP sequence was at the position of 313-318 of CSPG4, we considered that CSPG4 was AHSA-associated antigen. Double staining of CSPG4-postive MDA-MB-435S cells with AHSA and anti-CSPG4 rabbit antibody showed identical staining position, and reduced AHSA reactivity was observed in CSPG4-siRNA treated MDA-MB-435S cells. In conclusion, we retrieved a human Fab from antibody library of B-cell lymphoma patient, and identified CSPG4 as a recognizing antigen. AHSA may have potential benefits for development of CSPG4-targeting theranostics for B-cell lymphoma. © The Authors 2017. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved.

Desirability-based methods of multiobjective optimization and ranking for global QSAR studies. Filtering safe and potent drug candidates from combinatorial libraries.

PubMed

Cruz-Monteagudo, Maykel; Borges, Fernanda; Cordeiro, M Natália D S; Cagide Fajin, J Luis; Morell, Carlos; Ruiz, Reinaldo Molina; Cañizares-Carmenate, Yudith; Dominguez, Elena Rosa

2008-01-01

Up to now, very few applications of multiobjective optimization (MOOP) techniques to quantitative structure-activity relationship (QSAR) studies have been reported in the literature. However, none of them report the optimization of objectives related directly to the final pharmaceutical profile of a drug. In this paper, a MOOP method based on Derringer's desirability function that allows conducting global QSAR studies, simultaneously considering the potency, bioavailability, and safety of a set of drug candidates, is introduced. The results of the desirability-based MOOP (the levels of the predictor variables concurrently producing the best possible compromise between the properties determining an optimal drug candidate) are used for the implementation of a ranking method that is also based on the application of desirability functions. This method allows ranking drug candidates with unknown pharmaceutical properties from combinatorial libraries according to the degree of similarity with the previously determined optimal candidate. Application of this method will make it possible to filter the most promising drug candidates of a library (the best-ranked candidates), which should have the best pharmaceutical profile (the best compromise between potency, safety and bioavailability). In addition, a validation method of the ranking process, as well as a quantitative measure of the quality of a ranking, the ranking quality index (Psi), is proposed. The usefulness of the desirability-based methods of MOOP and ranking is demonstrated by its application to a library of 95 fluoroquinolones, reporting their gram-negative antibacterial activity and mammalian cell cytotoxicity. Finally, the combined use of the desirability-based methods of MOOP and ranking proposed here seems to be a valuable tool for rational drug discovery and development.

Cell-Based Selection Expands the Utility of DNA-Encoded Small-Molecule Library Technology to Cell Surface Drug Targets: Identification of Novel Antagonists of the NK3 Tachykinin Receptor.

PubMed

Wu, Zining; Graybill, Todd L; Zeng, Xin; Platchek, Michael; Zhang, Jean; Bodmer, Vera Q; Wisnoski, David D; Deng, Jianghe; Coppo, Frank T; Yao, Gang; Tamburino, Alex; Scavello, Genaro; Franklin, G Joseph; Mataruse, Sibongile; Bedard, Katie L; Ding, Yun; Chai, Jing; Summerfield, Jennifer; Centrella, Paolo A; Messer, Jeffrey A; Pope, Andrew J; Israel, David I

2015-12-14

DNA-encoded small-molecule library technology has recently emerged as a new paradigm for identifying ligands against drug targets. To date, this technology has been used with soluble protein targets that are produced and used in a purified state. Here, we describe a cell-based method for identifying small-molecule ligands from DNA-encoded libraries against integral membrane protein targets. We use this method to identify novel, potent, and specific inhibitors of NK3, a member of the tachykinin family of G-protein coupled receptors (GPCRs). The method is simple and broadly applicable to other GPCRs and integral membrane proteins. We have extended the application of DNA-encoded library technology to membrane-associated targets and demonstrate the feasibility of selecting DNA-tagged, small-molecule ligands from complex combinatorial libraries against targets in a heterogeneous milieu, such as the surface of a cell.

Application of QSAR and shape pharmacophore modeling approaches for targeted chemical library design.

PubMed

Ebalunode, Jerry O; Zheng, Weifan; Tropsha, Alexander

2011-01-01

Optimization of chemical library composition affords more efficient identification of hits from biological screening experiments. The optimization could be achieved through rational selection of reagents used in combinatorial library synthesis. However, with a rapid advent of parallel synthesis methods and availability of millions of compounds synthesized by many vendors, it may be more efficient to design targeted libraries by means of virtual screening of commercial compound collections. This chapter reviews the application of advanced cheminformatics approaches such as quantitative structure-activity relationships (QSAR) and pharmacophore modeling (both ligand and structure based) for virtual screening. Both approaches rely on empirical SAR data to build models; thus, the emphasis is placed on achieving models of the highest rigor and external predictive power. We present several examples of successful applications of both approaches for virtual screening to illustrate their utility. We suggest that the expert use of both QSAR and pharmacophore models, either independently or in combination, enables users to achieve targeted libraries enriched with experimentally confirmed hit compounds.

Establishment of hapten-specific monoclonal avian IgY by conversion of antibody fragments obtained from combinatorial libraries.

PubMed

Deckers, Susanne; Braren, Ingke; Greunke, Kerstin; Meyer, Nadine; Rühl, Dana; Bredehorst, Reinhard; Spillner, Edzard

2009-01-01

Nowadays, recombinant antibody and phage display technology enable the efficient generation of immunotools and a subsequent manipulation for optimized affinity, specificity or overall performance. Such advantages are of particular interest for haptenic target structures, such as TNT (2,4,6-trinitrotoluene). The toxicity of TNT and its breakdown products makes a reliable and fast detection of low levels in aqueous samples highly important. In the present study, we aimed for the generation of scFvs (single-chain antibody fragments) specific for the TNT-surrogate TNP (2,4,6-trinitrophenyl) and their subsequent production as monoclonal avian IgY immunoglobulins providing improved assay performance. Therefore we subjected a human synthetic scFv library to selection following different strategies. TNP-specific human antibody fragments could be identified, characterized for their primary structure and evaluated for production as soluble scFv in Escherichia coli. Additionally, a murine TNP-specific antibody fragment was obtained from the hybridoma 11B3; however, the prokaryotic expression level was found to be limited. To generate and evaluate immunoglobulin formats with superior characteristics, all recombinant antibody fragments then were converted into two different chimaeric bivalent IgY antibody formats. After expression in mammalian cells, the IgY antibodies were assessed for their reactivity towards TNT. The IgY antibodies generated on the basis of the combinatorial library proved to be useful for detection of TNT, thereby emphasizing the high potential of this approach for the development of detection devices for immunoassay-based techniques.

Synthesis and characterization of a novel inhibitor of C-reactive protein-mediated proinflammatory effects.

PubMed

Kumaresan, Pappanaicken R; Devaraj, Sridevi; Huang, Wenzhe; Lau, Edmond Y; Liu, Ruiwu; Lam, Kit S; Jialal, Ishwarlal

2013-06-01

Numerous studies have shown that high C-reactive protein (CRP) levels predict cardiovascular disease and augur a poor prognosis in patients with acute coronary syndromes. Much in vitro and in vivo data support of a role for CRP in atherogenesis. There is an urgent need to develop inhibitors that specifically block the biological effects of CRP in vivo. The one-bead-one-compound (OBOC) combinatorial library method has been used to discover ligands against several biological targets. In this study, we use a novel fluorescence-based screening method to screen an OBOC combinatorial library for the discovery of peptides against human CRP. Human CRP was labeled with fluorescein isothiocyanate (FITC) and human serum albumin (HuSA) was labeled with phycoerythrin (PE) and used for screening. The OBOC library LWH-01 was synthesized on TentaGel resin beads using a standard solid-phase "split/mix" approach. By subtraction screening, eight peptides that bind specifically to CRP and not to HuSA were identified. In human aortic endothelial cells (HAECs) incubated with CRP, inhibitors CRPi-2, CRPi-3, and CRPi-6 significantly inhibited CRP-induced superoxide, cytokine release, and nuclear factor-κB (NFκB) activity. Molecular docking studies demonstrate that CRPi-2 interacts with the two Ca(2+) ions in the single subunit of CRP. The binding of CRPi-2 is reminiscent of choline binding. Future studies will examine the utility of this inhibitor in animal models and clinical trials.

Discovery of DNA repair inhibitors by combinatorial library profiling

PubMed Central

Moeller, Benjamin J.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2011-01-01

Small molecule inhibitors of DNA repair are emerging as potent and selective anti-cancer therapies, but the sheer magnitude of the protein networks involved in DNA repair processes poses obstacles to discovery of effective candidate drugs. To address this challenge, we used a subtractive combinatorial selection approach to identify a panel of peptide ligands that bind DNA repair complexes. Supporting the concept that these ligands have therapeutic potential, we show that one selected peptide specifically binds and non-competitively inactivates DNA-PKcs, a protein kinase critical in double-strand DNA break repair. In doing so, this ligand sensitizes BRCA-deficient tumor cells to genotoxic therapy. Our findings establish a platform for large-scale parallel screening for ligand-directed DNA repair inhibitors, with immediate applicability to cancer therapy. PMID:21343400

Measuring and Specifying Combinatorial Coverage of Test Input Configurations

PubMed Central

Kuhn, D. Richard; Kacker, Raghu N.; Lei, Yu

2015-01-01

A key issue in testing is how many tests are needed for a required level of coverage or fault detection. Estimates are often based on error rates in initial testing, or on code coverage. For example, tests may be run until a desired level of statement or branch coverage is achieved. Combinatorial methods present an opportunity for a different approach to estimating required test set size, using characteristics of the test set. This paper describes methods for estimating the coverage of, and ability to detect, t-way interaction faults of a test set based on a covering array. We also develop a connection between (static) combinatorial coverage and (dynamic) code coverage, such that if a specific condition is satisfied, 100% branch coverage is assured. Using these results, we propose practical recommendations for using combinatorial coverage in specifying test requirements. PMID:28133442

DPN-Generated Combinatorial Libraries

DTIC Science & Technology

2012-02-29

µM final concentration) is added (Fig. 6). Control over deposition parameters was examined for two model proteins, cholera toxin β subunit (CTβ...conjugated anti- cholera toxin beta (anti-CTb). The wells in the mould are inverted pyramids with an average depth of 86 µm, edge length of 120 µm, and...Therapeutics,” (2011). 91. University of New Mexico , Chemistry Department Colloquium, Albuquerque, NM, “The Polyvalent Gold Nanoparticle Conjugate

Patient Stratification with a Novel Molecular Imaging Agent

DTIC Science & Technology

2014-12-01

policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188 Public reporting...ester of biotin and removed unreacted biotin with a desalting column. 0.7 biotins per protein were achieved as measured by matrix-assisted laser...translation to flow cytometry assays has been inconsistent. In addition to the next two advances (cellular panning and improved combinatorial library design

Constructing high complexity synthetic libraries of long ORFs using in vitro selection

NASA Technical Reports Server (NTRS)

Cho, G.; Keefe, A. D.; Liu, R.; Wilson, D. S.; Szostak, J. W.

2000-01-01

We present a method that can significantly increase the complexity of protein libraries used for in vitro or in vivo protein selection experiments. Protein libraries are often encoded by chemically synthesized DNA, in which part of the open reading frame is randomized. There are, however, major obstacles associated with the chemical synthesis of long open reading frames, especially those containing random segments. Insertions and deletions that occur during chemical synthesis cause frameshifts, and stop codons in the random region will cause premature termination. These problems can together greatly reduce the number of full-length synthetic genes in the library. We describe a strategy in which smaller segments of the synthetic open reading frame are selected in vitro using mRNA display for the absence of frameshifts and stop codons. These smaller segments are then ligated together to form combinatorial libraries of long uninterrupted open reading frames. This process can increase the number of full-length open reading frames in libraries by up to two orders of magnitude, resulting in protein libraries with complexities of greater than 10(13). We have used this methodology to generate three types of displayed protein library: a completely random sequence library, a library of concatemerized oligopeptide cassettes with a propensity for forming amphipathic alpha-helical or beta-strand structures, and a library based on one of the most common enzymatic scaffolds, the alpha/beta (TIM) barrel. Copyright 2000 Academic Press.

Compound Libraries: Recent Advances and Their Applications in Drug Discovery.

PubMed

Gong, Zhen; Hu, Guoping; Li, Qiang; Liu, Zhiguo; Wang, Fei; Zhang, Xuejin; Xiong, Jian; Li, Peng; Xu, Yan; Ma, Rujian; Chen, Shuhui; Li, Jian

2017-01-01

Hit identification is the starting point of small-molecule drug discovery and is therefore very important to the pharmaceutical industry. One of the most important approaches to identify a new hit is to screen a compound library using an in vitro assay. High-throughput screening has made great contributions to drug discovery since the 1990s but requires expensive equipment and facilities, and its success depends on the size of the compound library. Recent progress in the development of compound libraries has provided more efficient ways to identify new hits for novel drug targets, thereby helping to promote the development of the pharmaceutical industry, especially for firstin- class drugs. A multistage and systematic research of articles published between 1986 and 2017 has been performed, which was organized into 5 sections and discussed in detail. In this review, the sources and classification of compound libraries are summarized. The progress made in combinatorial libraries and DNA-encoded libraries is reviewed. Library design methods, especially for focused libraries, are introduced in detail. In the final part, the status of the compound libraries at WuXi is reported. The progress related to compound libraries, especially drug template libraries, DELs, and focused libraries, will help to identify better hits for novel drug targets and promote the development of the pharmaceutical industry. Moreover, these libraries can facilitate hit identification, which benefits most research organizations, including academics and small companies. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Quercetin derivatives as non-nucleoside inhibitors for dengue polymerase: molecular docking, molecular dynamics simulation, and binding free energy calculation.

PubMed

Anusuya, Shanmugam; Gromiha, M Michael

2017-10-01

Dengue is an important public health problem in tropical and subtropical regions of the world. Neither vaccine nor an antiviral medication is available to treat dengue. This insists the need of drug discovery for dengue. In order to find a potent lead molecule, RNA-dependent RNA polymerase which is essential for dengue viral replication is chosen as a drug target. As Quercetin showed antiviral activity against several viruses, quercetin derivatives developed by combinatorial library synthesis and mined from PubChem databases were screened for a potent anti-dengue viral agent. Our study predicted Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a dengue polymerase inhibitor. The results were validated by molecular dynamics simulation studies which reveal water bridges and hydrogen bonds as major contributors for the stability of the polymerase-lead complex. Interactions formed by this compound with residues Trp795, Arg792 and Glu351 are found to be essential for the stability of the polymerase-lead complex. Our study demonstrates Quercetin 3-(6″-(E)-p-coumaroylsophoroside)-7-rhamnoside as a potent non-nucleoside inhibitor for dengue polymerase.

"One-sample concept" micro-combinatory for high throughput TEM of binary films.

PubMed

Sáfrán, György

2018-04-01

Phases of thin films may remarkably differ from that of bulk. Unlike to the comprehensive data files of Binary Phase Diagrams [1] available for bulk, complete phase maps for thin binary layers do not exist. This is due to both the diverse metastable, non-equilibrium or instable phases feasible in thin films and the required volume of characterization work with analytical techniques like TEM, SAED and EDS. The aim of the present work was to develop a method that remarkably facilitates the TEM study of the diverse binary phases of thin films, or the creation of phase maps. A micro-combinatorial method was worked out that enables both preparation and study of a gradient two-component film within a single TEM specimen. For a demonstration of the technique thin Mn x Al 1- x binary samples with evolving concentration from x = 0 to x = 1 have been prepared so that the transition from pure Mn to pure Al covers a 1.5 mm long track within the 3 mm diameter TEM grid. The proposed method enables the preparation and study of thin combinatorial samples including all feasible phases as a function of composition or other deposition parameters. Contrary to known "combinatorial chemistry", in which a series of different samples are deposited in one run, and investigated, one at a time, the present micro-combinatorial method produces a single specimen condensing a complete library of a binary system that can be studied, efficiently, within a single TEM session. That provides extremely high throughput for TEM characterization of composition-dependent phases, exploration of new materials, or the construction of phase diagrams of binary films. Copyright © 2018 Elsevier B.V. All rights reserved.

Biomolecular Recognition of Semiconductors and Magnetic Materials to Assemble Nanoparticle Heterostructures

DTIC Science & Technology

2002-01-01

shown that engineered viruses can recognize specific semiconductor surfaces through the selection by combinatorial phage display method. These specific... phage display libraries. The screening method selected for binding affinity of a population of random peptides displayed as part of the pIII minor coat...shorter spacing than expected distance ( M13 phage length: 880 nm) corresponds to the length scale imposed by the phage which formed the tilted

Molecular Pathogenesis of Rickettsioses and Development of Novel Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2007-02-01

Rickettsiae are susceptible to few antibiotics including the tetracyclines and chloramphenicol and are highly resistant to beta - lactams and...agents available for rickettsiae and Orientia, emergence of chloramphenicol- and tetracycline -resistant strains of Orientia, and the possibility of...precipitation (RNAqueous-4 PCR, Ambion). RNA quality was verified by UV spectrophotometry and ribosomal RNA band integrity. The TrueLabeling-AMP™ 2.0 kit

Rare antibodies from combinatorial libraries suggests an S.O.S. component of the human immunological repertoire.

PubMed

Lerner, Richard A

2011-04-01

Convergence of observations from different sources is the norm in science. However, when convergence occurs in man for antibodies it is remarkable because the repertoire of possible immunoglobulin products is very large and diverse. Thus, one would not expect to see the same antibody twice from divergent populations unless there is special significance as to why the immune response is constrained. Now, broadly neutralizing antibodies isolated from combinatorial libraries from three separate populations have been shown to all use the same (V(H) 1-69) germ line gene and interact with the influenza virus in very similar ways. Here we discuss the reasons for this convergence in terms of how the immunological repertoire responds to emergency situations where time is short as occurs, for example, in potentially lethal infections. It is suggested that there is a first responder or S.O.S. component of the antibody repertoire that evolved to initiate rapid defense against infectious agents. The discovery of the homologies between these commonly produced antibodies may have significance for the design of novel vaccines. Finally, these convergent results may give much insight into why antibodies encoded by the V(H) 1-69 germ line gene are highly over represented in B-cell lymphomas.

Statistical molecular design of building blocks for combinatorial chemistry.

PubMed

Linusson, A; Gottfries, J; Lindgren, F; Wold, S

2000-04-06

The reduction of the size of a combinatorial library can be made in two ways, either base the selection on the building blocks (BB's) or base it on the full set of virtually constructed products. In this paper we have investigated the effects of applying statistical designs to BB sets compared to selections based on the final products. The two sets of BB's and the virtually constructed library were described by structural parameters, and the correlation between the two characterizations was investigated. Three different selection approaches were used both for the BB sets and for the products. In the first two the selection algorithms were applied directly to the data sets (D-optimal design and space-filling design), while for the third a cluster analysis preceded the selection (cluster-based design). The selections were compared using visual inspection, the Tanimoto coefficient, the Euclidean distance, the condition number, and the determinant of the resulting data matrix. No difference in efficiency was found between selections made in the BB space and in the product space. However, it is of critical importance to investigate the BB space carefully and to select an appropriate number of BB's to result in an adequate diversity. An example from the pharmaceutical industry is then presented, where selection via BB's was made using a cluster-based design.

A Versatile Microfluidic Device for Automating Synthetic Biology.

PubMed

Shih, Steve C C; Goyal, Garima; Kim, Peter W; Koutsoubelis, Nicolas; Keasling, Jay D; Adams, Paul D; Hillson, Nathan J; Singh, Anup K

2015-10-16

New microbes are being engineered that contain the genetic circuitry, metabolic pathways, and other cellular functions required for a wide range of applications such as producing biofuels, biobased chemicals, and pharmaceuticals. Although currently available tools are useful in improving the synthetic biology process, further improvements in physical automation would help to lower the barrier of entry into this field. We present an innovative microfluidic platform for assembling DNA fragments with 10× lower volumes (compared to that of current microfluidic platforms) and with integrated region-specific temperature control and on-chip transformation. Integration of these steps minimizes the loss of reagents and products compared to that with conventional methods, which require multiple pipetting steps. For assembling DNA fragments, we implemented three commonly used DNA assembly protocols on our microfluidic device: Golden Gate assembly, Gibson assembly, and yeast assembly (i.e., TAR cloning, DNA Assembler). We demonstrate the utility of these methods by assembling two combinatorial libraries of 16 plasmids each. Each DNA plasmid is transformed into Escherichia coli or Saccharomyces cerevisiae using on-chip electroporation and further sequenced to verify the assembly. We anticipate that this platform will enable new research that can integrate this automated microfluidic platform to generate large combinatorial libraries of plasmids and will help to expedite the overall synthetic biology process.

Production of a Human Antibody Library in the Phage-Display Vector pSEX81.

PubMed

Welschof, M; Little, M; Dörsam, H

1998-01-01

Human monoclonal antibodies (MAbs) are more suitable than MAbs of animal origin for clinical applications because of lower hypersensitivity reactions, less formation of circulating immune complexes and lower anti-immunoglobulin responses The classical production of human MAbs via the hybridoma technique or Epstein-Barr virus (EBV) transformation is limited by the instability of cell lines, low antibody production, and the problems of imununizing humans with certain antigens (1,2). A promising alternative 1s the production of human recombinant antibodies (3). Recombinant DNA technology has made it possible to clone human antibody genes in vectors and to generate antibody expression libraries (4-7). One approach has been to amplify and recombine the IgG repertoire of an "immunized" donor. This has been used to isolate several antibodies related to diseases (8,9). In order to obtain more universal antibody libraries the naive IgM repertoire of several "unimmunized" donors were pooled (10,12). The complexity of the combinatorial libraries has been further increased by creating the so-called "semisynthetic" antibody libraries (22-14).

A combinatorial framework to quantify peak/pit asymmetries in complex dynamics.

PubMed

Hasson, Uri; Iacovacci, Jacopo; Davis, Ben; Flanagan, Ryan; Tagliazucchi, Enzo; Laufs, Helmut; Lacasa, Lucas

2018-02-23

We explore a combinatorial framework which efficiently quantifies the asymmetries between minima and maxima in local fluctuations of time series. We first showcase its performance by applying it to a battery of synthetic cases. We find rigorous results on some canonical dynamical models (stochastic processes with and without correlations, chaotic processes) complemented by extensive numerical simulations for a range of processes which indicate that the methodology correctly distinguishes different complex dynamics and outperforms state of the art metrics in several cases. Subsequently, we apply this methodology to real-world problems emerging across several disciplines including cases in neurobiology, finance and climate science. We conclude that differences between the statistics of local maxima and local minima in time series are highly informative of the complex underlying dynamics and a graph-theoretic extraction procedure allows to use these features for statistical learning purposes.

Optimal protein library design using recombination or point mutations based on sequence-based scoring functions.

PubMed

Pantazes, Robert J; Saraf, Manish C; Maranas, Costas D

2007-08-01

In this paper, we introduce and test two new sequence-based protein scoring systems (i.e. S1, S2) for assessing the likelihood that a given protein hybrid will be functional. By binning together amino acids with similar properties (i.e. volume, hydrophobicity and charge) the scoring systems S1 and S2 allow for the quantification of the severity of mismatched interactions in the hybrids. The S2 scoring system is found to be able to significantly functionally enrich a cytochrome P450 library over other scoring methods. Given this scoring base, we subsequently constructed two separate optimization formulations (i.e. OPTCOMB and OPTOLIGO) for optimally designing protein combinatorial libraries involving recombination or mutations, respectively. Notably, two separate versions of OPTCOMB are generated (i.e. model M1, M2) with the latter allowing for position-dependent parental fragment skipping. Computational benchmarking results demonstrate the efficacy of models OPTCOMB and OPTOLIGO to generate high scoring libraries of a prespecified size.

Protein and Antibody Engineering by Phage Display.

PubMed

Frei, J C; Lai, J R

2016-01-01

Phage display is an in vitro selection technique that allows for the rapid isolation of proteins with desired properties including increased affinity, specificity, stability, and new enzymatic activity. The power of phage display relies on the phenotype-to-genotype linkage of the protein of interest displayed on the phage surface with the encoding DNA packaged within the phage particle, which allows for selective enrichment of library pools and high-throughput screening of resulting clones. As an in vitro method, the conditions of the binding selection can be tightly controlled. Due to the high-throughput nature, rapidity, and ease of use, phage display is an excellent technological platform for engineering antibody or proteins with enhanced properties. Here, we describe methods for synthesis, selection, and screening of phage libraries with particular emphasis on designing humanizing antibody libraries and combinatorial scanning mutagenesis libraries. We conclude with a brief section on troubleshooting for all stages of the phage display process. © 2016 Elsevier Inc. All rights reserved.

Targeting mammalian organelles with internalizing phage (iPhage) libraries

PubMed Central

Rangel, Roberto; Dobroff, Andrey S.; Guzman-Rojas, Liliana; Salmeron, Carolina C.; Gelovani, Juri G.; Sidman, Richard L.; Pasqualini, Renata; Arap, Wadih

2015-01-01

Techniques largely used for protein interaction studies and discovery of intracellular receptors, such as affinity capture complex purification and yeast two-hybrid, may produce inaccurate datasets due to protein insolubility, transient or weak protein interactions, or irrelevant intracellular context. A versatile tool to overcome these limitations as well as to potentially create vaccines and engineer peptides and antibodies as targeted diagnostic and therapeutic agents, is the phage display technique. We have recently developed a new technology for screening internalizing phage (iPhage) vectors and libraries utilizing a ligand/receptor-independent mechanism to penetrate eukaryotic cells. iPhage particles provide a unique discovery platform for combinatorial intracellular targeting of organelle ligands along with their corresponding receptors and to fingerprint functional protein domains in living cells. Here we explain the design, cloning, construction, and production of iPhage-based vectors and libraries, along with basic ligand-receptor identification and validation methodologies for organelle receptors. An iPhage library screening can be performed in ~8 weeks. PMID:24030441

A novel spiroindoline targets cell cycle and migration via modulation of microtubule cytoskeleton.

PubMed

Kumar, Naveen; Hati, Santanu; Munshi, Parthapratim; Sen, Subhabrata; Sehrawat, Seema; Singh, Shailja

2017-05-01

Natural product-inspired libraries of molecules with diverse architectures have evolved as one of the most useful tools for discovering lead molecules for drug discovery. In comparison to conventional combinatorial libraries, these molecules have been inferred to perform better in phenotypic screening against complicated targets. Diversity-oriented synthesis (DOS) is a forward directional strategy to access such multifaceted library of molecules. From a successful DOS campaign of a natural product-inspired library, recently a small molecule with spiroindoline motif was identified as a potent anti-breast cancer compound. Herein we report the subcellular studies performed for this molecule on breast cancer cells. Our investigation revealed that it repositions microtubule cytoskeleton and displaces AKAP9 located at the microtubule organization centre. DNA ladder assay and cell cycle experiments further established the molecule as an apoptotic agent. This work further substantiated the amalgamation of DOS-phenotypic screening-sub-cellular studies as a consolidated blueprint for the discovery of potential pharmaceutical drug candidates.

Sample size determination in combinatorial chemistry.

PubMed Central

Zhao, P L; Zambias, R; Bolognese, J A; Boulton, D; Chapman, K

1995-01-01

Combinatorial chemistry is gaining wide appeal as a technique for generating molecular diversity. Among the many combinatorial protocols, the split/recombine method is quite popular and particularly efficient at generating large libraries of compounds. In this process, polymer beads are equally divided into a series of pools and each pool is treated with a unique fragment; then the beads are recombined, mixed to uniformity, and redivided equally into a new series of pools for the subsequent couplings. The deviation from the ideal equimolar distribution of the final products is assessed by a special overall relative error, which is shown to be related to the Pearson statistic. Although the split/recombine sampling scheme is quite different from those used in analysis of categorical data, the Pearson statistic is shown to still follow a chi2 distribution. This result allows us to derive the required number of beads such that, with 99% confidence, the overall relative error is controlled to be less than a pregiven tolerable limit L1. In this paper, we also discuss another criterion, which determines the required number of beads so that, with 99% confidence, all individual relative errors are controlled to be less than a pregiven tolerable limit L2 (0 < L2 < 1). PMID:11607586

Searching Fragment Spaces with feature trees.

PubMed

Lessel, Uta; Wellenzohn, Bernd; Lilienthal, Markus; Claussen, Holger

2009-02-01

Virtual combinatorial chemistry easily produces billions of compounds, for which conventional virtual screening cannot be performed even with the fastest methods available. An efficient solution for such a scenario is the generation of Fragment Spaces, which encode huge numbers of virtual compounds by their fragments/reagents and rules of how to combine them. Similarity-based searches can be performed in such spaces without ever fully enumerating all virtual products. Here we describe the generation of a huge Fragment Space encoding about 5 * 10(11) compounds based on established in-house synthesis protocols for combinatorial libraries, i.e., we encode practically evaluated combinatorial chemistry protocols in a machine readable form, rendering them accessible to in silico search methods. We show how such searches in this Fragment Space can be integrated as a first step in an overall workflow. It reduces the extremely huge number of virtual products by several orders of magnitude so that the resulting list of molecules becomes more manageable for further more elaborated and time-consuming analysis steps. Results of a case study are presented and discussed, which lead to some general conclusions for an efficient expansion of the chemical space to be screened in pharmaceutical companies.

LAS0811: from combinatorial chemistry to activation of antioxidant response element.

PubMed

Zhu, Ming; Baek, Hyounggee; Liu, Ruiwu; Song, Aimin; Lam, Kit; Lau, Derick

2009-01-01

The antioxidant response element (ARE) and its transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), are potential targets for cancer chemoprevention. We sought to screen small molecules synthesized with combinatorial chemistry for activation of ARE. By high-throughput screening of 9400 small molecules from 10 combinatorial chemical libraries using HepG2 cells with an ARE-driven reporter, we have identified a novel small molecule, 1,2-dimethoxy-4,5-dinitrobenzene (LAS0811), as an activator of the ARE. LAS0811 upregulated the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1), a representative antioxidative enzyme regulated by ARE. It enhanced production of an endogenous reducing agent, glutathione (GSH). In addition, LAS0811 induced expression of heme oxygenase 1 (HO1), which is an ARE-regulated enzyme with anti-inflammatory activity. Furthermore, LAS0811 reduced cell death due to the cytotoxic stress of a strong oxidant, t-butyl hydroperoxide (t-BOOH). Mechanistically, LAS0811 upregulated the expression of Nrf2 and promoted its translocation into the nuclei leading to subsequent ARE activation. Taken together, LAS0811 is a novel activator of the ARE and its associated detoxifying genes and, thus, a potential agent for cancer chemoprevention.

LAS0811: From Combinatorial Chemistry to Activation of Antioxidant Response Element

PubMed Central

Zhu, Ming; Baek, Hyounggee; Liu, Ruiwu; Song, Aimin; Lam, Kit; Lau, Derick

2009-01-01

The antioxidant response element (ARE) and its transcription factor, nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), are potential targets for cancer chemoprevention. We sought to screen small molecules synthesized with combinatorial chemistry for activation of ARE. By high-throughput screening of 9400 small molecules from 10 combinatorial chemical libraries using HepG2 cells with an ARE-driven reporter, we have identified a novel small molecule, 1,2-dimethoxy-4,5-dinitrobenzene (LAS0811), as an activator of the ARE. LAS0811 upregulated the activity of NAD(P)H:quinone oxidoreductase 1 (NQO1), a representative antioxidative enzyme regulated by ARE. It enhanced production of an endogenous reducing agent, glutathione (GSH). In addition, LAS0811 induced expression of heme oxygenase 1 (HO1), which is an ARE-regulated enzyme with anti-inflammatory activity. Furthermore, LAS0811 reduced cell death due to the cytotoxic stress of a strong oxidant, t-butyl hydroperoxide (t-BOOH). Mechanistically, LAS0811 upregulated the expression of Nrf2 and promoted its translocation into the nuclei leading to subsequent ARE activation. Taken together, LAS0811 is a novel activator of the ARE and its associated detoxifying genes and, thus, a potential agent for cancer chemoprevention. PMID:19794825

Heteroleptic Copper(I)-Based Complexes for Photocatalysis: Combinatorial Assembly, Discovery, and Optimization.

PubMed

Minozzi, Clémentine; Caron, Antoine; Grenier-Petel, Jean-Christophe; Santandrea, Jeffrey; Collins, Shawn K

2018-05-04

A library of 50 copper-based complexes derived from bisphosphines and diamines was prepared and evaluated in three mechanistically distinct photocatalytic reactions. In all cases, a copper-based catalyst was identified to afford high yields, where new heteroleptic complexes derived from the bisphosphine BINAP displayed high efficiency across all reaction types. Importantly, the evaluation of the library of copper complexes revealed that even when photophysical data is available, it is not always possible to predict which catalyst structure will be efficient or inefficient in a given process, emphasizing the advantages for catalyst structures with high modularity and structural variability. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Site-directed protein recombination as a shortest-path problem.

PubMed

Endelman, Jeffrey B; Silberg, Jonathan J; Wang, Zhen-Gang; Arnold, Frances H

2004-07-01

Protein function can be tuned using laboratory evolution, in which one rapidly searches through a library of proteins for the properties of interest. In site-directed recombination, n crossovers are chosen in an alignment of p parents to define a set of p(n + 1) peptide fragments. These fragments are then assembled combinatorially to create a library of p(n+1) proteins. We have developed a computational algorithm to enrich these libraries in folded proteins while maintaining an appropriate level of diversity for evolution. For a given set of parents, our algorithm selects crossovers that minimize the average energy of the library, subject to constraints on the length of each fragment. This problem is equivalent to finding the shortest path between nodes in a network, for which the global minimum can be found efficiently. Our algorithm has a running time of O(N(3)p(2) + N(2)n) for a protein of length N. Adjusting the constraints on fragment length generates a set of optimized libraries with varying degrees of diversity. By comparing these optima for different sets of parents, we rapidly determine which parents yield the lowest energy libraries.

Solubility limits in quaternary SnTe-based alloys [Metastability and solubility limits in quaternary SnTe-based alloys guided by combinatorial sputtering

DOE PAGES

Siol, Sebastian; Holder, Aaron; Ortiz, Brenden R.; ...

2017-05-09

Here, the controlled decomposition of metastable alloys is an attractive route to form nanostructured thermoelectric materials with reduced thermal conductivity. The ternary SnTe–MnTe and SnTe–SnSe heterostructural alloys have been demonstrated as promising materials for thermoelectric applications. In this work, the quaternary Sn 1–yMnyTe 1–xSe x phase space serves as a relevant model system to explore how a combination of computational and combinatorial-growth methods can be used to study equilibrium and non-equilibrium solubility limits. Results from first principle calculations indicate low equilibrium solubility for x,y < 0.05 that are in good agreement with results obtained from bulk equilibrium synthesis experiments andmore » predict significantly higher spinodal limits. An experimental screening using sputtered combinatorial thin film sample libraries showed a remarkable increase in non-equilibrium solubility for x,y > 0.2. These theoretical and experimental results were used to guide the bulk synthesis of metastable alloys. The ability to reproduce the non-equilibrium solubility levels in bulk materials indicates that such theoretical calculations and combinatorial growth can inform bulk synthetic routes. Further, the large difference between equilibrium and non-equilibrium solubility limits in Sn 1–yMn yTe 1–xSe x indicates these metastable alloys are attractive in terms of nano-precipitate formation for potential thermoelectric applications.« less

Fragment based group QSAR and molecular dynamics mechanistic studies on arylthioindole derivatives targeting the α-β interfacial site of human tubulin

PubMed Central

2014-01-01

Background A number of microtubule disassembly blocking agents and inhibitors of tubulin polymerization have been elements of great interest in anti-cancer therapy, some of them even entering into the clinical trials. One such class of tubulin assembly inhibitors is of arylthioindole derivatives which results in effective microtubule disorganization responsible for cell apoptosis by interacting with the colchicine binding site of the β-unit of tubulin close to the interface with the α unit. We modelled the human tubulin β unit (chain D) protein and performed docking studies to elucidate the detailed binding mode of actions associated with their inhibition. The activity enhancing structural aspects were evaluated using a fragment-based Group QSAR (G-QSAR) model and was validated statistically to determine its robustness. A combinatorial library was generated keeping the arylthioindole moiety as the template and their activities were predicted. Results The G-QSAR model obtained was statistically significant with r2 value of 0.85, cross validated correlation coefficient q2 value of 0.71 and pred_r2 (r2 value for test set) value of 0.89. A high F test value of 65.76 suggests robustness of the model. Screening of the combinatorial library on the basis of predicted activity values yielded two compounds HPI (predicted pIC50 = 6.042) and MSI (predicted pIC50 = 6.001) whose interactions with the D chain of modelled human tubulin protein were evaluated in detail. A toxicity evaluation resulted in MSI being less toxic in comparison to HPI. Conclusions The study provides an insight into the crucial structural requirements and the necessary chemical substitutions required for the arylthioindole moiety to exhibit enhanced inhibitory activity against human tubulin. The two reported compounds HPI and MSI showed promising anti cancer activities and thus can be considered as potent leads against cancer. The toxicity evaluation of these compounds suggests that MSI is a promising therapeutic candidate. This study provided another stepping stone in the direction of evaluating tubulin inhibition and microtubule disassembly degeneration as viable targets for development of novel therapeutics against cancer. PMID:25521775

Balancing novelty with confined chemical space in modern drug discovery.

PubMed

Medina-Franco, José L; Martinez-Mayorga, Karina; Meurice, Nathalie

2014-02-01

The concept of chemical space has broad applications in drug discovery. In response to the needs of drug discovery campaigns, different approaches are followed to efficiently populate, mine and select relevant chemical spaces that overlap with biologically relevant chemical spaces. This paper reviews major trends in current drug discovery and their impact on the mining and population of chemical space. We also survey different approaches to develop screening libraries with confined chemical spaces balancing physicochemical properties. In this context, the confinement is guided by criteria that can be divided in two broad categories: i) library design focused on a relevant therapeutic target or disease and ii) library design focused on the chemistry or a desired molecular function. The design and development of chemical libraries should be associated with the specific purpose of the library and the project goals. The high complexity of drug discovery and the inherent imperfection of individual experimental and computational technologies prompt the integration of complementary library design and screening approaches to expedite the identification of new and better drugs. Library design approaches including diversity-oriented synthesis, biological-oriented synthesis or combinatorial library design, to name a few, and the design of focused libraries driven by target/disease, chemical structure or molecular function are more efficient if they are guided by multi-parameter optimization. In this context, consideration of pharmaceutically relevant properties is essential for balancing novelty with chemical space in drug discovery.

In situ click chemistry: a powerful means for lead discovery.

PubMed

Sharpless, K Barry; Manetsch, Roman

2006-11-01

Combinatorial chemistry and parallel synthesis are important and regularly applied tools for lead identification and optimisation, although they are often accompanied by challenges related to the efficiency of library synthesis and the purity of the compound library. In the last decade, novel means of lead discovery approaches have been investigated where the biological target is actively involved in the synthesis of its own inhibitory compound. These fragment-based approaches, also termed target-guided synthesis (TGS), show great promise in lead discovery applications by combining the synthesis and screening of libraries of low molecular weight compounds in a single step. Of all the TGS methods, the kinetically controlled variant is the least well known, but it has the potential to emerge as a reliable lead discovery method. The kinetically controlled TGS approach, termed in situ click chemistry, is discussed in this article.

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

Visco, Donald Patrick, Jr.; Faulon, Jean-Loup Michel; Roe, Diana C.

This report is a comprehensive review of the field of molecular enumeration from early isomer counting theories to evolutionary algorithms that design molecules in silico. The core of the review is a detail account on how molecules are counted, enumerated, and sampled. The practical applications of molecular enumeration are also reviewed for chemical information, structure elucidation, molecular design, and combinatorial library design purposes. This review is to appear as a chapter in Reviews in Computational Chemistry volume 21 edited by Kenny B. Lipkowitz.

Combinatorial and high-throughput screening of materials libraries: review of state of the art.

PubMed

Potyrailo, Radislav; Rajan, Krishna; Stoewe, Klaus; Takeuchi, Ichiro; Chisholm, Bret; Lam, Hubert

2011-11-14

Rational materials design based on prior knowledge is attractive because it promises to avoid time-consuming synthesis and testing of numerous materials candidates. However with the increase of complexity of materials, the scientific ability for the rational materials design becomes progressively limited. As a result of this complexity, combinatorial and high-throughput (CHT) experimentation in materials science has been recognized as a new scientific approach to generate new knowledge. This review demonstrates the broad applicability of CHT experimentation technologies in discovery and optimization of new materials. We discuss general principles of CHT materials screening, followed by the detailed discussion of high-throughput materials characterization approaches, advances in data analysis/mining, and new materials developments facilitated by CHT experimentation. We critically analyze results of materials development in the areas most impacted by the CHT approaches, such as catalysis, electronic and functional materials, polymer-based industrial coatings, sensing materials, and biomaterials.

A combinatorial screen of the CLOUD uncovers a synergy targeting the androgen receptor.

PubMed

Licciardello, Marco P; Ringler, Anna; Markt, Patrick; Klepsch, Freya; Lardeau, Charles-Hugues; Sdelci, Sara; Schirghuber, Erika; Müller, André C; Caldera, Michael; Wagner, Anja; Herzog, Rebecca; Penz, Thomas; Schuster, Michael; Boidol, Bernd; Dürnberger, Gerhard; Folkvaljon, Yasin; Stattin, Pär; Ivanov, Vladimir; Colinge, Jacques; Bock, Christoph; Kratochwill, Klaus; Menche, Jörg; Bennett, Keiryn L; Kubicek, Stefan

2017-07-01

Approved drugs are invaluable tools to study biochemical pathways, and further characterization of these compounds may lead to repurposing of single drugs or combinations. Here we describe a collection of 308 small molecules representing the diversity of structures and molecular targets of all FDA-approved chemical entities. The CeMM Library of Unique Drugs (CLOUD) covers prodrugs and active forms at pharmacologically relevant concentrations and is ideally suited for combinatorial studies. We screened pairwise combinations of CLOUD drugs for impairment of cancer cell viability and discovered a synergistic interaction between flutamide and phenprocoumon (PPC). The combination of these drugs modulates the stability of the androgen receptor (AR) and resensitizes AR-mutant prostate cancer cells to flutamide. Mechanistically, we show that the AR is a substrate for γ-carboxylation, a post-translational modification inhibited by PPC. Collectively, our data suggest that PPC could be repurposed to tackle resistance to antiandrogens in prostate cancer patients.

Dynamic Hierarchical Energy-Efficient Method Based on Combinatorial Optimization for Wireless Sensor Networks.

PubMed

Chang, Yuchao; Tang, Hongying; Cheng, Yongbo; Zhao, Qin; Yuan, Baoqing Li andXiaobing

2017-07-19

Routing protocols based on topology control are significantly important for improving network longevity in wireless sensor networks (WSNs). Traditionally, some WSN routing protocols distribute uneven network traffic load to sensor nodes, which is not optimal for improving network longevity. Differently to conventional WSN routing protocols, we propose a dynamic hierarchical protocol based on combinatorial optimization (DHCO) to balance energy consumption of sensor nodes and to improve WSN longevity. For each sensor node, the DHCO algorithm obtains the optimal route by establishing a feasible routing set instead of selecting the cluster head or the next hop node. The process of obtaining the optimal route can be formulated as a combinatorial optimization problem. Specifically, the DHCO algorithm is carried out by the following procedures. It employs a hierarchy-based connection mechanism to construct a hierarchical network structure in which each sensor node is assigned to a special hierarchical subset; it utilizes the combinatorial optimization theory to establish the feasible routing set for each sensor node, and takes advantage of the maximum-minimum criterion to obtain their optimal routes to the base station. Various results of simulation experiments show effectiveness and superiority of the DHCO algorithm in comparison with state-of-the-art WSN routing algorithms, including low-energy adaptive clustering hierarchy (LEACH), hybrid energy-efficient distributed clustering (HEED), genetic protocol-based self-organizing network clustering (GASONeC), and double cost function-based routing (DCFR) algorithms.

A dynamical systems model for combinatorial cancer therapy enhances oncolytic adenovirus efficacy by MEK-inhibition.

PubMed

Bagheri, Neda; Shiina, Marisa; Lauffenburger, Douglas A; Korn, W Michael

2011-02-01

Oncolytic adenoviruses, such as ONYX-015, have been tested in clinical trials for currently untreatable tumors, but have yet to demonstrate adequate therapeutic efficacy. The extent to which viruses infect targeted cells determines the efficacy of this approach but many tumors down-regulate the Coxsackievirus and Adenovirus Receptor (CAR), rendering them less susceptible to infection. Disrupting MAPK pathway signaling by pharmacological inhibition of MEK up-regulates CAR expression, offering possible enhanced adenovirus infection. MEK inhibition, however, interferes with adenovirus replication due to resulting G1-phase cell cycle arrest. Therefore, enhanced efficacy will depend on treatment protocols that productively balance these competing effects. Predictive understanding of how to attain and enhance therapeutic efficacy of combinatorial treatment is difficult since the effects of MEK inhibitors, in conjunction with adenovirus/cell interactions, are complex nonlinear dynamic processes. We investigated combinatorial treatment strategies using a mathematical model that predicts the impact of MEK inhibition on tumor cell proliferation, ONYX-015 infection, and oncolysis. Specifically, we fit a nonlinear differential equation system to dedicated experimental data and analyzed the resulting simulations for favorable treatment strategies. Simulations predicted enhanced combinatorial therapy when both treatments were applied simultaneously; we successfully validated these predictions in an ensuing explicit test study. Further analysis revealed that a CAR-independent mechanism may be responsible for amplified virus production and cell death. We conclude that integrated computational and experimental analysis of combinatorial therapy provides a useful means to identify treatment/infection protocols that yield clinically significant oncolysis. Enhanced oncolytic therapy has the potential to dramatically improve non-surgical cancer treatment, especially in locally advanced or metastatic cases where treatment options remain limited.

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry.

PubMed

Kim, Kyung Lock; Park, Kyeng Min; Murray, James; Kim, Kimoon; Ryu, Sung Ho

2018-05-23

Combinatorial post-translational modifications (PTMs), which can serve as dynamic "molecular barcodes", have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Direct Profiling the Post-Translational Modification Codes of a Single Protein Immobilized on a Surface Using Cu-free Click Chemistry

PubMed Central

2018-01-01

Combinatorial post-translational modifications (PTMs), which can serve as dynamic “molecular barcodes”, have been proposed to regulate distinct protein functions. However, studies of combinatorial PTMs on single protein molecules have been hindered by a lack of suitable analytical methods. Here, we describe erasable single-molecule blotting (eSiMBlot) for combinatorial PTM profiling. This assay is performed in a highly multiplexed manner and leverages the benefits of covalent protein immobilization, cyclic probing with different antibodies, and single molecule fluorescence imaging. Especially, facile and efficient covalent immobilization on a surface using Cu-free click chemistry permits multiple rounds (>10) of antibody erasing/reprobing without loss of antigenicity. Moreover, cumulative detection of coregistered multiple data sets for immobilized single-epitope molecules, such as HA peptide, can be used to increase the antibody detection rate. Finally, eSiMBlot enables direct visualization and quantitative profiling of combinatorial PTM codes at the single-molecule level, as we demonstrate by revealing the novel phospho-codes of ligand-induced epidermal growth factor receptor. Thus, eSiMBlot provides an unprecedentedly simple, rapid, and versatile platform for analyzing the vast number of combinatorial PTMs in biological pathways.

Jeffamine derivatized TentaGel beads and poly(dimethylsiloxane) microbead cassettes for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound combinatorial small molecule libraries.

PubMed

Townsend, Jared B; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S

2010-09-13

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated poly(dimethylsiloxane) (PDMS) cassette for ultrahigh-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting trifunctional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry resulting in beads with increased loading capacity, hydrophilicity, and porosity at the outer layer. We have found that such bead configuration can facilitate ultrahigh-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 min) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel were then layered over the microbead cassette to immobilize the compound-beads. After 24 h of incubation at 37 °C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were resynthesized and found to be cytotoxic (IC(50) 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultrahigh-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays.

Jeffamine Derivatized TentaGel Beads and PDMS Microbead Cassettes for Ultra-high Throughput in situ Releasable Solution-Phase Cell-based Screening of OBOC Combinatorial Small Molecule Libraries

PubMed Central

Townsend, Jared B.; Shaheen, Farzana; Liu, Ruiwu; Lam, Kit S.

2011-01-01

A method to efficiently immobilize and partition large quantities of microbeads in an array format in microfabricated polydimethylsiloxane (PDMS) cassette for high-throughput in situ releasable solution-phase cell-based screening of one-bead-one-compound (OBOC) combinatorial libraries is described. Commercially available Jeffamine triamine T-403 (∼440 Da) was derivatized such that two of its amino groups were protected by Fmoc and the remaining amino group capped with succinic anhydride to generate a carboxyl group. This resulting tri-functional hydrophilic polymer was then sequentially coupled two times to the outer layer of topologically segregated bilayer TentaGel (TG) beads with solid phase peptide synthesis chemistry, resulting in beads with increased loading capacity, hydrophilicity and porosity at the outer layer. We have found that such bead configuration can facilitate ultra high-throughput in situ releasable solution-phase screening of OBOC libraries. An encoded releasable OBOC small molecule library was constructed on Jeffamine derivatized TG beads with library compounds tethered to the outer layer via a disulfide linker and coding tags in the interior of the beads. Compound-beads could be efficiently loaded (5-10 minutes) into a 5 cm diameter Petri dish containing a 10,000-well PDMS microbead cassette, such that over 90% of the microwells were each filled with only one compound-bead. Jurkat T-lymphoid cancer cells suspended in Matrigel® were then layered over the microbead cassette to immobilize the compound-beads. After 24 hours of incubation at 37°C, dithiothreitol was added to trigger the release of library compounds. Forty-eight hours later, MTT reporter assay was used to identify regions of reduced cell viability surrounding each positive bead. From a total of about 20,000 beads screened, 3 positive beads were detected and physically isolated for decoding. A strong consensus motif was identified for these three positive compounds. These compounds were re-synthesized and found to be cytotoxic (IC50 50-150 μM) against two T-lymphoma cell lines and less so against the MDA-MB 231 breast cancer cell line. This novel ultra high-throughput OBOC releasable method can potentially be adapted to many existing 96- or 384-well solution-phase cell-based or biochemical assays. PMID:20593859

Combinatorial stresses kill pathogenic Candida species

PubMed Central

Kaloriti, Despoina; Tillmann, Anna; Cook, Emily; Jacobsen, Mette; You, Tao; Lenardon, Megan; Ames, Lauren; Barahona, Mauricio; Chandrasekaran, Komelapriya; Coghill, George; Goodman, Daniel; Gow, Neil A. R.; Grebogi, Celso; Ho, Hsueh-Lui; Ingram, Piers; McDonagh, Andrew; De Moura, Alessandro P. S.; Pang, Wei; Puttnam, Melanie; Radmaneshfar, Elahe; Romano, Maria Carmen; Silk, Daniel; Stark, Jaroslav; Stumpf, Michael; Thiel, Marco; Thorne, Thomas; Usher, Jane; Yin, Zhikang; Haynes, Ken; Brown, Alistair J. P.

2012-01-01

Pathogenic microbes exist in dynamic niches and have evolved robust adaptive responses to promote survival in their hosts. The major fungal pathogens of humans, Candida albicans and Candida glabrata, are exposed to a range of environmental stresses in their hosts including osmotic, oxidative and nitrosative stresses. Significant efforts have been devoted to the characterization of the adaptive responses to each of these stresses. In the wild, cells are frequently exposed simultaneously to combinations of these stresses and yet the effects of such combinatorial stresses have not been explored. We have developed a common experimental platform to facilitate the comparison of combinatorial stress responses in C. glabrata and C. albicans. This platform is based on the growth of cells in buffered rich medium at 30°C, and was used to define relatively low, medium and high doses of osmotic (NaCl), oxidative (H 2O2) and nitrosative stresses (e.g., dipropylenetriamine (DPTA)-NONOate). The effects of combinatorial stresses were compared with the corresponding individual stresses under these growth conditions. We show for the first time that certain combinations of combinatorial stress are especially potent in terms of their ability to kill C. albicans and C. glabrata and/or inhibit their growth. This was the case for combinations of osmotic plus oxidative stress and for oxidative plus nitrosative stress. We predict that combinatorial stresses may be highly signif cant in host defences against these pathogenic yeasts. PMID:22463109

Mapping quantum yield for (Fe-Zn-Sn-Ti)Ox photoabsorbers using a high throughput photoelectrochemical screening system.

PubMed

Xiang, Chengxiang; Haber, Joel; Marcin, Martin; Mitrovic, Slobodan; Jin, Jian; Gregoire, John M

2014-03-10

Combinatorial synthesis and screening of light absorbers are critical to material discoveries for photovoltaic and photoelectrochemical applications. One of the most effective ways to evaluate the energy-conversion properties of a semiconducting light absorber is to form an asymmetric junction and investigate the photogeneration, transport and recombination processes at the semiconductor interface. This standard photoelectrochemical measurement is readily made on a semiconductor sample with a back-side metallic contact (working electrode) and front-side solution contact. In a typical combinatorial material library, each sample shares a common back contact, requiring novel instrumentation to provide spatially resolved and thus sample-resolved measurements. We developed a multiplexing counter electrode with a thin layer assembly, in which a rectifying semiconductor/liquid junction was formed and the short-circuit photocurrent was measured under chopped illumination for each sample in a material library. The multiplexing counter electrode assembly demonstrated a photocurrent sensitivity of sub-10 μA cm(-2) with an external quantum yield sensitivity of 0.5% for each semiconductor sample under a monochromatic ultraviolet illumination source. The combination of cell architecture and multiplexing allows high-throughput modes of operation, including both fast-serial and parallel measurements. To demonstrate the performance of the instrument, the external quantum yields of 1819 different compositions from a pseudoquaternary metal oxide library, (Fe-Zn-Sn-Ti)Ox, at 385 nm were collected in scanning serial mode with a throughput of as fast as 1 s per sample. Preliminary screening results identified a promising ternary composition region centered at Fe0.894Sn0.103Ti0.0034Ox, with an external quantum yield of 6.7% at 385 nm.

A highly functional synthetic phage display library containing over 40 billion human antibody clones.

PubMed

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics.

A Highly Functional Synthetic Phage Display Library Containing over 40 Billion Human Antibody Clones

PubMed Central

Weber, Marcel; Bujak, Emil; Putelli, Alessia; Villa, Alessandra; Matasci, Mattia; Gualandi, Laura; Hemmerle, Teresa; Wulhfard, Sarah; Neri, Dario

2014-01-01

Several synthetic antibody phage display libraries have been created and used for the isolation of human monoclonal antibodies. The performance of antibody libraries, which is usually measured in terms of their ability to yield high-affinity binding specificities against target proteins of interest, depends both on technical aspects (such as library size and quality of cloning) and on design features (which influence the percentage of functional clones in the library and their ability to be used for practical applications). Here, we describe the design, construction and characterization of a combinatorial phage display library, comprising over 40 billion human antibody clones in single-chain fragment variable (scFv) format. The library was designed with the aim to obtain highly stable antibody clones, which can be affinity-purified on protein A supports, even when used in scFv format. The library was found to be highly functional, as >90% of randomly selected clones expressed the corresponding antibody. When selected against more than 15 antigens from various sources, the library always yielded specific and potent binders, at a higher frequency compared to previous antibody libraries. To demonstrate library performance in practical biomedical research projects, we isolated the human antibody G5, which reacts both against human and murine forms of the alternatively spliced BCD segment of tenascin-C, an extracellular matrix component frequently over-expressed in cancer and in chronic inflammation. The new library represents a useful source of binding specificities, both for academic research and for the development of antibody-based therapeutics. PMID:24950200

Temperature-programmed technique accompanied with high-throughput methodology for rapidly searching the optimal operating temperature of MOX gas sensors.

PubMed

Zhang, Guozhu; Xie, Changsheng; Zhang, Shunping; Zhao, Jianwei; Lei, Tao; Zeng, Dawen

2014-09-08

A combinatorial high-throughput temperature-programmed method to obtain the optimal operating temperature (OOT) of gas sensor materials is demonstrated here for the first time. A material library consisting of SnO2, ZnO, WO3, and In2O3 sensor films was fabricated by screen printing. Temperature-dependent conductivity curves were obtained by scanning this gas sensor library from 300 to 700 K in different atmospheres (dry air, formaldehyde, carbon monoxide, nitrogen dioxide, toluene and ammonia), giving the OOT of each sensor formulation as a function of the carrier and analyte gases. A comparative study of the temperature-programmed method and a conventional method showed good agreement in measured OOT.

Molecular computation: RNA solutions to chess problems.

PubMed

Faulhammer, D; Cukras, A R; Lipton, R J; Landweber, L F

2000-02-15

We have expanded the field of "DNA computers" to RNA and present a general approach for the solution of satisfiability problems. As an example, we consider a variant of the "Knight problem," which asks generally what configurations of knights can one place on an n x n chess board such that no knight is attacking any other knight on the board. Using specific ribonuclease digestion to manipulate strands of a 10-bit binary RNA library, we developed a molecular algorithm and applied it to a 3 x 3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine "bits" or placeholders in a combinatorial RNA library. We recovered a set of "winning" molecules that describe solutions to this problem.

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.

Distributed Drug Discovery, Part 2: Global Rehearsal of Alkylating Agents for the Synthesis of Resin-Bound Unnatural Amino Acids and Virtual D3 Catalog Construction

PubMed Central

2008-01-01

Distributed Drug Discovery (D3) proposes solving large drug discovery problems by breaking them into smaller units for processing at multiple sites. A key component of the synthetic and computational stages of D3 is the global rehearsal of prospective reagents and their subsequent use in the creation of virtual catalogs of molecules accessible by simple, inexpensive combinatorial chemistry. The first section of this article documents the feasibility of the synthetic component of Distributed Drug Discovery. Twenty-four alkylating agents were rehearsed in the United States, Poland, Russia, and Spain, for their utility in the synthesis of resin-bound unnatural amino acids 1, key intermediates in many combinatorial chemistry procedures. This global reagent rehearsal, coupled to virtual library generation, increases the likelihood that any member of that virtual library can be made. It facilitates the realistic integration of worldwide virtual D3 catalog computational analysis with synthesis. The second part of this article describes the creation of the first virtual D3 catalog. It reports the enumeration of 24 416 acylated unnatural amino acids 5, assembled from lists of either rehearsed or well-precedented alkylating and acylating reagents, and describes how the resulting catalog can be freely accessed, searched, and downloaded by the scientific community. PMID:19105725

Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space

PubMed Central

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R.; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J.

2013-01-01

For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding. PMID:23592960

Combinatorial pooling enables selective sequencing of the barley gene space.

PubMed

Lonardi, Stefano; Duma, Denisa; Alpert, Matthew; Cordero, Francesca; Beccuti, Marco; Bhat, Prasanna R; Wu, Yonghui; Ciardo, Gianfranco; Alsaihati, Burair; Ma, Yaqin; Wanamaker, Steve; Resnik, Josh; Bozdag, Serdar; Luo, Ming-Cheng; Close, Timothy J

2013-04-01

For the vast majority of species - including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.

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

Siol, Sebastian; Holder, Aaron; Ortiz, Brenden R.

Here, the controlled decomposition of metastable alloys is an attractive route to form nanostructured thermoelectric materials with reduced thermal conductivity. The ternary SnTe–MnTe and SnTe–SnSe heterostructural alloys have been demonstrated as promising materials for thermoelectric applications. In this work, the quaternary Sn 1–yMnyTe 1–xSe x phase space serves as a relevant model system to explore how a combination of computational and combinatorial-growth methods can be used to study equilibrium and non-equilibrium solubility limits. Results from first principle calculations indicate low equilibrium solubility for x,y < 0.05 that are in good agreement with results obtained from bulk equilibrium synthesis experiments andmore » predict significantly higher spinodal limits. An experimental screening using sputtered combinatorial thin film sample libraries showed a remarkable increase in non-equilibrium solubility for x,y > 0.2. These theoretical and experimental results were used to guide the bulk synthesis of metastable alloys. The ability to reproduce the non-equilibrium solubility levels in bulk materials indicates that such theoretical calculations and combinatorial growth can inform bulk synthetic routes. Further, the large difference between equilibrium and non-equilibrium solubility limits in Sn 1–yMn yTe 1–xSe x indicates these metastable alloys are attractive in terms of nano-precipitate formation for potential thermoelectric applications.« less

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage. lambda. immunoexpression library

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

Mullinax, R.L.; Gross, E.A.; Amberg, J.R.

1990-10-01

The authors have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and {kappa} light-chain variable and constant region domains, were inserted into modified bacteriophase {lambda} expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2{percent} in the library. These humanmore » antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. They estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.« less

Combinatorial synthesis and hydrogenation of Mg/Al libraries prepared by electron beam physical vapor deposition.

PubMed

Garcia, Gemma; Doménech-Ferrer, Roger; Pi, Francesc; Santiso, Josep; Rodríguez-Viejo, Javier

2007-01-01

We have grown thin film libraries of the Mg-Al system using a high-throughput synthesis methodology that combines the sequential deposition of pure elements (Mg and Al) by an electron-beam (e-beam) evaporation technique and the use of a special set of moving shadow masks. This novel mask has been designed to simultaneously prepare four identical arrays of different compositions that will permit the characterization of the same library after several treatments. Wavelength dispersive spectroscopy (WDS) and micro-X-ray diffraction have been used as high-throughput screening techniques for the determination of the composition and structure of every member of the library in the as-deposited state and after hydrogenation at 1 atm of H2 during 24 h at three different temperatures: 60, 80, and 110 degrees C. We have analyzed the influence of the Mg-Al ratio on the hydrogenation of magnesium, as well as on the appearance of complex hydride phases. We have also found that aluminum can act as a catalyzer for the hydrogenation reaction of magnesium.

Dynamic Hierarchical Energy-Efficient Method Based on Combinatorial Optimization for Wireless Sensor Networks

PubMed Central

Tang, Hongying; Cheng, Yongbo; Zhao, Qin; Li, Baoqing; Yuan, Xiaobing

2017-01-01

Routing protocols based on topology control are significantly important for improving network longevity in wireless sensor networks (WSNs). Traditionally, some WSN routing protocols distribute uneven network traffic load to sensor nodes, which is not optimal for improving network longevity. Differently to conventional WSN routing protocols, we propose a dynamic hierarchical protocol based on combinatorial optimization (DHCO) to balance energy consumption of sensor nodes and to improve WSN longevity. For each sensor node, the DHCO algorithm obtains the optimal route by establishing a feasible routing set instead of selecting the cluster head or the next hop node. The process of obtaining the optimal route can be formulated as a combinatorial optimization problem. Specifically, the DHCO algorithm is carried out by the following procedures. It employs a hierarchy-based connection mechanism to construct a hierarchical network structure in which each sensor node is assigned to a special hierarchical subset; it utilizes the combinatorial optimization theory to establish the feasible routing set for each sensor node, and takes advantage of the maximum–minimum criterion to obtain their optimal routes to the base station. Various results of simulation experiments show effectiveness and superiority of the DHCO algorithm in comparison with state-of-the-art WSN routing algorithms, including low-energy adaptive clustering hierarchy (LEACH), hybrid energy-efficient distributed clustering (HEED), genetic protocol-based self-organizing network clustering (GASONeC), and double cost function-based routing (DCFR) algorithms. PMID:28753962

Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags

PubMed Central

Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

2014-01-01

The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method “relay primer-dependent bypass” utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3′-terminus prior to ligation to adjacent oligonucleotides. The second reading method “repeat-dependent bypass” utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3′-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries. PMID:25483841

Bifurcations of 2-Periodic Nonautonomous Stunted Tent Systems

NASA Astrophysics Data System (ADS)

Silva, L.; Rocha, J. Leonel; Silva, M. T.

2017-06-01

In this paper, we will consider a family of 2-periodic nonautonomous dynamical systems, generated by the alternate iteration of two stunted tent maps and study its bifurcation skeleton. We will describe the bifurcation phenomena along and around the bones accomplished with the combinatorial data furnished by the respective symbolic dynamics.

PR01 Molecular Pathogenesis of Rickettsioses and Development of Anti-Rickettsial Treatment by Combinatorial Peptide-Based Libraries

DTIC Science & Technology

2006-02-01

likely reflecting similar cell death rates in all monolayers at late time points. By the end of the experiment at 120 hours, all monolayers showed a...50-55% increase in permeability when compared to the controls. 2. Cell death rates in rickettsiae-infected SV-HCEC monolayers In order to...necrotic cell death. Quantification of cell death was performed by determining the percent of total cells staining positive for PI. Cell death rates did

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

PubMed

Damnjanović, Jasmina; Takahashi, Rie; Suzuki, Atsuo; Nakano, Hideo; Iwasaki, Yugo

2012-08-01

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.

Molecular computation: RNA solutions to chess problems

PubMed Central

Faulhammer, Dirk; Cukras, Anthony R.; Lipton, Richard J.; Landweber, Laura F.

2000-01-01

We have expanded the field of “DNA computers” to RNA and present a general approach for the solution of satisfiability problems. As an example, we consider a variant of the “Knight problem,” which asks generally what configurations of knights can one place on an n × n chess board such that no knight is attacking any other knight on the board. Using specific ribonuclease digestion to manipulate strands of a 10-bit binary RNA library, we developed a molecular algorithm and applied it to a 3 × 3 chessboard as a 9-bit instance of this problem. Here, the nine spaces on the board correspond to nine “bits” or placeholders in a combinatorial RNA library. We recovered a set of “winning” molecules that describe solutions to this problem. PMID:10677471

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Real-time monitoring of surface-initiated atom transfer radical polymerization using silicon photonic microring resonators: implications for combinatorial screening of polymer brush growth conditions.

PubMed

Limpoco, F Ted; Bailey, Ryan C

2011-09-28

We directly monitor in parallel and in real time the temporal profiles of polymer brushes simultaneously grown via multiple ATRP reaction conditions on a single substrate using arrays of silicon photonic microring resonators. In addition to probing relative polymerization rates, we show the ability to evaluate the dynamic properties of the in situ grown polymers. This presents a powerful new platform for studying modified interfaces that may allow for the combinatorial optimization of surface-initiated polymerization conditions.

SPANG: a SPARQL client supporting generation and reuse of queries for distributed RDF databases.

PubMed

Chiba, Hirokazu; Uchiyama, Ikuo

2017-02-08

Toward improved interoperability of distributed biological databases, an increasing number of datasets have been published in the standardized Resource Description Framework (RDF). Although the powerful SPARQL Protocol and RDF Query Language (SPARQL) provides a basis for exploiting RDF databases, writing SPARQL code is burdensome for users including bioinformaticians. Thus, an easy-to-use interface is necessary. We developed SPANG, a SPARQL client that has unique features for querying RDF datasets. SPANG dynamically generates typical SPARQL queries according to specified arguments. It can also call SPARQL template libraries constructed in a local system or published on the Web. Further, it enables combinatorial execution of multiple queries, each with a distinct target database. These features facilitate easy and effective access to RDF datasets and integrative analysis of distributed data. SPANG helps users to exploit RDF datasets by generation and reuse of SPARQL queries through a simple interface. This client will enhance integrative exploitation of biological RDF datasets distributed across the Web. This software package is freely available at http://purl.org/net/spang .

Analytical validation of a psychiatric pharmacogenomic test.

PubMed

Jablonski, Michael R; King, Nina; Wang, Yongbao; Winner, Joel G; Watterson, Lucas R; Gunselman, Sandra; Dechairo, Bryan M

2018-05-01

The aim of this study was to validate the analytical performance of a combinatorial pharmacogenomics test designed to aid in the appropriate medication selection for neuropsychiatric conditions. Genomic DNA was isolated from buccal swabs. Twelve genes (65 variants/alleles) associated with psychotropic medication metabolism, side effects, and mechanisms of actions were evaluated by bead array, MALDI-TOF mass spectrometry, and/or capillary electrophoresis methods (GeneSight Psychotropic, Assurex Health, Inc.). The combinatorial pharmacogenomics test has a dynamic range of 2.5-20 ng/μl of input genomic DNA, with comparable performance for all assays included in the test. Both the precision and accuracy of the test were >99.9%, with individual gene components between 99.4 and 100%. This study demonstrates that the combinatorial pharmacogenomics test is robust and reproducible, making it suitable for clinical use.

Stepwise molding, etching, and imprinting to form libraries of nanopatterned substrates.

PubMed

Zhao, Zhi; Cai, Yangjun; Liao, Wei-Ssu; Cremer, Paul S

2013-06-04

Herein, we describe a novel colloidal lithographic strategy for the stepwise patterning of planar substrates with numerous complex and unique designs. In conjunction with colloidal self-assembly, imprint molding, and capillary force lithography, reactive ion etching was used to create complex libraries of nanoscale features. This combinatorial strategy affords the ability to develop an exponentially increasing number of two-dimensional nanoscale patterns with each sequential step in the process. Specifically, dots, triangles, circles, and lines could be assembled on the surface separately and in combination with each other. Numerous architectures are obtained for the first time with high uniformity and reproducibility. These hexagonal arrays were made from polystyrene and gold features, whereby each surface element could be tuned from the micrometer size scale down to line widths of ~35 nm. The patterned area could be 1 cm(2) or even larger. The techniques described herein can be combined with further steps to make even larger libraries. Moreover, these polymer and metal features may prove useful in optical, sensing, and electronic applications.

Identifying the preferred RNA motifs and chemotypes that interact by probing millions of combinations.

PubMed

Tran, Tuan; Disney, Matthew D

2012-01-01

RNA is an important therapeutic target but information about RNA-ligand interactions is limited. Here, we report a screening method that probes over 3,000,000 combinations of RNA motif-small molecule interactions to identify the privileged RNA structures and chemical spaces that interact. Specifically, a small molecule library biased for binding RNA was probed for binding to over 70,000 unique RNA motifs in a high throughput solution-based screen. The RNA motifs that specifically bind each small molecule were identified by microarray-based selection. In this library-versus-library or multidimensional combinatorial screening approach, hairpin loops (among a variety of RNA motifs) were the preferred RNA motif space that binds small molecules. Furthermore, it was shown that indole, 2-phenyl indole, 2-phenyl benzimidazole and pyridinium chemotypes allow for specific recognition of RNA motifs. As targeting RNA with small molecules is an extremely challenging area, these studies provide new information on RNA-ligand interactions that has many potential uses.

Identifying the Preferred RNA Motifs and Chemotypes that Interact by Probing Millions of Combinations

PubMed Central

Tran, Tuan; Disney, Matthew D.

2012-01-01

RNA is an important therapeutic target but information about RNA-ligand interactions is limited. Here we report a screening method that probes over 3,000,000 combinations of RNA motif-small molecule interactions to identify the privileged RNA structures and chemical spaces that interact. Specifically, a small molecule library biased for binding RNA was probed for binding to over 70,000 unique RNA motifs in a high throughput solution-based screen. The RNA motifs that specifically bind each small molecule were identified by microarray-based selection. In this library-versus-library or multidimensional combinatorial screening approach, hairpin loops (amongst a variety of RNA motifs) were the preferred RNA motif space that binds small molecules. Furthermore, it was shown that indole, 2-phenyl indole, 2-phenyl benzimidazole, and pyridinium chemotypes allow for specific recognition of RNA motifs. Since targeting RNA with small molecules is an extremely challenging area, these studies provide new information on RNA-ligand interactions that has many potential uses. PMID:23047683

Probing a 2-aminobenzimidazole library for binding to RNA internal loops via two-dimensional combinatorial screening.

PubMed

Velagapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P; French, Jonathan M; Disney, Matthew D

2012-11-16

There are many potential RNA drug targets in bacterial, viral, and human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition.

Interaction Analysis through Proteomic Phage Display

PubMed Central

2014-01-01

Phage display is a powerful technique for profiling specificities of peptide binding domains. The method is suited for the identification of high-affinity ligands with inhibitor potential when using highly diverse combinatorial peptide phage libraries. Such experiments further provide consensus motifs for genome-wide scanning of ligands of potential biological relevance. A complementary but considerably less explored approach is to display expression products of genomic DNA, cDNA, open reading frames (ORFs), or oligonucleotide libraries designed to encode defined regions of a target proteome on phage particles. One of the main applications of such proteomic libraries has been the elucidation of antibody epitopes. This review is focused on the use of proteomic phage display to uncover protein-protein interactions of potential relevance for cellular function. The method is particularly suited for the discovery of interactions between peptide binding domains and their targets. We discuss the largely unexplored potential of this method in the discovery of domain-motif interactions of potential biological relevance. PMID:25295249

[Peptide phage display in biotechnology and biomedicine].

PubMed

Kuzmicheva, G A; Belyavskaya, V A

2016-07-01

To date peptide phage display is one of the most common combinatorial methods used for identifying specific peptide ligands. Phage display peptide libraries containing billions different clones successfully used for selection of ligands with high affinity and selectivity toward wide range of targets including individual proteins, bacteria, viruses, spores, different kind of cancer cells and variety of nonorganic targets (metals, alloys, semiconductors etc.) Success of using filamentous phage in phage display technologies relays on the robustness of phage particles and a possibility to genetically modify its DNA to construct new phage variants with novel properties. In this review we are discussing characteristics of the most known non-commercial peptide phage display libraries of different formats (landscape libraries in particular) and their successful applications in several fields of biotechnology and biomedicine: discovery of peptides with diagnostic values against different pathogens, discovery and using of peptides recognizing cancer cells, trends in using of phage display technologies in human interactome studies, application of phage display technologies in construction of novel nano materials.

COMDECOM: predicting the lifetime of screening compounds in DMSO solution.

PubMed

Zitha-Bovens, Emrin; Maas, Peter; Wife, Dick; Tijhuis, Johan; Hu, Qian-Nan; Kleinöder, Thomas; Gasteiger, Johann

2009-06-01

The technological evolution of the 1990s in both combinatorial chemistry and high-throughput screening created the demand for rapid access to the compound deck to support the screening process. The common strategy within the pharmaceutical industry is to store the screening library in DMSO solution. Several studies have shown that a percentage of these compounds decompose in solution, varying from a few percent of the total to a substantial part of the library. In the COMDECOM (COMpound DECOMposition) project, the compound stability of screening compounds in DMSO solution is monitored in an accelerated thermal, hydrolytic, and oxidative decomposition program. A large database with stability data is collected, and from this database, a predictive model is being developed. The aim of this program is to build an algorithm that can flag compounds that are likely to decompose-information that is considered to be of utmost importance (e.g., in the compound acquisition process and when evaluation screening results of library compounds, as well as in the determination of optimal storage conditions).

Manipulating Tabu List to Handle Machine Breakdowns in Job Shop Scheduling Problems

NASA Astrophysics Data System (ADS)

Nababan, Erna Budhiarti; SalimSitompul, Opim

2011-06-01

Machine breakdowns in a production schedule may occur on a random basis that make the well-known hard combinatorial problem of Job Shop Scheduling Problems (JSSP) becomes more complex. One of popular techniques used to solve the combinatorial problems is Tabu Search. In this technique, moves that will be not allowed to be revisited are retained in a tabu list in order to avoid in gaining solutions that have been obtained previously. In this paper, we propose an algorithm to employ a second tabu list to keep broken machines, in addition to the tabu list that keeps the moves. The period of how long the broken machines will be kept on the list is categorized using fuzzy membership function. Our technique are tested to the benchmark data of JSSP available on the OR library. From the experiment, we found that our algorithm is promising to help a decision maker to face the event of machine breakdowns.

Wafer-scale growth of VO2 thin films using a combinatorial approach

PubMed Central

Zhang, Hai-Tian; Zhang, Lei; Mukherjee, Debangshu; Zheng, Yuan-Xia; Haislmaier, Ryan C.; Alem, Nasim; Engel-Herbert, Roman

2015-01-01

Transition metal oxides offer functional properties beyond conventional semiconductors. Bridging the gap between the fundamental research frontier in oxide electronics and their realization in commercial devices demands a wafer-scale growth approach for high-quality transition metal oxide thin films. Such a method requires excellent control over the transition metal valence state to avoid performance deterioration, which has been proved challenging. Here we present a scalable growth approach that enables a precise valence state control. By creating an oxygen activity gradient across the wafer, a continuous valence state library is established to directly identify the optimal growth condition. Single-crystalline VO2 thin films have been grown on wafer scale, exhibiting more than four orders of magnitude change in resistivity across the metal-to-insulator transition. It is demonstrated that ‘electronic grade' transition metal oxide films can be realized on a large scale using a combinatorial growth approach, which can be extended to other multivalent oxide systems. PMID:26450653

Quantitative Tracking of Combinatorially Engineered Populations with Multiplexed Binary Assemblies.

PubMed

Zeitoun, Ramsey I; Pines, Gur; Grau, Willliam C; Gill, Ryan T

2017-04-21

Advances in synthetic biology and genomics have enabled full-scale genome engineering efforts on laboratory time scales. However, the absence of sufficient approaches for mapping engineered genomes at system-wide scales onto performance has limited the adoption of more sophisticated algorithms for engineering complex biological systems. Here we report on the development and application of a robust approach to quantitatively map combinatorially engineered populations at scales up to several dozen target sites. This approach works by assembling genome engineered sites with cell-specific barcodes into a format compatible with high-throughput sequencing technologies. This approach, called barcoded-TRACE (bTRACE) was applied to assess E. coli populations engineered by recursive multiplex recombineering across both 6-target sites and 31-target sites. The 31-target library was then tracked throughout growth selections in the presence and absence of isopentenol (a potential next-generation biofuel). We also use the resolution of bTRACE to compare the influence of technical and biological noise on genome engineering efforts.

Coarse-grained molecular dynamics simulations of depletion-induced interactions for soft matter systems

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

Shendruk, Tyler N., E-mail: tyler.shendruk@physics.ox.ac.uk; Bertrand, Martin; Harden, James L.

2014-12-28

Given the ubiquity of depletion effects in biological and other soft matter systems, it is desirable to have coarse-grained Molecular Dynamics (MD) simulation approaches appropriate for the study of complex systems. This paper examines the use of two common truncated Lennard-Jones (Weeks-Chandler-Andersen (WCA)) potentials to describe a pair of colloidal particles in a thermal bath of depletants. The shifted-WCA model is the steeper of the two repulsive potentials considered, while the combinatorial-WCA model is the softer. It is found that the depletion-induced well depth for the combinatorial-WCA model is significantly deeper than the shifted-WCA model because the resulting overlap ofmore » the colloids yields extra accessible volume for depletants. For both shifted- and combinatorial-WCA simulations, the second virial coefficients and pair potentials between colloids are demonstrated to be well approximated by the Morphometric Thermodynamics (MT) model. This agreement suggests that the presence of depletants can be accurately modelled in MD simulations by implicitly including them through simple, analytical MT forms for depletion-induced interactions. Although both WCA potentials are found to be effective generic coarse-grained simulation approaches for studying depletion effects in complicated soft matter systems, combinatorial-WCA is the more efficient approach as depletion effects are enhanced at lower depletant densities. The findings indicate that for soft matter systems that are better modelled by potentials with some compressibility, predictions from hard-sphere systems could greatly underestimate the magnitude of depletion effects at a given depletant density.« less

Bacteriophages and medical oncology: targeted gene therapy of cancer.

PubMed

Bakhshinejad, Babak; Karimi, Marzieh; Sadeghizadeh, Majid

2014-08-01

Targeted gene therapy of cancer is of paramount importance in medical oncology. Bacteriophages, viruses that specifically infect bacterial cells, offer a variety of potential applications in biomedicine. Their genetic flexibility to go under a variety of surface modifications serves as a basis for phage display methodology. These surface manipulations allow bacteriophages to be exploited for targeted delivery of therapeutic genes. Moreover, the excellent safety profile of these viruses paves the way for their potential use as cancer gene therapy platforms. The merge of phage display and combinatorial technology has led to the emergence of phage libraries turning phage display into a high throughput technology. Random peptide libraries, as one of the most frequently used phage libraries, provide a rich source of clinically useful peptide ligands. Peptides are known as a promising category of pharmaceutical agents in medical oncology that present advantages such as inexpensive synthesis, efficient tissue penetration and the lack of immunogenicity. Phage peptide libraries can be screened, through biopanning, against various targets including cancer cells and tissues that results in obtaining cancer-homing ligands. Cancer-specific peptides isolated from phage libraries show huge promise to be utilized for targeting of various gene therapy vectors towards malignant cells. Beyond doubt, bacteriophages will play a more impressive role in the future of medical oncology.

Physicochemical Profiles of the Marketed Agrochemicals and Clues for Agrochemical Lead Discovery and Screening Library Development.

PubMed

Rao, Hanbing; Huangfu, Changxin; Wang, Yanying; Wang, Xianxiang; Tang, Tiansheng; Zeng, Xianyin; Li, Zerong; Chen, Yuzong

2015-05-01

Combinatorial chemistry, high-throughput and virtual screening technologies have been extensively used for discovering agrochemical leads from chemical libraries. The knowledge of the physicochemical properties of the marketed agrochemicals is useful for guiding the design and selection of such libraries. Since the earlier profiling of marketed agrochemicals, the number and types of marketed agrochemicals have significantly increased. Recent studies have shown the change of some physicochemical properties of oral drugs with time. There is a need to also profile the physicochemical properties of the marketed agrochemicals. In this work, we analyzed the key physicochemical properties of 1751 marketed agrochemicals in comparison with the previously-analyzed herbicides and insecticides, 106 391 natural products and 57 548 diverse synthetic libraries compounds. Our study revealed the distribution profiles and evolution trend of different types of agrochemicals that in many respects are broadly similar to the reported profiles for oral drugs, with the most marked difference being that agrochemicals have a lower number of hydrogen bond donors. The derived distribution patterns provided the rule of thumb guidelines for selecting potential agrochemical leads and also provided clues for further improving the libraries for agrochemical lead discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular scaffold analysis of natural products databases in the public domain.

PubMed

Yongye, Austin B; Waddell, Jacob; Medina-Franco, José L

2012-11-01

Natural products represent important sources of bioactive compounds in drug discovery efforts. In this work, we compiled five natural products databases available in the public domain and performed a comprehensive chemoinformatic analysis focused on the content and diversity of the scaffolds with an overview of the diversity based on molecular fingerprints. The natural products databases were compared with each other and with a set of molecules obtained from in-house combinatorial libraries, and with a general screening commercial library. It was found that publicly available natural products databases have different scaffold diversity. In contrast to the common concept that larger libraries have the largest scaffold diversity, the largest natural products collection analyzed in this work was not the most diverse. The general screening library showed, overall, the highest scaffold diversity. However, considering the most frequent scaffolds, the general reference library was the least diverse. In general, natural products databases in the public domain showed low molecule overlap. In addition to benzene and acyclic compounds, flavones, coumarins, and flavanones were identified as the most frequent molecular scaffolds across the different natural products collections. The results of this work have direct implications in the computational and experimental screening of natural product databases for drug discovery. © 2012 John Wiley & Sons A/S.

Designing Multi-target Compound Libraries with Gaussian Process Models.

PubMed

Bieler, Michael; Reutlinger, Michael; Rodrigues, Tiago; Schneider, Petra; Kriegl, Jan M; Schneider, Gisbert

2016-05-01

We present the application of machine learning models to selecting G protein-coupled receptor (GPCR)-focused compound libraries. The library design process was realized by ant colony optimization. A proprietary Boehringer-Ingelheim reference set consisting of 3519 compounds tested in dose-response assays at 11 GPCR targets served as training data for machine learning and activity prediction. We compared the usability of the proprietary data with a public data set from ChEMBL. Gaussian process models were trained to prioritize compounds from a virtual combinatorial library. We obtained meaningful models for three of the targets (5-HT2c , MCH, A1), which were experimentally confirmed for 12 of 15 selected and synthesized or purchased compounds. Overall, the models trained on the public data predicted the observed assay results more accurately. The results of this study motivate the use of Gaussian process regression on public data for virtual screening and target-focused compound library design. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution Non-Commercial NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

A Metagenome-Wide Association Study and Arrayed Mutant Library Confirm Acetobacter Lipopolysaccharide Genes Are Necessary for Association with Drosophila melanogaster.

PubMed

White, K Makay; Matthews, Melinda K; Hughes, Rachel C; Sommer, Andrew J; Griffitts, Joel S; Newell, Peter D; Chaston, John M

2018-03-28

A metagenome wide association (MGWA) study of bacterial host association determinants in Drosophila predicted that LPS biosynthesis genes are significantly associated with host colonization. We were unable to create site-directed mutants for each of the predicted genes in Acetobacter , so we created an arrayed transposon insertion library using Acetobacter fabarum DsW_054 isolated from Drosophila Creation of the A. fabarum DsW_054 gene knock-out library was performed by combinatorial mapping and Illumina sequencing of random transposon insertion mutants. Transposon insertion locations for 6,418 mutants were successfully mapped, including hits within 63% of annotated genes in the A. fabarum DsW_054 genome. For 45/45 members of the library, insertion sites were verified by arbitrary PCR and Sanger sequencing. Mutants with insertions in four different LPS biosynthesis genes were selected from the library to validate the MGWA predictions. Insertion mutations in two genes biosynthetically upstream of Lipid-A formation, lpxC and lpxB , show significant differences in host association, whereas mutations in two genes encoding LPS biosynthesis functions downstream of Lipid-A biosynthesis had no effect. These results suggest an impact of bacterial cell surface molecules on the bacterial capacity for host association. Also, the transposon insertion mutant library will be a useful resource for ongoing research on the genetic basis for Acetobacter traits. Copyright © 2018 White et al.

Automated phase mapping with AgileFD and its application to light absorber discovery in the V–Mn–Nb oxide system

DOE PAGES

Suram, Santosh K.; Xue, Yexiang; Bai, Junwen; ...

2016-11-21

Rapid construction of phase diagrams is a central tenet of combinatorial materials science with accelerated materials discovery efforts often hampered by challenges in interpreting combinatorial X-ray diffraction data sets, which we address by developing AgileFD, an artificial intelligence algorithm that enables rapid phase mapping from a combinatorial library of X-ray diffraction patterns. AgileFD models alloying-based peak shifting through a novel expansion of convolutional nonnegative matrix factorization, which not only improves the identification of constituent phases but also maps their concentration and lattice parameter as a function of composition. By incorporating Gibbs’ phase rule into the algorithm, physically meaningful phase mapsmore » are obtained with unsupervised operation, and more refined solutions are attained by injecting expert knowledge of the system. The algorithm is demonstrated through investigation of the V–Mn–Nb oxide system where decomposition of eight oxide phases, including two with substantial alloying, provides the first phase map for this pseudoternary system. This phase map enables interpretation of high-throughput band gap data, leading to the discovery of new solar light absorbers and the alloying-based tuning of the direct-allowed band gap energy of MnV 2O 6. Lastly, the open-source family of AgileFD algorithms can be implemented into a broad range of high throughput workflows to accelerate materials discovery.« less

Automated phase mapping with AgileFD and its application to light absorber discovery in the V–Mn–Nb oxide system

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

Suram, Santosh K.; Xue, Yexiang; Bai, Junwen

Rapid construction of phase diagrams is a central tenet of combinatorial materials science with accelerated materials discovery efforts often hampered by challenges in interpreting combinatorial X-ray diffraction data sets, which we address by developing AgileFD, an artificial intelligence algorithm that enables rapid phase mapping from a combinatorial library of X-ray diffraction patterns. AgileFD models alloying-based peak shifting through a novel expansion of convolutional nonnegative matrix factorization, which not only improves the identification of constituent phases but also maps their concentration and lattice parameter as a function of composition. By incorporating Gibbs’ phase rule into the algorithm, physically meaningful phase mapsmore » are obtained with unsupervised operation, and more refined solutions are attained by injecting expert knowledge of the system. The algorithm is demonstrated through investigation of the V–Mn–Nb oxide system where decomposition of eight oxide phases, including two with substantial alloying, provides the first phase map for this pseudoternary system. This phase map enables interpretation of high-throughput band gap data, leading to the discovery of new solar light absorbers and the alloying-based tuning of the direct-allowed band gap energy of MnV 2O 6. Lastly, the open-source family of AgileFD algorithms can be implemented into a broad range of high throughput workflows to accelerate materials discovery.« less

A noisy chaotic neural network for solving combinatorial optimization problems: stochastic chaotic simulated annealing.

PubMed

Wang, Lipo; Li, Sa; Tian, Fuyu; Fu, Xiuju

2004-10-01

Recently Chen and Aihara have demonstrated both experimentally and mathematically that their chaotic simulated annealing (CSA) has better search ability for solving combinatorial optimization problems compared to both the Hopfield-Tank approach and stochastic simulated annealing (SSA). However, CSA may not find a globally optimal solution no matter how slowly annealing is carried out, because the chaotic dynamics are completely deterministic. In contrast, SSA tends to settle down to a global optimum if the temperature is reduced sufficiently slowly. Here we combine the best features of both SSA and CSA, thereby proposing a new approach for solving optimization problems, i.e., stochastic chaotic simulated annealing, by using a noisy chaotic neural network. We show the effectiveness of this new approach with two difficult combinatorial optimization problems, i.e., a traveling salesman problem and a channel assignment problem for cellular mobile communications.

Synthetic antibodies: concepts, potential and practical considerations.

PubMed

Miersch, S; Sidhu, S S

2012-08-01

The last 100 years of enquiry into the fundamental basis of humoral immunity has resulted in the identification of antibodies as key molecular sentinels responsible for the in vivo surveillance, neutralization and clearance of foreign substances. Intense efforts aimed at understanding and exploiting their exquisite molecular specificity have positioned antibodies as a cornerstone supporting basic research, diagnostics and therapeutic applications [1]. More recently, efforts have aimed to circumvent the limitations of developing antibodies in animals by developing wholly in vitro techniques for designing antibodies of tailored specificity. This has been realized with the advent of synthetic antibody libraries that possess diversity outside the scope of natural immune repertoires and are thus capable of yielding specificities not otherwise attainable. This review examines the convergence of technologies that have contributed to the development of combinatorial phage-displayed antibody libraries. It further explores the practical concepts that underlie phage display, antibody diversity and the methods used in the generation of and selection from phage-displayed synthetic antibody libraries, highlighting specific applications in which design approaches gave rise to specificities that could not easily be obtained with libraries based upon natural immune repertories. Copyright © 2012 Elsevier Inc. All rights reserved.

Discovering Drugs with DNA-Encoded Library Technology: From Concept to Clinic with an Inhibitor of Soluble Epoxide Hydrolase.

PubMed

Belyanskaya, Svetlana L; Ding, Yun; Callahan, James F; Lazaar, Aili L; Israel, David I

2017-05-04

DNA-encoded chemical library technology was developed with the vision of its becoming a transformational platform for drug discovery. The hope was that a new paradigm for the discovery of low-molecular-weight drugs would be enabled by combining the vast molecular diversity achievable with combinatorial chemistry, the information-encoding attributes of DNA, the power of molecular biology, and a streamlined selection-based discovery process. Here, we describe the discovery and early clinical development of GSK2256294, an inhibitor of soluble epoxide hydrolase (sEH, EPHX2), by using encoded-library technology (ELT). GSK2256294 is an orally bioavailable, potent and selective inhibitor of sEH that has a long half life and produced no serious adverse events in a first-time-in-human clinical study. To our knowledge, GSK2256294 is the first molecule discovered from this technology to enter human clinical testing and represents a realization of the vision that DNA-encoded chemical library technology can efficiently yield molecules with favorable properties that can be readily progressed into high-quality drugs. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

A DNA-Encoded Library of Chemical Compounds Based on Common Scaffolding Structures Reveals the Impact of Ligand Geometry on Protein Recognition.

PubMed

Favalli, Nicholas; Biendl, Stefan; Hartmann, Marco; Piazzi, Jacopo; Sladojevich, Filippo; Gräslund, Susanne; Brown, Peter J; Näreoja, Katja; Schüler, Herwig; Scheuermann, Jörg; Franzini, Raphael; Neri, Dario

2018-06-01

A DNA-encoded chemical library (DECL) with 1.2 million compounds was synthesized by combinatorial reaction of seven central scaffolds with two sets of 343×492 building blocks. Library screening by affinity capture revealed that for some target proteins, the chemical nature of building blocks dominated the selection results, whereas for other proteins, the central scaffold also crucially contributed to ligand affinity. Molecules based on a 3,5-bis(aminomethyl)benzoic acid core structure were found to bind human serum albumin with a K d value of 6 nm, while compounds with the same substituents on an equidistant but flexible l-lysine scaffold showed 140-fold lower affinity. A 18 nm tankyrase-1 binder featured l-lysine as linking moiety, while molecules based on d-Lysine or (2S,4S)-amino-l-proline showed no detectable binding to the target. This work suggests that central scaffolds which predispose the orientation of chemical building blocks toward the protein target may enhance the screening productivity of encoded libraries. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Solubilization of a membrane protein by combinatorial supercharging.

PubMed

Hajduczki, Agnes; Majumdar, Sudipta; Fricke, Marie; Brown, Isola A M; Weiss, Gregory A

2011-04-15

Hydrophobic and aggregation-prone, membrane proteins often prove too insoluble for conventional in vitro biochemical studies. To engineer soluble variants of human caveolin-1, a phage-displayed library of caveolin variants targeted the hydrophobic intramembrane domain with substitutions to charged residues. Anti-selections for insolubility removed hydrophobic variants, and positive selections for binding to the known caveolin ligand HIV gp41 isolated functional, folded variants. Assays with several caveolin binding partners demonstrated the successful folding and functionality by a solubilized, full-length caveolin variant selected from the library. This caveolin variant allowed assay of the direct interaction between caveolin and cavin. Clustered along one face of a putative helix, the solubilizing mutations suggest a structural model for the intramembrane domain of caveolin. The approach provides a potentially general method for solubilization and engineering of membrane-associated proteins by phage display.

Genetic Constructor: An Online DNA Design Platform.

PubMed

Bates, Maxwell; Lachoff, Joe; Meech, Duncan; Zulkower, Valentin; Moisy, Anaïs; Luo, Yisha; Tekotte, Hille; Franziska Scheitz, Cornelia Johanna; Khilari, Rupal; Mazzoldi, Florencio; Chandran, Deepak; Groban, Eli

2017-12-15

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.

Peptide ligands targeting integrin alpha3beta1 in non-small cell lung cancer.

PubMed

Lau, Derick; Guo, Linlang; Liu, Ruiwu; Marik, Jan; Lam, Kit

2006-06-01

Lung cancer is one of the most common cancers and is the leading cause of cancer death. We wish to identify peptide ligands for unique cell surface receptors of non-small lung cancer with the hope of developing these ligands as diagnostic and therapeutic agents. Using the method of 'one-bead one-peptide' combinatorial chemistry, a library of random cyclic octapeptides was synthesized on polystyrene beads. This library was used to screen for peptides that promoted attachment of lung adenocarcinoma cells employing a 'cell-growth-on-bead' assay. Consensus peptide sequences of cNGXGXXc were identified. These peptides promoted cell adhesion by targeting integrin alpha3beta1 over-expressed in non-small lung cancer cells. These peptide beads can be applied to capture cancer cells in malignant pleural fluid for purpose of diagnosis of lung cancer.

Discovery of specific ligands for oral squamous carcinoma to develop anti-cancer drug loaded precise targeting nanotherapeutics.

PubMed

Yang, Fan; Liu, Ruiwu; Kramer, Randall; Xiao, Wenwu; Jordan, Richard; Lam, Kit S

2012-12-01

Oral squamous cell carcinoma has a low five-year survival rate, which may be due to late detection and a lack of effective tumor-specific therapies. Using a high throughput drug discovery strategy termed one-bead one-compound combinatorial library, the authors identified six compounds with high binding affinity to different human oral squamous cell carcinoma cell lines but not to normal cells. Current work is under way to develop these ligands to oral squamous cell carcinoma specific imaging probes or therapeutic agents.

Applications of SHAPES screening in drug discovery.

PubMed

Lepre, Christopher A; Peng, Jeffrey; Fejzo, Jasna; Abdul-Manan, Norzehan; Pocas, Jennifer; Jacobs, Marc; Xie, Xiaoling; Moore, Jonathan M

2002-12-01

The SHAPES strategy combines nuclear magnetic resonance (NMR) screening of a library of small drug-like molecules with a variety of complementary methods, such as virtual screening, high throughput enzymatic assays, combinatorial chemistry, X-ray crystallography, and molecular modeling, in a directed search for new medicinal chemistry leads. In the past few years, the SHAPES strategy has found widespread utility in pharmaceutical research. To illustrate a variety of different implementations of the method, we will focus in this review on recent applications of the SHAPES strategy in several drug discovery programs at Vertex Pharmaceuticals.

Stochastic dynamics and combinatorial optimization

NASA Astrophysics Data System (ADS)

Ovchinnikov, Igor V.; Wang, Kang L.

2017-11-01

Natural dynamics is often dominated by sudden nonlinear processes such as neuroavalanches, gamma-ray bursts, solar flares, etc., that exhibit scale-free statistics much in the spirit of the logarithmic Ritcher scale for earthquake magnitudes. On phase diagrams, stochastic dynamical systems (DSs) exhibiting this type of dynamics belong to the finite-width phase (N-phase for brevity) that precedes ordinary chaotic behavior and that is known under such names as noise-induced chaos, self-organized criticality, dynamical complexity, etc. Within the recently proposed supersymmetric theory of stochastic dynamics, the N-phase can be roughly interpreted as the noise-induced “overlap” between integrable and chaotic deterministic dynamics. As a result, the N-phase dynamics inherits the properties of the both. Here, we analyze this unique set of properties and conclude that the N-phase DSs must naturally be the most efficient optimizers: on one hand, N-phase DSs have integrable flows with well-defined attractors that can be associated with candidate solutions and, on the other hand, the noise-induced attractor-to-attractor dynamics in the N-phase is effectively chaotic or aperiodic so that a DS must avoid revisiting solutions/attractors thus accelerating the search for the best solution. Based on this understanding, we propose a method for stochastic dynamical optimization using the N-phase DSs. This method can be viewed as a hybrid of the simulated and chaotic annealing methods. Our proposition can result in a new generation of hardware devices for efficient solution of various search and/or combinatorial optimization problems.

Dynamical principles in neuroscience

NASA Astrophysics Data System (ADS)

Rabinovich, Mikhail I.; Varona, Pablo; Selverston, Allen I.; Abarbanel, Henry D. I.

2006-10-01

Dynamical modeling of neural systems and brain functions has a history of success over the last half century. This includes, for example, the explanation and prediction of some features of neural rhythmic behaviors. Many interesting dynamical models of learning and memory based on physiological experiments have been suggested over the last two decades. Dynamical models even of consciousness now exist. Usually these models and results are based on traditional approaches and paradigms of nonlinear dynamics including dynamical chaos. Neural systems are, however, an unusual subject for nonlinear dynamics for several reasons: (i) Even the simplest neural network, with only a few neurons and synaptic connections, has an enormous number of variables and control parameters. These make neural systems adaptive and flexible, and are critical to their biological function. (ii) In contrast to traditional physical systems described by well-known basic principles, first principles governing the dynamics of neural systems are unknown. (iii) Many different neural systems exhibit similar dynamics despite having different architectures and different levels of complexity. (iv) The network architecture and connection strengths are usually not known in detail and therefore the dynamical analysis must, in some sense, be probabilistic. (v) Since nervous systems are able to organize behavior based on sensory inputs, the dynamical modeling of these systems has to explain the transformation of temporal information into combinatorial or combinatorial-temporal codes, and vice versa, for memory and recognition. In this review these problems are discussed in the context of addressing the stimulating questions: What can neuroscience learn from nonlinear dynamics, and what can nonlinear dynamics learn from neuroscience?

Dynamical principles in neuroscience

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

Rabinovich, Mikhail I.; Varona, Pablo; Selverston, Allen I.

Dynamical modeling of neural systems and brain functions has a history of success over the last half century. This includes, for example, the explanation and prediction of some features of neural rhythmic behaviors. Many interesting dynamical models of learning and memory based on physiological experiments have been suggested over the last two decades. Dynamical models even of consciousness now exist. Usually these models and results are based on traditional approaches and paradigms of nonlinear dynamics including dynamical chaos. Neural systems are, however, an unusual subject for nonlinear dynamics for several reasons: (i) Even the simplest neural network, with only amore » few neurons and synaptic connections, has an enormous number of variables and control parameters. These make neural systems adaptive and flexible, and are critical to their biological function. (ii) In contrast to traditional physical systems described by well-known basic principles, first principles governing the dynamics of neural systems are unknown. (iii) Many different neural systems exhibit similar dynamics despite having different architectures and different levels of complexity. (iv) The network architecture and connection strengths are usually not known in detail and therefore the dynamical analysis must, in some sense, be probabilistic. (v) Since nervous systems are able to organize behavior based on sensory inputs, the dynamical modeling of these systems has to explain the transformation of temporal information into combinatorial or combinatorial-temporal codes, and vice versa, for memory and recognition. In this review these problems are discussed in the context of addressing the stimulating questions: What can neuroscience learn from nonlinear dynamics, and what can nonlinear dynamics learn from neuroscience?.« less

Combinatorial-topological framework for the analysis of global dynamics.

PubMed

Bush, Justin; Gameiro, Marcio; Harker, Shaun; Kokubu, Hiroshi; Mischaikow, Konstantin; Obayashi, Ippei; Pilarczyk, Paweł

2012-12-01

We discuss an algorithmic framework based on efficient graph algorithms and algebraic-topological computational tools. The framework is aimed at automatic computation of a database of global dynamics of a given m-parameter semidynamical system with discrete time on a bounded subset of the n-dimensional phase space. We introduce the mathematical background, which is based upon Conley's topological approach to dynamics, describe the algorithms for the analysis of the dynamics using rectangular grids both in phase space and parameter space, and show two sample applications.

Combinatorial-topological framework for the analysis of global dynamics

NASA Astrophysics Data System (ADS)

Bush, Justin; Gameiro, Marcio; Harker, Shaun; Kokubu, Hiroshi; Mischaikow, Konstantin; Obayashi, Ippei; Pilarczyk, Paweł

2012-12-01

We discuss an algorithmic framework based on efficient graph algorithms and algebraic-topological computational tools. The framework is aimed at automatic computation of a database of global dynamics of a given m-parameter semidynamical system with discrete time on a bounded subset of the n-dimensional phase space. We introduce the mathematical background, which is based upon Conley's topological approach to dynamics, describe the algorithms for the analysis of the dynamics using rectangular grids both in phase space and parameter space, and show two sample applications.

Seven Modeling Perspectives on Teaching and Learning: Some Interrelations and Cognitive Effects

ERIC Educational Resources Information Center

Easley, J. A., Jr.

1977-01-01

The categories of models associated with the seven perspectives are designated as combinatorial models, sampling models, cybernetic models, game models, critical thinking models, ordinary language analysis models, and dynamic structural models. (DAG)

A platform for rapid prototyping of synthetic gene networks in mammalian cells

PubMed Central

Duportet, Xavier; Wroblewska, Liliana; Guye, Patrick; Li, Yinqing; Eyquem, Justin; Rieders, Julianne; Rimchala, Tharathorn; Batt, Gregory; Weiss, Ron

2014-01-01

Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a well-defined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assembling and integrating different functional mammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines. PMID:25378321

Probing a 2-Aminobenzimidazole Library for Binding to RNA Internal Loops via Two-Dimensional Combinatorial Screening

PubMed Central

Velegapudi, Sai Pradeep; Pushechnikov, Alexei; Labuda, Lucas P.; French, Jonathan M.; Disney, Matthew D.

2012-01-01

There are many potential RNA drug targets in bacterial, viral, and the human transcriptomes. However, there are few small molecules that modulate RNA function. This is due, in part, to a lack of fundamental understanding about RNA-ligand interactions including the types of small molecules that bind to RNA structural elements and the RNA structural elements that bind to small molecules. In an effort to better understand RNA-ligand interactions, we diversified the 2-aminobenzimidazole core (2AB) and probed the resulting library for binding to a library of RNA internal loops. We chose the 2AB core for these studies because it is a privileged scaffold for binding RNA based on previous reports. These studies identified that N-methyl pyrrolidine, imidazole, and propylamine diversity elements at the R1 position increase binding to internal loops; variability at the R2 position is well tolerated. The preferred RNA loop space was also determined for five ligands using a statistical approach and identified trends that lead to selective recognition. PMID:22958065

Properties of anodic oxides grown on a hafnium–tantalum–titanium thin film library

PubMed Central

Mardare, Andrei Ionut; Ludwig, Alfred; Savan, Alan; Hassel, Achim Walter

2014-01-01

A ternary thin film combinatorial materials library of the valve metal system Hf–Ta–Ti obtained by co-sputtering was studied. The microstructural and crystallographic analysis of the obtained compositions revealed a crystalline and textured surface, with the exception of compositions with Ta concentration above 48 at.% which are amorphous and show a flat surface. Electrochemical anodization of the composition spread thin films was used for analysing the growth of the mixed surface oxides. Oxide formation factors, obtained from the potentiodynamic anodization curves, as well as the dielectric constants and electrical resistances, obtained from electrochemical impedance spectroscopy, were mapped along two dimensions of the library using a scanning droplet cell microscope. The semiconducting properties of the anodic oxides were mapped using Mott–Schottky analysis. The degree of oxide mixing was analysed qualitatively using x-ray photoelectron spectroscopy depth profiling. A quantitative analysis of the surface oxides was performed and correlated to the as-deposited metal thin film compositions. In the concurrent transport of the three metal cations during oxide growth a clear speed order of Ti > Hf > Ta was proven. PMID:27877648

Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

PubMed Central

Poreba, Marcin; Solberg, Rigmor; Rut, Wioletta; Lunde, Ngoc Nguyen; Kasperkiewicz, Paulina; Snipas, Scott J.; Mihelic, Marko; Turk, Dusan; Turk, Boris; Salvesen, Guy S.; Drag, Marcin

2018-01-01

SUMMARY Legumain (AEP) is a lysosomal cysteine protease that is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP up-regulation is linked to a number of diseases including inflammation, arteriosclerosis and tumorigenesis. Thus legumain is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling. PMID:27478158

Development and validation of novel AAV2 random libraries displaying peptides of diverse lengths and at diverse capsid positions.

PubMed

Naumer, Matthias; Ying, Ying; Michelfelder, Stefan; Reuter, Antje; Trepel, Martin; Müller, Oliver J; Kleinschmidt, Jürgen A

2012-05-01

Libraries based on the insertion of random peptide ligands into the capsid of adeno-associated virus type 2 (AAV2) have been widely used to improve the efficiency and selectivity of the AAV vector system. However, so far only libraries of 7-mer peptide ligands have been inserted at one well-characterized capsid position. Here, we expanded the combinatorial AAV2 display system to a panel of novel AAV libraries, displaying peptides of 5, 7, 12, 19, or 26 amino acids in length at capsid position 588 or displaying 7-mer peptides at position 453, the most prominently exposed region of the viral capsid. Library selections on two unrelated cell types-human coronary artery endothelial cells and rat cardiomyoblasts-revealed the isolation of cell type-characteristic peptides of different lengths mediating strongly improved target-cell transduction, except for the 26-mer peptide ligands. Characterization of vector selectivity by transduction of nontarget cells and comparative gene-transduction analysis using a panel of 44 human tumor cell lines revealed that insertion of different-length peptides allows targeting of distinct cellular receptors for cell entry with similar efficiency, but with different selectivity. The application of such novel AAV2 libraries broadens the spectrum of targetable receptors by capsid-modified AAV vectors and provides the opportunity to choose the best suited targeting ligand for a certain application from a number of different candidates.

Rapid Isolation of Antibody from a Synthetic Human Antibody Library by Repeated Fluorescence-Activated Cell Sorting (FACS)

PubMed Central

Yim, Sung Sun; Bang, Hyun Bae; Kim, Young Hwan; Lee, Yong Jae; Jeong, Gu Min; Jeong, Ki Jun

2014-01-01

Antibodies and their derivatives are the most important agents in therapeutics and diagnostics. Even after the significant progress in the technology for antibody screening from huge libraries, it takes a long time to isolate an antibody, which prevents a prompt action against the spread of a disease. Here, we report a new strategy for isolating desired antibodies from a combinatorial library in one day by repeated fluorescence-activated cell sorting (FACS). First, we constructed a library of synthetic human antibody in which single-chain variable fragment (scFv) was expressed in the periplasm of Escherichia coli. After labeling the cells with fluorescent antigen probes, the highly fluorescent cells were sorted by using a high-speed cell sorter, and these cells were reused without regeneration in the next round of sorting. After repeating this sorting, the positive clones were completely enriched in several hours. Thus, we screened the library against three viral antigens, including the H1N1 influenza virus, Hepatitis B virus, and Foot-and-mouth disease virus. Finally, the potential antibody candidates, which show KD values between 10 and 100 nM against the target antigens, could be successfully isolated even though the library was relatively small (∼106). These results show that repeated FACS screening without regeneration of the sorted cells can be a powerful method when a rapid response to a spreading disease is required. PMID:25303314

Mapping of Drug-like Chemical Universe with Reduced Complexity Molecular Frameworks.

PubMed

Kontijevskis, Aleksejs

2017-04-24

The emergence of the DNA-encoded chemical libraries (DEL) field in the past decade has attracted the attention of the pharmaceutical industry as a powerful mechanism for the discovery of novel drug-like hits for various biological targets. Nuevolution Chemetics technology enables DNA-encoded synthesis of billions of chemically diverse drug-like small molecule compounds, and the efficient screening and optimization of these, facilitating effective identification of drug candidates at an unprecedented speed and scale. Although many approaches have been developed by the cheminformatics community for the analysis and visualization of drug-like chemical space, most of them are restricted to the analysis of a maximum of a few millions of compounds and cannot handle collections of 10 8 -10 12 compounds typical for DELs. To address this big chemical data challenge, we developed the Reduced Complexity Molecular Frameworks (RCMF) methodology as an abstract and very general way of representing chemical structures. By further introducing RCMF descriptors, we constructed a global framework map of drug-like chemical space and demonstrated how chemical space occupied by multi-million-member drug-like Chemetics DNA-encoded libraries and virtual combinatorial libraries with >10 12 members could be analyzed and mapped without a need for library enumeration. We further validate the approach by performing RCMF-based searches in a drug-like chemical universe and mapping Chemetics library selection outputs for LSD1 targets on a global framework chemical space map.

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.

Virtual screening using combinatorial cyclic peptide libraries reveals protein interfaces readily targetable by cyclic peptides.

PubMed

Duffy, Fergal J; O'Donovan, Darragh; Devocelle, Marc; Moran, Niamh; O'Connell, David J; Shields, Denis C

2015-03-23

Protein-protein and protein-peptide interactions are responsible for the vast majority of biological functions in vivo, but targeting these interactions with small molecules has historically been difficult. What is required are efficient combined computational and experimental screening methods to choose among a number of potential protein interfaces worthy of targeting lead macrocyclic compounds for further investigation. To achieve this, we have generated combinatorial 3D virtual libraries of short disulfide-bonded peptides and compared them to pharmacophore models of important protein-protein and protein-peptide structures, including short linear motifs (SLiMs), protein-binding peptides, and turn structures at protein-protein interfaces, built from 3D models available in the Protein Data Bank. We prepared a total of 372 reference pharmacophores, which were matched against 108,659 multiconformer cyclic peptides. After normalization to exclude nonspecific cyclic peptides, the top hits notably are enriched for mimetics of turn structures, including a turn at the interaction surface of human α thrombin, and also feature several protein-binding peptides. The top cyclic peptide hits also cover the critical "hot spot" interaction sites predicted from the interaction crystal structure. We have validated our method by testing cyclic peptides predicted to inhibit thrombin, a key protein in the blood coagulation pathway of important therapeutic interest, identifying a cyclic peptide inhibitor with lead-like activity. We conclude that protein interfaces most readily targetable by cyclic peptides and related macrocyclic drugs may be identified computationally among a set of candidate interfaces, accelerating the choice of interfaces against which lead compounds may be screened.

Systemic combinatorial peptide selection yields a non-canonical iron-mimicry mechanism for targeting tumors in a mouse model of human glioblastoma

PubMed Central

Staquicini, Fernanda I.; Ozawa, Michael G.; Moya, Catherine A.; Driessen, Wouter H.P.; Barbu, E. Magda; Nishimori, Hiroyuki; Soghomonyan, Suren; Flores, Leo G.; Liang, Xiaowen; Paolillo, Vincenzo; Alauddin, Mian M.; Basilion, James P.; Furnari, Frank B.; Bogler, Oliver; Lang, Frederick F.; Aldape, Kenneth D.; Fuller, Gregory N.; Höök, Magnus; Gelovani, Juri G.; Sidman, Richard L.; Cavenee, Webster K.; Pasqualini, Renata; Arap, Wadih

2010-01-01

The management of CNS tumors is limited by the blood-brain barrier (BBB), a vascular interface that restricts the passage of most molecules from the blood into the brain. Here we show that phage particles targeted with certain ligand motifs selected in vivo from a combinatorial peptide library can cross the BBB under normal and pathological conditions. Specifically, we demonstrated that phage clones displaying an iron-mimic peptide were able to target a protein complex of transferrin and transferrin receptor (TfR) through a non-canonical allosteric binding mechanism and that this functional protein complex mediated transport of the corresponding viral particles into the normal mouse brain. We also showed that, in an orthotopic mouse model of human glioblastoma, a combination of TfR overexpression plus extended vascular permeability and ligand retention resulted in remarkable brain tumor targeting of chimeric adeno-associated virus/phage particles displaying the iron-mimic peptide and carrying a gene of interest. As a proof of concept, we delivered the HSV thymidine kinase gene for molecular-genetic imaging and targeted therapy of intracranial xenografted tumors. Finally, we established that these experimental findings might be clinically relevant by determining through human tissue microarrays that many primary astrocytic tumors strongly express TfR. Together, our combinatorial selection system and results may provide a translational avenue for the targeted detection and treatment of brain tumors. PMID:21183793

Soliton cellular automaton associated with Dn(1)-crystal B2,s

NASA Astrophysics Data System (ADS)

Misra, Kailash C.; Wilson, Evan A.

2013-04-01

A solvable vertex model in ferromagnetic regime gives rise to a soliton cellular automaton which is a discrete dynamical system in which site variables take on values in a finite set. We study the scattering of a class of soliton cellular automata associated with the U_q(D_n^{(1)})-perfect crystal B2, s. We calculate the combinatorial R matrix for all elements of B2, s ⊗ B2, 1. In particular, we show that the scattering rule for our soliton cellular automaton can be identified with the combinatorial R matrix for U_q(A_1^{(1)}) oplus U_q(D_{n-2}^{(1)})-crystals.

Synthesis and screening of one-bead-one-compound cyclic peptide libraries.

PubMed

Qian, Ziqing; Upadhyaya, Punit; Pei, Dehua

2015-01-01

Cyclic peptides have been a rich source of biologically active molecules. Herein we present a method for the combinatorial synthesis and screening of large one-bead-one-compound (OBOC) libraries of cyclic peptides against biological targets such as proteins. Up to ten million different cyclic peptides are rapidly synthesized on TentaGel microbeads by the split-and-pool synthesis method and subjected to a multistage screening protocol which includes magnetic sorting, on-bead enzyme-linked and fluorescence-based assays, and in-solution binding analysis of cyclic peptides selectively released from single beads by fluorescence anisotropy. Finally, the most active hit(s) is identified by the partial Edman degradation-mass spectrometry (PED-MS) method. This method allows a single researcher to synthesize and screen up to ten million cyclic peptides and identify the most active ligand(s) in ~1 month, without the time-consuming and expensive hit resynthesis or the use of any special equipment.

Optimum concentration gradient of the electrocatalyst, Nafion® and poly(tetrafluoroethylene) in a membrane-electrode-assembly for enhanced performance of direct methanol fuel cells.

PubMed

Liu, Jing Hua; Jeon, Min Ku; Lee, Ki Rak; Woo, Seong Ihl

2010-12-14

A combinatorial library of membrane-electrode-assemblies (MEAs) which consisted of 27 different compositions was fabricated to optimize the multilayer structure of direct methanol fuel cells. Each spot consisted of three layers of ink and a gradient was generated by employing different concentrations of the three components (Pt catalyst, Nafion® and polytetrafluoroethylene (PTFE)) of each layer. For quick evaluation of the library, a high-throughput optical screening technique was employed for methanol electro-oxidation reaction (MOR) activity. The screening results revealed that gradient layers could lead to higher MOR activity than uniform layers. It was found that the MOR activity was higher when the concentrations of Pt catalyst and Nafion ionomer decreased downward from the top layer to the bottom layer. On the other hand, higher MOR activity was observed when PTFE concentration increased downward from the top to the bottom layer.

Combinatorial Libraries of Arrayable Single-Chain Antibodies

NASA Astrophysics Data System (ADS)

Benhar, Itai

Antibodies that bind their respective targets with high affinity and specificity have proven to be essential reagents for biological research. Antibody phage display has become the leading tool for the rapid isolation of single-chain variable fragment (scFv) antibodies in vitro for research applications, but there is usually a gap between scFv isolation and its application in an array format suitable for high-throughput proteomics. In this chapter, we present our antibody phage display system where antibody isolation and scFv immobilization are facilitated by the design of the phagemid vector used as platform. In our system, the scFvs are fused at their C-termini to a cellulose-binding domain (CBD) and can be immobilized onto cellulose-based filters. This made it possible to develop a unique filter lift screen that allowed the efficient screen for multiple binding specificities, and to directly apply library-derived scFvs in an antibody spotted microarray.

Identification of Neurodegenerative Factors Using Translatome-Regulatory Network Analysis

PubMed Central

Brichta, Lars; Shin, William; Jackson-Lewis, Vernice; Blesa, Javier; Yap, Ee-Lynn; Walker, Zachary; Zhang, Jack; Roussarie, Jean-Pierre; Alvarez, Mariano J.; Califano, Andrea; Przedborski, Serge; Greengard, Paul

2016-01-01

For degenerative disorders of the central nervous system, the major obstacle to therapeutic advancement has been the challenge of identifying the key molecular mechanisms underlying neuronal loss. We developed a combinatorial approach including translational profiling and brain regulatory network analysis to search for key determinants of neuronal survival or death. Following the generation of transgenic mice for cell type-specific profiling of midbrain dopaminergic neurons, we established and compared translatome libraries reflecting the molecular signature of these cells at baseline or under degenerative stress. Analysis of these libraries by interrogating a context-specific brain regulatory network led to the identification of a repertoire of intrinsic upstream regulators that drive the dopaminergic stress response. The altered activity of these regulators was not associated with changes in their expression levels. This strategy can be generalized for the elucidation of novel molecular determinants involved in the degeneration of other classes of neurons. PMID:26214373

Design of a Selective Substrate and Activity Based Probe for Human Neutrophil Serine Protease 4

PubMed Central

Kasperkiewicz, Paulina; Poreba, Marcin; Snipas, Scott J.; Lin, S. Jack; Kirchhofer, Daniel; Salvesen, Guy S.; Drag, Marcin

2015-01-01

Human neutrophil serine protease 4 (NSP4), also known as PRSS57, is a recently discovered fourth member of the neutrophil serine proteases family. Although its biological function is not precisely defined, it is suggested to regulate neutrophil response and innate immune reactions. To create optimal substrates and visualization probes for NSP4 that distinguish it from other NSPs we have employed a Hybrid Combinatorial Substrate Library approach that utilizes natural and unnatural amino acids to explore protease subsite preferences. Library results were validated by synthesizing individual substrates, leading to the identification of an optimal substrate peptide. This substrate was converted to a covalent diphenyl phosphonate probe with an embedded biotin tag. This probe demonstrated high inhibitory activity and stringent specificity and may be suitable for visualizing NSP4 in the background of other NSPs. PMID:26172376

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli.

PubMed

Santos, Christine Nicole S; Xiao, Wenhai; Stephanopoulos, Gregory

2012-08-21

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations.

A combinatorial approach towards the design of nanofibrous scaffolds for chondrogenesis

NASA Astrophysics Data System (ADS)

Ahmed, Maqsood; Ramos, Tiago André Da Silva; Damanik, Febriyani; Quang Le, Bach; Wieringa, Paul; Bennink, Martin; van Blitterswijk, Clemens; de Boer, Jan; Moroni, Lorenzo

2015-10-01

The extracellular matrix (ECM) is a three-dimensional (3D) structure composed of proteinaceous fibres that provide physical and biological cues to direct cell behaviour. Here, we build a library of hybrid collagen-polymer fibrous scaffolds with nanoscale dimensions and screen them for their ability to grow chondrocytes for cartilage repair. Poly(lactic acid) and poly (lactic-co-glycolic acid) at two different monomer ratios (85:15 and 50:50) were incrementally blended with collagen. Physical properties (wettability and stiffness) of the scaffolds were characterized and related to biological performance (proliferation, ECM production, and gene expression) and structure-function relationships were developed. We found that soft scaffolds with an intermediate wettability composed of the highly biodegradable PLGA50:50 and collagen, in two ratios (40:60 and 60:40), were optimal for chondrogenic differentiation of ATDC5 cells as determined by increased ECM production and enhanced cartilage specific gene expression. Long-term cultures indicated a stable phenotype with minimal de-differentiation or hypertrophy. The combinatorial methodology applied herein is a promising approach for the design and development of scaffolds for regenerative medicine.

Rational, combinatorial, and genomic approaches for engineering L-tyrosine production in Escherichia coli

PubMed Central

Santos, Christine Nicole S.; Xiao, Wenhai; Stephanopoulos, Gregory

2012-01-01

Although microbial metabolic engineering has traditionally relied on rational and knowledge-driven techniques, significant improvements in strain performance can be further obtained through the use of combinatorial approaches exploiting phenotypic diversification and screening. Here, we demonstrate the combined use of global transcriptional machinery engineering and a high-throughput L-tyrosine screen towards improving L-tyrosine production in Escherichia coli. This methodology succeeded in generating three strains from two separate mutagenesis libraries (rpoA and rpoD) exhibiting up to a 114% increase in L-tyrosine titer over a rationally engineered parental strain with an already high capacity for production. Subsequent strain characterization through transcriptional analysis and whole genome sequencing allowed complete phenotype reconstruction from well-defined mutations and point to important roles for both the acid stress resistance pathway and the stringent response of E. coli in imparting this phenotype. As such, this study presents one of the first examples in which cell-wide measurements have helped to elucidate the genetic and biochemical underpinnings of an engineered cellular property, leading to the total restoration of metabolite overproduction from specific chromosomal mutations. PMID:22869698

A combinatorial approach towards the design of nanofibrous scaffolds for chondrogenesis.

PubMed

Ahmed, Maqsood; Ramos, Tiago André da Silva; Damanik, Febriyani; Quang Le, Bach; Wieringa, Paul; Bennink, Martin; van Blitterswijk, Clemens; de Boer, Jan; Moroni, Lorenzo

2015-10-07

The extracellular matrix (ECM) is a three-dimensional (3D) structure composed of proteinaceous fibres that provide physical and biological cues to direct cell behaviour. Here, we build a library of hybrid collagen-polymer fibrous scaffolds with nanoscale dimensions and screen them for their ability to grow chondrocytes for cartilage repair. Poly(lactic acid) and poly (lactic-co-glycolic acid) at two different monomer ratios (85:15 and 50:50) were incrementally blended with collagen. Physical properties (wettability and stiffness) of the scaffolds were characterized and related to biological performance (proliferation, ECM production, and gene expression) and structure-function relationships were developed. We found that soft scaffolds with an intermediate wettability composed of the highly biodegradable PLGA50:50 and collagen, in two ratios (40:60 and 60:40), were optimal for chondrogenic differentiation of ATDC5 cells as determined by increased ECM production and enhanced cartilage specific gene expression. Long-term cultures indicated a stable phenotype with minimal de-differentiation or hypertrophy. The combinatorial methodology applied herein is a promising approach for the design and development of scaffolds for regenerative medicine.

Rapid Scanning Structure-Activity Relationships in Combinatorial Data Sets: Identification of Activity Switches

PubMed Central

Medina-Franco, José L.; Edwards, Bruce S.; Pinilla, Clemencia; Appel, Jon R.; Giulianotti, Marc A.; Santos, Radleigh G.; Yongye, Austin B.; Sklar, Larry A.; Houghten, Richard A.

2013-01-01

We present a general approach to describe the structure-activity relationships (SAR) of combinatorial data sets with activity for two biological endpoints with emphasis on the rapid identification of substitutions that have a large impact on activity and selectivity. The approach uses Dual-Activity Difference (DAD) maps that represent a visual and quantitative analysis of all pairwise comparisons of one, two, or more substitutions around a molecular template. Scanning the SAR of data sets using DAD maps allows the visual and quantitative identification of activity switches defined as specific substitutions that have an opposite effect on the activity of the compounds against two targets. The approach also rapidly identifies single- and double-target R-cliffs, i.e., compounds where a single or double substitution around the central scaffold dramatically modifies the activity for one or two targets, respectively. The approach introduced in this report can be applied to any analogue series with two biological activity endpoints. To illustrate the approach, we discuss the SAR of 106 pyrrolidine bis-diketopiperazines tested against two formylpeptide receptors obtained from positional scanning deconvolution methods of mixture-based libraries. PMID:23705689

Combinatorial Approaches for the Identification of Brain Drug Delivery Targets

PubMed Central

Stutz, Charles C.; Zhang, Xiaobin; Shusta, Eric V.

2018-01-01

The blood-brain barrier (BBB) represents a large obstacle for the treatment of central nervous system diseases. Targeting endogenous nutrient transporters that transcytose the BBB is one promising approach to selectively and noninvasively deliver a drug payload to the brain. The main limitations of the currently employed transcytosing receptors are their ubiquitous expression in the peripheral vasculature and the inherent low levels of transcytosis mediated by such systems. In this review, approaches designed to increase the repertoire of transcytosing receptors which can be targeted for the purpose of drug delivery are discussed. In particular, combinatorial protein libraries can be screened on BBB cells in vitro or in vivo to isolate targeting peptides or antibodies that can trigger transcytosis. Once these targeting reagents are discovered, the cognate BBB transcytosis system can be identified using techniques such as expression cloning or immunoprecipitation coupled with mass spectrometry. Continued technological advances in BBB genomics and proteomics, membrane protein manipulation, and in vitro BBB technology promise to further advance the capability to identify and optimize peptides and antibodies capable of mediating drug transport across the BBB. PMID:23789958

Molecular computational elements encode large populations of small objects

NASA Astrophysics Data System (ADS)

Prasanna de Silva, A.; James, Mark R.; McKinney, Bernadine O. F.; Pears, David A.; Weir, Sheenagh M.

2006-10-01

Since the introduction of molecular computation, experimental molecular computational elements have grown to encompass small-scale integration, arithmetic and games, among others. However, the need for a practical application has been pressing. Here we present molecular computational identification (MCID), a demonstration that molecular logic and computation can be applied to a widely relevant issue. Examples of populations that need encoding in the microscopic world are cells in diagnostics or beads in combinatorial chemistry (tags). Taking advantage of the small size (about 1nm) and large `on/off' output ratios of molecular logic gates and using the great variety of logic types, input chemical combinations, switching thresholds and even gate arrays in addition to colours, we produce unique identifiers for members of populations of small polymer beads (about 100μm) used for synthesis of combinatorial libraries. Many millions of distinguishable tags become available. This method should be extensible to far smaller objects, with the only requirement being a `wash and watch' protocol. Our focus on converting molecular science into technology concerning analog sensors, turns to digital logic devices in the present work.

Molecular computational elements encode large populations of small objects.

PubMed

de Silva, A Prasanna; James, Mark R; McKinney, Bernadine O F; Pears, David A; Weir, Sheenagh M

2006-10-01

Since the introduction of molecular computation, experimental molecular computational elements have grown to encompass small-scale integration, arithmetic and games, among others. However, the need for a practical application has been pressing. Here we present molecular computational identification (MCID), a demonstration that molecular logic and computation can be applied to a widely relevant issue. Examples of populations that need encoding in the microscopic world are cells in diagnostics or beads in combinatorial chemistry (tags). Taking advantage of the small size (about 1 nm) and large 'on/off' output ratios of molecular logic gates and using the great variety of logic types, input chemical combinations, switching thresholds and even gate arrays in addition to colours, we produce unique identifiers for members of populations of small polymer beads (about 100 microm) used for synthesis of combinatorial libraries. Many millions of distinguishable tags become available. This method should be extensible to far smaller objects, with the only requirement being a 'wash and watch' protocol. Our focus on converting molecular science into technology concerning analog sensors, turns to digital logic devices in the present work.

Accelerated Combinatorial High Throughput Star Polymer Synthesis via a Rapid One-Pot Sequential Aqueous RAFT (rosa-RAFT) Polymerization Scheme.

PubMed

Cosson, Steffen; Danial, Maarten; Saint-Amans, Julien Rosselgong; Cooper-White, Justin J

2017-04-01

Advanced polymerization methodologies, such as reversible addition-fragmentation transfer (RAFT), allow unprecedented control over star polymer composition, topology, and functionality. However, using RAFT to produce high throughput (HTP) combinatorial star polymer libraries remains, to date, impracticable due to several technical limitations. Herein, the methodology "rapid one-pot sequential aqueous RAFT" or "rosa-RAFT," in which well-defined homo-, copolymer, and mikto-arm star polymers can be prepared in very low to medium reaction volumes (50 µL to 2 mL) via an "arm-first" approach in air within minutes, is reported. Due to the high conversion of a variety of acrylamide/acrylate monomers achieved during each successive short reaction step (each taking 3 min), the requirement for intermediary purification is avoided, drastically facilitating and accelerating the star synthesis process. The presented methodology enables RAFT to be applied to HTP polymeric bio/nanomaterials discovery pipelines, in which hundreds of complex polymeric formulations can be rapidly produced, screened, and scaled up for assessment in a wide range of applications. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure Based Library Design (SBLD) for new 1,4-dihydropyrimidine scaffold as simultaneous COX-1/COX-2 and 5-LOX inhibitors.

PubMed

Lokwani, Deepak; Azad, Rajaram; Sarkate, Aniket; Reddanna, Pallu; Shinde, Devanand

2015-08-01

The various scaffolds containing 1,4-dihydropyrimidine ring were designed by considering the environment of the active site of COX-1/COX-2 and 5-LOX enzymes. The structure-based library design approach, including the focused library design (Virtual Combinatorial Library Design) and virtual screening was used to select the 1,4-dihydropyrimidine scaffold for simultaneous inhibition of both enzyme pathways (COX-1/COX-2 and 5-LOX). The virtual library on each 1,4-dihydropyrimidine scaffold was enumerated in two alternative ways. In first way, the chemical reagents at R groups were filtered by docking of scaffold with single position substitution, that is, only at R1, or R2, or R3, … Rn on COX-2 enzyme using Glide XP docking mode. The structures that do not dock well were removed and the library was enumerated with filtered chemical reagents. In second alternative way, the single position docking stage was bypassed, and the entire library was enumerated using all chemical reagents by docking on the COX-2 enzyme. The entire library of approximately 15,629 compounds obtained from both ways after screening for drug like properties, were further screened for their binding affinity against COX-1 and 5-LOX enzymes using Virtual Screening Workflow. Finally, 142 hits were obtained and divided into two groups based on their binding affinity for COX-1/COX-2 and for both enzyme pathways (COX-1/COX-2 and 5-LOX). The ten molecules were selected, synthesized and evaluated for their COX-1, COX-2 and 5-LOX inhibiting activity. Copyright © 2015 Elsevier Ltd. All rights reserved.

Quantifying and resolving multiple vector transformants in S. cerevisiae plasmid libraries.

PubMed

Scanlon, Thomas C; Gray, Elizabeth C; Griswold, Karl E

2009-11-20

In addition to providing the molecular machinery for transcription and translation, recombinant microbial expression hosts maintain the critical genotype-phenotype link that is essential for high throughput screening and recovery of proteins encoded by plasmid libraries. It is known that Escherichia coli cells can be simultaneously transformed with multiple unique plasmids and thusly complicate recombinant library screening experiments. As a result of their potential to yield misleading results, bacterial multiple vector transformants have been thoroughly characterized in previous model studies. In contrast to bacterial systems, there is little quantitative information available regarding multiple vector transformants in yeast. Saccharomyces cerevisiae is the most widely used eukaryotic platform for cell surface display, combinatorial protein engineering, and other recombinant library screens. In order to characterize the extent and nature of multiple vector transformants in this important host, plasmid-born gene libraries constructed by yeast homologous recombination were analyzed by DNA sequencing. It was found that up to 90% of clones in yeast homologous recombination libraries may be multiple vector transformants, that on average these clones bear four or more unique mutant genes, and that these multiple vector cells persist as a significant proportion of library populations for greater than 24 hours during liquid outgrowth. Both vector concentration and vector to insert ratio influenced the library proportion of multiple vector transformants, but their population frequency was independent of transformation efficiency. Interestingly, the average number of plasmids born by multiple vector transformants did not vary with their library population proportion. These results highlight the potential for multiple vector transformants to dominate yeast libraries constructed by homologous recombination. The previously unrecognized prevalence and persistence of multiply transformed yeast cells have important implications for yeast library screens. The quantitative information described herein should increase awareness of this issue, and the rapid sequencing approach developed for these studies should be widely useful for identifying multiple vector transformants and avoiding complications associated with cells that have acquired more than one unique plasmid.

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach

PubMed Central

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions. PMID:29033978

Integrative Analysis of Transcription Factor Combinatorial Interactions Using a Bayesian Tensor Factorization Approach.

PubMed

Ye, Yusen; Gao, Lin; Zhang, Shihua

2017-01-01

Transcription factors play a key role in transcriptional regulation of genes and determination of cellular identity through combinatorial interactions. However, current studies about combinatorial regulation is deficient due to lack of experimental data in the same cellular environment and extensive existence of data noise. Here, we adopt a Bayesian CANDECOMP/PARAFAC (CP) factorization approach (BCPF) to integrate multiple datasets in a network paradigm for determining precise TF interaction landscapes. In our first application, we apply BCPF to integrate three networks built based on diverse datasets of multiple cell lines from ENCODE respectively to predict a global and precise TF interaction network. This network gives 38 novel TF interactions with distinct biological functions. In our second application, we apply BCPF to seven types of cell type TF regulatory networks and predict seven cell lineage TF interaction networks, respectively. By further exploring the dynamics and modularity of them, we find cell lineage-specific hub TFs participate in cell type or lineage-specific regulation by interacting with non-specific TFs. Furthermore, we illustrate the biological function of hub TFs by taking those of cancer lineage and blood lineage as examples. Taken together, our integrative analysis can reveal more precise and extensive description about human TF combinatorial interactions.

An efficient platform for genetic selection and screening of gene switches in Escherichia coli

PubMed Central

Muranaka, Norihito; Sharma, Vandana; Nomura, Yoko; Yokobayashi, Yohei

2009-01-01

Engineered gene switches and circuits that can sense various biochemical and physical signals, perform computation, and produce predictable outputs are expected to greatly advance our ability to program complex cellular behaviors. However, rational design of gene switches and circuits that function in living cells is challenging due to the complex intracellular milieu. Consequently, most successful designs of gene switches and circuits have relied, to some extent, on high-throughput screening and/or selection from combinatorial libraries of gene switch and circuit variants. In this study, we describe a generic and efficient platform for selection and screening of gene switches and circuits in Escherichia coli from large libraries. The single-gene dual selection marker tetA was translationally fused to green fluorescent protein (gfpuv) via a flexible peptide linker and used as a dual selection and screening marker for laboratory evolution of gene switches. Single-cycle (sequential positive and negative selections) enrichment efficiencies of >7000 were observed in mock selections of model libraries containing functional riboswitches in liquid culture. The technique was applied to optimize various parameters affecting the selection outcome, and to isolate novel thiamine pyrophosphate riboswitches from a complex library. Artificial riboswitches with excellent characteristics were isolated that exhibit up to 58-fold activation as measured by fluorescent reporter gene assay. PMID:19190095

CSBB-ConeExclusion, adapting structure based solution virtual screening to libraries on solid support.

PubMed

Shave, Steven; Auer, Manfred

2013-12-23

Combinatorial chemical libraries produced on solid support offer fast and cost-effective access to a large number of unique compounds. If such libraries are screened directly on-bead, the speed at which chemical space can be explored by chemists is much greater than that addressable using solution based synthesis and screening methods. Solution based screening has a large supporting body of software such as structure-based virtual screening tools which enable the prediction of protein-ligand complexes. Use of these techniques to predict the protein bound complexes of compounds synthesized on solid support neglects to take into account the conjugation site on the small molecule ligand. This may invalidate predicted binding modes, the linker may be clashing with protein atoms. We present CSBB-ConeExclusion, a methodology and computer program which provides a measure of the applicability of solution dockings to solid support. Output is given in the form of statistics for each docking pose, a unique 2D visualization method which can be used to determine applicability at a glance, and automatically generated PyMol scripts allowing visualization of protein atom incursion into a defined exclusion volume. CSBB-ConeExclusion is then exemplarically used to determine the optimum attachment point for a purine library targeting cyclin-dependent kinase 2 CDK2.

Intrinsic information carriers in combinatorial dynamical systems

NASA Astrophysics Data System (ADS)

Harmer, Russ; Danos, Vincent; Feret, Jérôme; Krivine, Jean; Fontana, Walter

2010-09-01

Many proteins are composed of structural and chemical features—"sites" for short—characterized by definite interaction capabilities, such as noncovalent binding or covalent modification of other proteins. This modularity allows for varying degrees of independence, as the behavior of a site might be controlled by the state of some but not all sites of the ambient protein. Independence quickly generates a startling combinatorial complexity that shapes most biological networks, such as mammalian signaling systems, and effectively prevents their study in terms of kinetic equations—unless the complexity is radically trimmed. Yet, if combinatorial complexity is key to the system's behavior, eliminating it will prevent, not facilitate, understanding. A more adequate representation of a combinatorial system is provided by a graph-based framework of rewrite rules where each rule specifies only the information that an interaction mechanism depends on. Unlike reactions, which deal with molecular species, rules deal with patterns, i.e., multisets of molecular species. Although the stochastic dynamics induced by a collection of rules on a mixture of molecules can be simulated, it appears useful to capture the system's average or deterministic behavior by means of differential equations. However, expansion of the rules into kinetic equations at the level of molecular species is not only impractical, but conceptually indefensible. If rules describe bona fide patterns of interaction, molecular species are unlikely to constitute appropriate units of dynamics. Rather, we must seek aggregate variables reflective of the causal structure laid down by the rules. We call these variables "fragments" and the process of identifying them "fragmentation." Ideally, fragments are aspects of the system's microscopic population that the set of rules can actually distinguish on average; in practice, it may only be feasible to identify an approximation to this. Most importantly, fragments are self-consistent descriptors of system dynamics in that their time-evolution is governed by a closed system of kinetic equations. Taken together, fragments are endogenous distinctions that matter for the dynamics of a system, which warrants viewing them as the carriers of information. Although fragments can be thought of as multisets of molecular species (an extensional view), their self-consistency suggests treating them as autonomous aspects cut off from their microscopic realization (an intensional view). Fragmentation is a seeded process that depends on the choice of observables whose dynamics one insists to describe. Different observables can cause distinct fragmentations, in effect altering the set of information carriers that govern the behavior of a system, even though nothing has changed in its microscopic constitution. In this contribution, we present a mathematical specification of fragments, but not an algorithmic implementation. We have described the latter elsewhere in rather technical terms that, although effective, were lacking an embedding into a more general conceptual framework, which we here provide.

Intrinsic information carriers in combinatorial dynamical systems.

PubMed

Harmer, Russ; Danos, Vincent; Feret, Jérôme; Krivine, Jean; Fontana, Walter

2010-09-01

Many proteins are composed of structural and chemical features--"sites" for short--characterized by definite interaction capabilities, such as noncovalent binding or covalent modification of other proteins. This modularity allows for varying degrees of independence, as the behavior of a site might be controlled by the state of some but not all sites of the ambient protein. Independence quickly generates a startling combinatorial complexity that shapes most biological networks, such as mammalian signaling systems, and effectively prevents their study in terms of kinetic equations-unless the complexity is radically trimmed. Yet, if combinatorial complexity is key to the system's behavior, eliminating it will prevent, not facilitate, understanding. A more adequate representation of a combinatorial system is provided by a graph-based framework of rewrite rules where each rule specifies only the information that an interaction mechanism depends on. Unlike reactions, which deal with molecular species, rules deal with patterns, i.e., multisets of molecular species. Although the stochastic dynamics induced by a collection of rules on a mixture of molecules can be simulated, it appears useful to capture the system's average or deterministic behavior by means of differential equations. However, expansion of the rules into kinetic equations at the level of molecular species is not only impractical, but conceptually indefensible. If rules describe bona fide patterns of interaction, molecular species are unlikely to constitute appropriate units of dynamics. Rather, we must seek aggregate variables reflective of the causal structure laid down by the rules. We call these variables "fragments" and the process of identifying them "fragmentation." Ideally, fragments are aspects of the system's microscopic population that the set of rules can actually distinguish on average; in practice, it may only be feasible to identify an approximation to this. Most importantly, fragments are self-consistent descriptors of system dynamics in that their time-evolution is governed by a closed system of kinetic equations. Taken together, fragments are endogenous distinctions that matter for the dynamics of a system, which warrants viewing them as the carriers of information. Although fragments can be thought of as multisets of molecular species (an extensional view), their self-consistency suggests treating them as autonomous aspects cut off from their microscopic realization (an intensional view). Fragmentation is a seeded process that depends on the choice of observables whose dynamics one insists to describe. Different observables can cause distinct fragmentations, in effect altering the set of information carriers that govern the behavior of a system, even though nothing has changed in its microscopic constitution. In this contribution, we present a mathematical specification of fragments, but not an algorithmic implementation. We have described the latter elsewhere in rather technical terms that, although effective, were lacking an embedding into a more general conceptual framework, which we here provide.

Synthetic and natural consensus design for engineering charge within an affibody targeting epidermal growth factor receptor.

PubMed

Case, Brett A; Hackel, Benjamin J

2016-08-01

Protein ligand charge can impact physiological delivery with charge reduction often benefiting performance. Yet neutralizing mutations can be detrimental to protein function. Herein, three approaches are evaluated to introduce charged-to-neutral mutations of three cations and three anions within an affibody engineered to bind epidermal growth factor receptor. These approaches-combinatorial library sorting or consensus design, based on natural homologs or library-sorted mutants-are used to identify mutations with favorable affinity, stability, and recombinant yield. Consensus design, based on 942 affibody homologs, yielded a mutant of modest function (Kd  = 11 ±4 nM, Tm  = 62°C, and yield = 4.0 ± 0.8 mg/L as compared to 5.3 ± 1.7 nM, 71°C, and 3.5 ± 0.3 mg/L for the parental affibody). Extension of consensus design to 10 additional mutants exhibited varied performance including a substantially improved mutant (Kd  = 6.9 ± 1.4 nM, Tm  = 71°C, and 12.7 ± 0.9 mg/L yield). Sorting a homolog-based combinatorial library of 7 × 10(5) mutants generated a distribution of mutants with lower stability and yield, but did identify one strongly binding variant (Kd  = 1.2 ± 0.3 nM, Tm  = 69°C, and 6.0 ± 0.4 mg/L yield). Synthetic consensus design, based on the amino acid distribution in functional library mutants, yielded higher affinities (P = 0.05) with comparable stabilities and yields. The best of four analyzed clones had Kd  = 1.7 ± 0.5 nM, Tm  = 68°C, and 7.0 ± 0.5 mg/L yield. While all three approaches were effective in creating targeted affibodies with six charged-to-neutral mutations, synthetic consensus design proved to be the most robust. Synthetic consensus design provides a valuable tool for ligand engineering, particularly in the context of charge manipulation. Biotechnol. Bioeng. 2016;113: 1628-1638. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Hydroxyapatite-binding peptides for bone growth and inhibition

DOEpatents

Bertozzi, Carolyn R [Berkeley, CA; Song, Jie [Shrewsbury, MA; Lee, Seung-Wuk [Walnut Creek, CA

2011-09-20

Hydroxyapatite (HA)-binding peptides are selected using combinatorial phage library display. Pseudo-repetitive consensus amino acid sequences possessing periodic hydroxyl side chains in every two or three amino acid sequences are obtained. These sequences resemble the (Gly-Pro-Hyp).sub.x repeat of human type I collagen, a major component of extracellular matrices of natural bone. A consistent presence of basic amino acid residues is also observed. The peptides are synthesized by the solid-phase synthetic method and then used for template-driven HA-mineralization. Microscopy reveal that the peptides template the growth of polycrystalline HA crystals .about.40 nm in size.

Lipid-based nanotubes as functional architectures with embedded fluorescence and recognition capabilities.

PubMed

John, George; Mason, Megan; Ajayan, Pulickel M; Dordick, Jonathan S

2004-11-24

A limited combinatorial strategy was used to synthesize a small library of soft lipid-based materials ranging from structurally unordered fibers to highly uniform nanotubes. The latter nanotubes are comprised of a bilayer structure with interdigitated alkyl chains associated through hydrophobic interactions. These tubes contain accessible 2,6-diaminopyridine linkers that can interact with thymidine and related nucleosides through multipoint hydrogen bonding, thereby quenching the intrinsic fluorescence of the aromatic linker. These results are the first example of a systematic strategy to design functional lipid nanotubes with precise structural and functional features.

Versatile microbial surface-display for environmental remediation and biofuels production

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

Wu, Cindy H.; Mulchandani, Ashok; Chen, wilfred

2008-02-14

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.

Human olfactory receptor responses to odorants

PubMed Central

Mainland, Joel D; Li, Yun R; Zhou, Ting; Liu, Wen Ling L; Matsunami, Hiroaki

2015-01-01

Although the human olfactory system is capable of discriminating a vast number of odors, we do not currently understand what chemical features are encoded by olfactory receptors. In large part this is due to a paucity of data in a search space covering the interactions of hundreds of receptors with billions of odorous molecules. Of the approximately 400 intact human odorant receptors, only 10% have a published ligand. Here we used a heterologous luciferase assay to screen 73 odorants against a clone library of 511 human olfactory receptors. This dataset will allow other researchers to interrogate the combinatorial nature of olfactory coding. PMID:25977809

Reproducibility of combinatorial peptide ligand libraries for proteome capture evaluated by selected reaction monitoring.

PubMed

Di Girolamo, Francesco; Righetti, Pier Giorgio; Soste, Martin; Feng, Yuehan; Picotti, Paola

2013-08-26

Systems biology studies require the capability to quantify with high precision proteins spanning a broad range of abundances across multiple samples. However, the broad range of protein expression in cells often precludes the detection of low-abundance proteins. Different sample processing techniques can be applied to increase proteome coverage. Among these, combinatorial (hexa)peptide ligand libraries (CPLLs) bound to solid matrices have been used to specifically capture and detect low-abundance proteins in complex samples. To assess whether CPLL capture can be applied in systems biology studies involving the precise quantitation of proteins across a multitude of samples, we evaluated its performance across the whole range of protein abundances in Saccharomyces cerevisiae. We used selected reaction monitoring assays for a set of target proteins covering a broad abundance range to quantitatively evaluate the precision of the approach and its capability to detect low-abundance proteins. Replicated CPLL-isolates showed an average variability of ~10% in the amount of the isolated proteins. The high reproducibility of the technique was not dependent on the abundance of the protein or the amount of beads used for the capture. However, the protein-to-bead ratio affected the enrichment of specific proteins. We did not observe a normalization effect of CPLL beads on protein abundances. However, CPLLs enriched for and depleted specific sets of proteins and thus changed the abundances of proteins from a whole proteome extract. This allowed the identification of ~400 proteins otherwise undetected in an untreated sample, under the experimental conditions used. CPLL capture is thus a useful tool to increase protein identifications in proteomic experiments, but it should be coupled to the analysis of untreated samples, to maximize proteome coverage. Our data also confirms that CPLL capture is reproducible and can be confidently used in quantitative proteomic experiments. Combinatorial hexapeptide ligand libraries (CPLLs) bound to solid matrices have been proposed to specifically capture and detect low-abundance proteins in complex samples. To assess whether the CPLL capture can be confidently applied in systems biology studies involving the precise quantitation of proteins across a broad range of abundances and a multitude of samples, we evaluated its reproducibility and performance features. Using selected reaction monitoring assays for proteins covering the whole range of abundances we show that the technique is reproducible and compatible with quantitative proteomic studies. However, the protein-to-bead ratio affects the enrichment of specific proteins and CPLLs depleted specific sets of proteins from a whole proteome extract. Our results suggest that CPLL-based analyses should be coupled to the analysis of untreated samples, to maximize proteome coverage. Overall, our data confirms the applicability of CPLLs in systems biology research and guides the correct use of this technique. Copyright © 2013 Elsevier B.V. All rights reserved.

Combinatorial investigation of rare-earth free permanent magnets

NASA Astrophysics Data System (ADS)

Fackler, Sean Wu

The combinatorial high throughput method allows one to rapidly study a large number of samples with systematically changing parameters. We apply this method to study Fe-Co-V alloys as alternatives to rare-earth permanent magnets. Rare-earth permanent magnets derive their unmatched magnetic properties from the hybridization of Fe and Co with the f-orbitals of rare-earth elements, which have strong spin-orbit coupling. It is predicted that Fe and Co may also have strong hybridization with 4d and 5d refractory transition metals with strong spin-orbit coupling. Refractory transition metals like V also have the desirable property of high temperature stability, which is important for permanent magnet applications in traction motors. In this work, we focus on the role of crystal structure, composition, and secondary phases in the origin of competitive permanent magnetic properties of a particular Fe-Co-V alloy. Fe38Co52V10, compositions are known as Vicalloys. Fe-CoV composition spreads were sputtered onto three-inch silicon wafers and patterned into discrete sample pads forming a combinatorial library. We employed highthroughput screening methods using synchrotron X-rays, wavelength dispersive spectroscopy, and magneto-optical Kerr effect (MOKE) to rapidly screen crystal structure, composition, and magnetic properties, respectively. We found that in-plane magnetic coercive fields of our Vicalloy thin films agree with known bulk values (300 G), but found a remarkable eight times increase of the out-of-plane coercive fields (˜2,500 G). To explain this, we measured the switching fields between in-plane and out-of-plane thin film directions which revealed that the Kondorsky model of 180° domain wall reversal was responsible for Vicalloy's enhanced out-of-plane coercive field and possibly its permanent magnetic properties. The Kondorsky model suggests that domain-wall pinning is the origin of Vicalloy's permanent magnetic properties, in contrast to strain, shape, or crystalline anisotropy mechanisms suggested in the literature. We also studied the thickness dependence of an Fe70Co30- V thin film library to consider the unique effects of our thin film libraries which are not found in bulk samples. We present results of data mining of synchrotron X-ray diffraction data using non-negative matrix factorization (NMF). NMF can automatically identify pure crystal phases that make up an unknown phase mixture. We found a strong correlation between magnetic properties and crystal phase quantity using this valuable visualization. In addition to the combinatorial study, this dissertation includes a study of strain controlled properties of magnetic thin films for future applications in random access memories. We investigated the local coupling between dense magnetic stripe domains in transcritical Permalloy (tPy) thin films and ferroelectric domains of BaTiO3 single crystals in a tPy/BaTiO3 heterostructure. Two distinct changes in the magnetic stripe domains of tPy were observed from the magnetic force microscopy images after cooling the heterostructure from above the ferroelectric Curie temperature of BaTiO3 (120°C) to room temperature. First, an abrupt break in the magnetic stripe domain direction was found at the ferroelectric a-c-domain boundaries due to an induced change in in-plane magnetic anisotropy. Second, the magnetic stripe domain period increased when coupled to a ferroelectric a-domain due to a change in out-of-plane magnetic anisotropy. Micromagnetic simulations reveal that local magnetic anisotropy energy from inverse magnetostriction is conserved between in-plane and out-of-plane components.

Engineering on-chip nanoporous gold material libraries via precision photothermal treatment

NASA Astrophysics Data System (ADS)

Chapman, Christopher A. R.; Wang, Ling; Biener, Juergen; Seker, Erkin; Biener, Monika M.; Matthews, Manyalibo J.

2015-12-01

Libraries of nanostructured materials on a single chip are a promising platform for high throughput and combinatorial studies of structure-property relationships in the fields of physics and biology. Nanoporous gold (np-Au), produced by an alloy corrosion process, is a nanostructured material specifically suited for such studies because of its self-similar thermally induced coarsening behavior. However, traditional heat application techniques for the modification of np-Au are bulk processes that cannot be used to generate a library of different pore sizes on a single chip. Here, laser micro-processing offers an attractive solution to this problem by providing a means to apply energy with high spatial and temporal resolution. In the present study we use finite element multiphysics simulations to predict the effects of laser mode (continuous-wave vs. pulsed) and thermal conductivity of the supporting substrate on the local np-Au film temperatures during photothermal annealing. Based on these results we discuss the mechanisms by which the np-Au network is coarsened. Thermal transport simulations predict that continuous-wave mode laser irradiation of np-Au thin films on a silicon substrate supports the widest range of morphologies that can be created through photothermal annealing of np-Au. Using the guidance provided by simulations, we successfully fabricate an on-chip material library consisting of 81 np-Au samples of 9 different morphologies for use in the parallel study of structure-property relationships.Libraries of nanostructured materials on a single chip are a promising platform for high throughput and combinatorial studies of structure-property relationships in the fields of physics and biology. Nanoporous gold (np-Au), produced by an alloy corrosion process, is a nanostructured material specifically suited for such studies because of its self-similar thermally induced coarsening behavior. However, traditional heat application techniques for the modification of np-Au are bulk processes that cannot be used to generate a library of different pore sizes on a single chip. Here, laser micro-processing offers an attractive solution to this problem by providing a means to apply energy with high spatial and temporal resolution. In the present study we use finite element multiphysics simulations to predict the effects of laser mode (continuous-wave vs. pulsed) and thermal conductivity of the supporting substrate on the local np-Au film temperatures during photothermal annealing. Based on these results we discuss the mechanisms by which the np-Au network is coarsened. Thermal transport simulations predict that continuous-wave mode laser irradiation of np-Au thin films on a silicon substrate supports the widest range of morphologies that can be created through photothermal annealing of np-Au. Using the guidance provided by simulations, we successfully fabricate an on-chip material library consisting of 81 np-Au samples of 9 different morphologies for use in the parallel study of structure-property relationships. Electronic supplementary information (ESI) available: Details of sample preparation, fabrication of material libraries, as well as further analysis and supporting scanning electron micrographs can be found in ESI. See DOI: 10.1039/c5nr04580k

DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

PubMed

Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

2014-04-15

DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target proteins and is likely to become a standard tool for pharmaceutical hit discovery, lead expansion, and Chemical Biology research. The introduction of new methodologies for library encoding and for compound synthesis in the presence of DNA is an exciting research field and will crucially contribute to the performance and the propagation of the technology.

Rapid and general profiling of protease specificity by using combinatorial fluorogenic substrate libraries

PubMed Central

Harris, Jennifer L.; Backes, Bradley J.; Leonetti, Francesco; Mahrus, Sami; Ellman, Jonathan A.; Craik, Charles S.

2000-01-01

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of proteases. The substrates contain the fluorogenic leaving group 7-amino-4-carbamoylmethylcoumarin (ACC). Substrates incorporating the ACC leaving group show kinetic profiles comparable to those with the traditionally used 7-amino-4-methylcoumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries by using 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase synthesis techniques. The approximately 3-fold-increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method, we profiled the substrate specificities of a diverse array of proteases, including the serine proteases thrombin, plasmin, factor Xa, urokinase-type plasminogen activator, tissue plasminogen activator, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases to aid in the design of selective substrates and potent inhibitors. PMID:10869434

Separation and characterization of metallosupramolecular libraries by ion mobility mass spectrometry.

PubMed

Li, Xiaopeng; Chan, Yi-Tsu; Casiano-Maldonado, Madalis; Yu, Jing; Carri, Gustavo A; Newkome, George R; Wesdemiotis, Chrys

2011-09-01

The self-assembly of Zn(II) ions and bis(terpyridine) (tpy) ligands carrying 120° or 180° angles between their metal binding sites was utilized to prepare metallosupramolecular libraries with the connectivity. These combinatorial libraries were separated and characterized by ion mobility mass spectrometry (IM MS) and tandem mass spectrometry (MS(2)). The 180°-angle building blocks generate exclusively linear complexes, which were used as standards to determine the architectures of the assemblies resulting from the 120°-angle ligands. The latter ligand geometry promotes the formation of macrocyclic hexamers, but other n-mers with smaller (n = 5) or larger ring sizes (n = 7-9) were identified as minor products, indicating that the angles in the bis(terpyridine) ligand and within the coordinative tpy-Zn(II)-tpy bonds are not as rigid, as previously believed. Macrocyclic and linear isomers were detected in penta- and heptameric assemblies; in the larger octa- and nonameric assemblies, ring-opened conformers with compact and folded geometries were observed in addition to linear extended and cyclic architectures. IM MS(2) experiments provided strong evidence that the macrocycles present in the libraries were already formed in solution, during the self-assembly process, not by dissociation of larger complexes in the gas phase. The IM MS/MS(2) methods provide a means to analyze, based on size and shape (architecture), supramolecular libraries that are not amenable to liquid chromatography, LC-MS, NMR, and/or X-ray techniques.

Combinatorial support vector machines approach for virtual screening of selective multi-target serotonin reuptake inhibitors from large compound libraries.

PubMed

Shi, Z; Ma, X H; Qin, C; Jia, J; Jiang, Y Y; Tan, C Y; Chen, Y Z

2012-02-01

Selective multi-target serotonin reuptake inhibitors enhance antidepressant efficacy. Their discovery can be facilitated by multiple methods, including in silico ones. In this study, we developed and tested an in silico method, combinatorial support vector machines (COMBI-SVMs), for virtual screening (VS) multi-target serotonin reuptake inhibitors of seven target pairs (serotonin transporter paired with noradrenaline transporter, H(3) receptor, 5-HT(1A) receptor, 5-HT(1B) receptor, 5-HT(2C) receptor, melanocortin 4 receptor and neurokinin 1 receptor respectively) from large compound libraries. COMBI-SVMs trained with 917-1951 individual target inhibitors correctly identified 22-83.3% (majority >31.1%) of the 6-216 dual inhibitors collected from literature as independent testing sets. COMBI-SVMs showed moderate to good target selectivity in misclassifying as dual inhibitors 2.2-29.8% (majority <15.4%) of the individual target inhibitors of the same target pair and 0.58-7.1% of the other 6 targets outside the target pair. COMBI-SVMs showed low dual inhibitor false hit rates (0.006-0.056%, 0.042-0.21%, 0.2-4%) in screening 17 million PubChem compounds, 168,000 MDDR compounds, and 7-8181 MDDR compounds similar to the dual inhibitors. Compared with similarity searching, k-NN and PNN methods, COMBI-SVM produced comparable dual inhibitor yields, similar target selectivity, and lower false hit rate in screening 168,000 MDDR compounds. The annotated classes of many COMBI-SVMs identified MDDR virtual hits correlate with the reported effects of their predicted targets. COMBI-SVM is potentially useful for searching selective multi-target agents without explicit knowledge of these agents. Copyright © 2011 Elsevier Inc. All rights reserved.

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

Isolation of a high-affinity Bet v 1-specific IgG-derived ScFv from a subject vaccinated with hypoallergenic Bet v 1 fragments.

PubMed

Gadermaier, E; Marth, K; Lupinek, C; Campana, R; Hofer, G; Blatt, K; Smiljkovic, D; Roder, U; Focke-Tejkl, M; Vrtala, S; Keller, W; Valent, P; Valenta, R; Flicker, S

2018-01-09

Recombinant hypoallergenic allergen derivatives have been used in clinical immunotherapy studies, and clinical efficacy seems to be related to the induction of blocking IgG antibodies recognizing the wild-type allergens. However, so far no treatment-induced IgG antibodies have been characterized. To clone, express, and characterize IgG antibodies induced by vaccination with two hypoallergenic recombinant fragments of the major birch pollen allergen, Bet v 1 in a nonallergic subject. A phage-displayed combinatorial single-chain fragment (ScFv) library was constructed from blood of the immunized subject and screened for Bet v 1-reactive antibody fragments. ScFvs were tested for specificity and cross-reactivity to native Bet v 1 and related pollen and food allergens, and epitope mapping was performed. Germline ancestor genes of the antibody were analyzed with the ImMunoGeneTics (IMGT) database. The affinity to Bet v 1 and cross-reactive allergens was determined by surface plasmon resonance measurements. The ability to inhibit patients' IgE binding to ELISA plate-bound allergens and allergen-induced basophil activation was assessed. A combinatorial ScFv library was obtained from the vaccinated donor after three injections with the Bet v 1 fragments. Despite being almost in germline configuration, ScFv (clone H3-1) reacted with high affinity to native Bet v 1 and homologous allergens, inhibited allergic patients' polyclonal IgE binding to Bet v 1, and partially suppressed allergen-induced basophil activation. Immunization with unfolded hypoallergenic allergen derivatives induces high-affinity antibodies even in nonallergic subjects which recognize the folded wild-type allergens and inhibit polyclonal IgE binding of allergic patients. © 2018 The Authors. Allergy Published by John Wiley & Sons Ltd.

Combinatorial Polymer Matrices Enhance In Vitro Maturation of Human Induced Pluripotent Cell Cell-Derived Cardiomyocytes

PubMed Central

Chun, Young Wook; Balikov, Daniel A.; Feaster, Tromondae K.; Williams, Charles H.; Sheng, Calvin C.; Lee, Jung-Bok; Boire, Timothy C.; Neely, M. Diana; Bellan, Leon M.; Ess, Kevin C.; Bowman, Aaron B.; Sung, Hak-Joon; Hong, Charles C.

2015-01-01

Cardiomyocytes derived from human induced pluripotent stem cells (iPSC-CMs) hold great promise for modeling human heart diseases. However, iPSC-CMs studied to date resemble immature embryonic myocytes and therefore do not adequately recapitulate native adult cardiomyocyte phenotypes. Since extracellular matrix plays an essential role in heart development and maturation in vivo, we sought to develop a synthetic culture matrix that could enhance functional maturation of iPSC-CMs in vitro. In this study, we employed a library of combinatorial polymers comprising of three functional subunits - poly-ε-caprolacton (PCL), polyethylene glycol (PEG), and carboxylated PCL (cPCL) - as synthetic substrates for culturing human iPSC-CMs. Of these, iPSC-CMs cultured on 4%PEG-96%PCL (each % indicates the corresponding molar ratio) exhibit the greatest contractility and mitochondrial function. These functional enhancements are associated with increased expression of cardiac myosin light chain-2v, cardiac troponin I and integrin alpha-7. Importantly, iPSC-CMs cultured on 4%PEG-95%PCL demonstrate troponin I (TnI) isoform switch from the fetal slow skeletal TnI (ssTnI) to the postnatal cardiac TnI (cTnI), the first report of such transition in vitro. Finally, culturing iPSC-CMs on 4%PEG-96%PCL also significantly increased expression of genes encoding intermediate filaments known to transduce integrin-mediated mechanical signals to the myofilaments. In summary, our study demonstrates that synthetic culture matrices engineered from combinatorial polymers can be utilized to promote in vitro maturation of human iPSC-CMs through the engagement of critical matrix-integrin interactions. PMID:26204225

LIBVERSIONINGCOMPILER: An easy-to-use library for dynamic generation and invocation of multiple code versions

NASA Astrophysics Data System (ADS)

Cherubin, S.; Agosta, G.

2018-01-01

We present LIBVERSIONINGCOMPILER, a C++ library designed to support the dynamic generation of multiple versions of the same compute kernel in a HPC scenario. It can be used to provide continuous optimization, code specialization based on the input data or on workload changes, or otherwise to dynamically adjust the application, without the burden of a full dynamic compiler. The library supports multiple underlying compilers but specifically targets the LLVM framework. We also provide examples of use, showing the overhead of the library, and providing guidelines for its efficient use.

Fidelity by design: Yoctoreactor and binder trap enrichment for small-molecule DNA-encoded libraries and drug discovery.

PubMed

Blakskjaer, Peter; Heitner, Tara; Hansen, Nils Jakob Vest

2015-06-01

DNA-encoded small-molecule library (DEL) technology allows vast drug-like small molecule libraries to be efficiently synthesized in a combinatorial fashion and screened in a single tube method for binding, with an assay readout empowered by advances in next generation sequencing technology. This approach has increasingly been applied as a viable technology for the identification of small-molecule modulators to protein targets and as precursors to drugs in the past decade. Several strategies for producing and for screening DELs have been devised by both academic and industrial institutions. This review highlights some of the most significant and recent strategies along with important results. A special focus on the production of high fidelity DEL technologies with the ability to eliminate screening noise and false positives is included: using a DNA junction called the Yoctoreactor, building blocks (BBs) are spatially confined at the center of the junction facilitating both the chemical reaction between BBs and encoding of the synthetic route. A screening method, known as binder trap enrichment, permits DELs to be screened robustly in a homogeneous manner delivering clean data sets and potent hits for even the most challenging targets. Copyright © 2015 Elsevier Ltd. All rights reserved.

Inventory management and reagent supply for automated chemistry.

PubMed

Kuzniar, E

1999-08-01

Developments in automated chemistry have kept pace with developments in HTS such that hundreds of thousands of new compounds can be rapidly synthesized in the belief that the greater the number and diversity of compounds that can be screened, the more successful HTS will be. The increasing use of automation for Multiple Parallel Synthesis (MPS) and the move to automated combinatorial library production is placing an overwhelming burden on the management of reagents. Although automation has improved the efficiency of the processes involved in compound synthesis, the bottleneck has shifted to ordering, collating and preparing reagents for automated chemistry resulting in loss of time, materials and momentum. Major efficiencies have already been made in the area of compound management for high throughput screening. Most of these efficiencies have been achieved with sophisticated library management systems using advanced engineering and data handling for the storage, tracking and retrieval of millions of compounds. The Automation Partnership has already provided many of the top pharmaceutical companies with modular automated storage, preparation and retrieval systems to manage compound libraries for high throughput screening. This article describes how these systems may be implemented to solve the specific problems of inventory management and reagent supply for automated chemistry.

Combinatorial synthesis and in vitro evaluation of a biaryl hydroxyketone library as antivirulence agents against MRSA.

PubMed

Yu, Guanping; Kuo, David; Shoham, Menachem; Viswanathan, Rajesh

2014-02-10

Antibiotic resistance coupled with decreased development of new antibiotics necessitates the search for novel antibacterial agents. Antivirulence agents offer an alternative to conventional antibiotics. In this work, we report on a family of small-molecule antivirulence agents against methicillin-resistant Staphylococcus aureus (MRSA), the most widespread bacterial pathogen. Structure-activity relationship studies led to the development of a concise synthesis of a 148-member biarylhydroxyketone library. An acylation bond-forming process afforded resorcinols (1) and aryloxy acetonitriles (2) as synthons. A Lewis-acid-activated Friedel-Crafts' acylation step involving a nitrile functionality of 2 by ZnCl2, followed by nucleophilic attack by 1 was executed to obtain biaryl hydroxyketones in excellent yields. A large number of products crystallized. This strategy affords a range of biarylhydroxyketones in a single step. This is the first collective synthetic study documenting access to this class of compounds through a single synthetic operation. In vitro efficacy of compounds in this library was evaluated by a rabbit erythrocyte hemolysis assay. The most efficacious compound, 4f-12, inhibits hemolysis by 98.1 ± 0.1% compared to control in the absence of the compound.

Hybrid Self-Adaptive Evolution Strategies Guided by Neighborhood Structures for Combinatorial Optimization Problems.

PubMed

Coelho, V N; Coelho, I M; Souza, M J F; Oliveira, T A; Cota, L P; Haddad, M N; Mladenovic, N; Silva, R C P; Guimarães, F G

2016-01-01

This article presents an Evolution Strategy (ES)--based algorithm, designed to self-adapt its mutation operators, guiding the search into the solution space using a Self-Adaptive Reduced Variable Neighborhood Search procedure. In view of the specific local search operators for each individual, the proposed population-based approach also fits into the context of the Memetic Algorithms. The proposed variant uses the Greedy Randomized Adaptive Search Procedure with different greedy parameters for generating its initial population, providing an interesting exploration-exploitation balance. To validate the proposal, this framework is applied to solve three different [Formula: see text]-Hard combinatorial optimization problems: an Open-Pit-Mining Operational Planning Problem with dynamic allocation of trucks, an Unrelated Parallel Machine Scheduling Problem with Setup Times, and the calibration of a hybrid fuzzy model for Short-Term Load Forecasting. Computational results point out the convergence of the proposed model and highlight its ability in combining the application of move operations from distinct neighborhood structures along the optimization. The results gathered and reported in this article represent a collective evidence of the performance of the method in challenging combinatorial optimization problems from different application domains. The proposed evolution strategy demonstrates an ability of adapting the strength of the mutation disturbance during the generations of its evolution process. The effectiveness of the proposal motivates the application of this novel evolutionary framework for solving other combinatorial optimization problems.

Reliability and mass analysis of dynamic power conversion systems with parallel of standby redundancy

NASA Technical Reports Server (NTRS)

Juhasz, A. J.; Bloomfield, H. S.

1985-01-01

A combinatorial reliability approach is used to identify potential dynamic power conversion systems for space mission applications. A reliability and mass analysis is also performed, specifically for a 100 kWe nuclear Brayton power conversion system with parallel redundancy. Although this study is done for a reactor outlet temperature of 1100K, preliminary system mass estimates are also included for reactor outlet temperatures ranging up to 1500 K.

Efficient search, mapping, and optimization of multi-protein genetic systems in diverse bacteria

PubMed Central

Farasat, Iman; Kushwaha, Manish; Collens, Jason; Easterbrook, Michael; Guido, Matthew; Salis, Howard M

2014-01-01

Developing predictive models of multi-protein genetic systems to understand and optimize their behavior remains a combinatorial challenge, particularly when measurement throughput is limited. We developed a computational approach to build predictive models and identify optimal sequences and expression levels, while circumventing combinatorial explosion. Maximally informative genetic system variants were first designed by the RBS Library Calculator, an algorithm to design sequences for efficiently searching a multi-protein expression space across a > 10,000-fold range with tailored search parameters and well-predicted translation rates. We validated the algorithm's predictions by characterizing 646 genetic system variants, encoded in plasmids and genomes, expressed in six gram-positive and gram-negative bacterial hosts. We then combined the search algorithm with system-level kinetic modeling, requiring the construction and characterization of 73 variants to build a sequence-expression-activity map (SEAMAP) for a biosynthesis pathway. Using model predictions, we designed and characterized 47 additional pathway variants to navigate its activity space, find optimal expression regions with desired activity response curves, and relieve rate-limiting steps in metabolism. Creating sequence-expression-activity maps accelerates the optimization of many protein systems and allows previous measurements to quantitatively inform future designs. PMID:24952589

A combinatorial approach to protein docking with flexible side chains.

PubMed

Althaus, Ernst; Kohlbacher, Oliver; Lenhof, Hans-Peter; Müller, Peter

2002-01-01

Rigid-body docking approaches are not sufficient to predict the structure of a protein complex from the unbound (native) structures of the two proteins. Accounting for side chain flexibility is an important step towards fully flexible protein docking. This work describes an approach that allows conformational flexibility for the side chains while keeping the protein backbone rigid. Starting from candidates created by a rigid-docking algorithm, we demangle the side chains of the docking site, thus creating reasonable approximations of the true complex structure. These structures are ranked with respect to the binding free energy. We present two new techniques for side chain demangling. Both approaches are based on a discrete representation of the side chain conformational space by the use of a rotamer library. This leads to a combinatorial optimization problem. For the solution of this problem, we propose a fast heuristic approach and an exact, albeit slower, method that uses branch-and-cut techniques. As a test set, we use the unbound structures of three proteases and the corresponding protein inhibitors. For each of the examples, the highest-ranking conformation produced was a good approximation of the true complex structure.

PNA-COMBO-FISH: From combinatorial probe design in silico to vitality compatible, specific labelling of gene targets in cell nuclei.

PubMed

Müller, Patrick; Rößler, Jens; Schwarz-Finsterle, Jutta; Schmitt, Eberhard; Hausmann, Michael

2016-07-01

Recently, advantages concerning targeting specificity of PCR constructed oligonucleotide FISH probes in contrast to established FISH probes, e.g. BAC clones, have been demonstrated. These techniques, however, are still using labelling protocols with DNA denaturing steps applying harsh heat treatment with or without further denaturing chemical agents. COMBO-FISH (COMBinatorial Oligonucleotide FISH) allows the design of specific oligonucleotide probe combinations in silico. Thus, being independent from primer libraries or PCR laboratory conditions, the probe sequences extracted by computer sequence data base search can also be synthesized as single stranded PNA-probes (Peptide Nucleic Acid probes) or TINA-DNA (Twisted Intercalating Nucleic Acids). Gene targets can be specifically labelled with at least about 20 probes obtaining visibly background free specimens. By using appropriately designed triplex forming oligonucleotides, the denaturing procedures can completely be omitted. These results reveal a significant step towards oligonucleotide-FISH maintaining the 3d-nanostructure and even the viability of the cell target. The method is demonstrated with the detection of Her2/neu and GRB7 genes, which are indicators in breast cancer diagnosis and therapy. Copyright © 2016. Published by Elsevier Inc.

A 5000-Fold Increase in the Specificity of a Bacterial Phosphotriesterase for Malathion through Combinatorial Active Site Mutagenesis

PubMed Central

Naqvi, Tatheer; Warden, Andrew C.; French, Nigel; Sugrue, Elena; Carr, Paul D.; Jackson, Colin J.; Scott, Colin

2014-01-01

Phosphotriesterases (PTEs) have been isolated from a range of bacterial species, including Agrobcaterium radiobacter (PTEAr), and are efficient enzymes with broad substrate ranges. The turnover rate of PTEAr for the common organophosphorous insecticide malathion is lower than expected based on its physical properties; principally the pka of its leaving group. In this study, we rationalise the turnover rate of PTEAr for malathion using computational docking of the substrate into a high resolution crystal structure of the enzyme, suggesting that malathion is too large for the PTEAr binding pocket. Protein engineering through combinatorial active site saturation testing (CASTing) was then used to increase the rate of malathion turnover. Variants from a CASTing library in which Ser308 and Tyr309 were mutated yielded variants with increased activity towards malathion. The most active PTEAr variant carried Ser308Leu and Tyr309Ala substitutions, which resulted in a ca. 5000-fold increase in k cat/K M for malathion. X-ray crystal structures for the PTEAr Ser308Leu\\Tyr309Ala variant demonstrate that the access to the binding pocket was enhanced by the replacement of the bulky Tyr309 residue with the smaller alanine residue. PMID:24721933

From globally coupled maps to complex-systems biology

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

Kaneko, Kunihiko, E-mail: kaneko@complex.c.u-tokyo.ac.jp

Studies of globally coupled maps, introduced as a network of chaotic dynamics, are briefly reviewed with an emphasis on novel concepts therein, which are universal in high-dimensional dynamical systems. They include clustering of synchronized oscillations, hierarchical clustering, chimera of synchronization and desynchronization, partition complexity, prevalence of Milnor attractors, chaotic itinerancy, and collective chaos. The degrees of freedom necessary for high dimensionality are proposed to equal the number in which the combinatorial exceeds the exponential. Future analysis of high-dimensional dynamical systems with regard to complex-systems biology is briefly discussed.

Discovery of high affinity anti-ricin antibodies by B cell receptor sequencing and by yeast display of combinatorial VH:VL libraries from immunized animals.

PubMed

Wang, Bo; Lee, Chang-Han; Johnson, Erik L; Kluwe, Christien A; Cunningham, Josephine C; Tanno, Hidetaka; Crooks, Richard M; Georgiou, George; Ellington, Andrew D

2016-01-01

Ricin is a toxin that could potentially be used as a bioweapon. We identified anti-ricin A chain antibodies by sequencing the antibody repertoire from immunized mice and by selecting high affinity antibodies using yeast surface display. These methods led to the isolation of multiple antibodies with high (sub-nanomolar) affinity. Interestingly, the antibodies identified by the 2 independent approaches are from the same clonal lineages, indicating for the first time that yeast surface display can identify native antibodies. The new antibodies represent well-characterized reagents for biodefense diagnostics and therapeutics development.

An Interactive Multiobjective Programming Approach to Combinatorial Data Analysis.

ERIC Educational Resources Information Center

Brusco, Michael J.; Stahl, Stephanie

2001-01-01

Describes an interactive procedure for multiobjective asymmetric unidimensional seriation problems that uses a dynamic-programming algorithm to generate partially the efficient set of sequences for small to medium-sized problems and a multioperational heuristic to estimate the efficient set for larger problems. Applies the procedure to an…

The Androgen-Regulated Protease TMPRSS2 Activates aProteolytic Cascade Involving Components of the Tumor Microenvironment and Promotes Prostate Cancer Metastasis

PubMed Central

Lucas, Jared M.; Heinlein, Cynthia; Kim, Tom; Hernandez, Susana A.; Malik, Muzdah S.; True, Lawrence D.; Morrissey, Colm; Corey, Eva; Montgomery, Bruce; Mostaghel, Elahe; Clegg, Nigel; Coleman, Ilsa; Brown, Christopher M.; Schneider, Eric L.; Craik, Charles; Simon, Julian; Bedalov, Tony; Nelson, Peter S.

2014-01-01

TMPRSS2 is an androgen-regulated cell surface serine protease expressed predominantly in prostate epithelium. TMPRSS2 is expressed highly in localized high-grade prostate cancers and in the majority of human prostate cancer metastasis. Through the generation of mouse models with a targeted deletion of Tmprss2, we demonstrate that the activity of this protease regulates cancer cell invasion and metastasis to distant organs. By screening combinatorial peptide libraries we identified a spectrum of TMPRSS2 substrates that include pro-hepatocyte growth factor (HGF). HGF activated by TMPRSS2 promoted c-Met receptor tyrosine kinase signaling, and initiated a pro-invasive EMT phenotype. Chemical library screens identified a potent bioavailable TMPRSS2 inhibitor that suppressed prostate cancer metastasis in vivo. Together, these findings provide a mechanistic link between androgen-regulated signaling programs and prostate cancer metastasis that operate via context-dependent interactions with extracellular constituents of the tumor microenvironment. PMID:25122198

Evolving artificial metalloenzymes via random mutagenesis

NASA Astrophysics Data System (ADS)

Yang, Hao; Swartz, Alan M.; Park, Hyun June; Srivastava, Poonam; Ellis-Guardiola, Ken; Upp, David M.; Lee, Gihoon; Belsare, Ketaki; Gu, Yifan; Zhang, Chen; Moellering, Raymond E.; Lewis, Jared C.

2018-03-01

Random mutagenesis has the potential to optimize the efficiency and selectivity of protein catalysts without requiring detailed knowledge of protein structure; however, introducing synthetic metal cofactors complicates the expression and screening of enzyme libraries, and activity arising from free cofactor must be eliminated. Here we report an efficient platform to create and screen libraries of artificial metalloenzymes (ArMs) via random mutagenesis, which we use to evolve highly selective dirhodium cyclopropanases. Error-prone PCR and combinatorial codon mutagenesis enabled multiplexed analysis of random mutations, including at sites distal to the putative ArM active site that are difficult to identify using targeted mutagenesis approaches. Variants that exhibited significantly improved selectivity for each of the cyclopropane product enantiomers were identified, and higher activity than previously reported ArM cyclopropanases obtained via targeted mutagenesis was also observed. This improved selectivity carried over to other dirhodium-catalysed transformations, including N-H, S-H and Si-H insertion, demonstrating that ArMs evolved for one reaction can serve as starting points to evolve catalysts for others.

Immune cell screening of a nanoparticle library improves atherosclerosis therapy

PubMed Central

Baxter, Samantha; Menon, Arjun; Alaarg, Amr; Sanchez-Gaytan, Brenda L.; Fay, Francois; Zhao, Yiming; Ouimet, Mireille; Braza, Mounia S.; Longo, Valerie A.; Abdel-Atti, Dalya; Duivenvoorden, Raphael; Calcagno, Claudia; Storm, Gert; Tsimikas, Sotirios; Moore, Kathryn J.; Swirski, Filip K.; Nahrendorf, Matthias; Fisher, Edward A.; Pérez-Medina, Carlos; Fayad, Zahi A.; Reiner, Thomas; Mulder, Willem J. M.

2016-01-01

Immunological complexity in atherosclerosis warrants targeted treatment of specific inflammatory cells that aggravate the disease. With the initiation of large phase III trials investigating immunomodulatory drugs for atherosclerosis, cardiovascular disease treatment enters a new era. We here propose a radically different approach: implementing and evaluating in vivo a combinatorial library of nanoparticles with distinct physiochemical properties and differential immune cell specificities. The library’s nanoparticles are based on endogenous high-density lipoprotein, which can preferentially deliver therapeutic compounds to pathological macrophages in atherosclerosis. Using the apolipoprotein E-deficient (Apoe−/−) mouse model of atherosclerosis, we quantitatively evaluated the library’s immune cell specificity by combining immunological techniques and in vivo positron emission tomography imaging. Based on this screen, we formulated a liver X receptor agonist (GW3965) and abolished its liver toxicity while still preserving its therapeutic function. Screening the immune cell specificity of nanoparticles can be used to develop tailored therapies for atherosclerosis and other inflammatory diseases. PMID:27791119

Toward automated biochemotype annotation for large compound libraries.

PubMed

Chen, Xian; Liang, Yizeng; Xu, Jun

2006-08-01

Combinatorial chemistry allows scientists to probe large synthetically accessible chemical space. However, identifying the sub-space which is selectively associated with an interested biological target, is crucial to drug discovery and life sciences. This paper describes a process to automatically annotate biochemotypes of compounds in a library and thus to identify bioactivity related chemotypes (biochemotypes) from a large library of compounds. The process consists of two steps: (1) predicting all possible bioactivities for each compound in a library, and (2) deriving possible biochemotypes based on predictions. The Prediction of Activity Spectra for Substances program (PASS) was used in the first step. In second step, structural similarity and scaffold-hopping technologies are employed. These technologies are used to derive biochemotypes from bioactivity predictions and the corresponding annotated biochemotypes from MDL Drug Data Report (MDDR) database. About a one million (982,889) commercially available compound library (CACL) has been tested using this process. This paper demonstrates the feasibility of automatically annotating biochemotypes for large libraries of compounds. Nevertheless, some issues need to be considered in order to improve the process. First, the prediction accuracy of PASS program has no significant correlation with the number of compounds in a training set. Larger training sets do not necessarily increase the maximal error of prediction (MEP), nor do they increase the hit structural diversity. Smaller training sets do not necessarily decrease MEP, nor do they decrease the hit structural diversity. Second, the success of systematic bioactivity prediction relies on modeling, training data, and the definition of bioactivities (biochemotype ontology). Unfortunately, the biochemotype ontology was not well developed in the PASS program. Consequently, "ill-defined" bioactivities can reduce the quality of predictions. This paper suggests the ways in which the systematic bioactivities prediction program should be improved.

High-throughput resistivity apparatus for thin-film combinatorial libraries

NASA Astrophysics Data System (ADS)

Hewitt, K. C.; Casey, P. A.; Sanderson, R. J.; White, M. A.; Sun, R.

2005-09-01

An apparatus, capable of measuring the dc resistance versus temperature of a 49-member library prepared by thin-film deposition techniques was designed and tested. The library is deposited by dc magnetron sputtering onto 10.16cm×10.16cm alumina substrates on which are placed aluminum masks consisting of 8mm diam holes cut on a 7×7 grid, the center-to-center spacing being 10.15mm. Electrical contact to the library is made in a standard van der Pauw geometry using 196 spring-loaded, gold-coated pins, four pins for each member of the library. The temperature is controlled using a helium refrigerator in combination with a liquid-nitrogen radiation shield that greatly reduces radiative heating of the sample stage. With the radiation shield, the cold finger is able to sustain a minimum temperature of 7K and the sample stage a minimum temperature of 27K. The temperature (27-291K) dependent dc resistivity of a thin-film silver library of varying thickness (48-639nm) is presented to highlight the capabilities of the apparatus. The thickness dependence of both the resistivity and the temperature coefficient of resistivity are quantitatively consistent with the literature. For thicknesses greater than about 100nm, the room-temperature resistivity (3.4μΩcm) are consistent with Matthiessen's rule for 1%-2% impurity content, and the temperature coefficient of resistivity is consistent with the bulk value. For thicknesses less than 100nm, an increase in resistivity by a factor of 8 is found, which may be due to surface and boundary scattering effects; a corresponding increase in the temperature coefficient of resistivity is consistent with a concomitant decrease in the magnitude of the elastic constants and surface scattering effects.

Effect of the Implicit Combinatorial Model on Combinatorial Reasoning in Secondary School Pupils.

ERIC Educational Resources Information Center

Batanero, Carmen; And Others

1997-01-01

Elementary combinatorial problems may be classified into three different combinatorial models: (1) selection; (2) partition; and (3) distribution. The main goal of this research was to determine the effect of the implicit combinatorial model on pupils' combinatorial reasoning before and after instruction. Gives an analysis of variance of the…

Stress-Driven Selection of Novel Phenotypes

NASA Technical Reports Server (NTRS)

Fox, George E.; Stepaov, Victor G.; Liu, Yamei

2011-01-01

A process has been developed that can confer novel properties, such as metal resistance, to a host bacterium. This same process can also be used to produce RNAs and peptides that have novel properties, such as the ability to bind particular compounds. It is inherent in the method that the peptide or RNA will behave as expected in the target organism. Plasmid-born mini-gene libraries coding for either a population of combinatorial peptides or stable, artificial RNAs carrying random inserts are produced. These libraries, which have no bias towards any biological function, are used to transform the organism of interest and to serve as an initial source of genetic variation for stress-driven evolution. The transformed bacteria are propagated under selective pressure in order to obtain variants with the desired properties. The process is highly distinct from in vitro methods because the variants are selected in the context of the cell while it is experiencing stress. Hence, the selected peptide or RNA will, by definition, work as expected in the target cell as the cell adapts to its presence during the selection process. Once the novel gene, which produces the sought phenotype, is obtained, it can be transferred to the main genome to increase the genetic stability in the organism. Alternatively, the cell line can be used to produce novel RNAs or peptides with selectable properties in large quantity for separate purposes. The system allows for easy, large-scale purification of the RNAs or peptide products. The process has been reduced to practice by imposing sub-inhibitory concentrations of NiCl2 on cells of the bacterium Escherichia coli that were transformed separately with the peptide library and RNA library. The evolved resistant clones were isolated, and sequences of the selected mini-gene variants were established. Clones resistant to NiCl2 were found to carry identical plasmid variants with a functional mini-gene that specifically conferred significant nickel tolerance on the host cells. Sequencing of the selected mini-gene revealed a propensity of the encoded peptide to bind transient metal ions. Expression of the mini-gene markedly improved growth parameters of the evolved clones at sub-inhibitory concentrations of NiCl2 while being slightly detrimental in the absence of stress. Similar results have been obtained with the RNA libraries. Overall, the results demonstrate a very natural outcome of the selection experiments in which the mini-genes were expected to be either successfully integrated into bacterial genetic networks, or rejected depending upon their effect on host fitness. This described approach can be useful as a laboratory model to study the dynamics of bacterial adaptive evolution on the molecular level. It can also provide a strategy for screening expressed DNA libraries in search of novel genes with desirable properties.

Selection of full-length IgGs by tandem display on filamentous phage particles and Escherichia coli fluorescence-activated cell sorting screening.

PubMed

Mazor, Yariv; Van Blarcom, Thomas; Carroll, Sean; Georgiou, George

2010-05-01

Phage display of antibody libraries is a powerful tool for antibody discovery and evolution. Recombinant antibodies have been displayed on phage particles as scFvs or Fabs, and more recently as bivalent F(ab')(2). We recently developed a technology (E-clonal) for screening of combinatorial IgG libraries using bacterial periplasmic display and selection by fluorescence-activated cell sorting (FACS) [Mazor Y et al. (2007) Nat Biotechnol 25, 563-565]. Although, as a single-cell analysis technique, FACS is very powerful, especially for the isolation of high-affinity binders, even with state of the art instrumentation the screening of libraries with diversity > 10(8) is technically challenging. We report here a system that takes advantage of display of full-length IgGs on filamentous phage particles as a prescreening step to reduce library size and enable subsequent rounds of FACS screening in Escherichia coli. For the establishment of an IgG phage display system, we utilized phagemid-encoded IgG with the fUSE5-ZZ phage as a helper phage. These phage particles display the Fc-binding ZZ protein on all copies of the phage p3 coat protein, and are exploited as both helper phages and anchoring surfaces for the soluble IgG. We demonstrate that tandem phage selection followed by FACS allows the selection of a highly diversified profile of binders from antibody libraries without undersampling, and at the same time capitalizes on the advantages of FACS for real-time monitoring and optimization of the screening process.

Biosynthesis of the Cyclotide MCoTI-II using an Engineered Intein

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

Cantor, J; Camarero, J A

2006-08-15

Cyclotides are an emerging family of naturally occurring circular mini-proteins ({approx}30-40 amino acids) characterized by six conserved Cys residues (forming 3 disulfide bridges) that create a topologically unique structure designated as a cyclic cysteine knot (CCK). The cysteine knot motif, which is embedded within the macrocylic backbone, is described as two disulfide bridges that form a ring that is penetrated by the third disulfide bridge. The cyclic backbone and CCK motif together confer cyclotides with a remarkable stability and resistance to proteolytic, chemical, and thermal degradation. Further, cyclotides are functionally diverse and display a wide range of functions including uterotonicmore » activity, trypsin inhibition, cytotoxicity, neurotensin binding, anti-HIV, antimicrobial, and insecticidal activity. Together, these characteristics make cyclotides attractive candidates for both drug design and agricultural applications, both in their native forms and as molecular scaffolds for the incorporation of novel bioactivities. [1] The ability to manipulate production of cyclotides within biological systems is critical for mutagenesis studies, production of grafted products, and the mass production of cyclotides with novel activities. My adviser's hope is to achieve this capability by employing recombinant DNA expression techniques to generate large combinatorial libraries of cyclotides. The advantage in creating a biosynthetic library (containing {approx}10{sup 6}-10{sup 10} members/library vs. chemically based libraries with typical values ranging from {approx}10{sup 3}-10{sup 5} members/library) is that it can be lead to the in vivo application of biological screening and selection methodologies based on a specific clone's ability to affect certain cellular processes.« less

Design and Characterization of a Human Monoclonal Antibody that Modulates Mutant Connexin 26 Hemichannels Implicated in Deafness and Skin Disorders

PubMed Central

Xu, Liang; Carrer, Andrea; Zonta, Francesco; Qu, Zhihu; Ma, Peixiang; Li, Sheng; Ceriani, Federico; Buratto, Damiano; Crispino, Giulia; Zorzi, Veronica; Ziraldo, Gaia; Bruno, Francesca; Nardin, Chiara; Peres, Chiara; Mazzarda, Flavia; Salvatore, Anna M.; Raspa, Marcello; Scavizzi, Ferdinando; Chu, Youjun; Xie, Sichun; Yang, Xuemei; Liao, Jun; Liu, Xiao; Wang, Wei; Wang, Shanshan; Yang, Guang; Lerner, Richard A.; Mammano, Fabio

2017-01-01

Background: Mutations leading to changes in properties, regulation, or expression of connexin-made channels have been implicated in 28 distinct human hereditary diseases. Eight of these result from variants of connexin 26 (Cx26), a protein critically involved in cell-cell signaling in the inner ear and skin. Lack of non-toxic drugs with defined mechanisms of action poses a serious obstacle to therapeutic interventions for diseases caused by mutant connexins. In particular, molecules that specifically modulate connexin hemichannel function without affecting gap junction channels are considered of primary importance for the study of connexin hemichannel role in physiological as well as pathological conditions. Monoclonal antibodies developed in the last three decades have become the most important class of therapeutic biologicals. Recombinant methods permit rapid selection and improvement of monoclonal antibodies from libraries with large diversity. Methods: By screening a combinatorial library of human single-chain fragment variable (scFv) antibodies expressed in phage, we identified a candidate that binds an extracellular epitope of Cx26. We characterized antibody action using a variety of biochemical and biophysical assays in HeLa cells, organotypic cultures of mouse cochlea and human keratinocyte-derived cells. Results: We determined that the antibody is a remarkably efficient, non-toxic, and completely reversible inhibitor of hemichannels formed by connexin 26 and does not affect direct cell-cell communication via gap junction channels. Importantly, we also demonstrate that the antibody efficiently inhibits hyperative mutant Cx26 hemichannels implicated in autosomal dominant non-syndromic hearing impairment accompanied by keratitis and hystrix-like ichthyosis-deafness (KID/HID) syndrome. We solved the crystal structure of the antibody, identified residues that are critical for binding and used molecular dynamics to uncover its mechanism of action. Conclusions: Although further studies will be necessary to validate the effect of the antibody in vivo, the methodology described here can be extended to select antibodies against hemichannels composed by other connexin isoforms and, consequently, to target other pathologies associated with hyperactive hemichannels. Our study highlights the potential of this approach and identifies connexins as therapeutic targets addressable by screening phage display libraries expressing human randomized antibodies. PMID:29018324

Dynamics of embedded curves by doubly-nonlocal reaction-diffusion systems

NASA Astrophysics Data System (ADS)

von Brecht, James H.; Blair, Ryan

2017-11-01

We study a class of nonlocal, energy-driven dynamical models that govern the motion of closed, embedded curves from both an energetic and dynamical perspective. Our energetic results provide a variety of ways to understand physically motivated energetic models in terms of more classical, combinatorial measures of complexity for embedded curves. This line of investigation culminates in a family of complexity bounds that relate a rather broad class of models to a generalized, or weighted, variant of the crossing number. Our dynamic results include global well-posedness of the associated partial differential equations, regularity of equilibria for these flows as well as a more detailed investigation of dynamics near such equilibria. Finally, we explore a few global dynamical properties of these models numerically.

Analysis of tasks for dynamic man/machine load balancing in advanced helicopters

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

Jorgensen, C.C.

1987-10-01

This report considers task allocation requirements imposed by advanced helicopter designs incorporating mixes of human pilots and intelligent machines. Specifically, it develops an analogy between load balancing using distributed non-homogeneous multiprocessors and human team functions. A taxonomy is presented which can be used to identify task combinations likely to cause overload for dynamic scheduling and process allocation mechanisms. Designer criteria are given for function decomposition, separation of control from data, and communication handling for dynamic tasks. Possible effects of n-p complete scheduling problems are noted and a class of combinatorial optimization methods are examined.

BASIC: A Simple and Accurate Modular DNA Assembly Method.

PubMed

Storch, Marko; Casini, Arturo; Mackrow, Ben; Ellis, Tom; Baldwin, Geoff S

2017-01-01

Biopart Assembly Standard for Idempotent Cloning (BASIC) is a simple, accurate, and robust DNA assembly method. The method is based on linker-mediated DNA assembly and provides highly accurate DNA assembly with 99 % correct assemblies for four parts and 90 % correct assemblies for seven parts [1]. The BASIC standard defines a single entry vector for all parts flanked by the same prefix and suffix sequences and its idempotent nature means that the assembled construct is returned in the same format. Once a part has been adapted into the BASIC format it can be placed at any position within a BASIC assembly without the need for reformatting. This allows laboratories to grow comprehensive and universal part libraries and to share them efficiently. The modularity within the BASIC framework is further extended by the possibility of encoding ribosomal binding sites (RBS) and peptide linker sequences directly on the linkers used for assembly. This makes BASIC a highly versatile library construction method for combinatorial part assembly including the construction of promoter, RBS, gene variant, and protein-tag libraries. In comparison with other DNA assembly standards and methods, BASIC offers a simple robust protocol; it relies on a single entry vector, provides for easy hierarchical assembly, and is highly accurate for up to seven parts per assembly round [2].

Hit discovery of 4-amino-N-(4-(3-(trifluoromethyl)phenoxy)pyrimidin-5-yl)benzamide: A novel EGFR inhibitor from a designed small library.

PubMed

Elkamhawy, Ahmed; Paik, Sora; Hassan, Ahmed H E; Lee, Yong Sup; Roh, Eun Joo

2017-12-01

Searching for hit compounds within the huge chemical space resembles the attempt to find a needle in a haystack. Cheminformatics-guided selection of few representative molecules of a rationally designed virtual combinatorial library is a powerful tool to confront this challenge, speed up hit identification and cut off costs. Herein, this approach has been applied to identify hit compounds with novel scaffolds able to inhibit EGFR kinase. From a generated virtual library, six 4-aryloxy-5-aminopyrimidine scaffold-derived compounds were selected, synthesized and evaluated as hit EGFR inhibitors. 4-Aryloxy-5-benzamidopyrimidines inhibited EGFR with IC 50 1.05-5.37 μM. Cell-based assay of the most potent EGFR inhibitor hit (10ac) confirmed its cytotoxicity against different cancerous cells. In spite of no EGFR, HER2 or VEGFR1 inhibition was elicited by 4-aryloxy-5-(thio)ureidopyrimidine derivatives, cell-based evaluation suggested them as antiproliferative hits acting by other mechanism(s). Molecular docking study provided a plausible explanation of incapability of 4-aryloxy-5-(thio)ureidopyrimidines to inhibit EGFR and suggested a reasonable binding mode of 4-aryloxy-5-benzamidopyrimidines which provides a basis to develop more optimized ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

Formation of Nitrogenase NifDK Tetramers in the Mitochondria of Saccharomyces cerevisiae

PubMed Central

2017-01-01

Transferring the prokaryotic enzyme nitrogenase into a eukaryotic host with the final aim of developing N2 fixing cereal crops would revolutionize agricultural systems worldwide. Targeting it to mitochondria has potential advantages because of the organelle’s high O2 consumption and the presence of bacterial-type iron–sulfur cluster biosynthetic machinery. In this study, we constructed 96 strains of Saccharomyces cerevisiae in which transcriptional units comprising nine Azotobacter vinelandii nif genes (nifHDKUSMBEN) were integrated into the genome. Two combinatorial libraries of nif gene clusters were constructed: a library of mitochondrial leading sequences consisting of 24 clusters within four subsets of nif gene expression strength, and an expression library of 72 clusters with fixed mitochondrial leading sequences and nif expression levels assigned according to factorial design. In total, 29 promoters and 18 terminators were combined to adjust nif gene expression levels. Expression and mitochondrial targeting was confirmed at the protein level as immunoblot analysis showed that Nif proteins could be efficiently accumulated in mitochondria. NifDK tetramer formation, an essential step of nitrogenase assembly, was experimentally proven both in cell-free extracts and in purified NifDK preparations. This work represents a first step toward obtaining functional nitrogenase in the mitochondria of a eukaryotic cell. PMID:28221768

Automatic design of synthetic gene circuits through mixed integer non-linear programming.

PubMed

Huynh, Linh; Kececioglu, John; Köppe, Matthias; Tagkopoulos, Ilias

2012-01-01

Automatic design of synthetic gene circuits poses a significant challenge to synthetic biology, primarily due to the complexity of biological systems, and the lack of rigorous optimization methods that can cope with the combinatorial explosion as the number of biological parts increases. Current optimization methods for synthetic gene design rely on heuristic algorithms that are usually not deterministic, deliver sub-optimal solutions, and provide no guaranties on convergence or error bounds. Here, we introduce an optimization framework for the problem of part selection in synthetic gene circuits that is based on mixed integer non-linear programming (MINLP), which is a deterministic method that finds the globally optimal solution and guarantees convergence in finite time. Given a synthetic gene circuit, a library of characterized parts, and user-defined constraints, our method can find the optimal selection of parts that satisfy the constraints and best approximates the objective function given by the user. We evaluated the proposed method in the design of three synthetic circuits (a toggle switch, a transcriptional cascade, and a band detector), with both experimentally constructed and synthetic promoter libraries. Scalability and robustness analysis shows that the proposed framework scales well with the library size and the solution space. The work described here is a step towards a unifying, realistic framework for the automated design of biological circuits.

A Barcoding Strategy Enabling Higher-Throughput Library Screening by Microscopy.

PubMed

Chen, Robert; Rishi, Harneet S; Potapov, Vladimir; Yamada, Masaki R; Yeh, Vincent J; Chow, Thomas; Cheung, Celia L; Jones, Austin T; Johnson, Terry D; Keating, Amy E; DeLoache, William C; Dueber, John E

2015-11-20

Dramatic progress has been made in the design and build phases of the design-build-test cycle for engineering cells. However, the test phase usually limits throughput, as many outputs of interest are not amenable to rapid analytical measurements. For example, phenotypes such as motility, morphology, and subcellular localization can be readily measured by microscopy, but analysis of these phenotypes is notoriously slow. To increase throughput, we developed microscopy-readable barcodes (MiCodes) composed of fluorescent proteins targeted to discernible organelles. In this system, a unique barcode can be genetically linked to each library member, making possible the parallel analysis of phenotypes of interest via microscopy. As a first demonstration, we MiCoded a set of synthetic coiled-coil leucine zipper proteins to allow an 8 × 8 matrix to be tested for specific interactions in micrographs consisting of mixed populations of cells. A novel microscopy-readable two-hybrid fluorescence localization assay for probing candidate interactions in the cytosol was also developed using a bait protein targeted to the peroxisome and a prey protein tagged with a fluorescent protein. This work introduces a generalizable, scalable platform for making microscopy amenable to higher-throughput library screening experiments, thereby coupling the power of imaging with the utility of combinatorial search paradigms.

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins

PubMed Central

Arvola, René M.

2017-01-01

ABSTRACT Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control. PMID:28318367

Combinatorial control of messenger RNAs by Pumilio, Nanos and Brain Tumor Proteins.

PubMed

Arvola, René M; Weidmann, Chase A; Tanaka Hall, Traci M; Goldstrohm, Aaron C

2017-11-02

Eukaryotes possess a vast array of RNA-binding proteins (RBPs) that affect mRNAs in diverse ways to control protein expression. Combinatorial regulation of mRNAs by RBPs is emerging as the rule. No example illustrates this as vividly as the partnership of 3 Drosophila RBPs, Pumilio, Nanos and Brain Tumor, which have overlapping functions in development, stem cell maintenance and differentiation, fertility and neurologic processes. Here we synthesize 30 y of research with new insights into their molecular functions and mechanisms of action. First, we provide an overview of the key properties of each RBP. Next, we present a detailed analysis of their collaborative regulatory mechanism using a classic example of the developmental morphogen, hunchback, which is spatially and temporally regulated by the trio during embryogenesis. New biochemical, structural and functional analyses provide insights into RNA recognition, cooperativity, and regulatory mechanisms. We integrate these data into a model of combinatorial RNA binding and regulation of translation and mRNA decay. We then use this information, transcriptome wide analyses and bioinformatics predictions to assess the global impact of Pumilio, Nanos and Brain Tumor on gene regulation. Together, the results support pervasive, dynamic post-transcriptional control.

Beyond Aztec Castles: Toric Cascades in the dP 3 Quiver

NASA Astrophysics Data System (ADS)

Lai, Tri; Musiker, Gregg

2017-12-01

Given one of an infinite class of supersymmetric quiver gauge theories, string theorists can associate a corresponding toric variety (which is a Calabi-Yau 3-fold) as well as an associated combinatorial model known as a brane tiling. In combinatorial language, a brane tiling is a bipartite graph on a torus and its perfect matchings are of interest to both combinatorialists and physicists alike. A cluster algebra may also be associated to such quivers and in this paper we study the generators of this algebra, known as cluster variables, for the quiver associated to the cone over the del Pezzo surface d P 3. In particular, mutation sequences involving mutations exclusively at vertices with two in-coming arrows and two out-going arrows are referred to as toric cascades in the string theory literature. Such toric cascades give rise to interesting discrete integrable systems on the level of cluster variable dynamics. We provide an explicit algebraic formula for all cluster variables that are reachable by toric cascades as well as a combinatorial interpretation involving perfect matchings of subgraphs of the d P 3 brane tiling for these formulas in most cases.

High-Throughput Synthesis and Characterization of Eu Doped Ba xSr2- xSiO4 Thin Film Phosphors.

PubMed

Frost, Sara; Guérin, Samuel; Hayden, Brian E; Soulié, Jean-Philippe; Vian, Chris

2018-06-20

High-throughput techniques have been employed for the synthesis and characterization of thin film phosphors of Eu-doped Ba x Sr 2- x SiO 4 . Direct synthesis from evaporation of the constituent elements under a flux of atomic oxygen on a sapphire substrate at 850 °C was used to directly produce thin film libraries (415 nm thickness) of the crystalline orthosilicate phase with the desired compositional variation (0.24 > x > 1.86). The orthosilicate phase could be synthesized as a pure, or predominantly pure, phase. Annealing the as synthesized library in a reducing atmosphere resulted in the reduction of the Eu while retaining the orthosilicate phase, and resulted in a materials thin film library where fluorescence excited by blue light (450 nm) was observable by the naked eye. Parallel screening of the fluorescence from the combinatorial libraries of Eu doped Ba x Sr 2- x SiO 4 has been implemented by imaging the fluorescent radiation over the library using a monochrome digital camera using a series of color filters. Informatics tools have been developed to allow the 1931 CIE color coordinates and the relative quantum efficiencies of the materials library to be rapidly assessed and mapped against composition, crystal structure and phase purity. The range of compositions gave values of CIE x between 0.17 and 0.52 and CIE y between 0.48 and 0.69 with relative efficiencies in the range 2.0 × 10 -4 -7.6 × 10 -4 . Good agreement was obtained between the thin film phosphors and the fluorescence characteristics of a number of corresponding bulk phosphor powders. The thermal quenching of fluorescence in the thin film libraries was also measured in the temperature range 25-130 °C: The phase purity of the thin film was found to significantly influence both the relative quantum efficiency and the thermal quenching of the fluorescence.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways.

PubMed

Wingler, Laura M; Cornish, Virginia W

2011-09-13

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop "Reiterative Recombination," a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 10(4) biosynthetic pathways. We anticipate that our system's simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo.

Reiterative Recombination for the in vivo assembly of libraries of multigene pathways

PubMed Central

Wingler, Laura M.; Cornish, Virginia W.

2011-01-01

The increasing sophistication of synthetic biology is creating a demand for robust, broadly accessible methodology for constructing multigene pathways inside of the cell. Due to the difficulty of rationally designing pathways that function as desired in vivo, there is a further need to assemble libraries of pathways in parallel, in order to facilitate the combinatorial optimization of performance. While some in vitro DNA assembly methods can theoretically make libraries of pathways, these techniques are resource intensive and inherently require additional techniques to move the DNA back into cells. All previously reported in vivo assembly techniques have been low yielding, generating only tens to hundreds of constructs at a time. Here, we develop “Reiterative Recombination,” a robust method for building multigene pathways directly in the yeast chromosome. Due to its use of endonuclease-induced homologous recombination in conjunction with recyclable markers, Reiterative Recombination provides a highly efficient, technically simple strategy for sequentially assembling an indefinite number of DNA constructs at a defined locus. In this work, we describe the design and construction of the first Reiterative Recombination system in Saccharomyces cerevisiae, and we show that it can be used to assemble multigene constructs. We further demonstrate that Reiterative Recombination can construct large mock libraries of at least 104 biosynthetic pathways. We anticipate that our system’s simplicity and high efficiency will make it a broadly accessible technology for pathway construction and render it a valuable tool for optimizing pathways in vivo. PMID:21876185

The disadvantage of combinatorial communication.

PubMed Central

Lachmann, Michael; Bergstrom, Carl T.

2004-01-01

Combinatorial communication allows rapid and efficient transfer of detailed information, yet combinatorial communication is used by few, if any, non-human species. To complement recent studies illustrating the advantages of combinatorial communication, we highlight a critical disadvantage. We use the concept of information value to show that deception poses a greater and qualitatively different threat to combinatorial signalling than to non-combinatorial systems. This additional potential for deception may represent a strategic barrier that has prevented widespread evolution of combinatorial communication. Our approach has the additional benefit of drawing clear distinctions among several types of deception that can occur in communication systems. PMID:15556886

The disadvantage of combinatorial communication.

PubMed

Lachmann, Michael; Bergstrom, Carl T

2004-11-22

Combinatorial communication allows rapid and efficient transfer of detailed information, yet combinatorial communication is used by few, if any, non-human species. To complement recent studies illustrating the advantages of combinatorial communication, we highlight a critical disadvantage. We use the concept of information value to show that deception poses a greater and qualitatively different threat to combinatorial signalling than to non-combinatorial systems. This additional potential for deception may represent a strategic barrier that has prevented widespread evolution of combinatorial communication. Our approach has the additional benefit of drawing clear distinctions among several types of deception that can occur in communication systems.

Preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens and their use for bacterial detection.

PubMed

Dykman, Lev A; Staroverov, Sergei A; Guliy, Olga I; Ignatov, Oleg V; Fomin, Alexander S; Vidyasheva, Irina V; Karavaeva, Olga A; Bunin, Viktor D; Burygin, Gennady L

2012-01-01

This article reports the first preparation of miniantibodies to Azospirillum brasilense Sp245 surface antigens by using a combinatorial phage library of sheep antibodies. The prepared phage antibodies were used for the first time for lipopolysaccharide and flagellin detection by dot assay, electro-optical analysis of cell suspensions, and transmission electron microscopy. Interaction of A. brasilense Sp245 with antilipopolysaccharide and antiflagellin phage-displayed miniantibodies caused the magnitude of the electro-optical signal to change considerably. The electro-optical results were in good agreement with the electron microscopic data. This is the first reported possibility of employing phage-displayed miniantibodies in bacterial detection aided by electro-optical analysis of cell suspensions.

MDTS: automatic complex materials design using Monte Carlo tree search.

PubMed

M Dieb, Thaer; Ju, Shenghong; Yoshizoe, Kazuki; Hou, Zhufeng; Shiomi, Junichiro; Tsuda, Koji

2017-01-01

Complex materials design is often represented as a black-box combinatorial optimization problem. In this paper, we present a novel python library called MDTS (Materials Design using Tree Search). Our algorithm employs a Monte Carlo tree search approach, which has shown exceptional performance in computer Go game. Unlike evolutionary algorithms that require user intervention to set parameters appropriately, MDTS has no tuning parameters and works autonomously in various problems. In comparison to a Bayesian optimization package, our algorithm showed competitive search efficiency and superior scalability. We succeeded in designing large Silicon-Germanium (Si-Ge) alloy structures that Bayesian optimization could not deal with due to excessive computational cost. MDTS is available at https://github.com/tsudalab/MDTS.

Defining the RNA Internal Loops Preferred by Benzimidazole Derivatives via Two-Dimensional Combinatorial Screening and Computational Analysis

PubMed Central

Velagapudi, Sai Pradeep; Seedhouse, Steven J.; French, Jonathan

2011-01-01

RNA is an important therapeutic target, however, RNA targets are generally underexploited due to a lack of understanding of the small molecules that bind RNA and the RNA motifs that bind small molecules. Herein, we describe the identification of the RNA internal loops derived from a 4096-member 3×3 nucleotide loop library that are the most specific and highest affinity binders to a series of four designer, drug-like benzimidazoles. These studies establish a potentially general protocol to define the highest affinity and most specific RNA motif targets for heterocyclic small molecules. Such information could be used to target functionally important RNAs in genomic sequence. PMID:21604752

MDTS: automatic complex materials design using Monte Carlo tree search

NASA Astrophysics Data System (ADS)

Dieb, Thaer M.; Ju, Shenghong; Yoshizoe, Kazuki; Hou, Zhufeng; Shiomi, Junichiro; Tsuda, Koji

2017-12-01

Complex materials design is often represented as a black-box combinatorial optimization problem. In this paper, we present a novel python library called MDTS (Materials Design using Tree Search). Our algorithm employs a Monte Carlo tree search approach, which has shown exceptional performance in computer Go game. Unlike evolutionary algorithms that require user intervention to set parameters appropriately, MDTS has no tuning parameters and works autonomously in various problems. In comparison to a Bayesian optimization package, our algorithm showed competitive search efficiency and superior scalability. We succeeded in designing large Silicon-Germanium (Si-Ge) alloy structures that Bayesian optimization could not deal with due to excessive computational cost. MDTS is available at https://github.com/tsudalab/MDTS.

Superposition-free comparison and clustering of antibody binding sites: implications for the prediction of the nature of their antigen

PubMed Central

Di Rienzo, Lorenzo; Milanetti, Edoardo; Lepore, Rosalba; Olimpieri, Pier Paolo; Tramontano, Anna

2017-01-01

We describe here a superposition free method for comparing the surfaces of antibody binding sites based on the Zernike moments and show that they can be used to quickly compare and cluster sets of antibodies. The clusters provide information about the nature of the bound antigen that, when combined with a method for predicting the number of direct antibody antigen contacts, allows the discrimination between protein and non-protein binding antibodies with an accuracy of 76%. This is of relevance in several aspects of antibody science, for example to select the framework to be used for a combinatorial antibody library. PMID:28338016

Novel Modeling of Combinatorial miRNA Targeting Identifies SNP with Potential Role in Bone Density

PubMed Central

Coronnello, Claudia; Hartmaier, Ryan; Arora, Arshi; Huleihel, Luai; Pandit, Kusum V.; Bais, Abha S.; Butterworth, Michael; Kaminski, Naftali; Stormo, Gary D.; Oesterreich, Steffi; Benos, Panayiotis V.

2012-01-01

MicroRNAs (miRNAs) are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting), a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD) in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential tool for miRNA-related studies. PMID:23284279

Scent Transmutation: A New Way to Teach on Chemical Equilibrium, Distillation, and Dynamic Combinatorial Chemistry

ERIC Educational Resources Information Center

Ji, Qing; El-Hamdi, Nadia S.; Miljanic´, Ognjen S?.

2014-01-01

Esters are volatile and pleasantly smelling compounds, commonly used as food additives. Using Ti(OBu)[subscript 4]-catalyzed acyl exchange, we demonstrate a scent transmutation experiment, in which two fragrant esters swap their acyl and alkoxy substituents and are, during the course of a reactive distillation, quantitatively converted into two…

Luminescence optimization of MBO 3:Eu 3+ (M=Y, Gd, Al) red phosphor by spray pyrolysis using combinatorial chemistry

NASA Astrophysics Data System (ADS)

Youl Jung, Kyeong

2010-08-01

Conventional solution-based combinatorial chemistry was combined with spray pyrolysis and applied to optimize the luminescence properties of (Y x, Gd y, Al z)BO 3:Eu 3+ red phosphor under vacuum ultraviolet (VUV) excitation. For the Y-Gd-Al ternary system, a compositional library was established to seek the optimal composition at which the highest luminescence under VUV (147 nm) excitation could be achieved. The Al content was found to mainly control the relative peak ratio (R/O) of red and orange colors due to the 5D 0→ 7F 2 to 5D 0→ 7F 1 transitions of Eu 3+. The substitution of Gd atoms in the place of Y sites did not contribute to change the R/O ratio, but was helpful to enhance the emission intensity. As a result, the 613 nm emission peak due to the 5D 0→ 7F 2 transitions of Eu 3+ was intensified by increasing the Al/Gd ratio at a fixed Y content, resulting in the improvement of the color coordinate. Finally, the optimized host composition was (Y 0.11, Gd 0.10, Al 0.79)BO 3 in terms of the emission intensity at 613 nm and the color coordinate.

Transient overexpression of DNA adenine methylase enables efficient and mobile genome engineering with reduced off-target effects

PubMed Central

Lennen, Rebecca M.; Nilsson Wallin, Annika I.; Pedersen, Margit; Bonde, Mads; Luo, Hao; Herrgård, Markus J.; Sommer, Morten O. A.

2016-01-01

Homologous recombination of single-stranded oligonucleotides is a highly efficient process for introducing precise mutations into the genome of E. coli and other organisms when mismatch repair (MMR) is disabled. This can result in the rapid accumulation of off-target mutations that can mask desired phenotypes, especially when selections need to be employed following the generation of combinatorial libraries. While the use of inducible mutator phenotypes or other MMR evasion tactics have proven useful, reported methods either require non-mobile genetic modifications or costly oligonucleotides that also result in reduced efficiencies of replacement. Therefore a new system was developed, Transient Mutator Multiplex Automated Genome Engineering (TM-MAGE), that solves problems encountered in other methods for oligonucleotide-mediated recombination. TM-MAGE enables nearly equivalent efficiencies of allelic replacement to the use of strains with fully disabled MMR and with an approximately 12- to 33-fold lower off-target mutation rate. Furthermore, growth temperatures are not restricted and a version of the plasmid can be readily removed by sucrose counterselection. TM-MAGE was used to combinatorially reconstruct mutations found in evolved salt-tolerant strains, enabling the identification of causative mutations and isolation of strains with up to 75% increases in growth rate and greatly reduced lag times in 0.6 M NaCl. PMID:26496947

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

Dynamical analysis of continuous higher-order hopfield networks for combinatorial optimization.

PubMed

Atencia, Miguel; Joya, Gonzalo; Sandoval, Francisco

2005-08-01

In this letter, the ability of higher-order Hopfield networks to solve combinatorial optimization problems is assessed by means of a rigorous analysis of their properties. The stability of the continuous network is almost completely clarified: (1) hyperbolic interior equilibria, which are unfeasible, are unstable; (2) the state cannot escape from the unitary hypercube; and (3) a Lyapunov function exists. Numerical methods used to implement the continuous equation on a computer should be designed with the aim of preserving these favorable properties. The case of nonhyperbolic fixed points, which occur when the Hessian of the target function is the null matrix, requires further study. We prove that these nonhyperbolic interior fixed points are unstable in networks with three neurons and order two. The conjecture that interior equilibria are unstable in the general case is left open.

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.

Towards an animal model of ovarian cancer: cataloging chicken blood proteins using combinatorial peptide ligand libraries coupled with shotgun proteomic analysis for translational research.

PubMed

Ma, Yingying; Sun, Zeyu; de Matos, Ricardo; Zhang, Jing; Odunsi, Kunle; Lin, Biaoyang

2014-05-01

Epithelial ovarian cancer is the most deadly gynecological cancer around the world, with high morbidity in industrialized countries. Early diagnosis is key in reducing its morbidity rate. Yet, robust biomarkers, diagnostics, and animal models are still limited for ovarian cancer. This calls for broader omics and systems science oriented diagnostics strategies. In this vein, the domestic chicken has been used as an ovarian cancer animal model, owing to its high rate of developing spontaneous epithelial ovarian tumors. Chicken blood has thus been considered a surrogate reservoir from which cancer biomarkers can be identified. However, the presence of highly abundant proteins in chicken blood has compromised the applicability of proteomics tools to study chicken blood owing to a lack of immunodepletion methods. Here, we demonstrate that a combinatorial peptide ligand library (CPLL) can efficiently remove highly abundant proteins from chicken blood samples, consequently doubling the number of identified proteins. Using an integrated CPLL-1DGE-LC-MSMS workflow, we identified a catalog of 264 unique proteins. Functional analyses further suggested that most proteins were coagulation and complement factors, blood transport and binding proteins, immune- and defense-related proteins, proteases, protease inhibitors, cellular enzymes, or cell structure and adhesion proteins. Semiquantitative spectral counting analysis identified 10 potential biomarkers from the present chicken ovarian cancer model. Additionally, many human homologs of chicken blood proteins we have identified have been independently suggested as diagnostic biomarkers for ovarian cancer, further triangulating our novel observations reported here. In conclusion, the CPLL-assisted proteomic workflow using the chicken ovarian cancer model provides a feasible platform for translational research to identify ovarian cancer biomarkers and understand ovarian cancer biology. To the best of our knowledge, we report here the most comprehensive survey of the chicken blood proteome to date.

Computational redesign of bacterial biotin carboxylase inhibitors using structure-based virtual screening of combinatorial libraries.

PubMed

Brylinski, Michal; Waldrop, Grover L

2014-04-02

As the spread of antibiotic resistant bacteria steadily increases, there is an urgent need for new antibacterial agents. Because fatty acid synthesis is only used for membrane biogenesis in bacteria, the enzymes in this pathway are attractive targets for antibacterial agent development. Acetyl-CoA carboxylase catalyzes the committed and regulated step in fatty acid synthesis. In bacteria, the enzyme is composed of three distinct protein components: biotin carboxylase, biotin carboxyl carrier protein, and carboxyltransferase. Fragment-based screening revealed that amino-oxazole inhibits biotin carboxylase activity and also exhibits antibacterial activity against Gram-negative organisms. In this report, we redesigned previously identified lead inhibitors to expand the spectrum of bacteria sensitive to the amino-oxazole derivatives by including Gram-positive species. Using 9,411 small organic building blocks, we constructed a diverse combinatorial library of 1.2×10⁸ amino-oxazole derivatives. A subset of 9×10⁶ of these compounds were subjected to structure-based virtual screening against seven biotin carboxylase isoforms using similarity-based docking by eSimDock. Potentially broad-spectrum antibiotic candidates were selected based on the consensus ranking by several scoring functions including non-linear statistical models implemented in eSimDock and traditional molecular mechanics force fields. The analysis of binding poses of the top-ranked compounds docked to biotin carboxylase isoforms suggests that: (1) binding of the amino-oxazole anchor is stabilized by a network of hydrogen bonds to residues 201, 202 and 204; (2) halogenated aromatic moieties attached to the amino-oxazole scaffold enhance interactions with a hydrophobic pocket formed by residues 157, 169, 171 and 203; and (3) larger substituents reach deeper into the binding pocket to form additional hydrogen bonds with the side chains of residues 209 and 233. These structural insights into drug-biotin carboxylase interactions will be tested experimentally in in vitro and in vivo systems to increase the potency of amino-oxazole inhibitors towards both Gram-negative as well as Gram-positive species.

Droplet microfluidic technology for single-cell high-throughput screening.

PubMed

Brouzes, Eric; Medkova, Martina; Savenelli, Neal; Marran, Dave; Twardowski, Mariusz; Hutchison, J Brian; Rothberg, Jonathan M; Link, Darren R; Perrimon, Norbert; Samuels, Michael L

2009-08-25

We present a droplet-based microfluidic technology that enables high-throughput screening of single mammalian cells. This integrated platform allows for the encapsulation of single cells and reagents in independent aqueous microdroplets (1 pL to 10 nL volumes) dispersed in an immiscible carrier oil and enables the digital manipulation of these reactors at a very high-throughput. Here, we validate a full droplet screening workflow by conducting a droplet-based cytotoxicity screen. To perform this screen, we first developed a droplet viability assay that permits the quantitative scoring of cell viability and growth within intact droplets. Next, we demonstrated the high viability of encapsulated human monocytic U937 cells over a period of 4 days. Finally, we developed an optically-coded droplet library enabling the identification of the droplets composition during the assay read-out. Using the integrated droplet technology, we screened a drug library for its cytotoxic effect against U937 cells. Taken together our droplet microfluidic platform is modular, robust, uses no moving parts, and has a wide range of potential applications including high-throughput single-cell analyses, combinatorial screening, and facilitating small sample analyses.

Beyond terrestrial biology: charting the chemical universe of α-amino acid structures.

PubMed

Meringer, Markus; Cleaves, H James; Freeland, Stephen J

2013-11-25

α-Amino acids are fundamental to biochemistry as the monomeric building blocks with which cells construct proteins according to genetic instructions. However, the 20 amino acids of the standard genetic code represent a tiny fraction of the number of α-amino acid chemical structures that could plausibly play such a role, both from the perspective of natural processes by which life emerged and evolved, and from the perspective of human-engineered genetically coded proteins. Until now, efforts to describe the structures comprising this broader set, or even estimate their number, have been hampered by the complex combinatorial properties of organic molecules. Here, we use computer software based on graph theory and constructive combinatorics in order to conduct an efficient and exhaustive search of the chemical structures implied by two careful and precise definitions of the α-amino acids relevant to coded biological proteins. Our results include two virtual libraries of α-amino acid structures corresponding to these different approaches, comprising 121 044 and 3 846 structures, respectively, and suggest a simple approach to exploring much larger, as yet uncomputed, libraries of interest.

GeNeDA: An Open-Source Workflow for Design Automation of Gene Regulatory Networks Inspired from Microelectronics.

PubMed

Madec, Morgan; Pecheux, François; Gendrault, Yves; Rosati, Elise; Lallement, Christophe; Haiech, Jacques

2016-10-01

The topic of this article is the development of an open-source automated design framework for synthetic biology, specifically for the design of artificial gene regulatory networks based on a digital approach. In opposition to other tools, GeNeDA is an open-source online software based on existing tools used in microelectronics that have proven their efficiency over the last 30 years. The complete framework is composed of a computation core directly adapted from an Electronic Design Automation tool, input and output interfaces, a library of elementary parts that can be achieved with gene regulatory networks, and an interface with an electrical circuit simulator. Each of these modules is an extension of microelectronics tools and concepts: ODIN II, ABC, the Verilog language, SPICE simulator, and SystemC-AMS. GeNeDA is first validated on a benchmark of several combinatorial circuits. The results highlight the importance of the part library. Then, this framework is used for the design of a sequential circuit including a biological state machine.

Acetylcholinesterase affinity-based screening assay on Lippia gracilis Schauer extracts.

PubMed

Vanzolini, K L; da F Sprenger, R; Leme, G M; de S Moraes, V R; Vilela, A F L; Cardoso, C L; Cass, Q B

2018-05-10

The use of affinity-based protein assay produced by covalently linking acetylcholinesterase to magnetic beads, followed by chemical characterization of the selective binders using Liquid Chromatography with tandem High-Resolution Mass Spectrometry (LC-HRMS) is herein described for profiling crude aqueous natural product extracts. The fishing assay was first modulated using galanthamine as a reference ligand and then, the assay condition was adjusted for the aqueous leaves extracts obtained from Lippia gracilis Schauer (genotype 201) that was used as the natural combinatory library. From the experiments, a selective binder has been undisclosed with an accurate mass of 449.1131 m/z and identified as eriodictyol 2'-O-glucoside or eriodictyol 3'-O-glucoside. The selectivity of the binding assay was demonstrated, as much as, that erydictiol 7-O-glucoside was not fished, although it was present in the crude aqueous extract. The binding assay platform exhibited high specificity and did not require any sample pretreatment, making it appropriate for profiling binders at natural libraries. Copyright © 2018 Elsevier B.V. All rights reserved.

Engineering modular ester fermentative pathways in Escherichia coli.

PubMed

Layton, Donovan S; Trinh, Cong T

2014-11-01

Sensation profiles are observed all around us and are made up of many different molecules, such as esters. These profiles can be mimicked in everyday items for their uses in foods, beverages, cosmetics, perfumes, solvents, and biofuels. Here, we developed a systematic 'natural' way to derive these products via fermentative biosynthesis. Each ester fermentative pathway was designed as an exchangeable ester production module for generating two precursors- alcohols and acyl-CoAs that were condensed by an alcohol acyltransferase to produce a combinatorial library of unique esters. As a proof-of-principle, we coupled these ester modules with an engineered, modular, Escherichia coli chassis in a plug-and-play fashion to create microbial cell factories for enhanced anaerobic production of a butyrate ester library. We demonstrated tight coupling between the modular chassis and ester modules for enhanced product biosynthesis, an engineered phenotype useful for directed metabolic pathway evolution. Compared to the wildtype, the engineered cell factories yielded up to 48 fold increase in butyrate ester production from glucose. Copyright © 2014 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

A salicylic acid-based small molecule inhibitor for the oncogenic Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2)

PubMed Central

Zhang, Xian; He, Yantao; Liu, Sijiu; Yu, Zhihong; Jiang, Zhong-Xing; Yang, Zhenyun; Dong, Yuanshu; Nabinger, Sarah C.; Wu, Li; Gunawan, Andrea M.; Wang, Lina; Chan, Rebecca J.; Zhang, Zhong-Yin

2010-01-01

The Src homology-2 domain containing protein tyrosine phosphatase-2 (SHP2) plays a pivotal role in growth factor and cytokine signaling. Gain-of-function SHP2 mutations are associated with Noonan syndrome, various kinds of leukemias and solid tumors. Thus there is considerable interest in SHP2 as a potential target for anti-cancer and anti-leukemia therapy. We report a salicylic acid-based combinatorial library approach aimed to bind both active site and unique nearby sub-pockets for enhanced affinity and selectivity. Screening of the library led to the identification of a SHP2 inhibitor II-B08 (compound 9) with highly efficacious cellular activity. Compound 9 blocks growth factor stimulated ERK1/2 activation and hematopoietic progenitor proliferation, providing supporting evidence that chemical inhibition of SHP2 may be therapeutically useful for anti-cancer and anti-leukemia treatment. X-ray crystallographic analysis of the structure of SHP2 in complex with 9 reveals molecular determinants that can be exploited for the acquisition of more potent and selective SHP2 inhibitors. PMID:20170098

Genomes to natural products PRediction Informatics for Secondary Metabolomes (PRISM)

PubMed Central

Skinnider, Michael A.; Dejong, Chris A.; Rees, Philip N.; Johnston, Chad W.; Li, Haoxin; Webster, Andrew L. H.; Wyatt, Morgan A.; Magarvey, Nathan A.

2015-01-01

Microbial natural products are an invaluable source of evolved bioactive small molecules and pharmaceutical agents. Next-generation and metagenomic sequencing indicates untapped genomic potential, yet high rediscovery rates of known metabolites increasingly frustrate conventional natural product screening programs. New methods to connect biosynthetic gene clusters to novel chemical scaffolds are therefore critical to enable the targeted discovery of genetically encoded natural products. Here, we present PRISM, a computational resource for the identification of biosynthetic gene clusters, prediction of genetically encoded nonribosomal peptides and type I and II polyketides, and bio- and cheminformatic dereplication of known natural products. PRISM implements novel algorithms which render it uniquely capable of predicting type II polyketides, deoxygenated sugars, and starter units, making it a comprehensive genome-guided chemical structure prediction engine. A library of 57 tailoring reactions is leveraged for combinatorial scaffold library generation when multiple potential substrates are consistent with biosynthetic logic. We compare the accuracy of PRISM to existing genomic analysis platforms. PRISM is an open-source, user-friendly web application available at http://magarveylab.ca/prism/. PMID:26442528

Self-sorting of dynamic metallosupramolecular libraries (DMLs) via metal-driven selection.

PubMed

Kocsis, Istvan; Dumitrescu, Dan; Legrand, Yves-Marie; van der Lee, Arie; Grosu, Ion; Barboiu, Mihail

2014-03-11

"Metal-driven" selection between finite mononuclear and polymeric metallosupramolecular species can be quantitatively achieved in solution and in a crystalline state via coupled coordination/stacking interactional algorithms within dynamic metallosupramolecular libraries - DMLs.

Optimal placement of excitations and sensors for verification of large dynamical systems

NASA Technical Reports Server (NTRS)

Salama, M.; Rose, T.; Garba, J.

1987-01-01

The computationally difficult problem of the optimal placement of excitations and sensors to maximize the observed measurements is studied within the framework of combinatorial optimization, and is solved numerically using a variation of the simulated annealing heuristic algorithm. Results of numerical experiments including a square plate and a 960 degrees-of-freedom Control of Flexible Structure (COFS) truss structure, are presented. Though the algorithm produces suboptimal solutions, its generality and simplicity allow the treatment of complex dynamical systems which would otherwise be difficult to handle.

Converting an intranet site to the cloud: using CampusGuides to refresh a library portal.

PubMed

Osterhaus Trzasko, Leah C; Farrell, Ann M; Rethlefsen, Melissa L

2012-01-01

After a major redesign project in 2002, Mayo Clinic Libraries' heavily used intranet portal remained largely static. Library staff were unable to make substantive design changes or introduce tools that would make the content more dynamic. CampusGuides offered a practical, user-friendly, web-based solution to add dynamic content to the library site. A task force was formed both to establish design and style guidelines that would integrate with the library site and to plan the conversion of content to CampusGuides. Converting intranet site content to CampusGuides gave the task force the opportunity to examine, re-imagine, and revitalize site content.

Synthesis of Chemiluminescent Esters: A Combinatorial Synthesis Experiment for Organic Chemistry Students

ERIC Educational Resources Information Center

Duarte, Robert; Nielson, Janne T.; Dragojlovic, Veljko

2004-01-01

A group of techniques aimed at synthesizing a large number of structurally diverse compounds is called combinatorial synthesis. Synthesis of chemiluminescence esters using parallel combinatorial synthesis and mix-and-split combinatorial synthesis is experimented.

Multi-Protein Dynamic Combinatorial Chemistry: A Novel Strategy that Leads to Simultaneous Discovery of Subfamily-Selective Inhibitors for Nucleic Acid Demethylases FTO and ALKBH3.

PubMed

Das, Mohua; Tianming, Yang; Jinghua, Dong; Prasetya, Fransisca; Yiming, Xie; Wong, Kendra; Cheong, Adeline; Woon, Esther C Y

2018-06-19

Dynamic combinatorial chemistry (DCC) is a powerful supramolecular approach for discovering ligands for biomolecules. To date, most, if not all, biologically-templated DCC employ only a single biomolecule in directing the self-assembly process. To expand the scope and potential of DCC, herein, we developed a novel multi-protein DCC strategy which combines the discriminatory power of zwitterionic 'thermal-tag' with the sensitivity of differential scanning fluorimetry. This strategy enables the discovery of ligands against several proteins of interest concurrently. It is remarkably sensitive and could differentiate the binding of ligands to structurally-similar subfamily members, which is extremely challenging to achieve. Through this approach, we were able to simultaneously identify subfamily-selective probes against two clinically important epigenetic enzymes, FTO (7; IC₅₀ = 2.6 µM) and ALKBH3 (8; IC₅₀ = 3.7 µM). To our knowledge, this is the first report of a subfamily-selective ALKBH3 inhibitor. The developed strategy could, in principle, be adapted to a broad range of proteins, thus it shall be of widespread scientific interest. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Lean and Efficient Software: Whole-Program Optimization of Executables

DTIC Science & Technology

2015-09-30

libraries. Many levels of library interfaces—where some libraries are dynamically linked and some are provided in binary form only—significantly limit...software at build time. The opportunity: Our objective in this project is to substantially improve the performance, size, and robustness of binary ...executables by using static and dynamic binary program analysis techniques to perform whole-program optimization directly on compiled programs

Asessing for Structural Understanding in Childrens' Combinatorial Problem Solving.

ERIC Educational Resources Information Center

English, Lyn

1999-01-01

Assesses children's structural understanding of combinatorial problems when presented in a variety of task situations. Provides an explanatory model of students' combinatorial understandings that informs teaching and assessment. Addresses several components of children's structural understanding of elementary combinatorial problems. (Contains 50…

Supramolecular assembly/reassembly processes: molecular motors and dynamers operating at surfaces.

PubMed

Ciesielski, Artur; Samorì, Paolo

2011-04-01

Among the many significant advances within the field of supramolecular chemistry over the past decades, the development of the so-called "dynamers" features a direct relevance to materials science. Defined as "combinatorial dynamic polymers", dynamers are constitutional dynamic systems and materials resulting from the application of the principles of supramolecular chemistry to polymer science. Like supramolecular materials in general, dynamers are reversible dynamic multifunctional architectures, capable of modifying their constitution by exchanging, recombining, incorporating components. They may exhibit a variety of novel properties and behave as adaptive materials. In this review we focus on the design of responsive switchable monolayers, i.e. monolayers capable to undergo significant changes in their physical or chemical properties as a result of external stimuli. Scanning tunneling microscopy studies provide direct evidence with a sub-nanometre resolution, on the formation and dynamic response of these self-assembled systems featuring controlled geometries and properties.

Rational design and synthesis of water-compatible molecularly imprinted polymers for selective solid phase extraction of amiodarone.

PubMed

Muhammad, Turghun; Cui, Liu; Jide, Wang; Piletska, Elena V; Guerreiro, Antonio R; Piletsky, Sergey A

2012-01-04

Novel water-compatible molecularly imprinted polymers (MIPs) selective for amiodarone (AD) were designed via a new methodology which relies on screening library of non-imprinted polymers (NIPs). The NIP library consisted of eighteen cross-linked co-polymers synthesized from monomers commonly used in molecular imprinting. The binding capacity of each polymer in the library was analyzed in two different solvents. Binding in water was used to assess non-specific (hydrophobic) interactions and binding in an appropriate organic solvent was used to assess specific interactions. A good correlation was found between the screening tests and modeling of monomer-template interactions performed using computational approach. Additionally, analysis of template-monomer interactions was performed using UV-vis spectroscopy. As the result, 4-vinylpyridine (4-VP) was selected as the best monomer for developing MIP for AD. The 4-VP-based polymers demonstrated imprinting factor equal 3.9. The polymers performance in SPE was evaluated using AD and its structural analogues. The recovery of AD was as high as 96% when extracted from spiked phosphate buffer (pH 4.5) solution and 82.1% from spiked serum samples. The developed MIP shown as a material with specific binding to AD, comparing to its structural analogues, 1-(2-diethylaminoethoxy)-2,6-diiodo-4-nitrobenzene and lidocaine, which shown 9.9% and 25.4% of recovery from the buffer solution, correspondingly. We believe that the screening of NIP library could be proposed as an alternative to commonly used computational and combinatorial approaches. Copyright © 2011 Elsevier B.V. All rights reserved.

Structure based re-design of the binding specificity of anti-apoptotic Bcl-xL

PubMed Central

Chen, T. Scott; Palacios, Hector; Keating, Amy E.

2012-01-01

Many native proteins are multi-specific and interact with numerous partners, which can confound analysis of their functions. Protein design provides a potential route to generating synthetic variants of native proteins with more selective binding profiles. Re-designed proteins could be used as research tools, diagnostics or therapeutics. In this work, we used a library screening approach to re-engineer the multi-specific anti-apoptotic protein Bcl-xL to remove its interactions with many of its binding partners, making it a high affinity and selective binder of the BH3 region of pro-apoptotic protein Bad. To overcome the enormity of the potential Bcl-xL sequence space, we developed and applied a computational/experimental framework that used protein structure information to generate focused combinatorial libraries. Sequence features were identified using structure-based modeling, and an optimization algorithm based on integer programming was used to select degenerate codons that maximally covered these features. A constraint on library size was used to ensure thorough sampling. Using yeast surface display to screen a designed library of Bcl-xL variants, we successfully identified a protein with ~1,000-fold improvement in binding specificity for the BH3 region of Bad over the BH3 region of Bim. Although negative design was targeted only against the BH3 region of Bim, the best re-designed protein was globally specific against binding to 10 other peptides corresponding to native BH3 motifs. Our design framework demonstrates an efficient route to highly specific protein binders and may readily be adapted for application to other design problems. PMID:23154169

Application of a novel design paradigm to generate general nonpeptide combinatorial templates mimicking beta-turns: synthesis of ligands for melanocortin receptors.

PubMed

Webb, Thomas R; Jiang, Luyong; Sviridov, Sergey; Venegas, Ruben E; Vlaskina, Anna V; McGrath, Douglas; Tucker, John; Wang, Jian; Deschenes, Alain; Li, Rongshi

2007-01-01

We report the further application of a novel approach to template and ligand design by the synthesis of agonists of the melanocortin receptor. This design method uses the conserved structural data from the three-dimensional conformations of beta-turn peptides to design rigid nonpeptide templates that mimic the orientation of the main chain C-alpha atoms in a peptide beta-turn. We report details on a new synthesis of derivatives of template 1 that are useful for the synthesis of exploratory libraries. The utility of this technique is further exemplified by several iterative rounds of high-throughput synthesis and screening, which result in new partially optimized nonpeptide agonists for several melanocortin receptors.

Hypergraph-Based Combinatorial Optimization of Matrix-Vector Multiplication

ERIC Educational Resources Information Center

Wolf, Michael Maclean

2009-01-01

Combinatorial scientific computing plays an important enabling role in computational science, particularly in high performance scientific computing. In this thesis, we will describe our work on optimizing matrix-vector multiplication using combinatorial techniques. Our research has focused on two different problems in combinatorial scientific…

Rediscovering natural products as a source of new drugs.

PubMed

Koehn, Frank E; Carter, Guy T

2005-04-01

Extract: Since the very beginnings of human medicine, physicians have relied on chemical compounds produced by animals, plants and microorganisms, so-called natural products, to treat diseases. Natural products are directly or indirectly responsible for roughly one-half of all drugs currently in use. Of the 877 small-molecule new drug molecules introduced between 1981 and 2002, 49% were natural products or natural product analogs. Despite the great success of the 70s and 80s, the pharmaceutical industry de-emphasized natural products research during the following decade. In this article, we examine the underlying reasons for the decline, and assess future prospects for natural products research in drug discovery. In the 1990s, major pharmaceutical companies moved to a lead-finding strategy based on High Throughput Screening (HTS) of very large collections (libraries) of synthetic compounds. The move arose from the belief that techniques such as combinatorial chemistry could produce larger, more cost-effective libraries with improved hit rates and quality. Additionally, advances in molecular biology, cellular biology and genomics dramatically increased the number of molecular targets, prompting shorter drug discovery timelines. In today's drug discovery environment, rapid screening and identification of potential drug molecules is essential for success. This puts traditional natural products-based programs, with their reliance on the lengthy processes of the screening of extracts library, bioassay-guided isolation of the active components, structure elucidation and subsequent production scale-up, at a competitive disadvantage.

Employing in vitro directed molecular evolution for the selection of α-amylase variant inhibitors with activity toward cotton boll weevil enzyme.

PubMed

da Silva, Maria Cristina Mattar; Del Sarto, Rafael Perseghini; Lucena, Wagner Alexandre; Rigden, Daniel John; Teixeira, Fabíola Rodrigues; Bezerra, Caroline de Andrade; Albuquerque, Erika Valéria Saliba; Grossi-de-Sa, Maria Fatima

2013-09-20

Numerous species of insect pests attack cotton plants, out of which the cotton boll weevil (Anthonomus grandis) is the main insect in Brazil and must be controlled to avert large economic losses. Like other insect pests, A. grandis secretes a high level of α-amylases in the midgut lumen, which are required for digestion of carbohydrates. Thus, α-amylase inhibitors (α-AIs) represent a powerful tool to apply in the control of insect pests. Here, we applied DNA shuffling and phage display techniques and obtained a combinatorial library containing 10⁸ α-AI variant forms. From this library, variants were selected exhibiting in vitro affinity for cotton boll weevil α-amylases. Twenty-six variant sequences were cloned into plant expression vectors and expressed in Arabidopsis thaliana. Transformed plant extracts were assayed in vitro to select specific and potent α-amylase inhibitors against boll weevil amylases. While the wild type inhibitors, used to create the shuffled library, did not inhibit the A. grandis α-amylases, three α-AI mutants, named α-AIC3, α-AIA11 and α-AIG4 revealed high inhibitory activities against A. grandis α-amylases in an in vitro assay. In summary, data reported here shown the potential biotechnology of new α-AI variant genes for cotton boll weevil control. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

Automatic Design of Synthetic Gene Circuits through Mixed Integer Non-linear Programming

PubMed Central

Huynh, Linh; Kececioglu, John; Köppe, Matthias; Tagkopoulos, Ilias

2012-01-01

Automatic design of synthetic gene circuits poses a significant challenge to synthetic biology, primarily due to the complexity of biological systems, and the lack of rigorous optimization methods that can cope with the combinatorial explosion as the number of biological parts increases. Current optimization methods for synthetic gene design rely on heuristic algorithms that are usually not deterministic, deliver sub-optimal solutions, and provide no guaranties on convergence or error bounds. Here, we introduce an optimization framework for the problem of part selection in synthetic gene circuits that is based on mixed integer non-linear programming (MINLP), which is a deterministic method that finds the globally optimal solution and guarantees convergence in finite time. Given a synthetic gene circuit, a library of characterized parts, and user-defined constraints, our method can find the optimal selection of parts that satisfy the constraints and best approximates the objective function given by the user. We evaluated the proposed method in the design of three synthetic circuits (a toggle switch, a transcriptional cascade, and a band detector), with both experimentally constructed and synthetic promoter libraries. Scalability and robustness analysis shows that the proposed framework scales well with the library size and the solution space. The work described here is a step towards a unifying, realistic framework for the automated design of biological circuits. PMID:22536398

ILP-2 modeling and virtual screening of an FDA-approved library:a possible anticancer therapy.

PubMed

Khalili, Saeed; Mohammadpour, Hemn; Shokrollahi Barough, Mahideh; Kokhaei, Parviz

2016-06-23

The members of the inhibitors of apoptosis protein (IAP) family inhibit diverse components of the caspase signaling pathway, notably caspase 3, 7, and 9. ILP-2 (BIRC-8) is the most recently identified member of the IAPs, mainly interacting with caspase 9. This interaction would eventually lead to death resistance in the case of cancerous cells. Therefore, structural modeling of ILP-2 and finding applicable inhibitors of its interaction with caspase 9 are a compelling challenge. Three main protein modeling approaches along with various model refinement measures were harnessed to achieve a reliable 3D model, using state-of-the-art software. Thereafter, the selected model was employed to perform virtual screening of an FDA approved library. A model built by a combinatorial approach (homology and ab initio approaches) was chosen as the best model. Model refinement processes successfully bolstered the model quality. Virtual screening of the compound library introduced several high affinity inhibitor candidates that interact with functional residues of ILP2. Given the 3D structure of the ILP2 molecule, we found promising inhibitory molecules. In addition to high affinity towards the ILP2 molecule, these molecules interact with residues that play pivotal rules in ILP2-caspase interaction. These molecules would inhibit ILP2-caspase interaction and consequently would lead to reactivated cell apoptosis through the caspases pathway.

Compositionally Dependent Nonlinear Optical Bandgap Behavior of Mixed Anodic Oxides in Niobium-Titanium System.

PubMed

Bleckenwegner, Petra; Mardare, Cezarina Cela; Cobet, Christoph; Kollender, Jan Philipp; Hassel, Achim Walter; Mardare, Andrei Ionut

2017-02-13

Optical bandgap mapping of Nb-Ti mixed oxides anodically grown on a thin film parent metallic combinatorial library was performed via variable angle spectroscopic ellipsometry (VASE). A wide Nb-Ti compositional spread ranging from Nb-90 at.% Ti to Nb-15 at.% Ti deposited by cosputtering was used for this purpose. The Nb-Ti library was stepwise anodized at potentials up to 10 V SHE, and the anodic oxides optical properties were mapped along the Nb-Ti library with 2 at.% resolution. The surface dissimilarities along the Nb-Ti compositional gradient were minimized by tuning the deposition parameters, thus allowing a description of the mixed Nb-Ti oxides based on a single Tauc-Lorentz oscillator for data fitting. Mapping of the Nb-Ti oxides optical bandgap along the entire compositional spread showed a clear deviation from the linear model based on mixing individual Nb and Ti electronegativities proportional to their atomic fractions. This is attributed to the strong amorphization and an in-depth compositional gradient of the mixed oxides. A systematic optical bandgap decrease toward values as low as 2.0 eV was identified at approximately 50 at.% Nb. Mixing of Nb 2 O 5 and TiO 2 with both amorphous and crystalline phases is concluded, whereas the possibility of complex Nb a Ti b O y oxide formation during anodization is unlikely.

Combinatorial structures to modeling simple games and applications

NASA Astrophysics Data System (ADS)

Molinero, Xavier

2017-09-01

We connect three different topics: combinatorial structures, game theory and chemistry. In particular, we establish the bases to represent some simple games, defined as influence games, and molecules, defined from atoms, by using combinatorial structures. First, we characterize simple games as influence games using influence graphs. It let us to modeling simple games as combinatorial structures (from the viewpoint of structures or graphs). Second, we formally define molecules as combinations of atoms. It let us to modeling molecules as combinatorial structures (from the viewpoint of combinations). It is open to generate such combinatorial structures using some specific techniques as genetic algorithms, (meta-)heuristics algorithms and parallel programming, among others.

Changing Libraries: Facilitating Self-Reflection and Action Research on Organizational Change in Academic Libraries

ERIC Educational Resources Information Center

Whitworth, Andrew; Torras I Calvo, Maria Carme; Moss, Bodil; Amlesom Kifle, Nazareth; Blåsternes, Terje

2014-01-01

Visualization and mapping techniques can build a dynamic picture of information practices, including action research, within libraries, raising awareness of how the information landscape at each library may both support and retard research into the library's information practices. These techniques have implications for researchers as they generate…

Fast Combinatorial Algorithm for the Solution of Linearly Constrained Least Squares Problems

DOEpatents

Van Benthem, Mark H.; Keenan, Michael R.

2008-11-11

A fast combinatorial algorithm can significantly reduce the computational burden when solving general equality and inequality constrained least squares problems with large numbers of observation vectors. The combinatorial algorithm provides a mathematically rigorous solution and operates at great speed by reorganizing the calculations to take advantage of the combinatorial nature of the problems to be solved. The combinatorial algorithm exploits the structure that exists in large-scale problems in order to minimize the number of arithmetic operations required to obtain a solution.

Generalised Spin Dynamics and Induced Bounds of Automorphic [A]nX, [AX]n NMR Systems via Dual Tensorial Sets: An Invariant Cardinality Role for CFP

NASA Astrophysics Data System (ADS)

Temme, Francis P.

For uniform spins and their indistinguishable point sets of tensorial bases defining automorphic group-based Liouvillian NMR spin dynamics, the role of recursively-derived coefficients of fractional parentage (CFP) bijections and Schur duality-defined CFP(0)(n) ≡ ¦GI¦(n) group invariant cardinality is central both to understanding the impact of time-reversal invariance(TRI) spin physics, and to analysis as density-matrix formalisms over democratic recoupled (DR) dual tensorial sets, {T{ṽ}k(11.1)(SU2 × ln)}. Over abstract spin space, these tensorial sets are (ṽ) invariant-theoretic forms which lie beyond the Liouvillian graph recoupling and Racah-forms envisaged by Sanctuary [1]. This is a direct consequence of the dominance of the ln group. It leads to new views on the value of projective group actions as mappings over specialised Liouvillian carrier spaces, and on the need for the replacement of Racah-Wigner (R-W) orthogonality for distinct point sets, by criteria based on explicit properties of invariants [J. Phys.: Math. & Theor. A 41, 015210 (2008)] for multiple invariant systems. Ũ × P group actions over disjoint (L) carrier subspaces, leading to exclusively combinatorial views of the nature of quantal completeness for indistinguishable point-based tensorial sets. Such generalised invariant-theoretic approaches lie beyond the range of Lévi-Civitá generator views, or of Lévy-Leblond and Lévy-Nahas [9] with its additional cyclic-commutators defining mono-invariant DR forms. Comparison of the latter with generalised multiple-invariant techniques provides an answer to the question of precisely why [A]n≥4(X) and [AX]n≥4 NMR system spin dynamics are not ameniable to conventional R-W analysis of recoupled discrete-point tensorial systems. Our work augments earlier Hilbert space views, both of Louck and Biedenharn [21] on boson pattern projective mapping, and of Corio [19]. The roles of recent ln group action and (λ ⊢ n)-Schur combinatorial concepts, as well as of polyhedral-combinatorial modelling over invariance algebras, contribute significantly to our understanding of invariant-based techniques of Liouville dual tensorial sets for automorphic NMR spin physics.1

Dynamic analysis of the combinatorial regulation involving transcription factors and microRNAs in cell fate decisions.

PubMed

Yan, Fang; Liu, Haihong; Liu, Zengrong

2014-01-01

P53 and E2F1 are critical transcription factors involved in the choices between different cell fates including cell differentiation, cell cycle arrest or apoptosis. Recent experiments have shown that two families of microRNAs (miRNAs), p53-responsive miR34 (miRNA-34 a, b and c) and E2F1-inducible miR449 (miRNA-449 a, b and c) are potent inducers of these different fates and might have an important role in sensitizing cancer cells to drug treatment and tumor suppression. Identifying the mechanisms responsible for the combinatorial regulatory roles of these two transcription factors and two miRNAs is an important and challenging problem. Here, based in part on the model proposed in Tongli Zhang et al. (2007), we developed a mathematical model of the decision process and explored the combinatorial regulation between these two transcription factors and two miRNAs in response to DNA damage. By analyzing nonlinear dynamic behaviors of the model, we found that p53 exhibits pulsatile behavior. Moreover, a comparison is given to reveal the subtle differences of the cell fate decision process between regulation and deregulation of miR34 on E2F1. It predicts that miR34 plays a critical role in promoting cell cycle arrest. In addition, a computer simulation result also predicts that the miR449 is necessary for apoptosis in response to sustained DNA damage. In agreement with experimental observations, our model can account for the intricate regulatory relationship between these two transcription factors and two miRNAs in the cell fate decision process after DNA damage. These theoretical results indicate that miR34 and miR449 are effective tumor suppressors and play critical roles in cell fate decisions. The work provides a dynamic mechanism that shows how cell fate decisions are coordinated by two transcription factors and two miRNAs. This article is part of a Special Issue entitled: Computational Proteomics, Systems Biology and Clinical Implications. Guest Editor: Yudong Cai. Crown Copyright © 2013. All rights reserved.

A verification library for multibody simulation software

NASA Technical Reports Server (NTRS)

Kim, Sung-Soo; Haug, Edward J.; Frisch, Harold P.

1989-01-01

A multibody dynamics verification library, that maintains and manages test and validation data is proposed, based on RRC Robot arm and CASE backhoe validation and a comparitive study of DADS, DISCOS, and CONTOPS that are existing public domain and commercial multibody dynamic simulation programs. Using simple representative problems, simulation results from each program are cross checked, and the validation results are presented. Functionalities of the verification library are defined, in order to automate validation procedure.

MARCC (Matrix-Assisted Reader Chromatin Capture): an antibody-free method to enrich and analyze combinatorial nucleosome modifications

PubMed Central

Su, Zhangli

2016-01-01

Combinatorial patterns of histone modifications are key indicators of different chromatin states. Most of the current approaches rely on the usage of antibodies to analyze combinatorial histone modifications. Here we detail an antibody-free method named MARCC (Matrix-Assisted Reader Chromatin Capture) to enrich combinatorial histone modifications. The combinatorial patterns are enriched on native nucleosomes extracted from cultured mammalian cells and prepared by micrococcal nuclease digestion. Such enrichment is achieved by recombinant chromatin-interacting protein modules, or so-called reader domains, which can bind in a combinatorial modification-dependent manner. The enriched chromatin can be quantified by western blotting or mass spectrometry for the co-existence of histone modifications, while the associated DNA content can be analyzed by qPCR or next-generation sequencing. Altogether, MARCC provides a reproducible, efficient and customizable solution to enrich and analyze combinatorial histone modifications. PMID:26131849

A New Approach for Proving or Generating Combinatorial Identities

ERIC Educational Resources Information Center

Gonzalez, Luis

2010-01-01

A new method for proving, in an immediate way, many combinatorial identities is presented. The method is based on a simple recursive combinatorial formula involving n + 1 arbitrary real parameters. Moreover, this formula enables one not only to prove, but also generate many different combinatorial identities (not being required to know them "a…

Novel fragment-based QSAR modeling and combinatorial design of pyrazole-derived CRK3 inhibitors as potent antileishmanials.

PubMed

Goyal, Sukriti; Dhanjal, Jaspreet K; Tyagi, Chetna; Goyal, Manisha; Grover, Abhinav

2014-07-01

The CRK3 cyclin-dependent kinase of Leishmania plays an important role in regulating the cell-cycle progression at the G2/M phase checkpoint transition, proliferation, and viability inside the host macrophage. In this study, a novel fragment-based QSAR model has been developed using 22 pyrazole-derived compounds exhibiting inhibitory activity against Leishmanial CRK3. Unlike other QSAR methods, this fragment-based method gives flexibility to study the relationship between molecular fragments of interest and their contribution for the variation in the biological response by evaluating cross-term fragment descriptors. Based on the fragment-based QSAR model, a combinatorial library was generated, and top two compounds were reported after predicting their activity. The QSAR model showed satisfactory statistical parameters for the data set (r(2) = 0.8752, q(2) = 0.6690, F-ratio = 30.37, and pred_r(2) = 0.8632) with four descriptors describing the nature of substituent groups and the environment of the substitution site. Evaluation of the model implied that electron-rich substitution at R1 position improves the inhibitory activity, while decline in inhibitory activity was observed in presence of nitrogen at R2 position. The analysis carried out in this study provides a substantial basis for consideration of the designed pyrazole-based leads as potent antileishmanial drugs. © 2014 John Wiley & Sons A/S.

Statistical theory for protein combinatorial libraries. Packing interactions, backbone flexibility, and the sequence variability of a main-chain structure.

PubMed

Kono, H; Saven, J G

2001-02-23

Combinatorial experiments provide new ways to probe the determinants of protein folding and to identify novel folding amino acid sequences. These types of experiments, however, are complicated both by enormous conformational complexity and by large numbers of possible sequences. Therefore, a quantitative computational theory would be helpful in designing and interpreting these types of experiment. Here, we present and apply a statistically based, computational approach for identifying the properties of sequences compatible with a given main-chain structure. Protein side-chain conformations are included in an atom-based fashion. Calculations are performed for a variety of similar backbone structures to identify sequence properties that are robust with respect to minor changes in main-chain structure. Rather than specific sequences, the method yields the likelihood of each of the amino acids at preselected positions in a given protein structure. The theory may be used to quantify the characteristics of sequence space for a chosen structure without explicitly tabulating sequences. To account for hydrophobic effects, we introduce an environmental energy that it is consistent with other simple hydrophobicity scales and show that it is effective for side-chain modeling. We apply the method to calculate the identity probabilities of selected positions of the immunoglobulin light chain-binding domain of protein L, for which many variant folding sequences are available. The calculations compare favorably with the experimentally observed identity probabilities.

Constellation Coverage Analysis

NASA Technical Reports Server (NTRS)

Lo, Martin W. (Compiler)

1997-01-01

The design of satellite constellations requires an understanding of the dynamic global coverage provided by the constellations. Even for a small constellation with a simple circular orbit propagator, the combinatorial nature of the analysis frequently renders the problem intractable. Particularly for the initial design phase where the orbital parameters are still fluid and undetermined, the coverage information is crucial to evaluate the performance of the constellation design. We have developed a fast and simple algorithm for determining the global constellation coverage dynamically using image processing techniques. This approach provides a fast, powerful and simple method for the analysis of global constellation coverage.