Mixture-based Synthetic Combinatorial Libraries Research Articles

Identification of B Cell and T Cell Epitopes Using Synthetic Peptide Combinatorial Libraries.

This article presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules to identify B and T cell epitopes. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches. © 2022 Wiley Periodicals LLC. Basic Protocol 1: Screening peptide library for antibody inhibition Basic Protocol 2: Screening a peptide library to identify CD4+ Or CD8+ T cell ligands Support Protocol 1: Optimizing antigen and antibody concentrations for screening assay Support Protocol 2: Preparing a positional scanning peptide library.

Abstract 2580: Screening of a mixture-based synthetic combinatorial library identifies small molecules that inhibit the ability of GTP to displace mant-GDP from mutant G12D KRas

Abstract Although mutant KRas significantly contributes to human oncogenesis and patient tumor resistance to therapy, there are no anticancer drugs directly targeting mt KRas available in clinic. A major effort in our laboratory is to identify novel compounds that bind mt KRas and inhibit potently and selectively KRas-driven malignant transformation. To this end, we have used a mixture-based synthetic combinatorial library (scaffold ranking library) containing over 6 million compounds in a mant-GDP assay to identify chemical scaffolds that inhibit the ability of GTP to displace mant-GDP from mt G12D KRas. Subsequent screening of the positional scanning libraries derived from the hit mixtures led to several individual compound hits with the most potent inhibiting the ability of GTP to displace mant-GDP from mt G12D KRas with IC50 of 61 μM. Current studies are focused on physical characterization of the hits to mt KRas and cellular activities of these hits in mt KRas-dependent and -independent human cancer cell lines. Citation Format: Perry C. Kennedy, Marc C. Guilanotti, Travis LsVoi, Said M. Sebti. Screening of a mixture-based synthetic combinatorial library identifies small molecules that inhibit the ability of GTP to displace mant-GDP from mutant G12D KRas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2580. doi:10.1158/1538-7445.AM2015-2580

Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries of peptide molecules. The protocols employ peptide libraries to identify peptides recognized by MAbs and T cells. The first protocol uses a positional scanning peptide library made up of hexapeptides to identify antigenic determinants recognized by MAbs. The 120 mixtures in the hexapeptide library are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide library to identify T cell peptide ligands. The 200 mixtures of the decapeptide library are tested for their ability to induce T cell activation. Support protocols cover optimization of the assay conditions for each MAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide library, along with deconvolution approaches.

Discovery of Novel Inhibitors of a Disintegrin and Metalloprotease 17 (ADAM17) Using Glycosylated and Non-glycosylated Substrates

A disintegrin and metalloprotease (ADAM) proteases are implicated in multiple diseases, but no drugs based on ADAM inhibition exist. Most of the ADAM inhibitors developed to date feature zinc-binding moieties that target the active site zinc, which leads to a lack of selectivity and off-target toxicity. We hypothesized that secondary binding site (exosite) inhibitors should provide a viable alternative to active site inhibitors. Potential exosites in ADAM structures have been reported, but no studies describing substrate features necessary for exosite interactions exist. Analysis of ADAM cognate substrates revealed that glycosylation is often present in the vicinity of the scissile bond. To study whether glycosylation plays a role in modulating ADAM activity, a tumor necrosis factor α (TNFα) substrate with and without a glycan moiety attached was synthesized and characterized. Glycosylation enhanced ADAM8 and -17 activities and decreased ADAM10 activity. Metalloprotease (MMP) activity was unaffected by TNFα substrate glycosylation. High throughput screening assays were developed using glycosylated and non-glycosylated substrate, and positional scanning was conducted. A novel chemotype of ADAM17-selective probes was discovered from the TPIMS library (Houghten, R. A., Pinilla, C., Giulianotti, M. A., Appel, J. R., Dooley, C. T., Nefzi, A., Ostresh, J. M., Yu, Y., Maggiora, G. M., Medina-Franco, J. L., Brunner, D., and Schneider, J. (2008) Strategies for the use of mixture-based synthetic combinatorial libraries. Scaffold ranking, direct testing in vivo, and enhanced deconvolution by computational methods. J. Comb. Chem. 10, 3-19; Pinilla, C., Appel, J. R., Borràs, E., and Houghten, R. A. (2003) Advances in the use of synthetic combinatorial chemistry. Mixture-based libraries. Nat. Med. 9, 118-122) that preferentially inhibited glycosylated substrate hydrolysis and spared ADAM10, MMP-8, and MMP-14. Kinetic studies revealed that ADAM17 inhibition occurred via a non-zinc-binding mechanism. Thus, modulation of proteolysis via glycosylation may be used for identifying novel, potentially exosite binding compounds. The newly described ADAM17 inhibitors represent research tools to investigate the role of ADAM17 in the progression of various diseases.

Integrating computational and mixture-based screening of combinatorial libraries

Mixture-based synthetic combinatorial library (MB-SCL) screening is a well-established experimental approach for rapidly retrieving structure-activity relationships (SAR) and identifying hits. Virtual screening is also a powerful approach that is increasingly being used in drug discovery programs and has a growing number of successful applications. However, limited efforts have been made to integrate both techniques. To this end, we combined experimental data from a MB-SCL of bicyclic guanidines screened against the κ-opioid receptor and molecular similarity methods. The activity data and similarity analyses were integrated in a biometric analysis-similarity map. Such a map allows the molecules to be categorized as actives, activity cliffs, low similarity to the reference compounds, or missed hits. A compound with IC(50) = 309 nM was found in the "missed hits" region, showing that active compounds can be retrieved from a MS-SCL via computational approaches. The strategy presented in this work is general and is envisioned as a general-purpose approach that can be applied to other MB-SCLs.

Strategies for the Use of Mixture-Based Synthetic Combinatorial Libraries: Scaffold Ranking, Direct Testing In Vivo, and Enhanced Deconvolution by Computational Methods

ADVERTISEMENT RETURN TO ISSUEPREVAccountsNEXTStrategies for the Use of Mixture-Based Synthetic Combinatorial Libraries: Scaffold Ranking, Direct Testing In Vivo, and Enhanced Deconvolution by Computational MethodsRichard A. Houghten*†, Clemencia Pinilla†, Marc A. Giulianotti†, Jon R. Appel†, Colette T. Dooley†, Adel Nefzi†, John M. Ostresh†, Yongping Yu⊥†, Gerald M. Maggiora‡, Jose L. Medina-Franco‡, Daniela Brunner§, and Jeff Schneider§∥View Author Information Torrey Pines Institute for Molecular Studies, 3550 General Atomics Court, San Diego, California 92121, BIO5 Institute and College of Pharmacy, University of Arizona, Tucson, Arizona 85721, PsychoGenics, Inc., 765 Old Saw Mill River Road, Tarrytown, New York 10591, and School of Computer Science, Carnegie Mellon University, 5000 Forbes Avenue, Pittsburgh, Pennsylvania 15213* To whom correspondence should be addressed. Phone: 858-455-3805 . Fax: 858-455-3804. E-mail: [email protected]†Torrey Pines Institute for Molecular Studies.‡University of Arizona.§PsychoGenics, Inc.∥Carnegie Mellon University.⊥Current address: College of Pharmaceutical Science, Zijin Campus, Zhejiang University, Hangzhou 310058, P. R. China.Cite this: J. Comb. Chem. 2008, 10, 1, 3–19Publication Date (Web):December 8, 2007Publication History Received19 July 2007Published online8 December 2007Published inissue 1 January 2008https://doi.org/10.1021/cc7001205Copyright © 2008 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views1078Altmetric-Citations98LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (2 MB) SUBJECTS:Assays,Drug discovery,Mixtures,Peptides and proteins,Scaffolds Get e-Alerts

Mixture‐Based Combinatorial Libraries: from Peptides and Peptidomimetics to Small Molecule Acyclic and Heterocyclic Compounds

AbstractFor Abstract see ChemInform Abstract in Full Text.

Successful identification of novel agents to control infectious diseases from screening mixture-based peptide combinatorial libraries in complex cell-based bioassays.

Mixture-based peptide synthetic combinatorial libraries (SCLs) represent a valuable source for the development of novel agents to control infectious diseases. Indeed, a number of studies have now proven the ability of identifying active peptides from libraries composed of thousands to millions of peptides in cell-based biosystems of varying complexity. Furthermore, progressing knowledge on the importance of endogenous peptides in various immune responses lead to a regain in importance for peptides as potential therapeutic agents. This article is aimed at providing recent studies in our laboratory for the development of antimicrobial or antiviral peptides derived from mixture-based SCLs using cell-based assays, as well as a short review of the importance of such peptides in the control of infectious diseases. Furthermore, the use of positional scanning (PS) SCL-based biometrical analyses for the identification of native optimal epitopes specific to HIV-1 proteins is also presented.

Toward targeted oral vaccine delivery systems: selection of lectin mimetics from combinatorial libraries.

Various lectins bind specifically to oligosaccharides on intestinal cells. Exploiting this specificity, Ulex europaeus agglutinin I (UEA1) has been used as a ligand for targeted oral vaccine delivery to M cells (antigen-presenting cells) in follicle-associated epithelium. In this study we characterized compounds identified from mixture-based positional scanning synthetic combinatorial libraries, which mimic UEA1 and, thus, may have properties applicable to targeted drug delivery. Two UEA1 mimetics were synthesized and their activity was verified on live cells. The ability of the lead compound, a tetragalloyl D-Lysine amide construct (4-copy gallic acid construct), to deliver dye-loaded polystyrene particles to M cells was assessed in an in situ mouse gut loop model. The 4-copy gallic acid construct inhibited UEA1 binding to Caco-2 cell membranes with an IC50 of 3 microM, a 650- to 5000-fold increase over the natural UEA1 substrate alpha-L-fucose. The biotin-labeled derivative of this construct demonstrated comparable binding activity as verified on live cells by fluorescence-activated cell sorting. Preclinical studies confirmed its ability to mediate M cell-specific delivery of streptavidin-coated particles in vivo. Polyphenolic compounds, D-Lysine scaffolds with multiple galloyl groups, can mimic functional activities of UEA1. Properties of such molecules, including low molecular weight, stability, ease of synthesis and low cost, highlight their potential for application in targeted vaccine delivery.

Mixture-Based Combinatorial Libraries: From Peptides and Peptidomimetics to Small Molecule Acyclic and Heterocyclic Compounds

Publisher Summary This chapter focuses on the mixture-based combinatorial libraries. The two major techniques are generally employed in solid-phase chemistry to generate mixture-based synthetic combinatorial libraries (SCL) of millions of compounds. The techniques include “divide-couple-recombine” (DCR) method and the “reagent mixture method.” Multiple functionalities at diverse positions within the library are incorporated either by mixing multiple resins or by using mixtures of incoming reagents. In contrast to the “reagent-mixture” method, the DCR method is more labor and cost intensive, because the amount of resin and work increases proportionally with the number of building blocks incorporated at a particular position. The generation of peptidomimetic libraries by chemical modification of an existing dipeptide library is shown diagrammatically. The two requirements that have to be fulfilled for the chemical modification of an existing library are: (1) one must begin with a well-defined library, and (ii) one must use a chemical reagent that can effectively alter chemical moieties, while leaving either all of the compound mixture on the resin or alternatively removing all of the mixture from the resin.

Identification of B cell and T cell epitopes using synthetic peptide combinatorial libraries.

This unit presents a combinatorial library method that consists of the synthesis and screening of mixture-based synthetic combinatorial libraries (SCL) of peptide molecules. The protocols employ peptide SCL to identify peptides recognized by mAbs and T cells. The first protocol uses a positional scanning SCL (PS-SCL) made up of hexapeptides to identify antigenic determinants recognized by mAbs. The 120 mixtures in the hexapeptide PS-SCL are tested for their inhibitory activity in a competitive ELISA. The second protocol uses a decapeptide PS-SCL to identify T cell peptide ligands. The 200 mixtures of the decapeptide PS-SCL are tested for their ability to induce proliferation. Support protocols cover optimization of the assay conditions for each mAb or T cell, to achieve the best level of sensitivity and reproducibility, and preparation of a hexapeptide PS-SCL, along with deconvolution approaches.

Stabilization of an alpha-helical conformation in an isolated hexapeptide inhibitor of calmodulin.

The conformational properties of two hexapeptides, Ac-LWRILW-NH(2) and its D-amino acid counterpart Ac-lwrilw-NH(2), identified as calmodulin inhibitors using mixture-based synthetic combinatorial library approaches, have been characterised by NMR and CD spectroscopy. The peptides fold into an alpha-helical conformation in aqueous solution. The observed short- and medium-range nuclear Overhauser effects were consistent with the formation of an alpha-helical structure and a reasonably well-defined set of structures was obtained by using restraints from the NMR data in simulated annealing calculations. Analysis of glycine-substitution analogues demonstrated that all the amino acids that make up the peptide sequence are important for the stabilization of the alpha-helical conformation. The results suggest that a well-defined set of interactions is indispensable to allow alpha-helix formation in this short hexapeptide.

Inhibition of β-Amyloid-Induced Neurotoxicity by Imidazopyridoindoles Derived from a Synthetic Combinatorial Library

Alzheimer's disease is a progressive neurodegenerative disorder characterized by the deposit of amyloid fibrils in the brain that result from the self-aggregative polymerization of the β-amyloid peptide (Aβ). Evidence of a direct correlation between the ability of Aβ to form stable aggregates in aqueous solution and its neurotoxicity has been reported. The cytotoxic effects of Aβ have been attributed to the aggregation properties of a domain corresponding to the peptide fragment Aβ25–35. In an effort to generate novel inhibitors of Aβ neurotoxicity and/or aggregation, a mixture-based synthetic combinatorial library composed of 23 375 imidazopyridoindoles was generated and screened for inhibition of Aβ25–35 neurotoxicity toward the rat pheochromocytoma PC-12 cell line. The effect of the identified lead compounds on Aβ25–35 aggregation was then evaluated by means of circular dichroism (CD) and thioflavin-T fluorescence spectroscopy. Their activity against Aβ1–42 neurotoxicity toward the PC-12 cell line was also determined. The most active imidazopyridoindoles inhibited both Aβ25–35 and Aβ1–42 neurotoxicity in the low- to mid-micromolar range. Furthermore, inhibition of the random coil to β-sheet transition and self-aggregation of Aβ25–35 was observed by CD and fluorescence spectroscopy, supporting the relationship between inhibition of the Aβ aggregation process and neurotoxicity.

Mixture-based synthetic combinatorial libraries.

Mixture-based synthetic combinatorial libraries.

Mixture-Based Heterocyclic Combinatorial Positional Scanning Libraries: Discovery of Bicyclic Guanidines Having Potent Antifungal Activities against Candida albicans and Cryptococcus neoformans

A mixture-based synthetic combinatorial library of more than 100,000 bicyclic guanidines was generated in a positional scanning format and assayed for activity against Candida albicans. Potent individual bicyclic guanidines were directly identified following the screening of the library. Time-kill curve studies indicated bactericidal activities for the individual bicyclic guanidines. These compounds also showed potent activity against Cryptococcus neoformans. These studies demonstrate the value of using mixture-based combinatorial positional scanning libraries made up of heterocyclic compounds for the rapid identification of novel classes of antifungal compounds.

Identification of novel antitumor agents from mixture-based synthetic combinatorial libraries using cell-based assays.

A new strategy is presented here which integrates combinatorial library technology with the antitumor in vitro screening system at the National Cancer Institute in the search for novel antitumor agents. Mixture-based synthetic combinatorial libraries (SCLs) representing hundreds of thousands to millions of individual compounds were screened against the cell-based assay, which evaluates compounds for their ability to inhibit the growth of 60 different human tumor cell lines. Five different SCLs, composed of peptides, peptidomimetics, polyamines or small molecules were first tested against three cell lines to identify the most active SCLs. Two SCLs, namely the N-perbenzylated pentamine and the N-acylated permethylated triamine, were deconvoluted to yield individual compounds having significant activities against the 60 tumor cell lines. Active compounds were tested in mice to determine the maximum tolerated dose, followed by in vivo testing in a hollow fiber assay. Using this strategy, three different compounds identified directly from SCLs are currently being evaluated in human tumor xenografts. This study demonstrates for the first time the use of in vitro cell-based assays to identify antitumor lead compounds from mixture-based combinatorial libraries.