Microsystem Technology: A Powerful Tool for Biomolecular Studies

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.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTGeneration and screening of combinatorial peptide libraries designed for rapid sequencing by mass spectrometryR. Scott Youngquist, Gary R. Fuentes, Martin P. Lacey, and Thomas KeoughCite this: J. Am. Chem. Soc. 1995, 117, 14, 3900–3906Publication Date (Print):April 1, 1995Publication History Published online1 May 2002Published inissue 1 April 1995https://pubs.acs.org/doi/10.1021/ja00119a002https://doi.org/10.1021/ja00119a002research-articleACS PublicationsRequest reuse permissionsArticle Views657Altmetric-Citations201LEARN 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 InRedditEmail Other access optionsGet e-Alertsclose Get e-Alerts

Screening of a synthetic peptide combinatorial library to identify inhibitors of the appressorium formation in Magnaporthe oryzae

Synthetic combinatorial libraries (SCLs) are collections of very large numbers of synthetic compounds, in which all possible combinations of the burlding blocks used are represented. The development and verification of the utility of combinatorial libraries represent a dramatic advance in the drug discovery process by greatly reducing the time needed to identify new drug leads. Positional scanning (PS) SCLs (,) represent a modified format of the origmal synthetic combinatorial libraries described by this laboratory (). In contrast to the original libraries, which required several iterative syntheses to identify individual active compounds, this library format provides mformation on the substituent responsible for activity at each varied position withm a structure. Therefore, only a single subsequent synthesis is required. The screening of PS-SCLs, in most instances, permits the identification of the most active substituents at each position of a compound in a single assay. Thus PS-SCLs serve to reduce further the time required to identify new drug leads. PS-SCLs are composed of individual positional SCLs, in which a single position is defined with one substituent while the remaining positions are composed of mixtures of substituents. The defined position is "walked" through the entire sequence of the PS-SCL. Therefore, the number of positional SCLs is equal to the number of residues in each compound of the PS-SCL. It should also be noted that each posrtional SCL, although addressing a single position of the sequence, represents the same collection of individual compounds For example, a hexapeptide, or a compound with six positions, can be represented as: O1XXXXX, XO2,XXXX, XXO3XXX, XXO4,XX, XXXXO5X, or XXXXXO6.

We describe methodologies for the generation and screening of combinatorial libraries of electrocatalyst materials for fuel cell applications, generated by cosputtering of three elements onto a Si substrate coated with a Ta adhesion underlayer. Screening was carried out via a fluorescence assay as well as by scanning electrochemical microscopy. Whereas the former provided rapid qualitative screening with limited spatial resolution, the latter provided high spatial resolution. The fluorescence screening method was tested on Pt, PtBi, PtPb, and PtRu nanoparticles, while both methods were tested on a film containing a Pt–Bi–Pb ternary composition spread.

Multispecific antibodies are exhibiting rapid growth in drug development due to their ability to engage multiple therapeutic targets and deliver unique functional properties inaccessible to conventional monoclonal antibodies. The development of these complex modalities, however, is limited by the large design space created by the numerous possible combinations associated with multiple binding moieties and molecular formats. Here, we present a high-throughput platform for the efficient production and screening of complex combinatorial multispecific antibody libraries using intein-mediated protein splicing. To validate the fidelity of this screening approach, we demonstrate that spliced bispecific molecules exhibit indistinguishable biochemical and functional properties compared to bispecific antibodies generated through conventional production processes. We further demonstrate that intein splicing can be combined with the DuetMab platform to create trispecific antibodies and perform dual-splicing reactions to generate bivalent bispecific and trispecific formats. This work establishes intein-based protein splicing as a powerful strategy for the efficient generation and screening of multispecific antibodies libraries, enabling the rapid identification of optimal binding configurations, combinations, and molecular formats that elicit the desired mechanism of action.

A new methodology for the solid-phase synthesis of peptide-based phosphine ligands has been developed. Solid supported peptide scaffolds possessing either primary or secondary amines were synthesised using commercially available Fmoc-protected amino acids and readily available Fmoc-protected amino aldehydes for reductive alkylation, in standard solid-phase peptide synthesis (SPPS). Phosphine moieties were introduced by phosphinomethylation of the free amines as the final solid-phase synthetic step, immediately prior to complexation with palladium(II), thus avoiding tedious protection/deprotection of the phosphine moieties during the synthesis of the ligands. The extensive use of commercial building blocks and standard SPPS makes this methodology well suited for the generation of solid-phase combinatorial libraries of novel ligands. Furthermore, it is possible to generate several different phosphine ligand libraries for every peptide scaffold library synthesised, by functionalising the scaffold libraries with different phosphine moieties. The synthesised ligands were characterised on solid support by conventional (31)P NMR spectroscopy and, cleaved from the support, as their phosphine oxides by HPLC, (1)H NMR, (31)P NMR and high resolution ESMS. Palladium(II) allyl complexes were generated from the resin bound ligands and to demonstrate their catalytic properties, palladium catalysed asymmetric allylic substitution reactions were performed. Good yields and moderate enantioselectivity was obtained for the selected combination of catalysts and substrate, but most importantly the concept of this new methodology was proven. Screening of ligand libraries should afford more selective catalysts.

Peptide aptamers are combinatorial recognition molecules that consist of a constant scaffold protein displaying a doubly constrained variable peptide loop. They bind specifically target proteins and interfere with their function. We have built a peptide aptamer library in a lentiviral expression system to isolate aptamers that inhibit cell proliferation in vitro. Using one of the isolated aptamers (R5G42) as a bait protein, we have performed yeast two-hybrid screening of cDNA libraries and identified calcineurin A as a target protein candidate. R5G42 bound calcineurin A in vitro and stimulated its phosphatase activity. When expressed transiently in human cells, R5G42 induced the dephosphorylation of BAD. We have identified an antiproliferative peptide aptamer that binds calcineurin and stimulates its activity. The use of this ligand may help elucidate the still elusive structural mechanisms of activation and inhibition of calcineurin. Our work illustrates the power of phenotypic screening of combinatorial protein libraries to interrogate the proteome and chart molecular regulatory networks.

Viral hemorrhagic fevers caused by the arenaviruses Lassa virus in Africa and Machupo, Guanarito, Junin, and Sabia virus in South America are among the most devastating emerging human diseases with fatality rates of 15-35% and a limited antiviral therapeutic repertoire available. Here we used high throughput screening of synthetic combinatorial small molecule libraries to identify inhibitors of arenavirus infection using pseudotyped virion particles bearing the glycoproteins (GPs) of highly pathogenic arenaviruses. Our screening efforts resulted in the discovery of a series of novel small molecule inhibitors of viral entry that are highly active against both Old World and New World hemorrhagic arenaviruses. We observed potent inhibition of infection of human and primate cells with live hemorrhagic arenaviruses (IC(50)=500-800 nm). Investigations of the mechanism of action revealed that the candidate compounds efficiently block pH-dependent fusion by the arenavirus GPs (IC(50) of 200-350 nm). Although our lead compounds were potent against phylogenetically distant arenaviruses, they did not show activity against other enveloped viruses with class I viral fusion proteins, indicating specificity for arenavirus GP-mediated membrane fusion.

Glycosyl sulfoxides have proven useful glycosylating agents in solution and on solid support. This chapter describes the application of glycosyl sulfoxides to the synthesis of oligosaccharides on polystyrene solid supports. The synthesis concept was the basis of a combinatorial library of trisaccharides that was screened for binding to a lectin while still attached to polystyrene beads.

For Abstract see ChemInform Abstract in Full Text.

A short and efficient access to functionalized tetrahydropyrimidines (IV) proceeds under mild conditions within a few minutes and is readily amenable to parallel synthesis and generation of combinatorial libraries.

This paper summarizes our recent activities to develop analytical spectroscopic tools for high-throughput screening (HTS) of combinatorial chemistry libraries and the adaptation of the developed techniques to more traditional, i.e., laboratory and manufacturing, scales. It is shown that, for a broad variety of applications, optical spectroscopic detection methods have a suite of attractive features that make them almost ideal HTS tools. Strategies for the development of new high-throughput screening tools are presented, followed by analysis of requirements for development of multivariate data analysis methods for prediction of properties of combinatorial materials, determination of contributing factors to combinatorial-scale chemical reactions using evolving factor analysis and multivariate curve resolution chemometric methods, high- throughput optimization of process parameters, and applicability of the developed HTS tools for in-line monitoring of scaled-up reactions.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The antibody repertoires of transgenic mice expressing human heavy chain only antibodies (HCAbs) can be retrieved from immune cells after antigen challenge. Compared with genetically modified rodents expressing conventional human antibodies (tetramers consisting of two heavy chains paired with two light chains), there is no chain pairing problem, since each antibody consists of a heavy chain dimer which is solely responsible for antigen binding. HCAbs can be obtained by classical hybridoma fusion, or the generation of phage libraries or eukaryotic cell libraries displaying or secreting HCAbs. Combined transcriptomic/serum proteomic approaches can also be used to determine the repertoire of antibodies, as well as single cell technologies such as the Beacon system that enable capture of immune cells of interest, analysis, and sequencing of antibodies in a short period of time. Here, we describe a protocol for obtaining monoclonal HCAbs from immunized Harbour transgenic mice through the generation and screening of HEK cell libraries of secreted antibodies. The method can be used routinely and is fast and affordable for everyone. Selected VH regions (single domains) are sequenced and individual HCAbs can be produced and purified from the same expression vector that is used for library generation (hIgG1 Fc). They can also be cloned into other expression plasmids and reformatted to equip them with a particular effector function, modify lifespan in serum, or optimize valency and avidity depending on the specific aim.

Chapter 16 - Generation, storage, and utilizations of mutant libraries