Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery

Houghten, Richard A.; Pinilla, Clemencia; Blondelle, Sylvie E.; Appel, Jon R.; Dooley, Colette T.; Cuervo, Julio H.

doi:10.1038/354084a0

Cited by 1,320 publications

(691 citation statements)

References 18 publications

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“…This antibody specifically binds with high affinity (K d 7.1 nM) to the N-terminal sequence YGGF (singleletter amino acid designation) of the ␤-endorphin protein (18). An octa-peptide library containing all 20 naturally occurring amino acids as building blocks was synthesized and screened for binding to the 3-E7 antibody by recursive deconvolution (19,20). In subsequent screening rounds the preferred amino acid at each position was determined and used to deduce an optimal binding motif.…”

Section: Resultsmentioning

confidence: 99%

Specificity and affinity motifs for Grb2 SH2-ligand interactions

Kessels

Ward

Schumacher

2002

Proc. Natl. Acad. Sci. U.S.A.

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Protein-protein interactions are often mediated by the recognition of short continuous amino acid stretches on target proteins by specific binding domains. Affinity-based selection strategies have successfully been used to define recognition motifs for a large series of such protein domains. However, in many biological systems specificity of interaction may be of equal or greater importance than affinity. To address this issue we have developed a peptide library screening technology that can be used to directly define ligands for protein domains based on both affinity and specificity of interaction. We demonstrate the value of this approach by the selection of peptide ligands that are either highly specific for the Grb2 Src homology 2 (SH2) domain or that are cross-reactive between a group of related SH2 domains. Examination of previously identified physiological ligands for the Grb2 SH2 domain suggests that for these ligands regulation of the specificity of interaction may be an important factor for in vivo ligand selection.T he intracellular organization that enables a cell to respond to external stimuli consists of a complex web of signal transduction pathways. Key factors in the regulation of cellular signaling are the protein binding domains-small, conserved protein modules that mediate intracellular protein-protein interactions. Many of these domains, such as the families of SH2 (Src homology 2), SH3 (Src homology 3), PTB (phosphotyrosine binding), or PDZ (postsynaptic density-95͞Discs large͞zona occludens-1) domains, use short peptide sequences for ligand recognition (1). For example, the binding of SH2 domains to target proteins involves the recognition of a phosphorylated tyrosine residue, and specificity of individual SH2 domains is mediated by the recognition of amino acid residues immediately C-terminal to the phospho-tyrosine (2). The binding preferences of SH2 domains have been studied extensively through the use of peptide libraries, and predictions for the optimal binding motifs for a large number of SH2 domains have been obtained in this manner (3,4). In addition, such binding motifs have been used extensively as lead structures for the design of selective small-molecular SH2 inhibitors (5, 6). Importantly, in traditional library screening strategies used to define SH2 ligand motifs the selection of ligands is exclusively based on the strength of the SH2-phospho-peptide interaction. Consequently, the motifs that are identified in this manner describe ligands with an optimal affinity for a given SH2 domain (here named affinity motifs). Because both affinity and specificity of protein interactions are controlled by the same thermodynamic factors (shape and charge complementarity in the ground state), the selection of high affinity ligands will often also result in the selection of highly specific ligands. However, it has previously been argued that for closely related targets (such as the families of SH2 domains and other signal transduction modules) affinity-based selections may result in the ide...

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Section: Resultsmentioning

confidence: 99%

Specificity and affinity motifs for Grb2 SH2-ligand interactions

Kessels

Ward

Schumacher

2002

Proc. Natl. Acad. Sci. U.S.A.

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“…15 In this approach one amino acid position is held constant while the others are varied. The Fmoc-Glu(EDANS)-Gly-Wang or Fmoc-Glu(EDANS)-Ala-Wang resin was divided into 20 equal portions, and each portion was coupled to an individual amino acid followed by coupling with an isokinetic mixture of eighteen amino acids.…”

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Substrate specificity and screening of the integral membrane protease Pla

Agarkov

Chauhan

Lory

et al. 2008

Bioorganic & Medicinal Chemistry Letters

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This paper reports a study to find small peptide substrates for the important virulence factor of Yersinia pestis, plasminogen activator, Pla. The method used to find small substrates for this protease is reported along with studies examining the ability of these peptides to inhibit activity of the enzyme. Through the use of parallel synthesis and positional scanning, small tripeptides were identified that are viable substrates for the protease. KeywordsYersinia pestis; plasminogen activator; protease substrate; peptide inhibitor; positional scan, parallel synthesis Yersinia pestis, the agent of plague, has been recognized as one of the most devastating, epidemic-causing bacteria experienced by mankind. 1 Typically, Y. pestis is transmitted to humans via a bite from a flea that previously fed on an infected rodent. From the initial site of infection, bacteria disseminate to the draining lymph node, causing swelling of this lymph node to form a bubo (bubonic plague).Y. pestis contains a unique, 9.5-kb plasmid pPCP that expresses plague plasminogen activator (Pla) which is responsible for fibrinolytic and coagulase activities. 2 Pla expression is associated with the marked ability of Y. pestis to colonize the vicera and thus cause lethal infection upon administration by peripheral (i.e. intradermal (i.d.), subcutaneous (s.c.) or intraperitoneal) routes of infection. 3 The importance of Pla for plague pathogenesis was verified with isogenic Pla mutants of epidemic Y. pestis strains KIM and CO92, which showed an up to 10 6 fold reduced virulence by the s.c. routes. 3,4 Recently, it was confirmed that bubonic plague, but not septicemic, depends on Pla to facilitate the rapid dissemination of bacteria from the site of i.d. infection. 5 Moreover, it was shown that Pla allows Y. pestis to replicate rapidly in the airways, thus playing the essential role in causing pneumonic plague. 6 The invasive properties of Pla which is a cell surface-located protease are likely due to the ability of this enzyme to induce fibrinolysis and degrade extracellular matrix and basement membranes. 7,8 The *Corresponding authors. E-mails: srgilber@utmb.edu and vlmotin@utmb.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. activation of plasminogen to plasmin while degrading the plasmin inhibitor α2-antiplasmin by Pla is the mechanism used by Y. pestis to progress from the peripheral sites into the circulation and systemic infection. 9,10 This paper reports work to identify inhibitors of Pla through parallel synthesis of small peptidic substrates for the enzyme. NIH Public AccessOne of the c...

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“…In contrast, the pool peptide libraries rely on the comparison of binding affinities of different pools of random peptides to obtain binding consensus motifs (12). Thus, the sequences of individual bound peptides do not need to be resolved.…”

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confidence: 99%

An Oriented Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions

Rodrı́guez

Harper

et al. 2004

Journal of Biological Chemistry

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One of the major questions in signal transduction is how the specificities of protein-protein interactions determine the assembly of distinct signaling complexes in response to stimuli. Several peptide library methods have been developed and widely used to study proteinprotein interactions. These approaches primarily rely on peptide or DNA sequencing to identify the peptide or consensus motif for binding and may prove too costly or difficult to accommodate high throughput applications. We report here an oriented peptide array library (OPAL) approach that should facilitate high throughput proteomic analysis of protein-protein interactions. OPAL integrates the principles of both the oriented peptide libraries and array technologies. Hundreds of pools of oriented peptide libraries are synthesized as amino acid scan arrays. We demonstrate that these arrays can be used to map the specificities of a variety of interactions, including antibodies, protein domains such Src homology 2 domains, and protein kinases.Signaling proteins are often composed of distinct protein domains or modules. Some of these serve as protein-protein interaction units, whereas others carry out enzymatic reactions (1). The elucidation of the genetic codes of various organisms, including humans, has presented an increasing number of protein domain families with unknown functions.To understand how signaling proteins interact with each other and maintain the fidelity of signal transduction, it is critical to determine the specificities of these protein domains. Protein domains (such as SH2, 1 SH3, and phosphotyrosine binding (PTB) domains) often mediate protein-protein interactions by recognizing short stretches of peptide sequences on target proteins (2, 3). In the past several years, many combinatorial peptide library based approaches have been developed to analyze the binding or substrate specificities of protein domains (reviewed in Ref. 4). These peptide libraries can be classified as either display or pool peptide libraries.The display libraries employ a variety of matrices for individual peptide presentation, including pin (5), phage (6 -8), beads (9), DNA binding proteins (10), or ribosomes (11). Following multiple rounds of affinity enrichment steps, the identities of the selected peptides can be ascertained by DNA or peptide sequencing (4). Display peptide libraries generally require isolation and sequencing of individual peptides to decode a binding motif, which is time-consuming and costly, limiting it to low throughput applications.In contrast, the pool peptide libraries rely on the comparison of binding affinities of different pools of random peptides to obtain binding consensus motifs (12). Thus, the sequences of individual bound peptides do not need to be resolved. A few years ago, we developed an oriented peptide library method to further simplify and facilitate screening (13). In this method, soluble pools of random peptides are oriented via a central "fixed" amino acid, which reduced the degeneracy of the library and prevented po...

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Generation and use of synthetic peptide combinatorial libraries for basic research and drug discovery

Cited by 1,320 publications

References 18 publications

Specificity and affinity motifs for Grb2 SH2-ligand interactions

Specificity and affinity motifs for Grb2 SH2-ligand interactions

Substrate specificity and screening of the integral membrane protease Pla

An Oriented Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions

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