Peptides as modulators of enzymes and regulatory proteins

Troitskaya, L. A.; Kodadek, Thomas

doi:10.1016/j.ymeth.2003.10.008

Cited by 12 publications

(14 citation statements)

References 55 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…These difficulties suggest that large, functionally diverse libraries might be essential for finding unique molecules that are capable of perturbing the intracellular levels of specific protein-protein interactions. The major challenge in sifting through such vast compound pools is the shortage of functional high-throughput assays for detection of the protein complex dissociation (4).…”

mentioning

confidence: 99%

A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill

Savinov

Benkovic

2004

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

107

159

View full text Add to dashboard Cite

Discovering small-molecule modulators of protein-protein interactions is a challenging task because of both the generally noncontiguous, large protein surfaces that form these interfaces and the shortage of high-throughput approaches capable of identifying such rare inhibitors. We describe here a robust and flexible methodology that couples disruption of protein-protein complexes to host cell survival. The feasibility of this approach was demonstrated through monitoring a small-molecule-mediated protein-protein association (FKBP12-rapamycin-FRAP) and two cases of dissociation (homodimeric HIV-1 protease and heterodimeric ribonucleotide reductase). For ribonucleotide reductase, we identified cyclic peptide inhibitors from genetically encoded libraries that dissociated the enzyme subunits. A solid-phase synthetic strategy and peptide ELISAs were developed to characterize these inhibitors, resulting in the discovery of cyclic peptides that operate in an unprecedented manner, thus highlighting the strengths of a functional approach. The ability of this method to process large libraries, coupled with the benefits of a genetic selection, allowed us to identify rare, uniquely active small-molecule modulators of protein-protein interactions at a frequency of less than one in 10 million.M any regulatory processes in living organisms are often a consequence of specific protein-protein contacts, and interference with such interactions provides a means to exert control over cellular events. The de novo discovery of small molecules capable of disrupting such protein-protein complexes has been fraught with challenges, yielding very few inhibitors at a low success rate (1-3). These difficulties suggest that large, functionally diverse libraries might be essential for finding unique molecules that are capable of perturbing the intracellular levels of specific protein-protein interactions. The major challenge in sifting through such vast compound pools is the shortage of functional high-throughput assays for detection of the protein complex dissociation (4).Genetic selection is uniquely capable of identifying individual molecules with desired properties from large libraries by using whole cells as reporters and correlating host growth to a desired functional property. Unlike recently popularized affinity-based selections (5), an intracellular genetic selection can directly assay for effects on enzymatic activity or the modulation of a proteinprotein complex, thus bypassing the inherent limitations of in vitro approaches. Additionally, library members must function within the context of the entire host proteome, requiring positive candidates to have an enhanced level of selectivity for their target. This feature represents an important advantage over traditional screen-based methods in drug discovery by allowing both target affinity and selectivity to be simultaneously optimized. The application of a genetic selection to the identification of small-molecule modulators may yield both potent and selective activities as well as uniq...

show abstract

mentioning

confidence: 99%

A systematic method for identifying small-molecule modulators of protein–protein interactions

Horswill

Savinov

Benkovic

2004

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

107

159

View full text Add to dashboard Cite

show abstract

“…Peptides show great promise as modulators of protein function, and are well suited to intracellular library generation [40] . The ability to cyclize peptides inside cells with inteins extends the toolbox of in vivo peptide modulators, which includes linear peptides and peptide aptamers.…”

Section: Cyclization Of Peptidesmentioning

confidence: 99%

The Cyclization of Peptides and Proteins with Inteins

Boles

Horswill

2008

Protein Engineering Handbook

View full text Add to dashboard Cite

“…Screening libraries of small molecules, peptides, and nucleic acids has been widely used to discover ligands that bind to proteins and modulate their functions [3,4]. Peptides represent a promising class of potential enzyme modulators [5] due to their large chemical diversity [6] and the existence of well-established approaches for library synthesis [7]. Peptides and their derivatives are found to inhibit many important enzymes [8], such as dehydrogenases [9], protein kinases [10], and proteases [11].…”

Section: Introductionmentioning

confidence: 99%

Microarray Selection of Cooperative Peptides for Modulating Enzyme Activities

2017

Microarrays

View full text Add to dashboard Cite

Recently, peptide microarrays have been used to distinguish proteins, antibodies, viruses, and bacteria based on their binding to random sequence peptides. We reported on the use of peptide arrays to identify enzyme modulators that involve screening an array of 10,000 defined and addressable peptides on a microarray. Primary peptides were first selected to inhibit the enzyme at low μM concentrations. Then, new peptides were found to only bind strongly with the enzyme–inhibitor complex, but not the native enzyme. These new peptides served as secondary inhibitors that enhanced the inhibition of the enzyme together with the primary peptides. Without the primary peptides, the secondary effect peptides had little effect on the enzyme activity. Conversely, we also selected peptides that recovered the activities of inhibited enzyme–peptide complex. The selection of cooperative peptide pairs will provide a versatile toolkit for modulating enzyme functions, which may potentially be applied to drug discovery and biocatalysis.

show abstract

Peptides as modulators of enzymes and regulatory proteins

Cited by 12 publications

References 55 publications

A systematic method for identifying small-molecule modulators of protein–protein interactions

A systematic method for identifying small-molecule modulators of protein–protein interactions

The Cyclization of Peptides and Proteins with Inteins

Microarray Selection of Cooperative Peptides for Modulating Enzyme Activities

Contact Info

Product

Resources

About