Journal
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
Volume 101, Issue 44, Pages 15591-15596Publisher
NATL ACAD SCIENCES
DOI: 10.1073/pnas.0406999101
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Funding
- NIGMS NIH HHS [R37 GM024129, R01 GM024129, GM 24129] Funding Source: Medline
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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.
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