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

Proc Natl Acad Sci U S A. 2004 Nov 2;101(44):15591-6. doi: 10.1073/pnas.0406999101. Epub 2004 Oct 21.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Carrier Proteins / chemistry
  • Carrier Proteins / genetics
  • Carrier Proteins / metabolism
  • Enzyme Inhibitors / pharmacology
  • Enzyme-Linked Immunosorbent Assay
  • HIV Protease / chemistry
  • HIV Protease / genetics
  • HIV Protease / metabolism
  • In Vitro Techniques
  • Peptide Library
  • Peptides, Cyclic / pharmacology
  • Phosphotransferases (Alcohol Group Acceptor)
  • Protein Binding
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonucleotide Reductases / antagonists & inhibitors
  • Ribonucleotide Reductases / chemistry
  • Ribonucleotide Reductases / genetics
  • Ribonucleotide Reductases / metabolism
  • Sirolimus / chemistry
  • Sirolimus / metabolism
  • TOR Serine-Threonine Kinases
  • Tacrolimus Binding Protein 1A / chemistry
  • Tacrolimus Binding Protein 1A / genetics
  • Tacrolimus Binding Protein 1A / metabolism
  • Two-Hybrid System Techniques

Substances

  • Carrier Proteins
  • Enzyme Inhibitors
  • Peptide Library
  • Peptides, Cyclic
  • Proteins
  • Recombinant Proteins
  • Ribonucleotide Reductases
  • Phosphotransferases (Alcohol Group Acceptor)
  • TOR Serine-Threonine Kinases
  • HIV Protease
  • Tacrolimus Binding Protein 1A
  • Sirolimus