A Systematic Method for Identifying Small-Molecule Modulators of Protein-Protein Interactions

• Published in: Proceedings of the National Academy of Sciences - PNAS 2004-11, Vol.101 (44), p.15591-15596
• Main Authors: Horswill, Alexander R., Savinov, Sergey N., Benkovic, Stephen J.
• Format: Article
• Language: English
• Subjects: Biochemistry; Biological Sciences; Carrier Proteins - chemistry; Carrier Proteins - genetics; Carrier Proteins - metabolism; Cell growth; Cyclic peptides; Enzyme Inhibitors - pharmacology; Enzyme-Linked Immunosorbent Assay; Enzymes; Genetic selection; HIV Protease - chemistry; HIV Protease - genetics; HIV Protease - metabolism; Human immunodeficiency virus 1; In Vitro Techniques; Journalism; Libraries; Methods; Molecules; Peptide Library; Peptides, Cyclic - pharmacology; Phosphotransferases (Alcohol Group Acceptor); Plasmids; Protein Binding; Proteins; Proteins - chemistry; Proteins - genetics; Proteins - metabolism; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Resins; Ribonucleotide Reductases - antagonists & inhibitors; Ribonucleotide Reductases - chemistry; Ribonucleotide Reductases - genetics; Ribonucleotide Reductases - metabolism; Sirolimus - chemistry; Sirolimus - metabolism; Tacrolimus Binding Protein 1A - chemistry; Tacrolimus Binding Protein 1A - genetics; Tacrolimus Binding Protein 1A - metabolism; TOR Serine-Threonine Kinases; Two-Hybrid System Techniques
• ISSN: 0027-8424; 1091-6490
• DOI: 10.1073/pnas.0406999101

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.