A systematic method for identifying small-molecule modulators of protein-protein interactions.
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.
Horswill
Alexander R
AR
Department of Chemistry, Pennsylvania State University, 414 Wartik Laboratory, University Park, PA 16802, USA.
Savinov
Sergey N
SN
Benkovic
Stephen J
SJ
D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004797:Enzyme-Linked Immunosorbent Assay; D016333:HIV Protease / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D019151:Peptide Library; D010456:Peptides, Cyclic / Q000494:pharmacology; D017853:Phosphotransferases (Alcohol Group Acceptor); D011485:Protein Binding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020123:Sirolimus / Q000737:chemistry / Q000378:metabolism; D058570:TOR Serine-Threonine Kinases; D022061:Tacrolimus Binding Protein 1A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D002352:Carrier Proteins; D004791:Enzyme Inhibitors; D019151:Peptide Library; D010456:Peptides, Cyclic; D011506:Proteins; D011994:Recombinant Proteins; D012264:Ribonucleotide Reductases; D017853:Phosphotransferases (Alcohol Group Acceptor); D058570:TOR Serine-Threonine Kinases; D016333:HIV Protease; D022061:Tacrolimus Binding Protein 1A; D020123:Sirolimus