High-throughput screen for inhibitors of protein–protein interactions in a reconstituted heat shock protein 70 (Hsp70) complex

• Published in: The Journal of biological chemistry Vol. 293; no. 11; pp. 4014 - 4025
• Main Authors: Taylor, Isabelle R., Dunyak, Bryan M., Komiyama, Tomoko, Shao, Hao, Ran, Xu, Assimon, Victoria A., Kalyanaraman, Chakrapani, Rauch, Jennifer N., Jacobson, Matthew P., Zuiderweg, Erik R.P., Gestwicki, Jason E.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.03.2018; American Society for Biochemistry and Molecular Biology
• Subjects: Adaptor Proteins, Signal Transducing - antagonists & inhibitors; Adenosine Triphosphatases - metabolism; Apoptosis Regulatory Proteins - antagonists & inhibitors; Binding Sites; chemical biology; Crystallography, X-Ray; Drug Discovery; Drug Evaluation, Preclinical; high-throughput screening (HTS); High-Throughput Screening Assays; HSP40 Heat-Shock Proteins - antagonists & inhibitors; HSP70 Heat-Shock Proteins - antagonists & inhibitors; Hsp70, heat shock protein; Humans; inhibition mechanism; inhibitor; molecular chaperone; Multiprotein Complexes - antagonists & inhibitors; Multiprotein Complexes - metabolism; Pharmaceutical Preparations - metabolism; Protein Binding; protein complex; Protein Interaction Maps - drug effects; Protein Structure and Folding; protein–protein interaction; inhibition mechanism; molecular chaperone; high-throughput screening (HTS); chemical biology; inhibitor; Hsp70, heat shock protein; protein–protein interaction; protein complex
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.RA117.001575

Protein–protein interactions (PPIs) are an important category of putative drug targets. Improvements in high-throughput screening (HTS) have significantly accelerated the discovery of inhibitors for some categories of PPIs. However, methods suitable for screening multiprotein complexes (e.g. those composed of three or more different components) have been slower to emerge. Here, we explored an approach that uses reconstituted multiprotein complexes (RMPCs). As a model system, we chose heat shock protein 70 (Hsp70), which is an ATP-dependent molecular chaperone that interacts with co-chaperones, including DnaJA2 and BAG2. The PPIs between Hsp70 and its co-chaperones stimulate nucleotide cycling. Thus, to re-create this ternary protein system, we combined purified human Hsp70 with DnaJA2 and BAG2 and then screened 100,000 diverse compounds for those that inhibited co-chaperone–stimulated ATPase activity. This HTS campaign yielded two compounds with promising inhibitory activity. Interestingly, one inhibited the PPI between Hsp70 and DnaJA2, whereas the other seemed to inhibit the Hsp70–BAG2 complex. Using secondary assays, we found that both compounds inhibited the PPIs through binding to allosteric sites on Hsp70, but neither affected Hsp70’s intrinsic ATPase activity. Our RMPC approach expands the toolbox of biochemical HTS methods available for studying difficult-to-target PPIs in multiprotein complexes. The results may also provide a starting point for new chemical probes of the Hsp70 system.