Abstract
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.
Highlights
Protein–protein interactions (PPIs) are an important category of putative drug targets
Pilot medicinal chemistry campaigns, combined with secondary assays, showed that one compound targeted the heat shock protein 70 (Hsp70) –DnaJA2 complex, and the other inhibited Hsp70 –BAG2. These findings suggest that reconstituted multiprotein complexes (RMPCs) present a tractable, biochemical approach for the discovery of inhibitors of even difficult PPIs within multiprotein systems, adding to the “toolbox” of methods for chemical biology
Our search for inhibitors of PPIs within the Hsp72–DnaJA2– BAG2 system was guided by three major design ideas as follows: (a) to avoid discovery of nucleotide-competitive molecules, we saturated the cleft using high concentrations of ATP (1 mM) in the screening buffer; (b) DnaJA2 and BAG2 were added at concentrations that produced half-maximal stimulation of ATP hydrolysis to improve the sensitivity of the assay to potential inhibitors and activators [17]; and (c) we combined all three proteins (Hsp72, DnaJA2, and BAG2) into the same wells to re-create native cycling conditions
Summary
Protein–protein interactions (PPIs) are an important category of putative drug targets. The interaction of Hsp with the BAG family of NEFs is relatively tight (mid-nanomolar), but the contact area is large and occurs over multiple subdomains of Hsp70’s NBD [9] This ternary system is emblematic of modern, multiprotein drug targets and includes multiple categories of difficult PPIs. We envisioned a strategy in which measuring the ATP cycling of this three-protein system might provide a readout for each of the PPIs. The intrinsic hydrolysis activity of Hsp in the absence of its co-chaperones is slow (5 ϫ 10Ϫ4 sϪ1) [6, 7], such that most of the measured signal would be expected to emerge as a direct result of co-chaperone interactions. It would be expected that only a full complement of co-chaperones would sample the full range of conformational states that normally occur under physiological conditions
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