High‐Throughput Platform for Discovery of Chemical Inhibitors of Heat Shock Protein 70 (Hsp70): Adaptation for the Specialized Bacterial HscA–HscB–IscU Complex

Adenosine triphosphatases (ATPases) power essential cellular processes, but they commonly achieve full activity only within multi-protein assemblies, where cofactors and partner proteins tune their function. Yet, most high-throughput screening (HTS) campaigns that target ATPases tend to use highly purified enzymes without these important partners. Here we present a high-throughput platform for discovering small-molecule modulators of the bacterial heat shock protein (Hsp70) system: HscA-HscB-IscU, a promising anti-bacterial target. In this multi-protein complex, HscA has ATPase activity that is stimulated by HscB and IscU, such that inhibitors might act at either the enzyme active site or at protein-protein interactions (PPIs). To enable discovery of such molecules, we reconstituted purified HscA, HscB, and IscU, optimized their ratios to favor the active complex and then miniaturized a quinaldine red-based, phosphate detection assay to 384-well plates (Z' = 0.68). A pilot screen of ~2000 bioactive compounds identified 253 primary hits, suggesting an abnormally high hit rate (13.6%); however, many of these signals were attributable to compound insolubility and could be triaged using aggregation-detection strategies. Collectively, this workflow establishes a scalable platform for discovering chemical probes of the HscA-HscB-IscU system. More broadly, this work provides the foundation for HTS campaigns targeting reconstituted, multi-protein complexes containing ATPase activity.