Identification of an Allosteric Small-Molecule Inhibitor Selective for the Inducible Form of Heat Shock Protein 70

Inducible Hsp70 (Hsp70i) is overexpressed in a wide spectrum of human tumors, and its expression correlates with metastasis, poor outcomes, and resistance to chemotherapy in patients. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. In this work, we used fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify HS-72, an allosteric inhibitor selective for Hsp70i. HS-72 displays the hallmarks of Hsp70 inhibition in cells, promoting substrate protein degradation and growth inhibition. Importantly, HS-72 is selective for Hsp70i over the closely related constitutively active Hsc70. Studies with purified protein show HS-72 acts as an allosteric inhibitor, reducing ATP affinity. In vivo HS-72 is well-tolerated, showing bioavailability and efficacy, inhibiting tumor growth and promoting survival in a HER2+ model of breast cancer. The HS-72 scaffold is amenable to resynthesis and iteration, suggesting an ideal starting point for a new generation of anticancer therapeutics targeting Hsp70i. [Display omitted] •HS-72 is an allosteric inhibitor selective for the inducible form of Hsp70•HS-72 discriminates Hsp70i from other Hsp70 family members including Hsc70•HS-72 shows the hallmarks of a selective Hsp70i inhibitor in multiple cell models•HS-72 is well tolerated, bioavailable, and shows efficacy in vivo Howe et al. identify a small-molecule inhibitor that is highly selective for inducible Hsp70. The inhibitor, called HS-72, shows hallmarks of Hsp70 inhibition in vitro as well as bioavailability and efficacy in a mouse model of HER2+ breast cancer.