Development of Isoform-Selective GRP78 Inhibitors from a Direct-to-Biology Strategy.

The development of isoform-selective HSP70 inhibitors remains challenging due to the high conservation of this chaperone family. Continuing our efforts to target the peptide-binding pocket within the substrate-binding domain (SBD) of GRP78 (BiP/HSPA5), we identified compound 12 using a Direct-to-Biology (D2B) strategy. Biochemical, biophysical, and cellular characterization showed that 12 directly engages the GRP78 peptide-binding pocket, selectively binds GRP78 over other tested HSP70 isoforms, and potently kills A549 lung cancer cells while showing minimal activity in other tested lung cancer cell lines. Cellular thermal shift assay (CETSA) demonstrated GRP78 engagement in cells, and unfolded protein response (UPR) signaling analysis further showed that 12 modulates GRP78-related stress-response pathways, supporting its functional engagement of GRP78. Collectively, compound 12 represents a well-characterized, isoform-selective, cell active, and cell selective GRP78 inhibitor, providing a valuable chemical probe for dissecting GRP78 biology and a promising starting point for the development of GRP78-targeted therapeutics.