GRP78 Selective Inhibitors From a Direct‐to‐Biology Strategy

Because cancer cells have heightened protein homeostasis (proteostasis) requirements, there is interest in targeting proteostasis machinery, including the 70 kDa heat shock proteins (HSP70s), as potential cancer therapeutics. However, studies have shown that the HSP70 family is differentially regulated across cancers, and global targeting may produce unwanted toxicities. For this reason, our lab has focused on isoform-selective targeting of HSP70s, including the endoplasmic reticulum-resident HSP70, GRP78 (HSPA5 or BiP). GRP78 is a central component of protein homeostasis in the secretory system and is the principal regulator of the unfolded protein response (UPR). Here, we report the use of a direct-to-biology (D2B) strategy to optimize a dipeptide-based scaffold that binds selectively to GRP78, relative to the other canonical HSP70s. We show that our lead compound, 12, potently and selectively inhibits GRP78, binds to the substrate binding pocket, kills A549 lung cancer cells in 2D (grown as a monolayer) and 3D (grown as spheroids) cultures, engages GRP78 in cells, and that GRP78 inhibition is responsible for the mode of action. This work represents the first GRP78-selective inhibitor that inhibits substrate binding.