Novel-smoothened inhibitors for therapeutic targeting of naïve and drug-resistant hedgehog pathway-driven cancers

The G protein-coupled receptor (GPCR) smoothened (SMO) is a key signaling component of the sonic hedgehog (Hh) pathway and a clinically validated target for cancer treatment. The FDA-approved SMO inhibitors GDC-0449/Vismodegib and LDE225/Sonidegib demonstrated clinical antitumor efficacy. Nevertheless, relatively high percentage of treated patients would eventually develop acquired cross resistance to both drugs. Here, based on published structure and activity of GDC-0449 inhibitor class, we replaced its amide core with benzimidazole which retained bulk of the SMO-targeting activity as measured in our Hh/SMO/Gli1-reporter system. Synthesis and screening of multiple series of benzimidazole derivatives identified HH-1, HH-13, and HH-20 with potent target suppression (IC₅₀: <0.1 μmol/L) in the reporter assays. In NIH3T3 cells stimulated with a secreted Hh (SHH), these inhibitors dose dependently reduced mRNA and protein expression of the endogenous pathway components PTCH-1, Gli1, and cyclin D1 resulting in growth inhibition via G₀/G₁ arrest. Mechanistically, the SMO-targeted growth inhibition involved downregulation of mTOR signaling inputs and readouts consistent with diminished mTORC1/mTORC2 functions and apoptosis. In mice, as with GDC-0449, orally administered HH inhibitors blocked paracrine activation of stromal Hh pathway in Calu-6 tumor microenvironment and attenuated growth of PTCH^+/−/P53^−/− medulloblastoma allograft tumors. Furthermore, HH-13 and HH-20 potently targeted the drug-resistant smoothened SMO-D473H (IC₅₀: <0.2 μmol/L) compared to the poor inhibition by GDC-0449 (IC₅₀: >60 μmol/L). These results identify HH-13 and HH-20 as potent inhibitors capable of targeting naïve and drug-resistant Hh/SMO-driven cancers. The current leads may be optimized to improve pharmaceutical property for potential development of new therapy for treatment of Hh pathway-driven cancers.