MRT-92 inhibits Hedgehog signaling by blocking overlapping binding sites in the transmembrane domain of the Smoothened receptor

The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [3H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmicproximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance