Development Of Tool Compounds For The Study Of The Smoothened Receptor

The Smoothened receptor(SMO)belongs to the Class Frizzled of the G proteincoupled receptor(GPCR)superfamily,constituting a key component of the Hedgehog(Hh)signaling pathway.Disruption of this pathway results in the disorder of embryonic development and tissue regeneration that are related to birth defects or cancers,especially basal cell carcinoma(BCC)and medulloblastoma(MB).Thus SMO has been a well pursued and now validated target for anticancer agent developments.Vismodegib(GDC-0449)and sonidegib(LDE-225)have been approved by FDA in 2012 and 2015 respectively for the treatment of BCC.However,the clinical application of these two drugs strictly limited to adult BCC patients due to many side effects and drug resistance.Therefore,our group aims to develop tool compounds for the further functional study and drug discovery of SMO.We designed a super-stabilizing tool ligand TC114 based on LY2940680 that further settle down the conformation and facilitate the crystalizing for structural study of multi-domain SMO at 2.9 ? using an X-ray free-electron laser source.This multidomain structure contains a 7-transmembrane helices domain(TMD),a hinge domain(HD),and an intact extracellular cysteine-rich domain(CRD).This architecture is important for the study of allosteric interactions and signaling pathway between the domains.Moreover,TC114 was identified as potent,with an IC50 value of 1.6 nM against the Hh signaling pathway.TC114 also showed inhibitory effect stimulated by ectopic expression of D473 H which may cause resistance to the clinically approved SMO antagonists.Due to the limited druggability of TC114 with a nitro group on aromatic ring,we further optimized LY2940680 guided with the co-crystallization structure of SMO and TC114.Several LY2940680 analogues were designed and synthesized for the study of structure activity relationship.The compound TC658 with phenyl ether extension on benzoyl was discovered that was identified as a pontent antagonist with IC50 value of 1.0 nM.This highly improved and more drug-like ligand showed enhanced stabilization and improved activity on SMO compared with LY2940680.This stragety can be applied to drug discovery for resistance by exploring the new interaction between TC658 and SMO.To further study the function of SMO,we designed a new fluorogenic probe TC618 combined with LY2940680 and a fluorescence group NBD that can selectively label K395 of SMO using ligand-directed chemistry.This method can also be applied in cell imaging to observe the motion and location of the fluoresence labeled SMO in living cells.In this project,we developed several tool compounds for the functional study of SMO and these compounds can also be applied in disease mechanism study and drug discovery.