Antagonists Against Drug-resistance On Smoothened Receptor: Mechanism Exploration And Optimization

Smoothened receptor(SMO)belongs to Class F G protein-coupled receptors(GPCRs)family.It plays a vital role in the Hedgehog(Hh)signaling pathway,and its dysregulation has been related to muptiple cancers.SMO has therefore been an important target for the development of anti-cancer therapeutics.Up to date,there have been three FDA approved drugs targeting SMO.However,soon after the clinical application of some drugs,severe drug resistance occurred,which mainly resulted from the mutation of some key residues near the drug binding site.To circonvent the drug resistance,it would be necessary to develop new antagonists based on new mechanism.Allo-1,an imidazolidinedione ligand,exhibits high inhibition on both wild-type and drug-reisitant mutant SMO with unknown mechanism.We developed a novel colocalization strategy using two complementary labeling photoaffinity probes derived from Allo-1.Combining the evidence from mass spectrometry,medicinal chemistry and computational biology,we identified an underneath binding pocket in the transmembrane domain of SMO,which is far away from the key residue responsible for the drug resistance on SMO.We then further rationally designed and synthesized three types of Allo-1 analogs by the “dissection-then-evolution” strategy.We explored the halogen bond interaction between the cholorine of Allo-1 and its adjacent residues.We also introduced a second halogen bond between Allo-1 and a residue at the bottom of the binding pocket.Finally,we obtained more potent ligands with 10-fold improvement.Same strategy was also applicable to other SMO anatagonists.In summary,we developed a novel colocalization photoaffinity labeling strategy,with which a new binding site was identified for SMO.Further optimization by rational introduction of halogen bond on Allo-1 and other ligands yielded improved antagonists.