Inhibition of constitutively active G s α by molecules targeted at R231

Activating mutations in G s α are responsible for a variety of human pathologies, including polyostotic fibrous dysplasia, McCune‐Albright Syndrome, and a variety of endocrine tumors. We hypothesized that designing a molecule that preferentially binds to the mutated form of G s α could provide the starting point for drug development and better therapy for these diseases. Previous work in our lab identified the region near R231 as a potential druggable target site on the mutated version of the protein, as mutations at this residue reduced the constitutive activity associated with mutations at R201. Utilizing GNAS E2−/E2− cells, R201H/R231C G s α was found to have similar basal activity and adenylyl cyclase coupling (forskolin‐mediated activity) as wild‐type G s α, and significantly reduced isoproterenol‐mediated cAMP production. In silico docking analysis provided a slate of candidate molecules to test for inhibition of the MAS protein's constitutive activation of adenylyl cyclase. Two molecules, 6‐oxo‐ N ‐{[2‐(pyrrolidin‐1‐yl)pyridin‐4‐yl]methyl}‐1,6‐dihydropyridazine‐3‐carboxamide and N ‐{[3‐(3‐methylphenyl)phenyl]methyl}‐6‐oxo‐1,6‐dihydropyridazine‐3‐carboxamide were able to significantly and in a dose‐dependent manner reduce the elevated basal cAMP levels in HEK cells transiently transfected with the constitutively active G s ‐R201H allele. Neither compound changed forskolin‐driven cAMP production, indicating that the effects are not downstream of the G‐protein. Maximum levels of cAMP production in response to luteinizing hormone receptor activation were also unchanged; however, a 10‐fold increase in the EC 50 for hormone was measured in the presence of both drugs. Support or Funding Information This work was supported in part by NIH grant 1R15ED020190‐01