Inhibition of adenylyl cyclase isoform 6 by cysteine nitrosylation in hypoxic pulmonary hypertension and rescue with introducing novel forskolin derivatives

Abstract

Disruption of adenylyl cyclase (AC)–cAMP signaling is a hallmark of hypoxic cardiopulmonary diseases, including persistent pulmonary hypertension of the newborn (PPHN). Among AC isoforms, AC6 is highly expressed in the pulmonary circuit, followed by AC3, AC7, and AC9. I hypothesized that [1] hypoxia inhibits AC6 via S-nitrosylation; [2] this inhibition can be rescued by novel forskolin derivatives that selectively target this isoform. I characterized activity and regulation of AC isoforms in pulmonary artery myocytes under chronic hypoxic conditions, using integrated in vitro, ex vivo, and in vivo models. The overarching objective of this research was to identify the AC isoform responsible for impaired cAMP signaling under hypoxia, determine redox-dependent mechanisms regulating AC6 activity, and develop novel AC6-selective forskolin derivatives to restore AC6 function. Hypoxia (10% O2; 72h) selectively inhibited AC6 activity, and decreased AC6-mediated cAMP. Forskolin partially restored hypoxic AC6 activity. Hypoxia promoted generation of nitrosylating species, which induce S-nitrosylation of AC6. Among multiple cysteine residues in AC6, we identified C1004, located at the docking interface with stimulatory protein Gαs, as the critical site mediating hypoxia-induced S-nitrosylation. Mutation of C1004 to alanine abolished hypoxic inhibition of AC6, by preventing its S-nitrosylation. Hypoxia increased myocyte superoxide and peroxynitrite production, enhanced xanthine oxidase activity, elevated the NADP⁺/NADPH ratio, and promoted eNOS uncoupling, collectively favoring conditions that drive protein S-nitrosylation over bioavailable NO production. Treatment with superoxide dismutase or peroxynitrite scavenger attenuated protein S-nitrosylation, partially restored AC activity and pulmonary vasoconstriction, confirming the link between redox imbalance and AC6 dysfunction. The non-specificity of forskolin highlighted the need for more selective AC6-targeted activators. To address AC6 impairment, I designed, synthesized and tested a series of novel forskolin derivatives, including a 1,9-cyclic thiocarbonate derivative (C13) which displayed favorable physicochemical properties, and selectively enhanced AC6 activity among all examined isoforms, increasing cAMP production and pulmonary vasodilation. Overall, this work delineates the mechanism by which hypoxia impairs AC6 activity via site-specific S-nitrosylation, and establishes an innovative pharmacological strategy for developing selective AC6-targeted therapeutics for cardiopulmonary diseases.