101 - Adenylyl Cyclase Isoform 6 Allosteric Binding Site Can Be Selectively Targeted by Forskolin Derivatives: A Potential Therapeutic Option for Pulmonary Hypertension

Monday, May 1, 2023

9:30 AM – 11:30 AM ET

Poster Number: 101
Publication Number: 101.426

VIKRAM BHATIA, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, WINNIPEG, MB, Canada; Alexander Pritchard, University of Manitoba, Winnipeg, MB, Canada; Saeid Maghsoudi, University of Manitoba, Winnipeg, MB, Canada; Martha Hinton, University of Manitoba, Winnipeg, MB, Canada; Nisha Singh, University of Manitoba, Winnipeg, MB, Canada; John L. Sorensen, University of Manitoba, Winnipeg, MB, Canada; Prashen Chelikani, University of Manitoba, Winnipeg, MB, Canada; Shyamala Dakshinamurti, Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba, Winnipeg, MB, Canada

Presenting Author(s)

VB

VIKRAM BHATIA, PhD (he/him/his)

Research Associate
Max Rady College of Medicine, Rady Faculty of Health Sciences, University of Manitoba
WINNIPEG, Manitoba, Canada

Background:

Adenylyl cyclase (AC) is crucial for pulmonary vasodilation in persistent pulmonary hypertension of the newborn (PPHN). Hypoxia inhibits AC activity due to cysteine nitrosylation of AC6. Forskolin (FSK) is a non-selective activator of AC isoforms that binds at the allosteric site to stimulate ATP catalysis. The only available three-dimension structure for AC is AC9, a forskolin-insensitive isoform. By computational modeling and mutational analysis, we sought to define the forskolin binding site and interacting residues of AC6, the main pulmonary AC isoform. We then analyzed the activity of a series of FSK derivatives against AC isoforms 3, 5, 6, 7, and 9 to determine whether selective activation of AC6 is feasible.

Objective:

We proposed to define the AC6 FSK allosteric binding site to determine if forskolin can be derivatized to optimize its interaction with this hydrophobic pocket, resulting in selective reactivation of this AC isoform and cAMP generation in PPHN pulmonary arteries.

Design/Methods: AC6 structure was built by a homology modeling approach. Using a site-directed mutagenesis approach, we validated the structural model and roles of the forskolin-interacting residues of the AC6 allosteric pocket identified by ligand docking. AC catalytic activity was measured using terbium norfloxacin fluorescence assay. Forskolin-dependent real-time cAMP generation was measured using a live cell cAMP biosensor assay. A library of forskolin derivatives was synthesized by modifications at C1, C6, C7, C9, C11, and/or C14/15 positions and screened against AC isoforms 3, 5, 6, 7, or 9 stably expressed in HEK293T. Compounds exhibiting AC6 selectivity underwent dose-response testing.

Results: AC6 catalytic activity is uniquely inhibited by hypoxia and by nitrosyl donor S-nitrosocysteine (CSNO). Homology modeling and docking revealed residues T500, N503, and S1035 interacting with forskolin; substitution mutation of T500, N503, or S1035 had decreased forskolin-stimulated AC activity compared to WT AC6 in all conditions. Forskolin site mutants were not further inhibited by hypoxia or CSNO. cAMP dynamics showed forskolin unresponsive in all mutants. Among forskolin derivatives tested, three offered selective activation of AC6.

Conclusion(s):

Hypoxia inhibits AC6, but forskolin stimulation of hypoxic AC6 catalytic activity is feasible. FSK-interacting amino acids are not involved in the hypoxic inhibition mechanism. Selective AC6 reactivation depends on unique features of the AC6 FSK binding site and is a viable therapeutic target in PPHN.