Development of a High-Throughput Screening Paradigm for the Discovery of Small-Molecule Modulators of Adenylyl Cyclase: Identification of an Adenylyl Cyclase 2 Inhibitor

Abstract

Adenylyl cyclase (AC) isoforms are implicated in several physiologic processes and disease states, but advancements in the therapeutic targeting of AC isoforms have been limited by the lack of potent and isoform-selective small-molecule modulators. The discovery of AC isoform-selective small molecules is expected to facilitate the validation of AC isoforms as therapeutic targets and augment the study of AC isoform function in vivo. Identification of chemical probes for AC2 is particularly important because there are no published genetic deletion studies and few small-molecule modulators. The present report describes the development and implementation of an intact-cell, small-molecule screening approach and subsequent validation paradigm for the discovery of AC2 inhibitors.The NIH clinical collections I and II were screened for inhibitors of AC2 activity using PMA-stimulated cAMP accumulation as a functional readout. Active compounds were subsequently confirmed and validated as direct AC2 inhibitors using orthogonal and counterscreening assays. The screening effort identified SKF-83566 [8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide] as a selective AC2 inhibitor with superior pharmacological properties for selective modulation of AC2 compared with currently available AC inhibitors. The utility of SKF-83566 as a small-molecule probe to study the function of endogenous ACs was demonstrated in C2C12 mouse skeletal muscle cells and human bronchial smooth muscle cells.

International Union of Basic and Clinical Pharmacology. CI. Structures and Small Molecule Modulators of Mammalian Adenylyl Cyclases

“…However, counterscreening against other AC isoforms and studies examining the potential role of G proteins (e.g., pertussis toxin treatment and sequestering Gbg subunits) are important considerations for intact cell studies. The results of intact cell studies can further be strengthened and supported by additional studies using cell membranes (Conley et al, 2013). Ultimately, one would like confirmation that pharmacological inhibition and genetic inhibition give rise to similar results.…”

“…For intact cells, various reliable immuno-based cAMP detection methods are available (Post et al, 2000), and high-throughput screening methods (with varying reliability) have been developed as well (Williams, 2004;Conley et al, 2013). However, formally, intact cell assays do not reflect AC activity assays but constitute cAMP accumulation assays.…”

“…pIC 50 values for sGC of the compounds listed in Table 7 are not available. A recent study reported on the identification of noncompetitive inhibitors with some selectivity for AC2 relative to ACs 1 and 5, compound 8-bromo-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol hydrobromide (SKF-83566) being a representative (Conley et al, 2013). However, SKF-83566 is also a potent dopamine D1 receptor antagonist (O'Boyle and Waddington, 1984).…”

“…AC2 and AC4 are the predominantly expressed AC isoforms in human airway smooth muscle cells, but AC2 expression is lacking in mouse bronchial smooth muscle cells, making confirmation of its function difficult (Bogard et al, 2011. Development of AC2-selective inhibitors may overcome the difficulties to elucidate the (patho)physiologic functions of AC2 (Pinto et al, 2008;Erdorf et al, 2011;Kinast et al, 2012;Conley et al, 2013). ADCY2 polymorphisms may be related to neuropsychiatric (Mühleisen et al, 2014;Suarez-Rama et al, 2015) and pulmonary diseases (Hardin et al, 2012;Panasevich et al, 2013).…”

Cyclic Adenosine 3′,5′-Monophosphate Elevation and Biological Signaling through a Secretin Family Gs-Coupled G Protein–Coupled Receptor Are Restricted to a Single Adenylate Cyclase Isoform

“…Identification of AC isoform-selective small molecule inhibitors is of translational interest for a number of disorders (Seifert et al, 2012), and indeed, compounds targeting several isoforms are currently undergoing rigorous characterization Conley et al, 2013). Our data show that PACAP-induced cAMP elevation is mediated mainly through a single isoform of adenylate cyclase, AC6.…”