Focused Library Screening for Isoform Selective Adenylyl Cyclase 1 Inhibitors as Non‐Opioid Alternatives for Chronic and Inflammatory Pain

Abstract

Due to the socioeconomic burden associated with chronic pain conditions and the fact that opioids have severe side effects, there is a need for new approaches and drug targets for the treatment of chronic pain. An increasing body of evidence suggests that inhibition of neuronal Adenylyl Cyclase 1 (AC1) may be one way to achieve this goal, as mice lacking AC1 are significantly less responsive in neuropathic and inflammatory pain models. ACs catalyze the formation of cAMP from ATP following stimulation by a variety of cellular inputs, like G protein subunits, kinases, calmodulin, and the exogenous small molecule forskolin. Here we used a recently developed cell model which lacks forskolin stimulated cAMP accumulation due to genetic ablation using CRISPR/Cas9 of AC3 and AC6, the major forskolin responsive AC isoforms in parental HEK293 cells. Following stable overexpression of recombinant human AC1, we screened over 20,000 compounds from the Eli Lilly Open Innovation Drug Discovery program against calcium‐calmodulin stimulated AC1 activity. The hit rate from the preliminary screen was less than 1%, highlighting the utility of this cell model as compared to parental HEK293 cells. Primary screen actives were retested against AC1 to confirm activity in dose‐response experiments. Counter‐screening was performed against representative AC isoforms AC2, AC5, and AC8, leading to the identification of two novel, promising scaffolds with distinct AC1 inhibition profiles. Subsequently, we evaluated the mechanism of inhibition of these lead compounds as compared to previously identified AC inhibitors in our cellular model, isolated membrane fractions, and mouse hippocampal homogenates in response to various stimuli, including GPCR‐Gαs, forskolin, and calcium‐calmodulin. Further, we have also initiated structure activity relationships for these two scaffolds, as well as begun examining their selectivity for AC1 over other unrelated neurological targets. Taken together, these scaffolds may hold promise as tool compounds for further interrogating the role of selective AC1 inhibition in pain neurobiology and for the development of AC1‐targeting therapeutics in the future.Support or Funding InformationThis work was supported by the Eli Lilly Open Innovation Drug Discovery program, Rick and Anne Borch Research Award, and Purdue University College of Pharmacy.