Targeting Adenylyl Cyclase 1 for the Treatment of Chronic Pain

<p dir="ltr">Chronic pain is a major public health concern affecting ~20% of U.S. adults. Treatment options remain limited, with opioids being the most effective analgesics. Opioids activate the µ-opioid receptor (MOR), which mediates analgesia partly through G protein-dependent inhibition of adenylyl cyclase 1 (AC1), an enzyme that converts ATP to cAMP. However, MOR signaling through additional pathways, including those mediated by Gβγ and β-arrestin, contributes to respiratory depression, tolerance, and dependence. Thus, directly targeting AC1 downstream of the MOR may provide analgesia while reducing opioid-associated adverse effects. Genetic and preclinical studies support this approach. AC1 activity increases following inflammatory stimuli, and AC1 knockout mice show reduced behavioral responses to inflammatory pain, whereas mice lacking the highly similar AC8 isoform respond similarly to wild-type mice. Here, we report two complementary strategies to identify selective AC1 inhibitors. First, we developed a fluorescence polarization assay to identify small molecules disrupting the AC1–CaM protein-protein interaction required for AC1 activation. Screening >23,000 compounds yielded 21 validated hits, with a dithiophene scaffold emerging as an AC1/AC8 dual inhibitor with tunable AC1/AC8 selectivity (analog <b>32</b> – AC1 IC₅₀ = 0.89 µM, AC8 IC₅₀ = 1.86 µM). Second, optimization of a high-throughput hit led to a series of pyrazolo-pyrimidinone amine-linked analogs. Lead compounds AC10084A, AC10136A, AC10142A, and AC10172A exhibited potent AC1 inhibition (IC₅₀ = 0.14–0.61 µM), complete selectivity over AC2, AC5, and AC8, and minimal cytotoxicity. Mechanistic studies revealed a Ca²⁺/CaM-dependent, Gαs-independent mechanism of inhibition, suggesting state-dependent targeting of active AC1. Lead compounds AC10084A and AC10142A demonstrated <i>in vivo</i> analgesic efficacy in the Complete Freund’s Adjuvant (CFA) model of inflammatory pain, with AC10142A showing sustained efficacy over 5 days in a repeated-dosing paradigm. These studies highlight two complementary approaches for AC1 inhibition and identify lead scaffolds with strong translational potential as non-opioid therapeutics for chronic pain.</p>