Ca2+/calmodulin-dependent inhibitors of adenylyl cyclase 1 as novel nonopioid analgesics for chronic inflammatory pain

Adenylyl cyclase 1 (AC1) plays an integral role in the excitatory signaling in the anterior cingulate cortex underlying chronic pain pathophysiology. Upon chronic nociceptive input, sustained Ca2+/calmodulin (CaM)-stimulated AC1 activity leads to the development of chronic pain. In this study, we characterize our recently reported series of pyrazolopyrimidinone amine analogs that selectively inhibit Ca2+/CaM-stimulated AC1 activity. Lead compounds AC10136A, AC10142A, and AC10172A exhibited potent AC1 inhibition (IC₅₀ = 140–290 nM) and complete selectivity over AC2, AC5, and AC8. All compounds showed high therapeutic indices (>300) and minimal cytotoxicity up to 100 μM. Lead compounds displayed no functional agonism at κ-opioid receptor. Further, compounds prevented and reversed μ-opioid receptor–mediated heterologous sensitization of AC1, highlighting potential utility in mitigating opioid-induced dependence. Further, chronic treatment with lead compounds failed to induce opioid-like adaptations, such as heterologous sensitization, and did not modulate expression of AC1. Mechanistic studies revealed that inhibition is Ca2+/CaM dependent but independent of Gα_s, suggesting preferential targeting of the active AC1 conformation. In mice, AC10142A demonstrated analgesic efficacy in the complete Freund adjuvant model of chronic inflammatory pain. Acute and repeated dosing at 48–120 h after complete Freund adjuvant treatment increased mechanical paw withdrawal thresholds and sustained analgesia over this repeated dosing. By targeting Ca2+/CaM-bound AC1, these inhibitors preferentially inhibit increased AC1 activity to its normal activity, demonstrating a state-dependent mechanism ideal for chronic pain treatment. Together, these findings establish AC10136A, AC10142A, and AC10172A as potent, selective, and well-tolerated AC1 inhibitors, with promising therapeutic potential for chronic pain and potentially opioid use disorder. Significance Statement: Adenylyl cyclase 1 (AC1) plays a key role in chronic pain sensitization and opioid use disorder, making it a promising therapeutic target. Selective pyrazolopyrimidinone amine inhibitors of AC1 potently block Ca2+/calmodulin-dependent activation (with IC₅₀ values of 140–290 nM), exhibit excellent in vitro safety profiles, prevent μ-opioid receptor–mediated AC1 sensitization in vitro, and demonstrate robust antiallodynic effects in vivo, positioning this compound series as a compelling nonopioid strategy for managing chronic pain and potentially addressing opioid use disorder.