Targeting Adenylyl Cyclase 1 for Development of Non-Opioid Pain Therapeutics

Abstract ID 92829 Poster Board 348 Treatment for chronic pain is limited, with opioids being the most efficacious analgesics for many types of chronic pain. Opioids activate the μ-opioid receptor (MOR), which in part, mediates analgesic activity via inhibition of adenylyl cyclase 1 (AC1), an enzyme that catalyzes conversion of ATP to cAMP, a second messenger. Additionally, MOR activation causes β-arrestin-mediated MOR internalization and subsequent tolerance. We hypothesize that targeting AC1 directly could avoid MOR-related side effects. AC1 has been implicated as a novel non-opioid target for treatment of chronic pain. AC1 is highly expressed in the brain and its activity is increased following inflammatory stimuli. Further, AC1 knockout (KO) mice had significantly reduced responses to inflammatory stimuli compared to wild-type (WT) mice. Knockout of AC8, a closely related isoform, had no effect compared to WT, whereas AC1/AC8 double KO caused memory deficits. Thus, selectively targeting AC1 represents an opportunity to develop non-opioid pain therapeutics with minimal side effects. Our group previously identified a pyrimidinone-pyrazolyl-benzamide scaffold that selectively inhibits AC1 signaling in HEK Δ3/6 KO cells expressing AC1. The benzamide series suffered from poor solubility, however, medicinal chemistry efforts developed a pyrimidinone-pyrazolyl-benzylamine (PBL) scaffold with markedly improved aqueous solubility. Through further SAR of the PBL series, inhibition of AC8 was eliminated to reach nearly complete AC1 versus AC8 selectivity. Our lead analogs maintain full AC1 inhibition, with improved potencies of ∼100 nM. Additionally, our lead analogs maintain selectivity versus other AC isoforms (AC2 and AC5), and display minimal cytotoxicity. For preclinical development, lead analogs have been investigated for metabolic stability and blood-brain barrier (BBB) penetration. In vivo plasma pharmacokinetics (PK) experiments in rats revealed moderate clearance. The in vivo BBB PK experiments in rats demonstrated that lead analogs accumulate to their IC₅₀ concentrations in the brain up to 1 hour, and also maintain in vivo anti-allodynic activity. Additional in vitro studies have explored the impact of our lead molecules on opioid receptor signaling in unique cellular models. The first model used CHO cells expressing the MOR and β-arrestin2 (βarr2), with or without AC1 (CHO-MOR-βarr2 or CHO-MOR-βarr2-AC1). Our second model used SH-SY5Y neuroblastoma cells, virally expressing AC1 and the MOR. In both models, AC1 expression was validated by selectively stimulating AC1 activity with A23817. This activity was then inhibited by an AC1-selective inhibitor, ST034307. MOR expression was validated by treating with the MOR agonist DAMGO, which dose-dependently inhibited cAMP accumulation. The CHO-MOR-βarr2-AC1 model was specifically used to assess the effects of the PBL analogs on MOR-mediated βarr2 recruitment. Both cellular models were then used to assess the effect of lead analogs on MOR-mediated cAMP inhibition, MOR-regulated Gβγ signaling, MOR desensitization, and MOR-induced heterologous sensitization. These experiments serve as validation that the PBL analogs do not modulate MOR-dependent signaling, but display similar efficacy to the MOR on AC1 inhibition and can prevent sensitization. This work provides in vitro translational cellular models from HEK293 and CHO cells to a model of neuronal function.