Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties

Abstract

Adenylyl cyclase 1 (AC1) belongs to a group of adenylyl cyclases (ACs) that are stimulated by calcium in a calmodulin-dependent manner. Studies with AC1 knockout mice suggest that inhibitors of AC1 may be useful for treating pain and opioid dependence. However, nonselective inhibition of AC isoforms could result in substantial adverse effects. We used chemical library screening to identify a selective AC1 inhibitor with a chromone core structure that may represent a new analgesic agent. After demonstrating that the compound (ST034307) inhibited Ca²⁺-stimulated adenosine 3',5'-monophosphate (cAMP) accumulation in human embryonic kidney (HEK) cells stably transfected with AC1 (HEK-AC1 cells), we confirmed selectivity for AC1 by testing against all isoforms of membrane-bound ACs. ST034307 also inhibited AC1 activity stimulated by forskolin- and Gα_s-coupled receptors in HEK-AC1 cells and showed inhibitory activity in multiple AC1-containing membrane preparations and mouse hippocampal homogenates. ST034307 enhanced μ-opioid receptor (MOR)-mediated inhibition of AC1 in short-term inhibition assays in HEK-AC1 cells stably transfected with MOR; however, the compound blocked heterologous sensitization of AC1 caused by chronic MOR activation in these cells. ST034307 reduced pain responses in a mouse model of inflammatory pain. Our data indicate that ST034307 is a selective small-molecule inhibitor of AC1 and suggest that selective AC1 inhibitors may be useful for managing pain.

Fig. 1

Chemical structures of AC1 inhibitors.

Fig. 2. Inhibitory activity of ST034307 and ST072383 for AC isoforms

(A) Inhibition of 3 µM A23187-stimulated cAMP accumulation in stably transfected HEK-AC1 cells. (B) Inhibition of 3 µM A23187-stimulated cAMP accumulation in stably transfected HEK-AC8 cells. (C) Stimulation of HEK cells transiently transfected with venus control plasmid or AC isoforms. AC1- and AC8-transfected cells were stimulated with 3 µM A23187; AC2-transfected cells were stimulated with 1 µM PMA; AC3-transfected cells were stimulated with 30 µM forskolin; AC4-trasfected cells were stimulated with 10 µM isoproterenol; AC5- and AC6-transfected cells were stimulated with 1 µM forskolin; AC7-transfected cells were stimulated with a combination of 1 µM PMA plus 1 µM forskolin in the presence of transfected Gα_s; AC9-transfected cells were stimulated with 100 nM isoproterenol in the presence of transfected Gα_s. The y-axis represents the cAMP accumulation when compared to the same transfection and stimulation condition in matched venus-transfected control cells. One sample t test with Bonferroni correction was carried out for statistical analyses. *p<0.05, **p<0.01, ****p<0.0001 compared to the same transfection and stimulation condition in matched venus-transfected control cells (n = 3–4). (D) Inhibition of cAMP accumulation in the transiently transfected HEK cells using the indicated concentration of inhibitor. Cells were transfected and stimulated as described in (C). The “venus”-transfected HEK cells were stimulated with 30 µM forskolin. One sample t test with Bonferroni correction was carried out for statistical analyses. *p<0.05, **p<0.01, ***p<0.001 compared to vehicle-treated cells (in the absence of inhibitor, n = 3–4). In A, B, and D data was normalized by defining the cAMP levels from activator treatment (without inhibitor) as 100% and baseline cAMP levels as 0%. All data shown represent the average and S.E.M. of at least three independent experiments conducted in duplicate or triplicate.

Fig. 3. Specificity and activity of ST034307 in cells and membrane preparations

(A) HEK-AC1 and nontransfected HEK cells (HEK-WT) were treated with 30 µM ST034307 and stimulated with 300 nM forskolin or 10 µM isoproterenol. (B) Cellular membranes from HEK-AC1 cells were isolated and cAMP production was stimulated with either 30 µM forskolin or 3 µM calmodulin in the presence of 10 µM free Ca²⁺ with or without either of the indicated AC inhibitors. (C) Cellular membranes from Sf9 cells expressing AC1, AC2, or AC5 were isolated and cAMP accumulation was stimulated with 50 nM Gα_s in the presence of 100 µM ST034307. The data was normalized by defining the cAMP levels from activator treatment (without inhibitor) as 100% and baseline cAMP levels as 0%.. All data shown represent the average and S.E.M. of at least three independent experiments conducted in duplicate or triplicate. One sample t test with Bonferroni correction was carried out for statistical analyses. *p<0.05, **p<0.01 all compared to vehicle-treated (in the absence of inhibitor).

Fig. 4. Mechanistic insights regarding ST034307

(A) Inhibition of A23187-stimulated cAMP accumulation in HEK-AC1 cells. (B) Inhibition of forskolin-stimulated cAMP accumulation in HEK-AC1 cells. The data was normalized by defining the cAMP levels from activator treatment (without inhibitor) as 100% and baseline cAMP levels as 0%. All data shown represent the average and S.E.M. of at least three independent experiments conducted in triplicate. Data were analyzed using two-way ANOVA followed by Tukey’s multiple comparisons test. *p<0.05, **p<0.01 between indicated concentrations.

Fig. 5. Inhibition of Ca²⁺/calmodulin-stimulated cAMP accumulation in hippocampal homogenates

(A) Mouse hippocampal homogenates were stimulated with 3 µM calmodulin in the presence of 10 µM free Ca²⁺ with basal cAMP activity in the absence of Ca²⁺/calmodulin set at 1. (B) Hippocampal homogenates were stimulated Ca²⁺/calmodulin in the presence of ST034307 or NKY80. Data are normalized to the response in the absence of the inhibitors (3 µM calmodulin plus 10 µM free Ca²⁺ was defined as 100% and baseline cAMP levels was defined as 0%) and represent the average and S.E.M. of six independent experiments conducted in triplicate. One sample t test for (A) and one sample t test with Bonferroni correction for (B), **p<0.01, ***p<0.001, ****p<0.0001 compared to basal or vehicle-treated (in the absence of inhibitor).

Fig. 6. Effects of ST034307 on MOR signaling

(A) Inhibition of A23187-stimulated cAMP accumulation in HEK-AC1/MOR cells. Approximate EC₂₀ concentrations of ST034307 (0.5 µM) and DAMGO (4 nM) or EC₅₀ concentrations of ST034307 (7.5 µM) and DAMGO (15 nM) were added alone or in combination as indicated and cAMP accumulation measured. Data was normalized by defining the inhibitory response to 1 µM DAMGO in the presence of 3 µM A23187 as 100% and the cAMP response to 3 µM A23187 as 0%. (B) β-arrestin 2 recruitiment in CHO-MOR cells that were treated with 10 µM DAMGO, 30 µM ST034307, or DAMGO + ST034307. (C) Heterologous sensitization of AC1 by the MOR was achieved by pre-treating the cells with 1 µM DAMGO for 2 h and subsequently stimulating cAMP accumulation with A23187 (3 µM) in the presence of naloxone (1 µM). (D) Effects of ST034307 on the development and maintenance of DAMGO-stimulated heterologous sensitization. Timeline of the drug treatments conducted for determining the effects of ST034307 on the development (top) and maintenance (bottom) of DAMGO-induced heterologous sensitization of AC1 (shown in graph below). Following drug pretreatment, heterologous sensitization was triggered by the addition of A23187 (3 µM) in the presence of naloxone (1 µM). The data in C and D were normalized to vehicle-treated cells under basal conditions (0%) or stimulated with A23187 in the presence of naloxone (100%). The data shown represent the average and S.E.M. of at least three independent experiments conducted in duplicate. One sample t test with Bonferroni correction was carried out for statistical analyses in (A), (B), and (C). For (A) *p<0.05, **p<0.01, and ***p<0.001 compared to respective DAMGO + ST034307 column; for (B) and (C) * p<0.05 and *** p<0.001 compared to vehicle-treated cells (in the absence of any drugs).

Fig. 7. Analgesic properties of ST034307 in a mouse model of inflammatory pain

(A) On day 0, baseline (BL) measurements of mechanical sensitivity of C57BL/6 mice to von Frey filaments were recorded and inflammatory hypersensitivity was induced by injection of CFA to the hind paw. On day 1, inflammatory hypersensitivity was measured by von Frey test. Animals received intrathecal injections with saline (n=10), 50 ng MOR-selective agonist DAMGO (n=11), or 0.5 µg ST034307 (n=11) and inflammatory hypersensitivity was measured again. *p<0.05, **p<0.01 compared to saline injection at 10 min, analyzed by one-way ANOVA. (B) Dose-response experiments with ST034307 (n=6 for each condition). Estimated ED₅₀ value for analgesia of 0.28 µg (95% CI = 0.13 – 0.43; n = 6). (C) At 1 day after CFA-induced inflammatory pain, animals received intrathecal injections with saline (n=9), 10 µg forskolin (n=8), 0.5 µg ST034307 (n=9), or 10 µg forskolin + 0.5 µg ST034307 (n=8) and mechanical hypersensitivity was measured by von Frey test 10 min after injections. *p<0.05, **p<0.01 compared to each corresponding treatment, analyzed by one-way ANOVA with Tukey’s test. The data shown represent the average and S.E.M. of each group of measurements to stimulation of the ipsilateral hind paw of the mice. All data was normalized by defining the baseline measurements as 100% and the CFA-induced hypersensitivity as 0%.