A Selective Adenylyl Cyclase 1 Inhibitor Relieves Pain Without Causing Tolerance

Abstract

Among the ten different adenylyl cyclase isoforms, studies with knockout animals indicate that inhibition of AC1 can relieve pain and reduce behaviors linked to opioid dependence. We previously identified ST034307 as a selective inhibitor of AC1. The development of an AC1-selective inhibitor now provides the opportunity to further study the therapeutic potential of inhibiting this protein in pre-clinical animal models of pain and related adverse reactions. In the present study we have shown that ST034307 relives pain in mouse models of formalin-induced inflammatory pain, acid-induced visceral pain, and acid-depressed nesting. In addition, ST034307 did not cause analgesic tolerance after chronic dosing. We were unable to detect ST034307 in mouse brain following subcutaneous injections but showed a significant reduction in cAMP concentration in dorsal root ganglia of the animals. Considering the unprecedented selectivity of ST034307, we also report the predicted molecular interaction between ST034307 and AC1. Our results indicate that AC1 inhibitors represent a promising new class of analgesic agents that treat pain and do not result in tolerance or cause disruption of normal behavior in mice. In addition, we outline a unique binding site for ST034307 at the interface of the enzyme's catalytic domain.

Keywords: AC1; adenylyl cyclase; analgesia; pain; tolerance.

FIGURE 1

ST034307 relieves inflammatory pain in mice. (A) Different doses of ST034307 reduce paw licking behavior caused by an intraplantar injection with 5% formalin. (B) Different doses of morphine reduce paw licking behavior caused by an intraplantar injection with 5% formalin. (C) Dose-response curves of the sum of time spent licking the paw during the first 10 min of the graphs in (A) and (B). Vehicle’s response was set as 0% and the maximal possible effect (0) to 100%. (D) Dose-response curves of the sum of time spent licking the paw during the period in between minute 16 and minute 40 of the graphs in (A) and (B). Vehicle’s response was set as 0% and the maximal possible effect (0) to 100%. (E) Reduction of time spent licking the injected paw in wild-type (WT) and in AC1-KO mice treated with vehicle (V), 30 mg/kg ST034307 (S), or 10 mg/kg morphine (M) during the first 10 min of the experiment. (F) Reduction of time spent licking the injected paw in wild-type (WT) and in AC1-KO mice treated with vehicle (V), 30 mg/kg ST034307 (S), or 10 mg/kg morphine (M) during the period in between minute 16 and minute 40 of the experiment. For E and F vehicle’s response in wild-type mice was set to 0% and zero to 100%. Data in all graphs represent the average ±S.E.M., N = 6–8. ****p < 0.0001 in one-way ANOVA with Dunnett’s test.

FIGURE 2

ST034307 relieves visceral pain in mice. (A) Different doses of ST034307 reduce the number of abdominal constrictions caused by an intraperitoneal injection with 0.75% acetic acid; N = 8–10. (B) Different doses of morphine reduce the number of abdominal constrictions caused by an intraperitoneal injection with 0.75% acetic acid; N = 8–10. (C) Dose-response curves of the total number of abdominal constrictions from the graphs in (A) and (B). Vehicle’s response was set as 0% and the maximal possible effect (0) to 100%. (D) Reduction of the total number of abdominal constrictions in wild-type (WT) and in AC1-KO mice treated with vehicle (V), 30 mg/kg ST034307 (S), or 10 mg/kg morphine (M). Vehicle’s response in wild-type mice was set as 0% and zero to 100%; N = 5. (E) Mice were injected daily with 30 mg/kg ST034307 or 100 mg/kg morphine and on day four or eight acid-induced writhing assays were performed. Mice that chronically received morphine displayed a decrease in efficacy of 3 mg/kg morphine on days four or eight, compared to a group of mice that received vehicle plus 3 mg/kg morphine on day zero. Mice that chronically received ST034307 did not display any changes in efficacy of 10 mg/kg ST034307; N = 5. (F) Mice were injected daily with 30 mg/kg ST034307 or 100 mg/kg morphine and on day four or eight acid-induced writhing assays were performed. Mice that chronically received morphine did not display any changes in efficacy with 10 mg/kg ST034307. Mice that chronically received ST034307 did not display any changes in efficacy with 3 mg/kg morphine; N = 5. Data in all graphs represent the average ±S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001 in one-way ANOVA with Dunnett’s test.

FIGURE 3

ST034307 rescues acid-depressed mouse nesting behavior. (A) ST034307 did not reduce mouse nesting behavior at doses up to 30 mg/kg. (B) At a dose of 3 mg/kg, morphine significantly reduced mouse nesting behavior at 30 and 60 min after nesting measurements were started. (C) Dose-response curves of the last experimental timepoints the graphs in (A) and (B). Vehicle’s response was set as 100% and the maximal possible effect (0) to 0%. (D) and (E) 3 mg/kg, 10 mg/kg, and 30 mg/kg ST034307 and 0.3 mg/kg and 1 mg/kg morphine significantly rescued mouse nesting behavior that was reduced by an intraperitoneal injection of 1% lactic acid. (F) Dose-response curves of the last experimental timepoints from the graphs in (D) and (E). Vehicle’s response was set as 0% and the maximal possible effect (5) to 100%. Data in all graphs represent the average ±S.E.M., N = 6–8. *p < 0.05, **p < 0.01, ***p < 0.001 in two-way ANOVA with Dunnett’s test.

FIGURE 4

ST034307 plasma concentration peaks at 60 min after subcutaneous injection and reduces cAMP concentration in the DRG. (A) Plasma ST0304307 concentration versus time profile after a single 10 mg/kg subcutaneous injection in mice. N = 2,3. (B) A 10 mg/kg subcutaneous injection of ST034307 causes a reduction in cAMP concentration in the DRG of mice. DRG were dissected 90 min after injection. Data was normalized to respective matched vehicle-injected controls. N = 6. **p < 0.01 in one sample T test compared to 100%.

FIGURE 5

Prediction of the interaction between AC1 and ST034307. (A) Cartoon representation of the AC1 model, showing its catalytic domain (C1a, in red, and C2a, in green) complexed to Gα_s (in blue), ST034307 (in cyan or purple), ATP (in yellow), and two magnesium ions (Mg²⁺, in green). Predicted poses of ST034307, using Gold (B) and Autodock Vina (C) programs, presenting hydrogen-bond (interrupted purple line) and steric interactions. The AC1 residue structures are shown as ball and stick models, ST034307 and ATP as stick models, and Mg²⁺ ions as sphere models using UCSF Chimera program (Pettersen et al., 2004). All the structures are colored by atom: the nitrogen atoms are shown in blue, the oxygen atoms in red, the chlorine atoms in green, the hydrogen atoms in white, and the carbon chain in gray, cyan, or purple. Non-polar hydrogens have been omitted for clarity.