Research ArticlePharmacology

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

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Sci. Signal.  21 Feb 2017:
Vol. 10, Issue 467, eaah5381
DOI: 10.1126/scisignal.aah5381
  • 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 1 μM PMA + 1 μM forskolin in the presence of transfected Gαs; AC9-transfected cells were stimulated with 100 nM isoproterenol in the presence of transfected Gαs. 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 to 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 to 4). In (A), (B), and (D), data were 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 SEM 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 Gαs in the presence of 100 μM ST034307. The data were 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 SEM 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 compared to vehicle-treated cells or membranes (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 (FSK)–stimulated cAMP accumulation in HEK-AC1 cells. The data were 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 SEM of at least three independent experiments conducted in triplicate. Data were analyzed using two-way analysis of variance (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 with Ca2+/calmodulin in the presence of ST034307 or NKY80. Data are normalized to the response in the absence of the inhibitors (3 μM calmodulin + 10 μM free Ca2+ was defined as 100%, and baseline cAMP levels were defined as 0%) and represent the average and SEM 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 homogenates (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 was measured. Data were 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 recruitment 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 pretreating the cells with 1 μM DAMGO for 2 hours 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). After 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 SEM 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 hindpaw. On day 1, inflammatory hypersensitivity was measured by von Frey test. Animals received intrathecal injections with saline (n = 10), 50 ng of MOR-selective agonist DAMGO (n = 11), or 0.5 μg of ST034307 (n = 11), and inflammatory hypersensitivity was measured again. *P < 0.05 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 to 0.43; n = 6). (C) One day after CFA-induced inflammatory pain, animals received intrathecal injections with saline (n = 9), 10 μg of forskolin (n = 8), 0.5 μg of ST034307 (n = 9), or 10 μg of forskolin + 0.5 μg of 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 SEM of each group of measurements to stimulation of the ipsilateral hindpaw of the mice. All data were normalized by defining the baseline measurements as 100% and the CFA-induced hypersensitivity as 0%.

  • Table 1 Inhibition of A23187-stimulated AC1 or AC8 activity and cell toxicity of hit compounds.

    Compounds were tested for inhibition of 3 μM A23187–stimulated cAMP accumulation in HEK-AC1 or HEK-AC8 cells. IC50 values are presented in μM with 95% confidence interval (CI), and percent inhibition (with 100% set to the basal cAMP) with SEM are reported. We also report the cell toxicity results as a percentage of the vehicle-treated cells. The data in the table represent the average of at least three independent experiments conducted in duplicate. NIO, no inhibition observed.

    HEK-AC1HEK-AC8Cell viability
    CompoundIC50
    (95% CI)
    % Inhibition
    (SEM)
    IC50
    (95% CI)
    % Inhibition
    (SEM)
    % Vehicle
    (SEM)
    ST0723830.3 (0.2–0.4)113 (±4)1.6 (0.5–5.7)55 (±9)97 (±3)
    ST0343072.3 (1.2–4.5)108 (±10)NIONIO85 (±6)

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/10/467/eaah5381/DC1

    Fig. S1. Inhibitory activity of ST034307 and NB001 against AC1.

    Fig. S2. Inhibitory activity of ST034307 after pertussis toxin treatment or sequestration of Gβγ.

    Fig. S3. A23187- and forskolin-stimulated cAMP accumulation in HEK-AC1 cells.

    Table S1. Inhibition of A23187-stimulated AC1 activity using two different methods of measuring cAMP accumulation.

    Data S1. NDL-3000 screen results.

  • Supplementary Materials for:

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

    Tarsis F. Brust, Doungkamol Alongkronrusmee, Monica Soto-Velasquez, Tanya A. Baldwin, Zhishi Ye, Mingji Dai, Carmen W. Dessauer, Richard M. van Rijn, Val J. Watts*

    *Corresponding author. Email: wattsv{at}purdue.edu

    This PDF file includes:

    • Fig. S1. Inhibitory activity of ST034307 and NB001 against AC1.
    • Fig. S2. Inhibitory activity of ST034307 after pertussis toxin treatment or sequestration of Gβγ.
    • Fig. S3. A23187- and forskolin-stimulated cAMP accumulation in HEK-AC1 cells.
    • Table S1. Inhibition of A23187-stimulated AC1 activity using two different methods of measuring cAMP accumulation.
    • Legend for data S1

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    Other Supplementary Material for this manuscript includes the following:

    • Data S1 (Microsoft Excel format). NDL-3000 screen results.

    Citation: T. F. Brust, D. Alongkronrusmee, M. Soto-Velasquez, T. A. Baldwin, Z. Ye, M. Dai, C. W. Dessauer, R. M. van Rijn, V. J. Watts, Identification of a selective small-molecule inhibitor of type 1 adenylyl cyclase activity with analgesic properties. Sci. Signal. 10, eaah5381 (2017).

    © 2017 American Association for the Advancement of Science