Isoform selectivity of adenylyl cyclase inhibitors: characterization of known and novel compounds

Abstract

Nine membrane-bound adenylyl cyclase (AC) isoforms catalyze the production of the second messenger cyclic AMP (cAMP) in response to various stimuli. Reduction of AC activity has well documented benefits, including benefits for heart disease and pain. These roles have inspired development of isoform-selective AC inhibitors, a lack of which currently limits exploration of functions and/or treatment of dysfunctions involving AC/cAMP signaling. However, inhibitors described as AC5- or AC1-selective have not been screened against the full panel of AC isoforms. We have measured pharmacological inhibitor profiles for all transmembrane AC isoforms. We found that 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22,536), 2-amino-7-(furanyl)-7,8-dihydro-5(6H)-quinazolinone (NKY80), and adenine 9-β-d-arabinofuranoside (Ara-A), described as supposedly AC5-selective, do not discriminate between AC5 and AC6, whereas the putative AC1-selective inhibitor 5-[[2-(6-amino-9H-purin-9-yl)ethyl]amino]-1-pentanol (NB001) does not directly target AC1 to reduce cAMP levels. A structure-based virtual screen targeting the ATP binding site of AC was used to identify novel chemical structures that show some preference for AC1 or AC2. Mutation of the AC2 forskolin binding pocket does not interfere with inhibition by SQ22,536 or the novel AC2 inhibitor, suggesting binding to the catalytic site. Thus, we show that compounds lacking the adenine chemical signature and targeting the ATP binding site can potentially be used to develop AC isoform-specific inhibitors, and discuss the need to reinterpret literature using AC5/6-selective molecules SQ22,536, NKY80, and Ara-A.

Fig. 1.

AC inhibition profile of SQ22,536. (A) Chemical structure of SQ22,536. (B) Virtual docking of SQ22,536 to a P-site inhibitor-bound conformation of AC (PDB code 1CJT). SQ22,536 is shown with cyan carbons; the crystallized position of the 2′,3′ddATP ligand is shown with gray carbons for reference. The C1 domain is yellow; the C2 domain is silver. (C) Inhibition profile of SQ22,536 for all nine AC isoforms. Inhibition curves for each AC are shown as a calculated fit to the means of each concentration (indicated by symbols and grouped with colors by AC family; n = 3, performed in duplicate; error bars removed for clarity). Membranes from Sf9 cells expressing AC1–7 were stimulated by 50 µM forskolin. Membranes from HEK293 cells expressing AC8 and AC9 were stimulated by 300 µM calmodulin and 300 nM Gα_s, respectively. Ctrl, control.

Fig. 2.

AC inhibition profile of NKY80. (A) Chemical structure of NKY80. (B) Virtual docking of NKY80 to the 2′,3′ddATP-bound conformation of AC as was described for Fig. 1B. (C) Complete AC isoform inhibition profile of NKY80. Membranes and stimulation conditions were as described in Fig. 1C. Experimental data indicated by symbols (n = 3, performed in duplicate); lines are the calculated fits to experimental means for the dose-response curves. Ctrl, control.

Fig. 3.

Ara-A does not discriminate between AC5 and AC6. (A) Chemical structure of Ara-A. (B) Virtual docking of Ara-A to the 2′3′ddATP-bound conformation of AC as described for Fig. 1B. (C) Complete AC isoform inhibition profile of Ara-A. Inhibition curves for each AC isoform shown as fit to means of AC activity assays (n = 3). Membranes and stimulation conditions were as described in Fig. 1C (n = 3, performed in duplicate). Ctrl, control.

Fig. 4.

NB001 decreases AC1-dependent cAMP accumulation but does not directly inhibit AC1. (A) Chemical structure of NB001. (B) HEK293 cells expressing AC1 or pcDNA control were incubated with NB001 (10 or 100 µM) or vehicle prior to stimulation with 10 µM forskolin and 10 µM ionomycin. Statistics (t test) for NB001 inhibition: *P < 0.05. (C) NB001 (100 µM) or vehicle control were incubated with membranes from HEK293 cells expressing AC1 and stimulated as indicated. Experiments (n = 3) for (B) and (C) were performed in duplicate. Representative experiments with SD are shown.

Fig. 5.

Identification of novel AC1 inhibitors. Chemical structure of CB-6673567 (A) and CB-7921220 (D). Virtual local docking of CB-6673567 (B) and CB-7921220 (E) to the catalytic site of 2′3′ddATP-bound AC. Inhibitor carbons are cyan, C1 domain is yellow, and C2 domain is silver. Inhibition of AC5 (blue) at 1 mM CB-6673567 (C) and CB-7921220 (F) and inhibition profile at 100 µM for AC5, AC1 (red), AC2 (green), and AC6 (cyan). All Sf9 membranes were stimulated by 50 µM forskolin. Statistics (paired t test) for inhibition of indicated AC isoform (n = 3–4, performed in duplicate): *P < 0.05; **P < 0.01; ***P < 0.001. Ctrl, control.

Fig. 6.

Identification of a novel AC2 inhibitor, CB-7833407. (A) Chemical structure of CB-7833407. (B) Virtual local docking of CB-7833407 to the catalytic site of 2′3′ddATP-bound AC. (C) Inhibition of AC5 (blue) at 1 mM CB-7833407 and inhibitory profile at 100 µM for AC5, AC1 (red), AC2 (green), and AC6 (cyan). All Sf9 membranes were stimulated by 50 µM forskolin. Statistics (paired t test) for inhibition of indicated AC isoform (n = 3): *P < 0.05; **P < 0.01. Ctrl, control.

Fig. 7.

Mutation of AC2 forskolin binding pocket does not impair inhibition by SQ22,536 or CB-7833407. (A) Basal AC activity of COS-7 membranes expressing AC2 wild type (WT), AC2 S942P mutant, or a pcDNA control. (B) COS-7 membranes expressing AC2 wild type, AC2 S942P mutant, or a pcDNA control were incubated with 10 µM forskolin and/or 50 nM activated Gα_s, and AC2 activity was measured. (C) COS-7 membranes expressing AC2 wild type, AC2 S942P mutant, or a pcDNA control were preincubated in the absence or presence of the indicated inhibitor (100 µM SQ22,536 or 500 µM CB-7833407) and then stimulated with 50 nM Gα_s. Experiments (n = 3–4) were performed in duplicate; representative experiments with SD are shown. Statistics (paired t test) for inhibition of AC2 WT/S942P by the indicated AC inhibitor: *P < 0.05; **P < 0.01. For differences between inhibition of AC2 WT versus S942P: n.s., not significant.