BACKGROUND AND PURPOSE: Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH: Whole cell cGMP concentrations or ³²P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS: Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t(½) for Gö6976 inhibition was 7 s and IC₅₀ was 380 nM. Gö6976 increased the EC₅₀ for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.
BACKGROUND AND PURPOSE:Atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) decrease vascular volume and pressure by activating guanylyl cyclase-A (GC-A). C-type natriuretic peptide (CNP) activation of guanylyl cyclase-B (GC-B) stimulates long bone growth. This study investigated the effects of the indolocarbazole, Gö6976, on the guanylyl cyclase activity of GC-A and GC-B as a first step towards developing small molecule regulators of these enzymes. EXPERIMENTAL APPROACH: Whole cell cGMP concentrations or ³²P-cGMP accumulation in membrane preparations measured the effects of indolocarbazoles on the enzymatic activity GC-A and GC-B from transfected 293T or endogenously expressing 3T3-L1 cells. KEY RESULTS: Gö6976 blocked cellular CNP-dependent cGMP elevations in 293T-GC-B cells. The t(½) for Gö6976 inhibition was 7 s and IC₅₀ was 380 nM. Gö6976 increased the EC₅₀ for CNP 4.5-fold, but increasing the CNP concentration did not overcome the inhibition. Half of the inhibition was lost 1 h after removal of Gö6976 from the medium. Cellular exposure to Gö6976 reduced basal and natriuretic peptide-dependent, but not detergent-dependent, GC-A and GC-B activity. Inhibition was also observed when Gö6976 was added directly to the cyclase assay. A constitutively phosphorylated form of GC-B was similarly inhibited. CONCLUSIONS AND IMPLICATIONS: These data demonstrate that Gö6976 potently, rapidly and reversibly inhibited GC-A and GC-B via a process that did not require intact cells, known phosphorylation sites or inactivation of all catalytic sites. This is the first report of an intracellular inhibitor of a transmembrane guanylyl cyclase and the first report of a non-kinase target for Gö6976.
Authors: Bernhard F Becker; Matthias Jacob; Stephanie Leipert; Andrew H J Salmon; Daniel Chappell Journal: Br J Clin Pharmacol Date: 2015-05-22 Impact factor: 4.335