Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation‐induced degradation

Abstract

Background and purpose:In endothelial dysfunction, signalling by nitric oxide (NO) is impaired because of the oxidation and subsequent loss of the soluble guanylyl cyclase (sGC) haem. The sGC activator 4-[((4-carboxybutyl){2- [(4-phenethylbenzyl) oxy]phenethyl}amino)methyl[benzoic]acid (BAY 58-2667) is a haem-mimetic able to bind with high affinity to sGC when the native haem (the NO binding site) is removed and it also protects sGC from ubiquitin-triggered degradation. Here we investigate whether this protection is a unique feature of BAY 58-2667 or a general characteristic of haem-site ligands such as the haem-independent sGC activator 5-chloro-2-(5-chloro-thiophene-2-sulphonylamino-N-(4-(morpholine-4-sulphonyl)-phenyl)-benzamide sodium salt (HMR 1766), the haem-mimetic Zn-protoporphyrin IX (Zn-PPIX) or the haem-dependent sGC stimulatorExperimental approach: The sGC inhibitor 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was used to induce oxidation-induced degradation of sGC. Activity and protein levels of sGC were measured in a Chinese hamster ovary cell line as well as in primary porcine endothelial cells. Cells expressing mutant sGC were used to elucidate the molecular mechanism underlying the effects observed. Key results: Oxidation-induced sGC degradation was prevented by BAY 58-2667 and Zn-PPIX in both cell types. In contrast, the structurally unrelated sGC activator, HMR 1766, and the sGC stimulator, BAY 41-2272, did not protect. Similarly, the constitutively haem-free sGC mutant b1H105F was stabilized by BAY 58-2667 and Zn-PPIX. Conclusions:The ability of BAY 58-2667 not only to activate but also to stabilize oxidized/haem-free sGC represents a unique example of bimodal target interaction and distinguishes this structural class from non-stabilizing sGC activators and sGC stimulators such as HMR 1766 and BAY 41-2272, respectively.

Activation of Haem-Oxidized Soluble Guanylyl Cyclase with BAY 60-2770 in Human Platelets Lead to Overstimulation of the Cyclic GMP Signaling Pathway

“…Previous studies indicated that oxidation-induced degradation becomes prominent when the incubation time exceeds 2 h [11], [32]. Therefore, washed platelets were incubated with ODQ (10 µM) in the absence and in the presence of BAY 60-2770 (10 µM) for 2.5 h, after which levels of sGC protein for α1 and β1 sGC subunits were determined.…”

Regulation of soluble guanylyl cyclase redox state by hydrogen sulfide

“…Moreover, it has been demonstrated that sGC with oxidized heme has a propensity to lose the heme moiety [11, 12]. The pool of ferric or heme-free sGC is much more susceptible to degradation [12, 13]. About fifteen years ago, NO-independent sGC activators and stimulators were discovered [14, 15]; these agents enhance sGC activity in a heme-dependent or -independent manner.…”

Soluble Guanylate Cyclase as an Emerging Therapeutic Target in Cardiopulmonary Disease

“…20 It activates oxidized/heme-free sGC by binding in the sGC heme pocket and mimicking the heme group; it also protects heme-free sGC from oxidation-induced proteasomal degradation. Cinaciguat therefore opens up the possibility of new mechanism-based therapies for cardiovascular diseases associated with oxidative stress, 8,23 and is currently in clinical development for the treatment of acute decompensated heart failure. 24–26 A more recently discovered sGC activator, the anthranilic acid derivative ataciguat (HMR 1766), 27 has also been studied in clinical trials in healthy volunteers, 28 in patients with intermittent claudication due to peripheral arterial disease and in patients with neuropathic pain.…”