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

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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 stimulator 5‐cyclopropyl‐2‐[1‐(2‐fluoro‐benzyl)‐1H‐pyrazolo[3,4‐b]pyridin‐3‐yl]‐pyrimidin‐4‐ylamine (BAY 41‐2272).Experimental 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 β1H105F 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.