Runcaciguat activates soluble guanylyl cyclase via the histidine essential for heme binding and nitric oxide activation

Soluble guanylyl cyclase (sGC) is a well-established pharmacological target for the treatment of acute angina pectoris, pulmonary hypertension and heart failure. Histidine 105 in the heme binding pocket of sGC is a crucial residue for heme binding and natural enzyme activation by NO. It was assumed that the heme-free sGC mutants α₁/β₁H105F and α₁/β₁H105A were valuable research tools for studying NO independent sGC activators. These mutants have been used in drug screening and animal models. We confirm that the first generation of sGC activators cinaciguat and BAY 60-2770 activate the α₁/β₁H105F and α₁/β₁H105A mutants. In contrast, we show that the second generation sGC activators runcaciguat and BAY 543 only activate heme-free sGC when the β₁H105 residue is present. By testing runcaciguat in β₁ H105F knock-in mice, we confirm this histidine-dependency in vivo. We propose a novel classification of sGC activators, distinguishing between the histidine-dependent activators runcaciguat and BAY 543 and the histidine-independent activators cinaciguat, BAY 60-2770 and BI 703704. The histidine-dependency of some of the sGC activators provides a compelling rationale for a re-evaluation of previous research and drug development programs based on sGC histidine mutants. Whether the classification of sGC activators based on the activation mechanism also makes a therapeutic difference needs to be clarified in the future.