Soluble guanylate cyclase is a heterodimeric enzyme of 150 kDa molecular mass which consists of two subunits—alpha (α) and beta (β) [
1]. Of several isoforms of α and β subunits, the α1β1 and α2β1 are predominantly expressed heterodimers of sGC in mammalian cells [
2]. Starting from N-terminus, the β subunit folds into H-NOX, PAS, coiled coil (CC) and catalytic domains; the α subunit also follows the same architecture, however its N-terminal does not bind with heme and istherefore termed as pseudo-H-NOX domain [
3]. The β H-NOX harbors a histidine bound heme molecule which is capable of bindingnitric oxide (NO) with femtomolar sensitivity and has been also been found to bind with other gaseous ligands such as O
2 and CO, though with much lesser sensitivity than that with NO [
4,
5,
6,
7]. The sensitivity of sGC is high when its heme moiety is reduced i.e., when the heme iron is in Fe
2+ state. High concentration of cellular reactive oxygen species oxidize the heme (Fe
3+) disabling it from capturing small gaseous ligands which in turn leads to sGC inactivation [
2]. sGC is known as the only receptor of NO in mammals, which regulates many physiological responses such as vasodilation, smooth muscle relaxation, thrombosis, platelet aggregation and inhibition of inflammation [
8,
9]. In mammals, cellular NO is produced by nitric oxide synthases (NOS). On NO binding, H-NOX gets activated which in-turn communicates with PAS and CC domains to activate the cyclase domain for the conversion of a GTP molecule into cGMP. The cGMP serves as second messenger playing significant role in the regulation of many downstream. sGC is therefore a hot target for designing novel drugs to cure the disorders associated to cGMP signaling pathway such as peripheral hypertension, pulmonary arterial hypertension (PAH), heart failure and liver fibrosis [
10,
11].
Experimental and clinical studies have shown that impaired bioavailability of NO contributes to cardiovascular, endothelial, hepatic and pulmonary dysfunctions [
12]. Organic nitrates (sodium nitroprusside, nitroglycerin) have been used as nitrovasodilators for the treatment of cardiovascular disorders, but their utilization has some limitations such as severe hemodynamic effects including reduced bioavailability, lack of selectivity, tolerance and insufficient metabolism [
13]. Therefore, therapeutic approaches sought for discovering novel modulators which could trigger sGC to enhance cGMP production. The sGC modulators have two classes, stimulators and activators. The sGC stimulators stimulate sGC directly i.e., when its heme iron is reduced (Fe
2+) but NO synthesis is impaired or enzyme’s NO sensitivity is compromised. These include YC-1, BAY41-8543, BAY63-2521 (riociguat) and BAY41-2272 [
2,
14,
15]. Among sGC stimulators, riociguat has already been approved by FDA for the treatment of pulmonary hypertension [
16,
17,
18]. sGC activators, such as HMR-1766, BAY58-2667 and BAY60-2770, bind to sGC and activate it in a NO- and heme-independent manner i.e., when heme iron of sGC has been oxidized (Fe
3+) or heme has been lost by the enzyme [
2,
19]. Recently sGC activators have drawn attention of researchers as during oxidative stress, when the enzyme becomes inactive i.e., its heme iron is oxidized by ROS or RNS (Reactive Nitrogen Species), stimulators like riociguat cannot elicit the cyclase activity [
20,
21]. Among the activators, BAY58-2667 (cinaciguat) is already in clinical development by Bayer AG and has been found to elicit vasodilation while preserving the kidney function, although it has been reported to cause hypotension [
22,
23,
24]. The drug has been also found to result a long-lasting antihypertensive effect and inhibit platelet aggregation and ischemia [
25,
26]. The therapeutic potential of BAY60-2770 has been also demonstrated against hypertension in rat, erectile dysfunction in obese mice, platelet aggregation in humans, and asthma in mice [
27,
28,
29,
30,
31,
32]. Both BAY58-2667 and BAY60-2770 mimic the heme moiety, (
Figure 1). Both BAY activators have two charged hydrophilic carboxylate groups which are crucial to interact with the Y-S-R motif of H-NOX like heme carboxylate group. The only difference between the two activators is their hydrophobic tail. These trifurcated activators behave somewhat like heme moiety. Cinaciguat’s tri-benzyl tail establishes hydrophobic binding modes with L4, W74, T78, K83, F97, L101, L104, V108, L148, and L152 residues in bacteria. In contrast to BAY58-2667, the hydrophobic tail of BAY60-2770 extends with fluoro and trifluoromethyl-biphenyl moieties which contribute to extensive interactions with binding pocket residues such as Y2, Y83, S111 and F112 [
11,
22].
In this study, we seek to reveal the dynamics effects of sGC activators BAY58-2667 and BAY60-2770 on bacterial and human H-NOX domains (bH-NOX and hH-NOX). The study aims to enhance our understanding of binding pocket behavior against the activator compounds in both the biological systems (bacteria and humans) and observe the most prominent dynamic events that might be involved in signal transduction for cGMP production. Stability of activator bound bH-NOX and hH-NOX complexes was studied by 50 ns MD simulations. We have observed that there are some non-conserved residue positions in the binding pocket that may affect the dynamics of H-NOX in both systems. This study would therefore provide further insight for designing drug screening projects against H-NOX.