Soluble guanylate cyclase and NO/cGMP signalling in vascular disease

Soluble guanylate cyclase (sGC) is the main receptor for the vascular signal molecule nitric oxide (NO). sGC plays an important role in cardiovascular physiology via the production of the cellular messenger molecule, cGMP. The native structure of sGC is heterodimeric consisting of an a- and a heme-containing b-subunit. Upon binding of NO to Fe(II) heme, sGC-catalysed conversion of GTP to cGMP is activated approximately 200-fold.

The therapeutic importance of this NO-cGMP pathway has been well recognised for many years with NO and NO donors being used for the treatment of systemic (angina, hypertension), pulmonary (pulmonary hypertension) and cerebral (stroke) vascular disorders. However, the development of nitrate tolerance or potential for cyanide toxicity (eg. from glyceryl trinitrate or sodium nitroprusside, respectively) limit their clinical use. In addition, aberrant sGC-dependent signalling and an associated loss in potency of NO-based therapeutics is observed in a number of cardiovascular disorders such as hypertension and atherosclerosis. This may arise as a consequence of the high degree of oxidative stress associated with these cardiovascular pathologies since the bioavailability of NO is reduced under oxidative stress due to its scavenging by reactive oxygen species (ROS). This also results in the formation of the powerful oxidant peroxynitrite (ONOO^-).

Figure 1. NO-Synthase (NOS) generated NO leads to the activation of the reduced heme-containing sGC and to the subsequent formation of the second messenger cGMP. Reactive oxygen species (ROS) impair this pathway by scavenging NO and by oxidizing sGC to its ferric NO-insensitive state. BAY 58-2667 is able to bypass the impaired NO-sGC-cGMP pathway by activation of the oxidized and heme-free sGC.

Furthermore, sGC activity itself may be reduced as a consequence of oxidative modification by ROS since one major prerequisite for the NO-induced activation of sGC is the presence of the reduced Fe²⁺ heme moiety. The oxidation of the sGC heme strongly reduces its affinity for the sGC heme binding site, often resulting in a subsequent loss of the prosthetic heme group. This leads to the formation of an NO-insensitive form of the enzyme. Such changes in the redox state of sGC can be induced by ROS such as superoxide (O₂^-) and peroxynitrite (ONOO^-) generated under oxidative stress conditions. Indeed, in hypertension and cerebrovascular pathologies such as subarachnoid haemorrhage (SAH), alterations in the redox state of sGC may underlie the attenuated and lack of responsiveness to nitrovasodilators.

 A new series of NO-independent activators of sGC (BAY 58-2667, HMR1766) have recently been developed which preferentially target NO-insensitive oxidised (Fe³⁺) or heme-free (apo) sGC and thus offer a new therapeutic avenue in the treatment of vascular disease. BAY 58-2667, a porphyrin mimetic, replaces the oxidised sGC heme moiety and has the intriguing ability to activate purified sGC more potently once the heme group is oxidised (Fe³⁺) or rendered heme-deficient. Thus, sGC activators, which activate heme-oxidised and heme-free sGC, provide the unique opportunity to preferentially target arteries more susceptible to the effects of oxidative stress and selectively dilate the diseased versus non-diseased vasculature.

Indeed, the vasorelaxant and sGC stimulating activity of BAY 58-2667 is enhanced following oxidation of sGC with oxidants such as ODQ and ONOO^-. Furthermore, under pathological conditions an increase in vasodilator potency of porphyrin mimetics such as BAY 58-2667 appears to correlate with an increase in oxidative stress. Importantly, we have made similar observations in human vessels such that the vasorelaxant potency of BAY 58-2667 is increased in mesocolon arteries from patients with type 2 diabetes versus non-diabetic patients. However, whether a disease-induced increase in the pool of oxidised/heme-free sGC holds true for every blood vessel, vascular bed and species is not yet known.

 These findings with BAY 58-2667 have recently been reproduced with the structurally unrelated sGC activator HMR1766. HMR1766 is an anthranilic acid derivative and preferentially activates the NO-insensitive heme-oxidised (Fe³⁺) form of sGC and possibly heme-free sGC, albeit with a lower potency and efficacy as compared to BAY 58-2667. Thus, BAY 58-2667 and HMR1766 represent a new class of NO-independent sGC activators, which specifically target the oxidised and heme-free forms of the enzyme. Consequently,  these compounds may not only serve as effective therapies for the treatment of cardiovascular disorders associated with oxidative stress such as hypertension, stroke and atherosclerosis but also as unique diagnostic tools to identify oxidative stress and dysfunctional blood vessels. 

Until now, it was impossible to quantify the pool of oxidised sGC within living cells. Due to the amount of expressed heme proteins (1.5nmol per mg protein), biophysical approaches for the identification of the oxidation state of single enzyme species have failed. Moreover, biochemical methods based on the lysis of cells and subsequent purification of sGC combined with spectroscopic determination of the heme oxidation state do not conserve the oxidation state of the native tissue. Therefore, an enzyme specific and non-invasive quantification of the intracellular sGC oxidation offers the only feasible approach. With BAY 58-2667, which interacts specifically with the oxidized form of sGC, this goal has become achievable.

To date only indirect BAY 58-2667-based quantification of the intracellular sGC oxidation state has been achieved via measuring its enzymatic activity or downstream effects such as vessel relaxation.

 Therefore, the Vascular Drug Discovery group will elucidate the mechanisms leading to NO-insensitive sGC and characterise the unique profile of the drug candidates currently in clinical development.