br Introduction Soluble guanylyl cyclase GC maintains vascul

Introduction Soluble guanylyl cyclase (GC-1) maintains vascular function through the NO/GC-1/cGMP pathway [1,2] by catalyzing the conversion of GTP into cGMP (Fig. 1). The GC-1 heme prosthetic group binds NO with picomolar affinity, resulting in a 100- to 200-fold increase in catalytic activity. cGMP activates protein kinase G, which phosphorylates a myriad of targets to induce, among other effects, vasodilation and inhibition of platelet aggregation and adhesion to the arterial wall. Dysfunction in the NO/GC-1/cGMP pathway through reactive oxygen species (ROS) has been linked to a variety of vascular diseases [3,4]. ROS can disrupt this pathway by reacting with free NO, thus reducing NO bioavailability, or oxidizing the ferrous heme group in GC-1, causing it to lose its sensitivity toward NO. GC-1 has a significantly lower affinity for oxidized heme [5]. Once the heme is lost, apo-GC-1 is targeted for degradation [6]. Cardiovascular diseases are responsible for the highest mortality rate globally with approximately 1 in 3 deaths being attributed to a cardio-related illness. The role of GC-1 in maintaining vascular health makes it an ideal target to improve cardiovascular function. Pharmaceuticals that elevate GC-1 activity are classified as either heme-dependent (stimulators) or heme-independent (activators; reviewed in Ref. [7]). In 2013, riociguat became the first FDA-approved stimulator to target GC-1 for the treatment of pulmonary arterial kasugamycin and chronic thromboembolic pulmonary hypertension (CTEPH). Clinical trials in CTEPH patients showed treatment with riociguat improved 6-min walking distances and reduced pulmonary blood pressure over the placebo group [8]. Cinaciguat and ataciguat are known GC-1 activators. Structural evidence using a bacterial homolog of the NO-sensor domain with bound cinaciguat suggested the activator rescues GC-1 activity under oxidative stress by occupying the empty heme pocket and making key interactions with nearby residues while mimicking the NO-severed His ligand [9]. This hypothesis was supported by studies showing that cinaciguat improved cGMP production in endothelial cells after oxidative damage [6]. Further clinical trials with cinaciguat found hypotensive side-effects when treating patients for acute heart failure [10] and were ceased by the FDA in 2011. Treatment of rat aortic smooth muscle cells with ataciguat under oxidative stress improved basal and NO-stimulated GC-1 activity [11]. Ongoing clinical trials are using ataciguat as a treatment for aortic stenosis due to calcification. More recently, studies using the biotinylated IWP-854 GC-1 stimulator helped to identify a conserved binding site for other GC-1 stimulators in the βHNOX NO-sensor domain [12]. Elucidating this binding region could provide the foundation for a class of novel GC-1 stimulators. A dysfunctional NO/GC-1/cGMP pathway has been implicated in other vasculature disorders. Glaucoma is a leading cause of blindness in the United States with approximately 2 million people afflicted and is characterized by increased intraocular pressure (IOP) and damage to the optic nerve. Primary open-angle glaucoma (POAG) is a subtype of glaucoma and has no known underlying etiology. Treatment for POAG manages symptoms through beta-blockers to reduce aqueous humor inflow and eye surgery to relieve IOP [13]. Multiple groups have identified a dysfunctional NO/GC-1/cGMP pathway as an alternate target for POAG treatment (reviewed in Ref. [4]). NO-donors and cGMP have been used in POAG animal studies to lower IOP [14,15]. Older mice lacking the αGC-1 polypeptide had typical POAG symptoms including reduced aqueous humor outflow, increased IOP, and damage to the optic nerve [16], directly implicating GC-1 in the disease. More recently, treatment of mouse eyes with elevated IOP and reduced aqueous humor outflow using a novel GC-1 stimulator improved ocular flow rate over the vehicle-treated group; similar results were observed using an NO-donor [17]. Targeting the NO/GC-1/cGMP pathway for improved optic blood flow may prove useful in the treatment of POAG.