A diseasome cluster-based drug repurposing of soluble guanylate cyclase activators from smooth muscle relaxation to direct neuroprotection

Abstract

Network medicine utilizes common genetic origins, markers and co-morbidities to uncover mechanistic links between diseases. These links can be summarized in the diseasome, a comprehensive network of disease-disease relationships and clusters. The diseasome has been influential during the past decade, although most of its links are not followed up experimentally. Here, we investigate a high prevalence unmet medical need cluster of disease phenotypes linked to cyclic GMP. Hitherto, the central cGMP-forming enzyme, soluble guanylate cyclase (sGC), has been targeted pharmacologically exclusively for smooth muscle modulation in cardiology and pulmonology. Here, we examine the disease associations of sGC in a non-hypothesis based manner in order to identify possibly previously unrecognized clinical indications. Surprisingly, we find that sGC, is closest linked to neurological disorders, an application that has so far not been explored clinically. Indeed, when investigating the neurological indication of this cluster with the highest unmet medical need, ischemic stroke, pre-clinically we find that sGC activity is virtually absent post-stroke. Conversely, a heme-free form of sGC, apo-sGC, was now the predominant isoform suggesting it may be a mechanism-based target in stroke. Indeed, this repurposing hypothesis could be validated experimentally in vivo as specific activators of apo-sGC were directly neuroprotective, reduced infarct size and increased survival. Thus, common mechanism clusters of the diseasome allow direct drug repurposing across previously unrelated disease phenotypes redefining them in a mechanism-based manner. Specifically, our example of repurposing apo-sGC activators for ischemic stroke should be urgently validated clinically as a possible first-in-class neuroprotective therapy.

Conflict of interest statement

H.H.H.W.S. receive a research grant from Bayer Healthcare, the patent owner of BAY58-2667 and BAY60-2770. The remaining authors declare no competing financial interests.

Figures

Fig. 1

A cGMP-related phenotype cluster within the human diseasome suggests a predominant neurological relevance. a shows the human disease network where nodes represent disease phenotypes that are linked if they share a genetic association. Different colors indicate different organ systems. Within the network a local cluster contains several diseases phenotypes therapeutically amenable to drugs modulating cGMP forming or cGMP metabolizing enzymes. This is magnified in b and reveals additional metabolic (blue: DM, diabetes mellitus; OB, obesity), pulmonary (brown: AB, asthma) and neurological (red: ST, stroke; AD, Alzheimer’s disease; MG, migraine; DE, dementia; PD, Parkinson’s disease) linked disease phenotypes. For each disease we included in the squares those drugs that are either in the clinic or in late stage clinical development (sGCa, apo-sGC activators; sGCs, sGC stimulators; NOd, NO donors; ARNi; neprilysin inhibitors; PDEi, phosphodiesterase inhibitors). Panels indicate common associated genes c, physical interactions between affected proteins d, shared symptoms e and elevated comorbidity f. When analyzing the interactome of sGC g, its subunits (orange) are significantly closer to neurological diseases (red) than random expectation and surprisingly more distant to cardiovascular applications (dark blue) that current clinical practice (Table 2)

Fig. 2

Decreased sGC expression and activity upon ischemic stroke. We tested the primary role of sGC in stroke in a mouse model of transient middle cerebral artery occlusion (tMCAO). a Collection of the right brain hemispheres 24 h after tMCAO (stroke) or from control animals (non-stroke) and subsequent cryo-homogenization in sGC assay buffer. Subsequently, the expression of sGCα and sGCβ subunits were measured by Western blot b sGC activity as cGMP formation c, d, in the absence or presence of the NO donor, DEA/NO, which stimulates the Fe(II) heme-containing native sGC, or apo-sGC activator (sGCa), BAY58-2667, which activates the heme-free apo-sGC (see Methods). sGCα expression (n = 5, p < 0,05), in b and NO-Fe(II)sGC activity (n = 5) were significantly reduced after stroke. c, d This loss of NO-Fe(II)sGC activity resulted in a near complete shift in the cGMP response being post-stroke largely dependent on apo-sGC activation (n = 3, p < 0,05). e Loss of heme in the NO-responsive Fe(II)sGC upon stroke, which may possibly involve ROS formation (19). The resulting heme-free apo-sGC is prone to degradation of its α subunit. cGMP formation can be partially recovered by apo-sGC activation, which binds to the empty heme pocket of apo-sGC opening up a mechanism-based therapeutic option

Fig. 3

Post-stroke treatment with either of two apo-sGC activators reduces infarct size, prevents blood-brain barrier disruption, improves two of three neurological outcomes and increases survival in a reperfusion-dependent manner. a 24 h after transient (with reperfusion) but not permanent (without reperfusion) occlusion of the middle cerebral artery (tMCAO and pMCAO, resp.) infarct size is reduced in mice treated with BAY58-2667 (30 μg/kg) 1 h post-stroke (n = 10, p < 0,001) and BAY60-2770 (10 μg/kg) 1 h (n = 19, p < 0,001) and 4 h (n = 10, p < 0,001). Infarct volume was also significantly reduced in middle-aged animals treated with BAY60-2770 (10 μg/kg) 1 h post-stroke (n = 8, p < 0,01). b Mechanistically, BAY60-2770 treatment (5 and 10 μg/kg) reduced blood- brain barrier leakage in a dose dependent manner (n = 10, p < 0,0001). c Apo-sGC activation treatment increased survival as soon as after day 1, which persisted until day 8, when 100% of control mice had died (n = 10, p < 0.05). d Post-stroke cerebral blood flow was increased in response to BAY60-2770 (n = 5, p < 0.05) while e systemic blood pressure was not modified at the chosen dose (10 μg/kg) (n = 4). f–h With respect to the neurological outcome of BAY60-2770 treatment in surviving mice, the elevated body swing test indicated a significant increase for the right swing number/total swing number ratio both in adult (n = 19, p < 0.01; panel f) and middle-aged mice (n = 8, p < 0.05; panel f) while significantly improved motor outcome after four limb hanging test was only seen in the adult model (n = 19, p < 0.05, panel g). Similarly, the grip-test was significantly improved in both mice models (n = 19, n = 8, p < 0.05, panel h)

Fig. 4

Both in vitro and in vivo sGC activation is directly neuroprotective in a cGMP-PKG-CREB dependent manner. a Improved outcome in treated mice (see Fig. 3) was associated with decreased cortical neuronal apoptosis (n = 4, p < 0.001) suggesting a novel neuroprotective mechanism. b shows an in vitro ischemia model free of vascular and blood-brain-barrier components. After dissection, hippocampal brain slices were subjected to oxygen and glucose deprivation (OGD)/re-oxygenation (ReOx) in the absence or presence of the sGC activators, BAY58-2667 or BAY60-2770, with and without the cGMP-dependent protein kinase inhibitor, KT5823. Both BAY58-2667 (n = 7, p < 0.05) or BAY60-2667 (n = 13, p < 0.05) increased cell viability after OGD/Re-Ox. Adding the PKG-inhibitor KT5823 reversed this effect for both BAY58-2667 (n = 8, p < 0.05) and BAY60-2770 (n = 7, p < 0.05). c BAY60-2770 treatment increased the ratio of the PKG substrate p-CREB/t-CREB upon OGD/ReOx (n = 8, p < 0.05), an effect was completely prevented in the presence of the PKG-inhibitor KT5823 (n = 4, p < 0.05), suggesting a neuroprotective link. d Similarly, p-CREB/t-CREB ratio was significantly increased in vivo 24 h post-stroke (n = 4, p < 0.05). e Schematic representation of the suggested neuroprotective signaling events: BAY58-2667/60-2770 specifically activates the predominant post-stroke form of sGC (heme-free apo-sGC) and thereby recovers lost sGC-dependent cGMP formation, which leads to CREB phosphorylation and direct neuroprotection