Search

Soluble guanylyl cyclase (sGC) is the receptor for nitric oxide and a highly sought-after therapeutic target for the management of cardiovascular diseases. New compounds that stimulate sGC show clinical promise, but where these stimulator compounds bind and how they function remains unknown. Here, using a photolyzable diazirine derivative of a novel stimulator compound, IWP-051, and MS analysis, we localized drug binding to the β1 heme domain of sGC proteins from the hawkmoth Manduca sexta and from human. Covalent attachments to the stimulator were also identified in bacterial homologs of the sGC heme domain, referred to as H-NOX domains, including those from Nostoc sp. PCC 7120, Shewanella oneidensis, Shewanella woodyi, and Clostridium botulinum, indicating that the binding site is highly conserved. The identification of photoaffinity-labeled peptides was aided by a signature MS fragmentation pattern of general applicability for unequivocal identification of covalently attached compounds. Using NMR, we also examined stimulator binding to sGC from M. sexta and bacterial H-NOX homologs. These data indicated that stimulators bind to a conserved cleft between two subdomains in the sGC heme domain. L12W/T48W substitutions within the binding pocket resulted in a 9-fold decrease in drug response, suggesting that the bulkier tryptophan residues directly block stimulator binding. The localization of stimulator binding to the sGC heme domain reported here resolves the longstanding question of where stimulators bind and provides a path forward for drug discovery.

Soluble guanylyl/guanylate cyclase (sGC) is the primary receptor for nitric oxide (NO) and is central to the physiology of blood pressure regulation, wound healing, memory formation, and other key physiological activities. sGC is increasingly implicated in disease and is targeted by novel therapeutic compounds. The protein displays a rich evolutionary history and a fascinating signal transduction mechanism, with NO binding to an N-terminal heme-containing domain, which activates the C-terminal cyclase domains. Recent Advances: Crystal structures of individual sGC domains or their bacterial homologues coupled with small-angle x-ray scattering, electron microscopy, chemical cross-linking, and Förster resonance energy transfer measurements are yielding insight into the overall structure for sGC, which is elongated and likely quite dynamic. Transient kinetic measurements reveal a role for individual domains in lowering NO affinity for heme. New sGC stimulatory drugs are now in the clinic and appear to function through binding near or directly to the sGC heme domain, relieving inhibitory contacts with other domains. New sGC-activating drugs show promise for recovering oxidized sGC in diseases with high inflammation by replacing lost heme.Despite the many recent advances, sGC regulation, NO activation, and mechanisms of drug binding remain unclear. Here, we describe the molecular evolution of sGC, new molecular models, and the linked equilibria between sGC NO binding, drug binding, and catalytic activity.Recent results and ongoing studies lay the foundation for a complete understanding of structure and mechanism, and they open the door for new drug discovery targeting sGC. Antioxid. Redox Signal. 26, 107-121.