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  • Spatial localization of zGC and zGCAP

    2021-11-19

    Spatial localization of zGC and zGCAP transcripts by in situ hybridization was detected in the ONL. No transcripts were detected in the outermost periphery of the photoreceptor cell layer adjacent to the marginal zone. This region is described as the area of cone genesis and maturation [29], which further indicates that zGCs and zGCAPs are mainly expressed in mature and functional cones.
    Acknowledgement
    Introduction The heterodimeric enzyme soluble guanylate cyclase (sGC) is an endogenous receptor for nitric oxide (NO). NO binds to a haem prosthetic group resulting in a conformational change which activates the enzyme. Upon activation, sGC converts guanosine triphosphate (GTP) into cyclic guanosine monophosphate (cGMP). NO-induced activation of sGC is key to maintaining cardiovascular homeostasis and in the brain, NO—sGC acts as a neurotransmitter—receptor system.1, 2, 3 NO-induced signalling has been implicated in the modulation of synaptic transmission and to act in long-term potentiation, one of the major cellular mechanisms that underlie the processes of learning and memory. In rats, high cGMP levels promote neural stem Nucleozin differentiation to neurons whilst reduced cGMP levels promote differentiation to non-neuronal (mainly glial) cells, which consequently leads to impaired cognitive function. NO can mediate neurotoxicity and cause neuronal cell death. The rapid on–off-kinetics and desensitization profile of NO, combined with variations in the rate of cGMP breakdown, provide fundamental mechanisms for shaping cellular cGMP responses and is important in decoding NO signals under physiological and pathological conditions. The neurotransmitter is associated with pathogenic mechanisms involved in multiple neurodegenerative diseases, including Parkinson’s Disease (PD). The NO—sGC system is also involved in the etiology of migraine. Recent research has suggested the involvement of NO in PD is due to activation of sGC. Disruption of striatal NO-sGC-cGMP signalling cascades resulted in profound changes in behavioural, electrophysiological, and molecular responses to pharmacological manipulations of dopamine and glutamate transmission. Studies performed in animal models of PD with a sGC inhibitor, ODQ 1 (Fig. 1), have shown that the enzyme could be a new drug target for restoring basal ganglia dysfunction and attenuating motor symptoms associated with PD. ODQ 1 has been widely used to study the function of the NO-sGC-cGMP signal transduction pathway and it has been a valuable tool to distinguish signalling events mediated by sGC from those involving other nucleotide cyclases. The small molecule binds to the ferrous haem in the β-subunit of the enzyme, yielding ferric haem which cannot bind NO.11, 12 Haem-binding compounds such as ODQ 1 and its 8-bromo-analogue NS2028, show activity against other haem containing proteins as such as haemoglobin, albeit at high concentrations.13, 14 ODQ may also not be able to block cGMP signalling in all circumstances. Other known ways of inhibiting sGC activity include block of the catalytic site with ATP and GTP analogues16, 17, 18, 19, 20 though these have weak inhibitory potency. Inhibitors such as LY-83583 act indirectly by generating superoxide which reacts rapidly with NO. Previously we demonstrated that surface plasmon resonance (SPR) allied to biochemical screening was an effective way of discovering new sGC ligands. In this study we identified a new small molecule inhibitor of sGC, which does not act through oxidation of the haem.
    Results
    Discussion The sGC heterodimer is composed of a α-subunit and a shorter β-subunit which presents in the N-terminal the prosthetic haem group bound to histidine-105. Each subunit is composed of a haem-nitric oxide binding domain, a PAS-like domain, a coiled-coil bundle, and the C-terminal catalytic domain where turnover of GTP into cGMP occurs. The catalytic domain of sGC shows two sites at the interface of the α- and β-subunits that can accommodate small molecules: the GTP binding site, and an allosteric regulatory binding site.29, 30, 31 The two sites are different, and small molecules can bind to one or to both sites, thus showing a 1:1 or 1:2 binding ratio. We have previously shown by surface plasmon resonance (SPR) that ATP binds to both sites and may inhibit sGC activity via competition with GTP, highlighting the potential for allosteric regulation of the enzyme. GC domains are also present in the membrane-bound natriuretic peptide receptors type-A and type-B. There receptors, also referred to as particulate GCs (pGCs), are activated by binding of natriuretic peptides to the extracellular domain of the receptors, leading to an increase in GTP turnover at the intracellular GC domain.32, 33, 34 In this study we observed by SPR that compound 10 bound to the catalytic domain of sGC in a 1:1 ratio. Inhibition of particulate GC was also observed and supported the hypothesis of interaction at the catalytic domain. Binding of compound 10 to an allosteric site that is also capable of binding ATP could suggest these compounds might also act on other ATP- and cyclic-nucleotide—binding domains. We tested compound 10 against adenylyl cyclase, an enzyme that converts ATP into cAMP, and it showed no activity on basal or forskolin-stimulated adenylyl cyclase (data not shown). However, activity against kinases or phosphodiesterases was not investigated.