Hills, Daniel Richard (2017) In vitro characterisation of biological activity of novel nitric oxide donor compounds. (PhD thesis), Kingston University, .

Abstract

Nitric oxide (NO), being a free radical, has a unique biological chemistry that, since its elucidation as a signaling molecule in the cardiovascular system, has been implicated in a range of diverse biological functions. Both cardiovascular disease, exacerbated by hypertension, and antibiotic-resistant bacterial infections are major yet unavoidable public health concerns. Atypical NO synthesis may be a factor these pathologies and exogenous NO may provide an alternative therapeutic strategy when endogenous NO synthesis is atypically low. Furoxans are an old, yet largely untapped resource of nitric oxide donor compounds. A panel of twelve such compounds were developed through heterocyclic synthetic chemistry and assessed for their potential biological activity. The compounds have had their NO release characterised via the Griess assay and have been investigated for both novel antibacterial and vasodilatory effects. Direct toxicity was explored using an optimised MTT assay to assess cell viability post incubation in three bacterial species: Staphylococcus epidermis, Escherichia coli, and Pseudomonas aeruginosa. The lead compound RJP06A was then exposed to bacterial biofilms, investigating its ability to prevent initial biofilm formation and promote its dispersal before determining its mechanism. Vasodilatory potential of the compounds was studied using rat thoracic aorta, pulmonary artery and renal artery. Results demonstrate that these compounds elicit a toxic effect against all three bacterial species with IC50 values ranging from 31-745 uM, which was enhanced in the presence of S-nitrosoglutathione, despite having negligible toxicity itself. Biofilm investigations of RJP06A demonstrate its ability to disperse mature single-species bacterial biofilms in a dose-dependent manner. Isometric tension data show that three quarters of the compounds produced strong, sustained relaxation of contracted aorta, and two in particular caused almost complete reversal of contraction at concentrations of 250 and 350 nM respectively. Mechanism studies suggest that the lead furoxan, RJP06A exerts its effects primarily through a cGMP-mediated NO-activated pathway, but may also have a small component whereby thiols can bypass this step and activate calcium channels directly. RJP06A has been shown to be better at inducing relaxation of resistance vessels, such as aorta, rather than pulmonary or renal artery. In both investigations, nitric oxide from RJP06A appears to be the key molecule involved in the observed effects, with scavenging evolved NO using haemoglobin attenuating its effect, and importantly the two mechanisms identified in very different models imply an evolutionarily conserved pathway of nitric oxide biology.

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