BeyondSNO: Signalling beyond protein S-nitrosylation - determining the roles of nitroxyl and hydroxylamine

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Nitroxyl (HNO) is formed endogenously in cells, with drugs that release it in clinical development for heart failure. Despite this, it remains relatively unclear how this chemical relative of nitric oxide (NO) exerts its biological and therapeutic actions - an important issue we will address here. We will define the protein cysteines that nitroxyl post-translationally modifies to modulate cardiovascular function using redox proteomics. An underappreciated and yet undefined consequence of HNO modifying protein thiols is formation of hydroxylamine (NH2OH), which removes modifications such as S-palmitoylation from proteins. Consequently, we will also identify the proteins in heart and arteries that are removed by hydroxylamine formed when HNO is present. Identifying the target proteins modified by HNO and hydroxylamine will enable the cellular consequences of nitroxyl-driven signalling to be determined by making and functionally characterising 'redox dead' mutant proteins with the target cysteine mutated. Generation of novel 'redox dead' mice that lack these specific regulatory cysteines will allow the role of HNO and hydroxylamine signaling in cardiovascular health and disease to be established in vivo. We will leverage our findings therapeutically by identifying thiol reactive drugs, from our electrophilic compounds library, that target and alter the activity of the proteins modified by HNO or hydroxylamine. Such drugs are likely to have less side effects than nitroxyl donors that cause widespread off-target oxidations in addition to those that mediate their therapeutic actions. In contrast, the electrophilic drugs we identify will selectively modify the protein cysteine residue that mediates the therapeutic actions of HNO. The 'redox dead' mice we will generate will also be invaluable for drug discovery, as they will be resistant to the electrophilic drug because they lack the critical cysteine, providing robust therapeutic target validation.