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

一氧化氮（HNO）是在细胞中内源性形成的，在心力衰竭的临床开发中释放它的药物。尽管如此，它仍然是相对不清楚的化学相对的一氧化氮（NO）如何发挥其生物和治疗作用-一个重要的问题，我们将在这里解决。我们将使用氧化还原蛋白质组学来定义蛋白质半胱氨酸，硝酰基后修饰以调节心血管功能。HNO修饰蛋白质硫醇的一个未被充分认识和尚未确定的结果是形成羟胺（NH 2 OH），其从蛋白质中去除修饰，如S-棕榈酰化。因此，我们还将确定心脏和动脉中的蛋白质，这些蛋白质在HNO存在时被形成的羟胺去除。鉴定由HNO和羟胺修饰的靶蛋白将使硝酰基驱动的信号传导的细胞后果能够通过制备和功能表征具有靶半胱氨酸突变的“氧化还原死亡”突变蛋白来确定。缺乏这些特异性调节性半胱氨酸的新型“氧化还原死亡”小鼠的产生将允许在体内建立HNO和羟胺信号传导在心血管健康和疾病中的作用。我们将利用我们的研究结果，从我们的亲电子化合物库中鉴定巯基反应性药物，这些药物靶向并改变由HNO或羟胺修饰的蛋白质的活性。这类药物可能比硝酰基供体具有更少的副作用，除了介导其治疗作用的那些之外，硝酰基供体还引起广泛的脱靶氧化。相比之下，我们确定的亲电子药物将选择性地修饰介导HNO治疗作用的蛋白质半胱氨酸残基。我们将产生的“氧化还原死亡”小鼠对于药物发现也将是非常宝贵的，因为它们将对亲电药物产生抗性，因为它们缺乏关键的半胱氨酸，提供了强大的治疗靶点验证。

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.