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

BeyondSNO：蛋白质 S-亚硝基化之外的信号传导 - 确定硝酰基和羟胺的作用

• 批准号: EP/Y027698/1
• 负责人: Philip Eaton
• 金额: $ 269.79万
• 依托单位: Queen Mary University of London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2024
• 资助国家: 英国
• 起止时间: 2024 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=EP%2FY027698%2F1
• 关键词: BeyondSNO; Signalling; beyond; protein; nitrosylation

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.

硝氧基（HNO）在细胞中内源形成，并在心力衰竭的临床发育中释放它。尽管如此，仍然尚不清楚一氧化氮（NO）的化学亲属如何发挥其生物学和治疗作用 - 我们将在这里解决的一个重要问题。我们将定义硝氧基翻译后修饰以使用氧化还原蛋白质组学调节心血管功能的蛋白质半胱氨酸。 HNO修饰蛋白硫醇的结果不足但未定义的后果是形成羟胺（NH2OH），它去除了诸如蛋白质中S-甲米酰化之类的修饰。因此，我们还将鉴定出存在HNO时形成的羟胺去除的心脏和动脉中的蛋白质。鉴定通过HNO和羟胺修饰的靶蛋白将使硝基驱动信号传导的细胞后果能够通过制造和功能表征与靶半胱氨酸突变的“氧化还原死”突变蛋白来确定。缺乏这些特定调节性半胱氨酸的新型“氧化还原死”小鼠的产生将使HNO和羟胺信号传导在体内建立心血管健康和疾病中的作用。我们将通过从我们的亲电化合物文库中鉴定出硫醇反应性药物来利用我们的发现，这些药物靶向和改变了HNO或羟胺修饰的蛋白质的活性。此类药物可能比硝酸供体具有较少的副作用，而硝基供体除了介导其治疗作用的那些外，这些药物还引起广泛的脱靶氧化。相反，我们识别的亲电药物将有选择地修改介导HNO治疗作用的蛋白质半胱氨酸残基。我们将生成的“氧化还原死”小鼠对于药物发现也将是无价的，因为它们将对亲电药物具有抗药性，因为它们缺乏关键的半胱氨酸，从而提供了强大的治疗靶标验证。