A chemical cousin of nitric oxide called nitroxyl is being developed into heart failure drugs, but no one knows exactly which proteins it grabs hold of inside cells—and that blind spot is now being tackled head-on. Nitroxyl forms naturally in the body, and drug companies are already testing nitroxyl-releasing compounds for heart failure. But without knowing which proteins nitroxyl modifies, or how it removes other chemical tags from those proteins, researchers cannot predict side effects or design better drugs. This project will use redox proteomics to identify the specific cysteine amino acids that nitroxyl targets in heart and artery proteins, and will also map the proteins from which nitroxyl-generated hydroxylamine strips S-palmitoylation modifications. If successful, the team will create genetically engineered “redox dead” mice that lack those specific cysteines, allowing them to confirm which effects are truly due to nitroxyl signalling. They will then screen a library of electrophilic compounds to find drugs that hit only those same cysteines—potentially offering more selective treatments with fewer off-target oxidations than current nitroxyl donors. The redox dead mice will also serve as a validation tool, because any drug that fails to work in those animals must be acting through the intended cysteine. This is fundamental science with a clear therapeutic pipeline: understanding a precise chemical handshake inside cells, then exploiting it.
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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.
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