<h3>Title</h3>
<h3>Small Molecule Tools for Studying Cellular Redox Homeostasis</h3>
<p><strong><span style="text-decoration: underline;">Abstract</span> :</strong></p>
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<p class="MsoNormal" style="text-align: justify;"><span style="font-size: 11.0pt;">It has long been recognized that maintenance of redox homeostasis is crucial for cellular survival and growth. However, precise cellular responses to redox stress, where cells loses capacity to counteract excess of reducing or oxidizing equivalents, remain poorly characterized. An important sub-set of various cellular components useful for maintenance of redox homeostasis is gaseous reactive species of nitrogen, oxygen and sulfur. Altered levels of these gases are associated with various pathophysiological conditions and disease states underscoring the importance of regulating intracellular levels of these species. Owing to their high reactivity and diffusible nature, the use of chemical tools for this purpose has become indispensable. Our laboratory has developed several small molecule tools to reliably enhance gaseous redox-active reactive species including superoxide (O<sub>2</sub></span><sup><span style="font-size: 11.0pt; font-family: Symbol; mso-ascii-font-family: 'Times New Roman'; mso-hansi-font-family: 'Times New Roman'; mso-char-type: symbol; mso-symbol-font-family: Symbol;"><span style="mso-char-type: symbol; mso-symbol-font-family: Symbol;">·</span></span></sup><sup><span style="font-size: 11.0pt;"></span></sup><span style="font-size: 11.0pt;">), nitric oxide (NO), sulfur dioxide (SO<sub>2</sub>) and hydrogen sulfide (H<sub>2</sub>S). Our design strategy offers both scope for spatiotemporal control as well as cell-type specificity. Here, we present examples of tools developed in our laboratory that can reliably enhance reactive oxygen, nitrogen and sulfur species and the progress towards studying cellular responses. For example, we have developed bis(4-nitrobenzyl)sulfane, a class of organic sources of H<sub>2</sub>S, that is specifically activated by the bacterial enzyme nitroreductase to generate H<sub>2</sub>S. We provide evidence for the suitability of (4-nitrobenzyl)sulfanes for enhancement of intracellular H<sub>2</sub>S levels in a range of bacteria including mycobacteria. Next, we have developed HyPR-1, a small molecule containing a superoxide generator, strategically linked to a diazeniumdiolate-based nitric oxide donor. HyPR-1 produces nearly temporally concurrent fluxes of superoxide and NO in physiological pH when triggered by DT-diaphorase (DT-D), an enzyme that is commonly found in mammalian cells. We provide unequivocal evidence for HyPR-1’s ability to generate ONOO</span><sup><span style="font-size: 11.0pt; mso-fareast-font-family: Symbol;"></span></sup><span style="font-size: 11.0pt;"> in cell-free systems in the presence of DT-D as well as reliably enhance ONOO</span><sup><span style="font-size: 11.0pt; mso-fareast-font-family: Symbol;"></span></sup><span style="font-size: 11.0pt;"> within cells. Using HyPR-1 in colorectal cancer cells as a case study, we present evidence that ONOO</span><sup><span style="font-size: 11.0pt; mso-fareast-font-family: Symbol;"></span></sup><span style="font-size: 11.0pt;"> mediates epithelial-mesenchymal transition (EMT), which is a key process in metastasis and tumour progression. Together, these studies lay the foundation for understanding mechanisms of antibiotic resistance and cancer progression and metastasis.</span></p>