SYNTHESIS AND EVALUATION OF ENZYME-ACTIVATED NITROXYL (HNO) GENERATORS

Abstract

Nitroxyl (HNO), which is a one electron reduced sibling of nitric oxide (NO), activates intracellular signalling pathways, and has vasodilatory and anti-oxidant properties. Recently, a small molecule that generates HNO has emerged as a safe and effective candidate in the treatment of acute heart failure (AHF). Spontaneous generation of HNO is required for cardiovascular applications. Given that HNO is a short-lived and reactive towards biomolecules, controlled and site-directed generation is a prerequisite for other potential therapeutic applications. HNO donors that can be activated by light, chemical or enzyme are reported but have limitations including uncontrolled HNO release, diminished HNO yields, the generation of toxic by-product(s), and concurrent NO release that may complicate mechanistic interpretations. To overcome these limitations, we designed and synthesized a focussed library of N,N,O-protected hydroxylamines that can be activated by enzymes to produce HNO. First, we synthesized compounds that are candidates for activation by esterase to generate HNO. Using a variety of assay, we demonstrate that this class of compounds produce HNO and the HNO generating profile can be modulated by structural modifications. Using these results as the foundation, we designed a donor that produces HNO as well as a fluorescent signal that can act as a reporter for HNO release. The release of HNO within cells was demonstrated using imaging studies. A nearly concomitant increase in fluorescence due to the reporter was also observed. Consistent with the intermediacy of HNO, these donors were also capable of enhancing sulfane sulfur pool in the presence of hydrogen sulfide suggesting possible antioxidant activity for this class of compounds. Next, to enhance the aqueous solubility of donors, we developed glucosidase and galactosidase-activated nitroxyl donors. Upon activation by -glycosidase, controlled and nearly quantitative HNO release was observed. Lastly, the -galactosidase-activated donor was found to mitigate reactive oxygen species in senescent cells, which are known to over-express -galactosidase. Together, we have developed a series of triggerable and tuneable HNO donors that can permeate cells to enhance HNO, and the design allows for incorporating a fluorescence reporter.