title: Enzymatic generation of the NO/HNO-releasing IPA/NO anion at controlled rates in physiological media using β-galactosidase.
abstract: We introduce a strategy for generating mixtures of nitric oxide (NO) and nitroxyl (HNO) at tunable rates in physiological media. The approach involves converting a spontaneously HNO/NO-generating ion to a caged (prodrug) form that is essentially stable in neutral media, but that can be activated for HNO/NO release by adding an enzyme capable of efficiently opening the cage to regenerate the ion. By judiciously choosing the enzyme, substrate, and reaction conditions, unwanted scavenging of the HNO and NO by the protein can be minimised and the catalytic efficiency of the enzyme can be maintained. We illustrate this approach with a proof-of-concept study wherein the prodrug is Gal-IPA/NO, a diazeniumdiolate of structure iPrHN-N(O)NOR, with R=β-d-galactosyl. Escherichia coli-derived β-d-galactosidase at concentrations of 1.9-15nM hydrolysed 56μM substrate with half-lives of 140-19min, respectively, producing the IPA/NO anion (iPrHN-N(O)NO(-), half-life ∼3min), which in turn spontaneously hydrolysed to mixtures of HNO with NO. Using saturating substrate concentrations furnished IPA/NO generation rates that were directly proportional to enzyme concentration. Consistent with these data, the enzyme/substrate combination applied to ventricular myocytes isolated from wild-type mouse hearts resulted not only in a significant positive inotropic effect, but also rescued the cells from the negative inotropy, hypercontractions, and occasional cell death seen with the enzyme alone. This mechanism represents an alternate approach for achieving controlled fluxes of NO/HNO to investigate their biological actions.
authors.affiliation: Drug Design Section, Chemical Biology Laboratory, National Cancer Institute at Frederick, Frederick, MD 21702, USA. Electronic address: hollandrj@mail.nih.gov.
authors.forename: Ryan J
authors.initials: RJ
authors.lastname: Holland
authors.forename: Rika
authors.initials: R
authors.lastname: Paulisch
authors.forename: Zhao
authors.initials: Z
authors.lastname: Cao
authors.forename: Larry K
authors.initials: LK
authors.lastname: Keefer
authors.forename: Joseph E
authors.initials: JE
authors.lastname: Saavedra
authors.forename: Sonia
authors.initials: S
authors.lastname: Donzelli
mesh_terms: D000818:Animals; D000838:Anions; D001391:Azo Compounds / Q000378:metabolism; D006868:Hydrolysis; D051379:Mice; D032342:Muscle Cells / Q000378:metabolism; D009206:Myocardium / Q000166:cytology / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D009589:Nitrogen Oxides / Q000378:metabolism*; D001616:beta-Galactosidase / Q000378:metabolism*
chemical_list: D000838:Anions; D001391:Azo Compounds; D009589:Nitrogen Oxides; C452539:diazeniumdiolate; D009569:Nitric Oxide; D001616:beta-Galactosidase; C039900:nitroxyl
keywords: Diazeniumdiolate; Gal-IPA/NO; HNO; IPA/NO; NO; Nitroxyl; Prodrug; nitric oxide; nitroxyl; sodium 1-(isopropylamino)diazen-1-ium-1,2-diolate; β-Galactosidase