Jenna F. DuMond

Nitroxyl-Mediated Disulfide Bond Formation Between Cardiac Myofilament Cysteines Enhances Contractile Function

Rationale: In the myocardium, redox/cysteine modification of proteins regulating Ca2+ cycling can affect contraction and may have therapeutic value. Nitroxyl (HNO), the one-electron-reduced form of nitric oxide, enhances cardiac function in a manner that suggests reversible cysteine modifications of the contractile machinery. Objective: To determine the effects of HNO modification in cardiac myofilament proteins. Methods and Results: The HNO-donor, 1-nitrosocyclohexyl acetate, was found to act directly on the myofilament proteins, increasing maximum force (Fmax) and reducing the concentration of Ca2+ for 50% activation (Ca50) in intact and skinned cardiac muscles. The effects of 1-nitrosocyclohexyl acetate are reversible by reducing agents and distinct from those of another HNO donor, Angeli salt, which was previously reported to increase Fmax without affecting Ca50. Using a new mass spectrometry capture technique based on the biotin switch assay, we identified and characterized the formation by HNO of a disulfide-linked actin–tropomyosin and myosin heavy chain–myosin light chain 1. Comparison of the 1-nitrosocyclohexyl acetate and Angeli salt effects with the modifications induced by each donor indicated the actin–tropomyosin and myosin heavy chain–myosin light chain 1 interactions independently correlated with increased Ca2+ sensitivity and force generation, respectively. Conclusions: HNO exerts a direct effect on cardiac myofilament proteins increasing myofilament Ca2+ responsiveness by promoting disulfide bond formation between critical cysteine residues. These findings indicate a novel, redox-based modulation of the contractile apparatus, which positively impacts myocardial function, providing further mechanistic insight for HNO as a therapeutic agent.

The Chemistry of Nitroxyl-Releasing Compounds

Nitroxyl (HNO) demonstrates a diverse and unique biological profile compared to nitric oxide, a redox-related compound. Although numerous studies support the use of HNO as a therapeutic agent, the inherent chemical reactivity of HNO requires the use of donor molecules. Two general chemical strategies currently exist for HNO generation from nitrogen-containing molecules: (i) the disproportionation of hydroxylamine derivatives containing good leaving groups attached to the nitrogen atom and (ii) the decomposition of nitroso compounds (X-N=O, where X represents a good leaving group). This review summarizes the synthesis and structure, the HNO-releasing mechanisms, kinetics and by-product formation, and alternative reactions of six major groups of HNO donors: Angelis salt, Pilotys acid and its derivatives, cyanamide, diazenium diolate-derived compounds, acyl nitroso compounds, and acyloxy nitroso compounds. A large body of work exists defining these six groups of HNO donors and the overall chemistry of each donor requires consideration in light of its ability to produce HNO. The increasing interest in HNO biology and the potential of HNO-based therapeutics presents exciting opportunities to further develop HNO donors as both research tools and potential treatments.

Ethanesulfohydroxamic acid ester prodrugs of nonsteroidal anti-inflammatory drugs (NSAIDs): synthesis, nitric oxide and nitroxyl release, cyclooxygenase inhibition, anti-inflammatory, and ulcerogenicity index studies.

The carboxylic acid group of the anti-inflammatory (AI) drugs indo-methacin, (S)-naproxen and ibuprofen was covalently linked via a two-carbon ethyl spacer to a sulfohydroxamic acid moiety (CH(2)CH(2)SO(2)NHOH) to furnish a group of hybrid ester prodrugs that release nitric oxide (NO) and nitroxyl (HNO). Biological data acquired for this hitherto unknown class of ethanesulfohydroxamic acid ester prodrugs showed (i) all compounds exhibited superior NO, but similar HNO, release properties relative to arylsulfohydroxamic acids, (ii) the (S)-naproxen and ibuprofen prodrug esters are more potent AI agents than their parent NSAID, (iii) the indomethacin prodrug ester, in contrast to indomethacin which is highly ulcerogenic, showed no visible stomach lesions [ulcer index (UI) = 0 for a 80 μmol/kg oral dose] while retaining potent AI activity, and iv) that the indomethacin prodrug ester, unlike indomethacin which is an ulcerogenic selective COX-1 inhibitor, is a selective COX-2 inhibitor (COX-2 selectivity index = 184) devoid of ulcerogenicity that is attributed to its high COX-2 SI and/or ability to release cytoprotective NO.

Acyloxy Nitroso Compounds as Nitroxyl (HNO) Donors: Kinetics, Reactions with Thiols and Vasodilation Properties

Acyloxy nitroso compounds hydrolyze to nitroxyl (HNO), a nitrogen monoxide with distinct chemistry and biology. Ultraviolet-visible spectroscopy and mass spectrometry show hydrolysis rate depends on pH and ester group structure with the observed rate being trifluoroacetate (3) > acetate (1) > pivalate (2). Under all conditions, 3 rapidly hydrolyzes to HNO. A combination of spectroscopic, kinetic, and product studies show that addition of thiols increases the decomposition rate of 1 and 2, leading to hydrolysis and HNO. Under conditions that favor thiolates, the thiolate directly reacts with the nitroso group, yielding oximes without HNO formation. Biologically, 3 behaves like Angelis salt, demonstrating thiol-sensitive nitric oxide-mediated soluble guanylate cyclase-dependent vasorelaxation, suggesting HNO-mediated vasorelaxation. The slow HNO-donor 1 demonstrates weak thiol-insensitive vasorelaxation, indicating HNO release kinetics determine HNO bioavailability and activity. These results show that acyloxy nitroso compounds represent new HNO donors capable of vasorelaxation depending on HNO release kinetics.

O2-Sulfonylethyl Protected Isopropylamine Diazen-1-ium-1,2-diolates as Nitroxyl (HNO) Donors: Synthesis, β-Elimination Fragmentation, HNO Release, Positive Inotropic Properties, and Blood Pressure Lowering Studies

New types of nonexplosive O(2)-sulfonylethyl protected (-CH(2)CH(2)SO(2)R; R = OMe, NHOMe, NHOBn, Me) derivatives of isopropylamine diazen-1-ium-1,2-diolate (IPA/NO) (2-5) were developed that are designed to act as novel HNO donors. These compounds, with suitable half-lives (6.6-17.1 h) at pH 7.4, undergo a base-induced β-elimination reaction that releases a methyl vinyl sulfone product and the parent IPA/NO anion which subsequently preferentially releases HNO (46-61% range). Importantly, the O(2)-methylsulfonylethyl compound 5 exhibited a significant in vitro inotropic effect up to 283% of the baseline value and increased the rates of contraction and relaxation but did not induce a chronotropic effect. Furthermore, compound 5 (22.5 mg/kg po dose) provided a significant reduction in blood pressure up to 6 h after drug administration. All these data suggest that O(2)-sulfonylethyl protected derivatives of IPA/NO, which are efficient HNO donors, could have potential applications to treat cardiovascular disease(s) such as congestive heart failure.

The new HNO donor, 1-nitrosocyclohexyl acetate, increases contractile force in normal and β-adrenergically desensitized ventricular myocytes

Contractile dysfunction and diminished response to β-adrenergic agonists are characteristics for failing hearts. Chemically donated nitroxyl (HNO) improves contractility in failing hearts and thus may have therapeutic potential. Yet, there is a need for pharmacologically suitable donors. In this study we tested whether the pure and long acting HNO donor, 1-nitrosocyclohexyl acetate (NCA), affects contractile force in normal and pathological ventricular myocytes (VMs) as well as in isolated hearts. VMs were isolated from mice either subjected to isoprenaline-infusion (ISO; 30 μg/g per day) or to vehicle (0.9% NaCl) for 5 days. Sarcomere shortening and Ca2+ transients were simultaneously measured using the IonOptix system. Force of contraction of isolated hearts was measured by a Langendorff-perfusion system. NCA increased peak sarcomere shortening by+40-200% in a concentration-dependent manner (EC50 ∼55 μM). Efficacy and potency did not differ between normal and chronic ISO VMs, despite the fact that the latter displayed a markedly diminished inotropic response to acute β-adrenergic stimulation with ISO (1 μM). NCA (60 μM) increased peak sarcomere shortening and Ca2+ transient amplitude by ∼200% and ∼120%, respectively, suggesting effects on both myofilament Ca2+ sensitivity and sarcoplasmic reticulum (SR) Ca2+ cycling. Importantly, NCA did not affect diastolic Ca2+ or SR Ca2+ content, as assessed by rapid caffeine application. NCA (45 μM) increased force of contraction by 30% in isolated hearts. In conclusion, NCA increased contractile force in normal and β-adrenergically desensitized VMs as well as in isolated mouse hearts. This profile warrants further investigations of this HNO donor in the context of heart failure.

Pharmacological Characterization of 1-Nitrosocyclohexyl Acetate, a Long-Acting Nitroxyl Donor That Shows Vasorelaxant and Antiaggregatory Effects

Nitroxyl (HNO) donors have potential benefit in the treatment of heart failure and other cardiovascular diseases. 1-Nitrosocyclohexyl acetate (NCA), a new HNO donor, in contrast to the classic HNO donors Angeli’s salt and isopropylamine NONOate, predominantly releases HNO and has a longer half-life. This study investigated the vasodilatative properties of NCA in isolated aortic rings and human platelets and its mechanism of action. NCA was applied on aortic rings isolated from wild-type mice and apolipoprotein E–deficient mice and in endothelial-denuded aortae. The mechanism of action of HNO was examined by applying NCA in the absence and presence of the HNO scavenger glutathione (GSH) and inhibitors of soluble guanylyl cyclase (sGC), adenylyl cyclase (AC), calcitonin gene-related peptide receptor (CGRP), and K+ channels. NCA induced a concentration-dependent relaxation (EC50, 4.4 µM). This response did not differ between all groups, indicating an endothelium-independent relaxation effect. The concentration-response was markedly decreased in the presence of excess GSH; the nitric oxide scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide had no effect. Inhibitors of sGC, CGRP, and voltage-dependent K+ channels each significantly impaired the vasodilator response to NCA. In contrast, inhibitors of AC, ATP-sensitive K+ channels, or high-conductance Ca2+-activated K+ channels did not change the effects of NCA. NCA significantly reduced contractile response and platelet aggregation mediated by the thromboxane A2 mimetic 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α in a cGMP-dependent manner. In summary, NCA shows vasoprotective effects and may have a promising profile as a therapeutic agent in vascular dysfunction, warranting further evaluation.

Water soluble acyloxy nitroso compounds: HNO release and reactions with heme and thiol containing proteins

Nitroxyl (HNO) has gained interest as a potential treatment of congestive heart failure through the ability of the HNO donor, Angelis salt (AS), to evoke positive inotropic effects in canine cardiac muscle. The release of nitrite during decomposition limits the use of AS requiring other HNO sources. Acyloxy nitroso compounds liberate HNO and small amounts of nitrite upon hydrolysis and the synthesis of the water-soluble 4-nitrosotetrahydro-2H-pyran-4-yl acetate and pivalate allows for pig liver esterase (PLE)-catalysis increasing the rate of decomposition and HNO release. The pivalate derivative does not release HNO, but the addition of PLE catalyzes hydrolysis (t(1/2)=39 min) and HNO formation (65% after 30 min). In the presence of PLE, this compound converts metmyoglobin (MetMb) to iron nitrosyl Mb and oxyMb to metMb indicating that these compounds only react with heme proteins as HNO donors. The pivalate in the presence and the absence of PLE inhibits aldehyde dehydrogenase (ALDH) with IC(50) values of 3.5 and 3.3 μM, respectively, in a time-dependent manner. Reversibility assays reveal reversible inhibition of ALDH in the absence of PLE and partially irreversible inhibition with PLE. Liquid chromatography-mass spectrometry (LC-MS) reveals formation of a disulfide upon incubation of an ALDH peptide without PLE and a mixture of disulfide and sulfinamide in the presence of PLE. A dehydroalanine residue forms upon incubation of this peptide with excess AS. These results identify acyloxy nitroso compounds as unique HNO donors capable of thiol modification through direct electrophilic reaction or HNO release.

Acyclic triaryl olefins possessing a sulfohydroxamic acid pharmacophore: synthesis, nitric oxide/nitroxyl release, cyclooxygenase inhibition, and anti-inflammatory studies

Nitric oxide (NO) and its reduced form nitroxyl (HNO), effective vasodilation agents that can inhibit platelet aggregation and adhesion, could suppress adverse cardiovascular effects associated with the use of selective COX-2 inhibitors. In this regard, a sulfohydroxamic acid (SO(2)NHOH) substituent, that can act as a dual NO/HNO donor moiety, was inserted at the para-position of the C2 phenyl ring of acyclic 2-alkyl-1,1,2-triaryl olefins previously shown to be potent and highly selective COX-2 inhibitors. Although this new group of 1,1-diaryl-2-(4-hydroxyaminosulfonylphenyl)alk-1-enes exhibited weak inhibition of the constitutive cyclooxygenase-1 (COX-1) and inducible COX-2 isozymes, in vivo studies showed anti-inflammatory potencies that were generally intermediate between that of the reference drugs aspirin and ibuprofen. All compounds released NO (5.6-13.5% range) upon incubation with phosphate buffer which was increased further (8.3-25.6% range) in the presence of the oxidant K(3)(FeCN(6)).The low release of HNO in MeOH-buffer (< 2% at 24 h incubation) was much higher at alkaline pH (11-37% range). The concept of designing better anti-inflammatory drugs possessing either an effective HNO, or dual NO/HNO, donor moiety that are devoid of adverse ulcerogenic and/or cardiovascular side effects warrants further investigation.

Direct and nitroxyl (HNO)-mediated reactions of acyloxy nitroso compounds with the thiol-containing proteins glyceraldehyde 3-phosphate dehydrogenase and alkyl hydroperoxide reductase subunit C.

Nitroxyl (HNO) reacts with thiols, and this reactivity requires the use of donors with 1-nitrosocyclohexyl acetate, pivalate, and trifluoroacetate, forming a new group. These acyloxy nitroso compounds inhibit glyceraldehyde 3-phosphate dehydrogenase (GAPDH) by forming a reduction reversible active site disulfide and a reduction irreversible sulfinic acid or sulfinamide modification at Cys244. Addition of these acyloxy nitroso compounds to AhpC C165S yields a sulfinic acid and sulfinamide modification. A potential mechanism for these transformations includes nucleophilic addition of the protein thiol to a nitroso compound to yield an N-hydroxysulfenamide, which reacts with thiol to give disulfide or rearranges to sulfinamides. Known HNO donors produce the unsubstituted protein sulfinamide as the major product, while the acetate and pivalate give substituted sulfinamides that hydrolyze to sulfinic acids. These results suggest that nitroso compounds form a general class of thiol-modifying compounds, allowing their further exploration.