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

Abstract

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 Cys 244. 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 un-substituted 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.

New acyloxy nitroso compounds with improved water solubility and nitroxyl (HNO) release kinetics and inhibitors of platelet aggregation

“…Presumably, under both buffered and basic conditions, some or all of the thiol exists as the thiolate (an enhanced nucleophile) that adds to the nitroso group of 4-7 , faster than hydrolysis and HNO release, to yield the corresponding oxime (Scheme 3). [31,32] GC-MS measurements of the reaction mixture of 6-7 in the presence of 2-fold GSH in PBS, support this route revealing only dihydro-2 H -pyran-4(3 H )-one oxime ( 8 ) and no dihydro-2 H -pyran-4(3 H )-one as the product. [27] …”

“…[ [32] ] The higher activity of 4 may suggest a non-HNO mediated mechanism for GAPDH inhibition that includes the direct addition of the nitroso group of the acyloxy nitroso compound to the cysteine thiol of GAPDH as previously observed. [32] …”

“…[38,42] Earlier work shows that acyloxy nitroso compounds ( 1-3 ) inhibit GAPDH by modification of various protein thiols as determined by high resolution mass spectrometry (HRMS) suggesting that 4 , 6 and 7 would also inhibit GAPDH. [32] …”

“…[32] High-resolution mass spectra were obtained using a Thermo Scientific LTQ Orbitrap mass spectrometer equipped with a heated electrospray ionization source operated in positive ion mode.…”

“…[30] While capable of releasing HNO, these compounds demonstrate limited water solubility and 1 directly reacts with thiols before HNO release occurs. [31,32] Other acyloxy nitroso compounds ( 4-5 ) that contain a pyran core have been prepared and demonstrate improved water solubility that allows porcine liver esterase (PLE) catalyzed decomposition that greatly increases the rate of HNO release. [31,33] Despite this advance, the use of PLE may not be compatible in all circumstances and the present work describes the synthesis and evaluation of new acyloxy nitroso compounds ( 6-7 , Figure 1) as HNO donors.…”