Jean-Luc Boucher

Neuronal nitric oxide synthase (nNOS) catalyzes single-electron reduction of quinones (Q), nitroaromatic compounds (ArNO₂) and aromatic N-oxides (ArN → O), and is partly responsible for their oxidative stress-type cytotoxicity. In order to expand a limited knowledge on the enzymatic mechanisms of these processes, we aimed to disclose the specific features of nNOS in the reduction of such xenobiotics. In the absence or presence of calmodulin (CAM), the reactivity of Q and ArN → O increases with their single-electron reduction midpoint potential (E¹₇). ArNO₂ form a series with lower reactivity. The calculations according to an “outer-sphere” electron transfer model show that the binding of CAM decreases the electron transfer distance from FMNH₂ to quinone by 1–2 Å. The effects of ionic strength point to the interaction of oxidants with a negatively charged protein domain close to FMN, and to an increase in accessibility of the active center induced by high ionic strength. The multiple turnover experiments of nNOS show that, in parallel with reduced FAD-FMN, duroquinone reoxidizes the reduced heme, in particular its Fe²⁺-NO form. This finding may help to design the heme-targeted bioreductively activated agents and contribute to the understanding of the role of P-450-type heme proteins in the bioreduction of quinones and other prooxidant xenobiotics.

Specific inhibition of NADPH oxidases (NOX) and NO-synthases (NOS), two enzymes associated with redox stress in tumor cells, has aroused great pharmacological interest. Here, we show how these enzymes distinguish between isomeric 2′- and 3′-phosphate derivatives, a difference used to improve the specificity of inhibition by isolated 2′- and 3′-phosphate isomers of our NADPH analogue NS1. Both isomers become fluorescent upon binding to their target proteins as observed by in vitro assay and in vivo imaging. The 2′-phosphate isomer of NS1 exerted more pronounced effects on NOS and NOX-dependent physiological responses than the 3′-phosphate isomer did. Docking and molecular dynamics simulations explain this specificity at the level of the NADPH site of NOX and NOS, where conserved arginine residues distinguished between the 2′-phosphate over the 3′-phosphate group, in favor of the 2′-phosphate.

It has been reported that formation of H2S from the metabolism of anetholedithiolethione (ADT, Sulfarlem) and anetholedithiolone (ADO) by liver microsomes mainly derived from their oxidation by cytochrome P450 (CYP)-dependent monooxygenases, and that ADO was a better H2S-donor than ADT under these conditions. This article compares the H2S-donor abilities of 18 dithiolethione and dithiolone analogs of ADT and ADO in the presence of rat liver microsomes. It shows that, for all the studied compounds, maximal H2S formation was obtained in microsomal incubations in the presence of NADPH, and that this formation greatly decreased in the presence of N-benzyl imidazole (Bz-ImH), an usual inhibitor of CYP. This indicates that H2S formation from all the studied compounds requires, as previously observed in the case of ADT and ADO, oxidations catalyzed by CYP-dependent monooxygenases. Under these conditions, the studied dithiolones were almost always better H2S-donors than the corresponding dithiolethiones. Interestingly, the best H2S yields (up to 75%) were observed in microsomal oxidation of ADO and its close analogs, pCl-Ph-DO and Ph-DO, in the presence of glutathione (GSH), whereas only small amounts of H2S were formed in microsomal incubations of those compounds in the presence of GSH but in the absence of NADPH. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with ADOSO (ADO sulfoxide), the ADO metabolite involved in H2S formation in microsomal oxidation of ADO. SIGNIFICANCE STATEMENT A series of 18 dithiolethiones and dithiolones was compared for their ability to form hydrogen sulphide (H2S) in oxidations catalyzed by microsomal monooxygenases. The studied dithiolones were better H2S-donors than the corresponding dithiolethiones and the addition of glutathione to the incubations strongly increased H2S formation. A possible mechanism for this effect of GSH is proposed on the basis of results obtained from reactions of GSH with 5-(p-methoxyphenyl)-3H-1,2-dithiole-3-one-1-sulfoxide, a metabolite of the choleretic and sialologic drug Sulfarlem.

The drug anetholedithiolethione, ADT, and its analogs have been extensively used as H2S donors. However, the mechanism of H2S formation from ADT under biological conditions remains almost completely unknown. This article shows that only small amounts of H2S are formed during incubation of ADT and of its metabolite anetholedithiolone, ADO, with rat liver cytosol or with rat liver microsomes (RLM) in the absence of NADPH, indicating that H2S formation under these conditions is only to a minor extent of hydrolytic origin. By contrast, much greater amounts of H2S are formed upon incubation of ADT and ADO with RLM in the presence of NADPH and dioxygen with a concomitant formation of H2S and para-methoxy-acetophenone, pMA. Moreover, H2S and pMA formation under those conditions are greatly inhibited in the presence of N-benzyl-imidazole indicating the involvement of cytochrome P450-dependent monooxygenases. Mechanistic studies show the intermediate formation of the ADT-derived 1,2-dithiolium cation and of the ADO sulfoxide during microsomal metabolism of ADT and ADO, respectively. This article proposes first detailed mechanisms for the formation of H2S from microsomal metabolism of ADT and ADO in agreement with those data and with previously published data on the metabolism of compounds involving a C=S bond. Finally, this article shows for the first time that ADO is a better H2S donor than ADT under those conditions. SIGNIFICANCE STATEMENT Incubation of anetholedithiolethione, ADT, or its metabolite anetholedithiolone, ADO, in the presence of rat liver microsomes, NADPH and O2, leads to H2S. This article shows for the first time that this H2S formation involves several steps catalyzed by microsomal monooxygenases and that ADO is a better H2S donor than ADT. We propose first detailed mechanisms for the formation of H2S from the microsomal metabolism of ADT and ADO.

A study of the metabolism of anethole dithiolethione (ADT) by rat and human liver microsomes showed the formation of the corresponding S-oxide and the S-oxide of desmethyl-ADT (dmADT), and of p-methoxy-acetophenone (pMA) and p-hydroxy-acetophenone (pHA), in addition to the previously described metabolites, dmADT, anethole dithiolone (ADO) and its demethylated derivative, dmADO. The microsomal metabolism of ADO under identical conditions led to dmADO and to pMA and pHA. The metabolites of ADT derive from two competing oxidative pathways: an O-demethylation catalyzed by cytochromes P450 (CYPs) and an S-oxidation mainly catalyzed by flavin-dependent monooxygenases (FMOs) and, to a minor extent, by CYPs. The most active human CYPs for ADT demethylation appeared to be CYP1A1, 1A2, 1B1, 2C9, 2C19 and 2E1. ADT S-oxidation is catalyzed by FMOs 1 and 3, and, to a minor extent, by CYPs such as CYP3A4.

Cytochrome P450 2U1 (CYP2U1) exhibits several distinctive characteristics among the 57 human CYPs, such as its presence in almost all living organisms with a highly conserved sequence, its particular gene organization with only five exons, its major location in thymus and brain, and its protein sequence involving an unusually long N-terminal region containing 8 proline residues and an insert of about 20 amino acids containing 5 arginine residues after the transmembrane helix. Few substrates, including fatty acids, N-arachidonoylserotonin (AS), and some drugs, have been reported so far. However, its biological roles remain largely unknown, even though CYP2U1 mutations have been involved in some pathological situations, such as complicated forms of hereditary spastic paraplegia. These data together with its ability to hydroxylate some fatty acids and AS suggest its possible role in lipid metabolism.