Conjugates of plumbagin and phenyl-2-amino-1-thioglucoside inhibit MshB, a deacetylase involved in the biosynthesis of mycothiol.

N-Acetylglucosaminylinositol (GlcNAc-Ins)-deacetylase (MshB) and mycothiol-S-conjugate amidase (Mca), structurally related amidases present in mycobacteria and other Actinomycetes, are involved in the biosynthesis of mycothiol and in the detoxification of xenobiotics as their mycothiol-S-conjugates, respectively. With substrate analogs of GlcNAc-Ins, MshB showed a marked preference for inositol as the aglycon present in GlcNAc-Ins. The inhibition of MshB and Mca by 10 thioglycosides, 7 cyclohexyl-2-deoxy-2-C-alkylglucosides, and 4 redox cyclers was evaluated. The latter contained plumbagin tethered via 2 to 5 methylene carbons and an amide linkage to phenyl-2-deoxy-2-amino-1-thio-alpha-d-glucopyranoside. These proved to be the most potent amongst the 21 compounds tested as inhibitors of MshB. Their inhibitory potency varied with the length of the spacer, with the compound with longest spacer being the most effective.

The metabolism of nitrosothiols in the Mycobacteria: identification and characterization of S-nitrosomycothiol reductase.

When grown in culture Mycobacterium smegmatis metabolized S-nitrosoglutathione to oxidized glutathione and nitrate, which suggested a possible involvement of an S-nitrosothiol reductase and mycobacterial haemoglobin. The mycothiol-dependent formaldehyde dehydrogenase from M. smegmatis was purified by a combination of Ni2+-IMAC (immobilized metal ion affinity chromatography), hydrophobic interaction, anion-exchange and affinity chromatography. The enzyme had a subunit molecular mass of 38263 kDa. Steady-state kinetic studies indicated that the enzyme catalyses the NAD+-dependent conversion of S-hydroxymethylmycothiol into formic acid and mycothiol by a rapid-equilibrium ordered mechanism. The enzyme also catalysed an NADH-dependent decomposition of S-nitrosomycothiol (MSNO) by a sequential mechanism and with an equimolar stoichiometry of NADH:MSNO, which indicated that the enzyme reduces the nitroso group to the oxidation level of nitroxyl. Vmax for the MSNO reductase reaction indicated a turnover per subunit of approx. 116700 min(-1), which was 76-fold faster than the formaldehyde dehydrogenase activity. A gene, Rv2259, annotated as a class III alcohol dehydrogenase in the Mycobacterium tuberculosis genome was cloned and expressed in M. smegmatis as the C-terminally His6-tagged product. The purified recombinant enzyme from M. tuberculosis also catalysed both activities. M. smegmatis S-nitrosomycothiol reductase converted MSNO into the N -hydroxysulphenamide, which readily rearranged to mycothiolsulphinamide. In the presence of MSNO reductase, M. tuberculosis HbN (haemoglobin N) was converted with low efficiency into metHbN [HbN(Fe3+)] and this conversion was dependent on turnover of MSNO reductase. These observations suggest a possible route in vivo for the dissimilation of S-nitrosoglutathione.

Trypanosomal antioxidants and emerging aspects of redox regulation in the trypanosomatids.

Leishmania and Trypanosoma are two genera of the protozoal Order Kinetoplastida that cause widespread diseases of humans and their livestock. The production of reactive oxygen and nitrogen intermediates by the host plays an important role in the control of infections by these organisms. Signal transduction and its redox regulation have not been studied in any depth in trypanosomatids, but homologs of the redox-sensitive signal transduction machinery of other eukaryotes have been recognized. These include homologs of activator protein-1, human apurinic endonuclease 1 (Ref-1) endonuclease, iron-responsive protein, protein kinases, and phosphatases. The detoxification of peroxide is catalyzed by a trypanothione-dependent system that has no counterpart in mammals, and thus ranks as one of the biochemical peculiarities of trypanosomatids. There is substantial evidence that trypanothione is essential for the survival of Trypanosoma brucei and for the virulence of Leishmania spp. Apart from trypanothione and its precursors, trypanosomatids also possess significant amounts of N(1)-methyl-4-mercaptohistidine or ovothiol A, but its function in the trypanosomatids is not presently understood. The biosynthesis of ovothiol A in Crithidia fasciculata proceeds by addition of sulfur from cysteine to histidine to form 4-mercaptohistidine. S-(4'-L-Histidyl)-L-cysteine sulfoxide is the transsulfuration intermediate. 4-Mercaptohistidine is subsequently methylated with S-adenosylmethionine as the likely methyl donor.

Thiol metabolism of the trypanosomatids as potential drug targets.

Trypanosomatids produce significant amounts of four major low molecular mass thiols, trypanothione, glutathionylspermidine, glutathione, and ovothiol A. Of these, only glutathione is present in cells of the host. All four low molecular mass thiols are directly or indirectly maintained in a reduced state by trypanothione reductase. Available evidence, from gene disruption studies, indicate that this is an essential enzyme. Attempts to exploit trypanothione reductase as a chemotherapeutic target lead to the design of competitive and irreversible inhibitors of the enzyme. A promising route involves the design of redox cyclers interacting specifically with trypanothione reductase as subversive substrates. Progress in studies on the biosynthesis of ovothiol A is summarized.

Preparation and utilization of a reagent for the isolation and purification of low-molecular-mass thiols.

Problems inherent in the isolation of thiols from natural sources, such as oxidation, undesirable addition reactions, and low concentration of thiol species in cell-free extracts, can be circumvented by reversible derivatization to a less labile form which can be concentrated selectively. These objectives are realized by converting thiols to heterodisulfides in which the thiol partner is an apolar thiol with strong affinity for hydrophobic stationary phases. When reacted with 2-S-(2(')-thiopyridyl)-6-hydroxynaphthyldisulfide at pH<5, where most thiol species are relatively stable to atmospheric oxidation, mixed disulfides with 2-mercapto-6-hydroxynaphthalene as the apolar partner are obtained in good yield and can be concentrated onto a hydrophobic stationary phase. Such heterodisulfides exhibit excellent chromatographic properties when separated on reversed-phase media and the derivatization reaction can, therefore, be conveniently monitored. Following their isolation as the heterodisulfides the thiol species of interest are recovered by reduction and facile separation from the apolar 2-mercapto-6-hydroxynaphthalene partner.

The metabolism of S-nitrosothiols in the trypanosomatids: the role of ovothiol A and trypanothione.

It has recently been established that nitrosoglutathione is the preferred substrate of the glutathione-dependent formaldehyde dehydrogenase from divergent organisms. Trypanosomatids produce not only glutathione, but also glutathionylspermidine, trypanothione and ovothiol A. The formaldehyde dehydrogenase activity of Crithidia fasciculata was independent of these thiols and extracts possessed very low levels of nitrosothiol reductase activity with glutathione or its spermidine conjugates as the thiol component. Although ovothiol A did not form a stable nitrosothiol, it decomposed the S -nitroso groups of nitrosoglutathione (GSNO) and dinitrotrypanothione [T(SNO)(2)] with second-order rate constants of 19.12 M(-1) x s(-1) and 8.67 M(-1) x s(-1) respectively. The reaction of T(SNO)(2) with ovothiol A, however, accelerated to a rate similar to that seen with GSNO. Ovothiol A can act catalytically to decompose these nitrosothiols, although non-productive mechanisms exist. The catalytic phase of the reaction was dependent on the production of thiyl radicals, since it was abolished in the presence of 5,5-dimethyl-1-pyrroline- N -oxide and the formation of nitric oxide could be detected by means of the conversion of oxyhaemoglobin into methaemoglobin. The rate-limiting step in the catalytic process was the reduction of oxidized ovothiol species and, in this respect, T(SNO)(2) is a more efficient substrate than GSNO. Trypanothione decomposed GSNO with a second-order rate constant of 0.786 M(-1) x s(-1) and the major nitrogenous end product changed from nitrite to ammonia as the ratio of thiol to nitrosothiol increased. The results indicate that ovothiol A acts in synergy with trypanothione in the decomposition of T(SNO)(2).

Synthesis of 2-deoxy-2-C-alkylglucosides of myo-inositol as possible inhibitors of a N-deacetylase enzyme in the biosynthesis of mycothiol.

Two new analogues of 1-D-1-O-(2-acetamido-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol, a biosynthetic intermediate in the production of mycothiol in the Mycobacteria have been synthesized. Both the 2-deoxy-2-C-(2'-hydroxypropyl)-D-glucoside 5, and the 2-deoxy-2-C-(2'-oxopropyl)-D-glucoside 6, are derived from fully benzylated 1-D-1-O-(2-C-allyl-2-deoxy)-D-glucopyranosyl)-myo-inositol 20, readily assembled via a protected 2-C-allyl-2-deoxyglucosyl fluoride. Both 5 and 6 inhibit the incorporation of [3H]inositol by whole cells of Mycobacterium smegmatis into a number of metabolites which contain inositol.

Synthesis of mycothiol, 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol, principal low molecular mass thiol in the actinomycetes.

Members of the actinomycetes produce 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol or mycothiol 1 as principal low molecular mass thiol. Chemical synthesis of a biosynthetic precursor of mycothiol, the pseudodisaccharide 1D-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 was achieved by the following steps: (1) Enantioselective synthesis gave the glycosyl acceptors (-)-2,3,4,5,6-penta-O-acetyl-D-myo-inositol D-7 and the corresponding L-isomer L-7. (2) Condensation of D-7 and L-7 with the glycosyl donor 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-alpha-D-glucopyranosylbromide afforded the corresponding alpha and beta anomeric products, which could be resolved by silica gel chromatography. (3) Deprotection of these by hydrolysis using an anion exchange resin gave 1D- and 1L-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 and 15 and the corresponding beta-coupled anomers 14 and 16. Only 13, and to a much lesser extent 15, were used by enzymes present in an ammonium sulphate fraction of a cellfree extract of Mycobacterium smegmatis for the enzymatic synthesis of mycothiol. In the absence of acetyl-SCoA, the immediate biosynthetic precursor of 1, desacetylmycothiol, was the major product.