1,3-Diarylpyrazolyl-acylsulfonamides Target HadAB/BC Complex in Mycobacterium tuberculosis.

Alternative mode-of-inhibition of clinically validated targets is an effective strategy for circumventing existing clinical drug resistance. Herein, we report 1,3-diarylpyrazolyl-acylsulfonamides as potent inhibitors of HadAB/BC, a 3-hydroxyl-ACP dehydratase complex required to iteratively elongate the meromycolate chain of mycolic acids in Mycobacterium tuberculosis (Mtb). Mutations in compound 1-resistant Mtb mutants mapped to HadC (Rv0637; K157R), while chemoproteomics confirmed the compound's binding to HadA (Rv0635), HadB (Rv0636), and HadC. The compounds effectively inhibited the HadAB and HadBC enzyme activities and affected mycolic acid biosynthesis in Mtb, in a concentration-dependent manner. Unlike known 3-hydroxyl-ACP dehydratase complex inhibitors of clinical significance, isoxyl and thioacetazone, 1,3-diarylpyrazolyl-acylsulfonamides did not require activation by EthA and thus are not liable to EthA-mediated resistance. Further, the crystal structure of a key compound in a complex with Mtb HadAB revealed unique binding interactions within the active site of HadAB, providing a useful tool for further structure-based optimization of the series.

Further Insights into the Oxidative Pathway of Thiocarbonyl-Type Antitubercular Prodrugs: Ethionamide, Thioacetazone, and Isoxyl.

A chemical activation study of the thiocarbonyl-type antitubercular prodrugs, ethionamide (ETH), thioacetazone (TAZ), and isoxyl (ISO), was performed. Biomimetic oxidation of ethionamide using H2O2 (1 equiv) led to ETH-SO as the only stable S-oxide compound, which was found to occur in solution in the preferential form of a sulfine (ETH═S═O vs the sulfenic acid tautomer ETH-S-OH), as previously observed in the crystal state. It was also demonstrated that ETH-SO is capable of reacting with amines, as the putative sulfinic derivative (ETH-SO2H) was supposed to do. Unlike ETH, oxidation of TAZ did not allow observation of the mono-oxygenated species (TAZ-SO), leading directly to the more stable sulfinic acid derivative (TAZ-SO2H), which can then lose a SOxH group after further oxidation or when placed in a basic medium. It was also noticed that the unstable TAZ-SO intermediate can lead to the carbodiimide derivative as another electrophilic species. It is suggested that TAZ-SOH, TAZ-SO2H, and the carbodiimide compound can also react with NH2-containing nucleophilic species, and therefore be involved in toxic effects. Finally, ISO showed a very complex reactivity, here assigned to the coexistence of two mono-oxygenated structures, the sulfine and sulfenic acid tautomers. The mono- and dioxygenated derivatives of ISO are also highly unstable, leading to a panel of multiple metabolites, which are still reactive and likely contribute to the toxicity of this prodrug.

Simultaneous determination of a new antituberculosis agent KBF-611 and its de-acetylated metabolite in mouse and rabbit plasma by HPLC.

KBF-611 is a new thiosemicarbazone derivative which has demonstrated significant antituberculosis effect. A sensitive and specific HPLC method was established and validated for the determination of KBF-611 and its deacetylated metabolite (KM) in mouse and rabbit plasma. Chromatographic separation was achieved on a Eurospher-100 C8 column using acetonitrile, methanol, phosphate buffer (pH 7) and TEA (25:5:70:0.1, v/v), as mobile phase at a flow rate of 1 mL/min. KBF-611, KM and internal standard (4-acetamido-3-chlorobenzaldehyde thiosemicarbazone) were detected at the wavelength of 323 nm. The calibration curves were linear within the concentration range from 0.02-5 microg/mL and 0.02-1 microg/mL for KBF-611 and KM respectively. The limit of detection and the limit of quantitation were 6 ng/mL and 20 ng/mL respectively for both KBF-611 and KM. The relative standard deviation for intra- and inter-day precision was less than 7.5%. Average recoveries were 70.8% and 75.0% for KBF-611 and KM respectively. The established HPLC method was validated to be a simple, rapid and reliable procedure and successfully applied to study the preclinical pharmacokinetics of KBF-611 and KM in mice and rabbits.

Human flavin-containing monooxygenase 2.1 catalyzes oxygenation of the antitubercular drugs thiacetazone and ethionamide.

The second-line antitubercular drugs thiacetazone (TAZ) and ethionamide (ETA) are bioactivated by the mycobacterial enzyme EtaA. We report here that human flavin-containing monooxygenase 2.1 (FMO2.1), which is expressed predominantly in the lung, catalyzes oxygenation of TAZ. The metabolites generated, the sulfenic acid, sulfinic acid, and carbodiimide derivatives, are the same as those produced by EtaA and human FMO1 and FMO3. Two of the metabolites, the sulfenic acid and carbodiimide, are known to be harmful to mammalian cells. FMO2.1 also catalyzes oxygenation of ETA, producing the S-oxide. We have developed a novel spectrophotometric assay for TAZ oxygenation. The assay was used to determine kinetic parameters for TAZ oxygenation catalyzed by human FMO1, FMO2.1, and FMO3 and by EtaA. Although the K(M) values for the four enzyme-catalyzed reactions are similar, k(cat) and, consequently, k(cat)/K(M) (the specificity constant) for FMO2.1-catalyzed TAZ oxygenation are much higher than those of FMO1, FMO3, or EtaA. This indicates that FMO2.1 is more effective in catalyzing TAZ oxygenation than are the other three enzymes and thus is likely to contribute substantially to the metabolism of TAZ, decreasing the availability of the prodrug to mycobacteria and producing toxic metabolites. Because of a genetic polymorphism, Europeans and Asians lack FMO2.1. However, in sub-Saharan Africa, a region in which tuberculosis is a major health problem, a substantial proportion of individuals express FMO2.1. Thus, our results may explain some of the observed interindividual differences in response to TAZ and ETA and have implications for the treatment of tuberculosis in sub-Saharan Africa.

Oxidative activation of thiacetazone by the Mycobacterium tuberculosis flavin monooxygenase EtaA and human FMO1 and FMO3.

Thiacetazone (TAZ) and ethionamide (ETA) are, respectively, thiourea- and thioamide-containing second line antitubercular prodrugs for which there is an extensive clinical history of cross-resistance in Mycobacterium tuberculosis. EtaA, a recently identified flavin-containing monooxygenase (FMO), is responsible for the oxidative activation of ETA in M. tuberculosis. We report here that EtaA also oxidizes TAZ and identify a sulfinic acid and a carbodiimide as the isolable metabolites. Both of these metabolites are derived from an initial sulfenic acid intermediate. Oxidation of TAZ by EtaA at basic pH favors formation of the carbodiimide, whereas neutral or acidic conditions favor formation of the sulfinic acid. The same metabolites are formed from TAZ by human FMO1 and FMO3. The sulfenic acid and carbodiimide metabolites, but not the sulfinic acid product, readily react with glutathione, the first to regenerate the parent drug and the second to give a glutathione adduct. These reactions may contribute to the antitubercular activity and/or toxicity of TAZ.

Thiacetazone--avoid like poison or use with care?

Recent 5 reports of severe cutaneous hypersensitivity reactions in patients infected with human immunodeficiency virus (HIV) and with tuberculosis treated with thiacetazone have prompted the World Health Organization to advise against the use of thiacetazone in patients known, or suspected, to be infected with HIV. Because the poorest countries will have great difficulty in replacing thiacetazone, the history, metabolism and possible mechanisms underlying the toxicity of this inexpensive, but problematic, drug are reviewed. Guidelines for National Tuberculosis Control Programme policies in response to thiacetazone toxicity are discussed, taking into account the differing levels of resources available to developing countries.

Characteristics of a cytosolic arylacylamidase metabolizing thiacetazone.

The N-deacetylation of thiacetazone, an antitubercular drug possessing hepatotoxic side effects, by an exclusively cytosolic arylacylamidase has been identified in the liver and kidney of rat by monitoring the appearance of its metabolite p-aminobenzaldehydethiosemicarbazone spectrophotometrically. Studies toward its characterization in liver cytosol revealed that the hydrolase possesses a broad pH optimum ranging from 6.0 to 9.0. The Km and Vmax values for the N-deacetylation of thiacetazone are 5.7 x 10(-4) M and 0.123 nmol of p-aminobenzaldehydethiosemicarbazone formed/min/mg cytosolic protein, respectively. The ability to metabolize thiacetazone was the same in the livers of cat, mouse and human, but lagged significantly in that of rat. Among the biodegradable esters examined as potential rivals of thiacetazone, only aspirin competitively inhibited thiacetazone hydrolysis (Ki = 2.1 x 10(-4) M). Discrimination of cytosolic thiacetazone N-deacetylase from nonspecific p-nitrophenylacetate esterase on the basis of their differential reactivity toward various inhibitors and activators disclosed that low concentrations of p-chloromercuribenzoate, AgNO3 and CuSO4 selectively undermine the activity of thiacetazone N-deacetylase, whereas SKF 525-A, ZnSO4 and FeCl3 are effective inhibitors of p-nitrophenylacetate esterase. However, divalent ions (Ca++ and Mg++) and EDTA failed to alter the activity of the enzyme. Besides, thiacetazone metabolism was significantly retarded upon exposure to malathion. Notably, Nal/Kl stimulated the N-deacetylase activity as a function of iodide concentration. The hydrolysis of thiacetazone in the liver and kidney remained uninduced by phenobarbital, 3-methylcholanthrene or benzo(a)pyrene (80 mg/kg, p.o., 8 days).(ABSTRACT TRUNCATED AT 250 WORDS)