Residue analysis, dissipation patterns of chlorfenapyr, diafenthiuron and their corresponding metabolites in tea trees, and dietary intake risk assessment.

BACKGROUND: Recently, chlorfenapyr and diafenthiuron have been widely used to prevent and control diseases and pests in tea production. However, rare studies have investigated the dissipation patterns of chlorfenapyr, diafenthiuron and their metabolites simultaneously in tea matrices. Here, we established an analytical method to investigate the degradation patterns of five target compounds in tea shoots and made tea samples. Moreover, the dietary intake risk assessment of chlorfenapyr-diafenthiuron mixture among Chinese populations was evaluated based on the supervised field experiment. RESULTS: The mean recoveries of the primary analytes at five spiking levels were between 95.6% and 112.6% in tea shoots and made tea, respectively, and the values of RSD (relative standard deviation) were lower than 9.7% for all the target analytes. The field trial results showed that the half-lives of chlorfenapyr and diafenthiuron based on the residue definition were 10.0-12.4 days and 4.3-5.9 days, respectively, in tea shoots. For the dietary intake risk assessment, the risk quotient (RQ) values in made tea ranged from 30.4% to 73.9% at the pre-harvest interval of 14 days, which were significantly less than 100%. CONCLUSION: The accuracy and precision of the developed method were satisfied by the measurement requirements according to the validation results. The dynamic dissipation experiments suggested that diafenthiuron was much easier to dissipate than chlorfenapyr. Moreover, the existence of tralopyril made the half-life of chlorfenapyr significantly increase, indicating that practical application of chlorfenapyr should take careful consideration of its metabolite. Finally, the potential chronic dietary risks of the chlorfenapyr-diafenthiuron mixture to human communities were within the acceptable range. © 2022 Society of Chemical Industry.

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.

Development of phenylthiourea derivatives as allosteric inhibitors of pyoverdine maturation enzyme PvdP tyrosinase.

Infections caused by Pseudomonas aeruginosa become increasingly difficult to treat because these bacteria have acquired various mechanisms for antibiotic resistance, which creates the need for mechanistically novel antibiotics. Such antibiotics might be developed by targeting enzymes involved in the iron uptake mechanism because iron is essential for bacterial survival. For P. aeruginosa, pyoverdine has been described as an important virulence factor that plays a key role in iron uptake. Therefore, inhibition of enzymes involved in the pyoverdine synthesis, such as PvdP tyrosinase, can open a new window for the treatment of P. aeruginosa infections. Previously, we reported phenylthiourea as the first allosteric inhibitor of PvdP tyrosinase with high micromolar potency. In this report, we explored structure-activity relationships (SAR) for PvdP tyrosinase inhibition by phenylthiourea derivatives. This enables identification of a phenylthiourea derivative (3c) with a potency in the submicromolar range (IC50 = 0.57 + 0.05 µM). Binding could be rationalized by molecular docking simulation and 3c was proved to inhibit the bacterial pyoverdine production and bacterial growth in P. aeruginosa PA01 cultures.

Effect of Treatment With 3-Octylthio-1,1,1-Trifluoropropan-2-One in the Diamondback Moth (Lepidoptera: Plutellidae) to the Toxicity of Diafenthiuron, Indoxacarb, and Bacillus thuringiensis.

The diamondback moth, Plutella xylostella (L.), is a worldwide insect pest of cruciferous crops. Although insecticides have long been used for its control, diamondback moth rapidly evolves resistance to almost any insecticide. In insects, juvenile hormone (JH) is critically involved in almost all biological processes. The correct activity of JH depends on the precise regulation of its titer, and juvenile hormone esterase (JHE) is the key regulator. Thus, JH and JHE have become important targets for new insecticide development. Trifluoromethyl ketones are specific JHE inhibitors, among which 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP) has the highest activity. The interaction effects between pretreatment with or combination of OTFP and the insecticides diafenthiuron, indoxacarb, and Bacillus thuringiensis (Bt) were investigated in diamondback moth larvae to determine OTFP's potential as an insecticide synergist. In third-instar larvae, both pretreatment and combination treatment with OTFP decreased or antagonized the toxicities of diafenthiuron, indoxacarb, and Bt at all set concentrations. In fourth-instar larvae, combination treatment with OTFP decreased or antagonized the toxicities of diafenthiuron and indoxacarb at all set concentrations. However, it increased or synergized the toxicity of Bt at lower concentrations despite the limited effect at higher concentrations. Our results indicated that the effect of OTFP on the toxicities of insecticides varied with the type and concentration, larval stage, and treatment method. These findings contribute to the better use of OTFP in diamondback moth control.

Sublethal effects of methylthio-diafenthiuron on the life table parameters and enzymatic properties of the diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae).

The diamondback moth (DBM), Plutella xylostella (L.), is a major pest affecting cruciferous vegetables, and seriously affects the quality and yield of these vegetables. Diafenthiuron is a traditional thiourea-based insecticide, but it is rarely used to control pests on cruciferous vegetables due to its phytotoxicity on these vegetables under high temperature and light conditions. Thus, there is an ongoing need for more effective pesticides that can be used on cruciferous vegetables, possibly including new formulations of diafenthiuron. A new thiourea insecticide, methylthio-diafenthiuron, is intended to optimize the structure of diafenthiuron not only to preserve its insecticidal bioactivity but also to overcome its phytotoxicity to cruciferous vegetables, aiming to control insect pests on cruciferous vegetables. In this study, we compared the toxicity of methylthio-diafenthiuron to some frequently used insecticides on the third-instar larvae of DBM. The parental pupal duration was significantly longer under the treatment than in the control, but the pupal weight, fecundity, and hatching rate significantly decreased. By studying the changes in three detoxifying enzymes within 72 h after treatment with a sublethal concentration, the activity of CarE and ODM in the treatment group significantly increased at first and then decreased. In addition, methylthio-diafenthiuron clearly inhibited three kinds of ATPases in the DBM and significantly reduced the eclosion rate of the pupae. This research provides valuable information for the assessment and rational application of methylthio-diafenthiuron for the control of pests on cruciferous vegetables.

Disubstituted 4-Chloro-3-nitrophenylthiourea Derivatives: Antimicrobial and Cytotoxic Studies.

4-Chloro-3-nitrophenylthioureas 1⁻30 were synthesized and tested for their antimicrobial and cytotoxic activities. Compounds exhibited high to moderate antistaphylococcal activity against both standard and clinical strains (MIC values 2⁻64 µg/mL). Among them derivatives with electron-donating alkyl substituents at the phenyl ring were the most promising. Moreover, compounds 1⁻6 and 8⁻19 were cytotoxic against MT-4 cells and various other cell lines derived from human hematological tumors (CC50 ≤ 10 µM). The influence of derivatives 11, 13 and 25 on viability, mortality and the growth rate of immortalized human keratinocytes (HaCaT) was observed.

Histone deacetylase 8 protects human proximal tubular epithelial cells from hypoxia-mimetic cobalt- and hypoxia/reoxygenation-induced mitochondrial fission and cytotoxicity.

Cell death by hypoxia followed by reoxygenation (H/R) is responsible for tissue injury in multiple pathological conditions. Recent studies found that epigenetic reprogramming mediated by histone deacetylases (HDACs) is implicated in H/R-induced cell death. However, among 18 different isoforms comprising 4 classes (I-IV), the role of each HDAC in cell death is largely unknown. This study examined the role of HDAC8, which is the most distinct isoform of class I, in the hypoxia mimetic cobalt- and H/R-induced cytotoxicity of human proximal tubular HK-2 cells. Using the HDAC8-specific activator TM-2-51 (TM) and inhibitor PCI34051, we found that HDAC8 played a protective role in cytotoxicity. TM or overexpression of wild-type HDAC8, but not a deacetylase-defective HDAC8 mutant, prevented mitochondrial fission, loss of mitochondrial transmembrane potential and release of cytochrome C into the cytoplasm. TM suppressed expression of dynamin-related protein 1 (DRP1) which is a key factor required for mitochondrial fission. Suppression of DRP1 by HDAC8 was likely mediated by decreasing the level of acetylated histone H3 lysine 27 (a hallmark of active promoters) at the DRP1 promoter. Collectively, this study shows that HDAC8 inhibits cytotoxicity induced by cobalt and H/R, in part, through suppressing DRP1 expression and mitochondrial fission.

Effect of Diafenthiuron exposure under short and long term experimental conditions on hematology, serum biochemical profile and elemental composition of a non-target organism, Labeo rohita.

Diafenthiuron is a thiourea compound that has a novel mode of action as it inhibits mitochondrial functioning in insect pests. It has been reported in local newspapers that this pesticide is entering in our fresh water bodies on regular basis and it is a potential threat for aquatic life. The present study was designed to determine effect of Diafenthiuron, a commonly used pesticide in Pakistan, on the hematology, serum biochemical profile and elemental composition of a non-target organism, Labeo rohita (L. rohita). A sub-lethal dose (0.0075 mg L-1) of Diafenthiuron was applied under short (2, 4 and 8 days) and long term (16, 32 and 64 days) experimental conditions. Our results indicated that the pesticide exposed fish had significantly higher white blood count, lymphocyte, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, red cell distribution width than the control group. However, platelets count, plateletcrit and platelet distribution width were significantly reduced in Diafenthiuron treatments than their respective control groups. Concentration of total serum proteins, albumin, globulin, cholesterol, triglycerides and AST were disturbed in pesticide exposed treatments compared to control groups. Comparison of elemental concentrations revealed that calcium, potassium and cadmium concentration varied significantly when compared between Diafenthiuron treated and untreated L. rohita. In conclusion, we are reporting that Diafenthiuron can adversely affect the hematological, serum and elemental concentrations of a non-target organism like L. rohita and may therefore pose a threat to the food web.

Susceptibility of field populations of the diamondback moth, Plutella xylostella, to a selection of insecticides in Central China.

The diamondback moth (DBM), Plutella xylostella (L.) (Lepidoptera: Plutellidae), is a globally distributed and important economic pest. Chemical control is the primary approach to regulate populations of this pest. However, resistance to insecticides evolves following heavy and frequent use. Therefore, the insecticide resistance in field populations of P. xylostella collected from Central China from 2013 to 2014 was determined with a leaf-dipping method. Based on the results of the monitoring, P. xylostella has developed high levels of resistance to beta-cypermethrin (resistance ratio=69.76-335.76-fold), Bt (WG-001) (RR=35.43-167.36), and chlorfluazuron (RR=13.60-104.95) and medium levels of resistance to chlorantraniliprole (RR=1.19-14.26), chlorfenapyr (RR=4.22-13.44), spinosad (RR=5.89-21.45), indoxacarb (RR=4.01-34.45), and abamectin (RR=23.88-95.15). By contrast, the field populations of P. xylostella remained susceptible to or developed low levels of resistance to diafenthiuron (RR=1.61-8.05), spinetoram (RR=0.88-2.35), and cyantraniliprole (RR=0.4-2.15). Moreover, the LC50 values of field populations of P. xylostella were highly positively correlated between chlorantraniliprole and cyantraniliprole (r=0.88, P=0.045), chlorantraniliprole and spinosad (r=0.66, P=0.039), spinosad and diafenthiuron (r=0.57, P=0.0060), and chlorfenapyr and diafenthiuron (r=0.51, P=0.016). Additionally, the activities of detoxification enzymes in field populations of P. xylostella were significantly positively correlated with the log LC50 values of chlorantraniliprole and spinosad. The results of this study provide an important base for developing effective and successful strategies to manage insecticide resistance in P. xylostella.

Targeting of Chk2 as a countermeasure to dose-limiting toxicity triggered by topoisomerase-II (TOP2) poisons.

The DNA damage response (DDR) gene cell cycle checkpoint kinase 2 (Chk2) triggers programmed cell death and lethal radiation-induced toxicity in mice in vivo. However, it is not well established to what extent targeting of Chk2 may protect from dose-limiting toxicities (DLT) inflicted by mainstay cancer chemotherapy. We screened different classes of chemotherapy in wild type and Chk2-deficient cells. Here we show that loss of Chk2 protect from cell death in vitro and lethal toxicity in vivo following treatment with topoisomerase II (TOP2)-inhibitors whereas no such protection was observed following treatment with topoisomerase I (TOP1) inhibitors. Furthermore, through combined in silico and functional screens of the Diversity Set II (NCI/NTP) chemical library we identified the carbanilide-derivative NSC105171, also known as ptu-23, as a novel Chk2 inhibitor (Chk2i). Indeed, NSC105171 can be administered safely to mice to countermeasure etoposide-induced toxicity. Incorporation of Chk2i into chemotherapy protocols employing TOP2-inhibitors may be an effective strategy to prevent DLT's without interfering with treatment.

Cis-vaccenic acid induces differentiation and up-regulates gamma globin synthesis in K562, JK1 and transgenic mice erythroid progenitor stem cells.

Gamma globin induction remains a promising pharmacological therapeutic treatment mode for sickle cell anemia and beta thalassemia, however Hydroxyurea remains the only FDA approved drug which works via this mechanism. In this regard, we assayed the γ-globin inducing capacity of Cis-vaccenic acid (CVA). CVA induced differentiation of K562, JK1 and transgenic mice primary bone marrow hematopoietic progenitor stem cells. CVA also significantly up-regulated γ-globin gene expression in JK-1 and transgenic mice bone marrow erythroid progenitor stem cells (TMbmEPSCs) but not K562 cells without altering cell viability. Increased γ-globin expression was accompanied by KLF1 suppression in CVA induced JK-1 cells. Erythropoietin induced differentiation of JK-1 cells 24h before CVA induction did not significantly alter CVA induced differentiation and γ-globin expression in JK-1 cells. Inhibition of JK-1 and Transgenic mice bone marrow erythroid progenitor stem cells Fatty acid elongase 5 (Elovl5) and Δ(9) desaturase suppressed the γ-globin inductive effects of CVA. CVA treatment failed to rescue γ-globin expression in Elovl5 and Δ(9)-desaturase inhibited cells 48 h post inhibition in JK-1 cells. The data suggests that CVA directly modulates differentiation of JK-1 and TMbmEPSCs, and indirectly modulates γ-globin gene expression in these cells. Our findings provide important clues for further evaluations of CVA as a potential fetal hemoglobin therapeutic inducer.

Palladium(II) complexes of OS donor N-(di(butyl/phenyl)carbamothioyl)benzamide and their antiamoebic activity.

Two promising palladium(II) compounds of general formula, cis-[Pd(L-O,S)2] [where HL-O,S = N-(di(butyl/phenyl)carbamothioyl)benzamide] as metal based antiamoebic drug candidates, have been synthesized. Both complexes are characterized in the solid state by FT-IR spectroscopy, TGA and single crystal X-ray study, as well as in solution by other spectroscopic techniques such as (1)H and (13)C NMR, and UV-visible. All these studies confirm the coordination of ligands through oxygen and sulphur atoms upon thioenolization induced delocalization. Complexes adopt cis-configuration in the solid state. Both the complexes and their respective ligands were screened in vitro for antiamoebic activity against HM1:1MSS strain of Entamoeba histolytica by microdilution method and cell viability in response to drugs was checked by using MTT assay. The IC50 values in the range 0.30-0.80 µM for ligands as well as complexes compared to 1.40 for metronidazole along with their similar inhibitory effect on cell viability of HEK293 cells like metronidazole make them promising future antiamoebic drugs.

Synthesis, X-ray, NMR, FT-IR, UV/vis, DFT and TD-DFT studies of N-(4-chlorobutanoyl)-N'-(2-, 3- and 4-methylphenyl)thiourea derivatives.

A new isomers of thiourea derivatives, namely N-(4-chlorobutanoyl)-N'-(2-methylphenyl)-thiourea (1a), N-(4-chlorobutanoyl)-N'-(3-methylphenyl)thiourea (1b) and N-(4-chlorobutanoyl)-N'-(4-methylphenyl)thiourea (1c) have been synthesized by refluxing mixture of equimolar amounts of 4-chlorobutanoylisothiocyanate with 2, 3 or 4-toluidine, respectively. The three isomers were characterized by spectroscopic (UV/vis, FT-IR and NMR) and X-ray crystallography techniques. To investigate the isomerization effect on spectroscopic data, DFT and TD-DFT calculations have been carried out using five hybrid functionals (B3LYP, B3P86, CAM-B3LYP, M06-2X and PBE0) to predict UV/vis absorption bands (n→π∗ and π→π∗), (1)H and (13)C NMR chemical shifts, FT-IR vibration modes and X-ray parameters (bonds, bond angles and torsion angles) for 1a, 1b and 1c isomers. The results showed that the isomerization effect is significant on λ(MAX) absorption bands, while for IR and NMR the effect is negligible. In accordance with previous studies, B3LYP, B3P86 and PBE0 gave the most reliable to predict the excitation energies of thiourea derivatives.

Mechanism of N-Acylthiourea-mediated activation of human histone deacetylase 8 (HDAC8) at molecular and cellular levels.

We reported previously that an N-acylthiourea derivative (TM-2-51) serves as a potent and isozyme-selective activator for human histone deacetylase 8 (HDAC8). To probe the molecular mechanism of the enzyme activation, we performed a detailed account of the steady-state kinetics, thermodynamics, molecular modeling, and cell biology studies. The steady-state kinetic data revealed that TM-2-51 binds to HDAC8 at two sites in a positive cooperative manner. Isothermal titration calorimetric and molecular modeling data conformed to the two-site binding model of the enzyme-activator complex. We evaluated the efficacy of TM-2-51 on SH-SY5Y and BE(2)-C neuroblastoma cells, wherein the HDAC8 expression has been correlated with cellular malignancy. Whereas TM-2-51 selectively induced cell growth inhibition and apoptosis in SH-SY5Y cells, it showed no such effects in BE(2)-C cells, and this discriminatory feature appears to be encoded in the p53 genotype of the above cells. Our mechanistic and cellular studies on HDAC8 activation have the potential to provide insight into the development of novel anticancer drugs.

Compromised structure and function of HDAC8 mutants identified in Cornelia de Lange Syndrome spectrum disorders.

Cornelia de Lange Syndrome (CdLS) is a multiple congenital anomaly disorder resulting from mutations in genes that encode the core components of the cohesin complex, SMC1A, SMC3, and RAD21, or two of its regulatory proteins, NIPBL and HDAC8. HDAC8 is the human SMC3 lysine deacetylase required for cohesin recycling in the cell cycle. To date, 16 different missense mutations in HDAC8 have recently been identified in children diagnosed with CdLS. To understand the molecular effects of these mutations in causing CdLS and overlapping phenotypes, we have fully characterized the structure and function of five HDAC8 mutants: C153F, A188T, I243N, T311M, and H334R. X-ray crystal structures reveal that each mutation causes local structural changes that compromise catalysis and/or thermostability. For example, the C153F mutation triggers conformational changes that block acetate product release channels, resulting in only 2% residual catalytic activity. In contrast, the H334R mutation causes structural changes in a polypeptide loop distant from the active site and results in 91% residual activity, but the thermostability of this mutant is significantly compromised. Strikingly, the catalytic activity of these mutants can be partially or fully rescued in vitro by the HDAC8 activator N-(phenylcarbamothioyl)benzamide. These results suggest that HDAC8 activators might be useful leads in the search for new therapeutic strategies in managing CdLS.

Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor.

To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950), boceprevir (Sch503034), simeprevir (TMC435350), danoprevir (ITMN-191), and vaniprevir (MK-7009), to alpha interferon 2b, and to the putative NS4A inhibitor ACH-806. The efficacy of ACH-806 was lower than that of protease inhibitors and was not influenced by changes at amino acids 1042 and 1065 (in the NS3 protease), which have been suggested to mediate resistance to ACH-806 in replicons. Genotype 1a, 1b, and 2a recombinants showed viral spread under long-term treatment with ACH-806, without acquisition of resistance mutations in the NS3-NS4A region. Relatively high concentrations of ACH-806 inhibited viral assembly, but not replication, in a single-cycle production assay. The developed HCV culture systems will facilitate studies benefitting from expression of genotype-specific NS4A in a constant backbone in the context of the complete viral replication cycle, including functional studies and evaluations of the efficacy of antivirals.

ACH-806, an NS4A antagonist, inhibits hepatitis C virus replication by altering the composition of viral replication complexes.

Treatment of hepatitis C patients with direct-acting antiviral drugs involves the combination of multiple small-molecule inhibitors of distinctive mechanisms of action. ACH-806 (or GS-9132) is a novel, small-molecule inhibitor specific for hepatitis C virus (HCV). It inhibits viral RNA replication in HCV replicon cells and was active in genotype 1 HCV-infected patients in a proof-of-concept clinical trial (1). Here, we describe a potential mechanism of action (MoA) wherein ACH-806 alters viral replication complex (RC) composition and function. We found that ACH-806 did not affect HCV polyprotein translation and processing, the early events of the formation of HCV RC. Instead, ACH-806 triggered the formation of a homodimeric form of NS4A with a size of 14 kDa (p14) both in replicon cells and in Huh-7 cells where NS4A was expressed alone. p14 production was negatively regulated by NS3, and its appearance in turn was associated with reductions in NS3 and, especially, NS4A content in RCs due to their accelerated degradation. A previously described resistance substitution near the N terminus of NS3, where NS3 interacts with NS4A, attenuated the reduction of NS3 and NS4A conferred by ACH-806 treatment. Taken together, we show that the compositional changes in viral RCs are associated with the antiviral activity of ACH-806. Small molecules, including ACH-806, with this novel MoA hold promise for further development and provide unique tools for clarifying the functions of NS4A in HCV replication.

Recycling and refurbishing old antitubercular drugs: the encouraging case of inhibitors of mycolic acid biosynthesis.

One of the first approaches undertaken in the quest for antitubercular compounds was that of understanding the mechanism of action of old drugs and proposing chemical modifications or other strategies to improve their activity, generally lost to the mechanisms of resistance developed by Mycobacterium tuberculosis. A leading case was the work carried out on a set of compounds with proven activity on the essential pathway of the synthesis of mycolic acids. As a result, different solutions were presented, improving the activity of those inhibitors or producing novel compounds acting on the same molecular target(s), but avoiding the most common resistance strategies developed by the tubercle bacilli. This review focuses on the activity of those compounds, developed following the completion of the studies on several of the classic antitubercular drugs.

Point mutations within the fatty acid synthase type II dehydratase components HadA or HadC contribute to isoxyl resistance in Mycobacterium tuberculosis.

The mechanism by which the antitubercular drug isoxyl (ISO) inhibits mycolic acid biosynthesis has not yet been reported. We found that point mutations in either the HadA or HadC component of the type II fatty acid synthase (FAS-II) are associated with increased levels of resistance to ISO in Mycobacterium tuberculosis. Overexpression of the HadAB, HadBC, or HadABC heterocomplex also produced high-level resistance. These results show that the FAS-II dehydratases are involved in ISO resistance.

A common mechanism of inhibition of the Mycobacterium tuberculosis mycolic acid biosynthetic pathway by isoxyl and thiacetazone.

Isoxyl (ISO) and thiacetazone (TAC), two prodrugs once used in the clinical treatment of tuberculosis, have long been thought to abolish Mycobacterium tuberculosis (M. tuberculosis) growth through the inhibition of mycolic acid biosynthesis, but their respective targets in this pathway have remained elusive. Here we show that treating M. tuberculosis with ISO or TAC results in both cases in the accumulation of 3-hydroxy C(18), C(20), and C(22) fatty acids, suggestive of an inhibition of the dehydratase step of the fatty-acid synthase type II elongation cycle. Consistently, overexpression of the essential hadABC genes encoding the (3R)-hydroxyacyl-acyl carrier protein dehydratases resulted in more than a 16- and 80-fold increase in the resistance of M. tuberculosis to ISO and TAC, respectively. A missense mutation in the hadA gene of spontaneous ISO- and TAC-resistant mutants was sufficient to confer upon M. tuberculosis high level resistance to both drugs. Other mutations found in hypersusceptible or resistant M. tuberculosis and Mycobacterium kansasii isolates mapped to hadC. Mutations affecting the non-essential mycolic acid methyltransferases MmaA4 and MmaA2 were also found in M. tuberculosis spontaneous ISO- and TAC-resistant mutants. That MmaA4, at least, participates in the activation of the two prodrugs as proposed earlier is not supported by our biochemical evidence. Instead and in light of the known interactions of both MmaA4 and MmaA2 with HadAB and HadBC, we propose that mutations affecting these enzymes may impact the binding of ISO and TAC to the dehydratases.