Identification of novel antimicrobial compounds targeting Mycobacterium tuberculosis shikimate kinase using in silico hierarchical structure-based drug screening.

The development of new anti-TB drugs to prevent the spread of multidrug-resistant Mycobacterium tuberculosis (Mtb) strains is imperative. Mtb shikimate kinase (MtSK) was selected as the target protein to screen for new anti-TB drugs. We performed hierarchical in silico screening using a library of 154,118 compounds to search for novel compounds that could bind to the active site of MtSK. The growth-inhibitory effects of the candidate compounds on Mycobacterium smegmatis were evaluated in vitro. Nine of the 11 candidate compounds exhibited inhibitory effects against mycobacteria in vitro. The inhibitory activity of Compound 2 (IC50 = 1.39 µM) was higher than that of isoniazid, the first-line drug for TB treatment. Moreover, Compound 2 did not exhibit toxicity against mammalian cells and Escherichia coli. Molecular dynamics simulations using the MtSK-Compound 2 complex structure in a timeframe of 100 ns suggested that Compound 2 could stably bind to MtSK. The binding free energy of Compound 2 was estimated to be -37.96 kcal/mol using the MM/PBSA method, demonstrating that Compound 2 can stably bind to MtSK. These in silico and in vitro results indicated that Compound 2 is a promising hit compound for the development of novel anti-TB drugs.

Quantitative analysis of the bioenergetics of Mycobacterium tuberculosis along with Glyoxylate cycle as a drug target under inhibition of enzymes using Petri net.

The bacteria Mycobacterium tuberculosis is responsible for the infectious disease Tuberculosis. Targeting the tubercule bacteria is an important challenge in developing the antimycobacterials. The glyoxylate cycle is considered as a potential target for the development of anti-tuberculosis agents, due to its absence in the humans. Humans only possess tricarboxylic acid cycle, while this cycle gets connected to glyoxylate cycle in microbes. Glyoxylate cycle is essential to the Mycobacterium for its growth and survival. Due to this reason, it is considered as a potential therapeutic target for the development of anti-tuberculosis agents. Here, we explore the effect on the behavior of the tricarboxylic acid cycle, glyoxylate cycle and their integrated pathway with the bioenergetics of the Mycobacterium, under the inhibition of key glyoxylate cycle enzymes using Continuous Petri net. Continuous Petri net is a special Petri net used to perform the quantitative analysis of the networks. We first study the tricarboxylic acid cycle and glyoxylate cycle of the tubercule bacteria by simulating its Continuous Petri net model under different scenarios. Both the cycles are then integrated with the bioenergetics of the bacteria and the integrated pathway is again simulated under different conditions. The simulation graphs show the metabolic consequences of inhibiting the key glyoxylate cycle enzymes and adding the uncouplers on the individual as well as integrated pathway. The uncouplers that inhibit the synthesis of adenosine triphosphate, play an important role as anti-mycobacterials. The simulation study done here validates the proposed Continuous Petri net model as compared with the experimental outcomes and also explains the consequences of the enzyme inhibition on the biochemical reactions involved in the metabolic pathways of the mycobacterium.

Lactobacillus casei Improve Anti-Tuberculosis Drugs-Induced Intestinal Adverse Reactions in Rat by Modulating Gut Microbiota and Short-Chain Fatty Acids.

The adverse effects of anti-tuberculosis (TB) drugs in the intestines were related to alteration of the intestinal microbiota. However, there was less information about microbial metabolism on the adverse reactions. This study aimed to explore whether Lactobacillus casei could regulate gut microbiota or short-chain fatty acids (SCFAs) disorders to protect intestinal adverse reactions induced by isoniazid (H) and rifampicin (R). Male Wistar rats were given low and high doses of Lactobacillus casei two hours before daily administration of anti-TB drugs. After 42 days, colon tissue and blood were collected for analysis. The feces at two-week and six-week were collected to analyze the microbial composition and the content of SCFAs in colon contents was determined. Supplementation of Lactobacillus casei increased the proportion of intestinal goblet cells induced by H and R (p < 0.05). In addition, HR also reduced the level of mucin-2 (p < 0.05), and supplementation of Lactobacillus casei restored. After two weeks of HR intervention, a decrease in OTUs, diversity index, the abundance of Bacteroides, Akkermansia, and Blautia, and an increase of the abundance of Lacetospiraceae NK4A136 group and Rumencoccus UCG-005, were observed compared with the control group (p all < 0.05). These indices in Lactobacillus casei intervention groups were similar to the HR group. Six-week intervention resulted in a dramatic reduction of Lacetospiraceae NK4A136 group, butyric acid, valeric acid and hexanoic acid, while an increase of Bacteroides and Blautia (p all < 0.05). Pretreatment with Lactobacillus casei significantly increased the content of hexanoic acid compared with HR group (p < 0.05). Lactobacillus casei might prevent intestinal injury induced by anti-tuberculosis drugs by regulating gut microbiota and SCFAs metabolism.

Traditional Medicinal Plants as a Source of Antituberculosis Drugs: A System Review.

Medicinal plants are the chief components in the different oriental formulations in different traditional medical systems worldwide. As a thriving source of medicine, the medicinal plants with antituberculosis (TB) properties inspire the pharmacists to develop new drugs based on their active components or semimetabolites. In the present review, the anti-TB medicinal plants were screened from the scientific literatures, based on the botanical classification and the anti-TB activity. The obtained anti-TB medicinal plants were categorized into three different categories, viz., 159 plants critically examined with a total 335 isolated compounds, 131 plants with their crude extracts showing anti-TB activity, and 27 plants in literature with the prescribed formula by the traditional healers. Our systemic analysis on the medicinal plants can assist the discovery of novel and more efficacious anti-TB drugs.

A sensitive EZMTT method provides microscale, quantitative and high-throughput evaluation of drug efficacy in the treatment of Mycobacterium tuberculosis infectious diseases.

Drug resistance has become a serious public health problem in mycobacterial infectious diseases. Here, we investigated a water soluble tetrazolium salt (EZMTT)-based detection method to provide an easy, safe and quantitative antimycobacterial susceptibility test (AMST) method, especially for targeting early detection of loss of drug susceptibility in mycobacteria. After a single addition of the EZMTT detection reagent at the inoculation of mycobacteria culture, the AMST was continuously analyzed in a sealed 96-well plate (100 µl), or a sealed tube to ensure biosafety. Using Mycobacterium tuberculosis H37Ra as the model strain, the EZMTT assay was developed with high reproducibility (Z factor of 0.64) for facile measurements of growth and drug susceptibility. In the comparative AMST study, the 7-day EZMTT method identified not only the same set of drug resistance as the other two methods (the 30-day traditional Löwenstein Jensen solid medium assay and the 10-14 day 8 ml Mycobacteria Growth Indicator Tube liquid method), but also additional strains with loss of drug susceptibility. In conclusion, we demonstrated that the EZMTT-based AMST assay in a sealed microtiter plate has great potential for routine use in medical diagnosis and drug screening to battle the unmet medical need in the treatment of multi- and extensive-drug resistant mycobacteria.

Ameliorative potential of Adhatoda vasica against anti-tubercular drugs induced hepatic impairments in female Wistar rats in relation to oxidative stress and xeno-metabolism.

ETHNOPHARMACOLOGICAL RELEVANCE: Adhatoda vasica Nees is widely used herb of indigenous system to treat various ailments especially upper respiratory tract infections. Not only, anti-tubercular efficacy of crude extract and phytoconstituents of A. vasica has been documented but its hepatoprotective role against various drugs mediated hepatic alterations in different animal models has also been observed. BACKGROUND AND PURPOSE: Isoniazid, rifampicin and pyrazinamide (H-R-Z) are anti-tubercular drugs normally prescribed by health professionals for the treatment of tuberculosis, however along with their medical effectiveness these drugs also exhibit hepatotoxicity among TB patients. Unexpectedly, substantial toxicological data on the metabolism of anti-TB drugs are available but the mystery behind these xenobiotics is too complex and partly implicit. In this study, we further explored the hepatotoxic effects of these xeno-metabolic products and their amelioration by Adhatoda vasica Nees by elucidating its mechanistic action. METHODS: We generated a hepatotoxic rodent model by oral administration of H, R and Z (30.85, 61.7 and 132.65 mg/kg body weight) drugs for 25 days in Wistar rats. Additionally, to achieve hepatoprotection two different doses of Adhatoda vasica Nees ethanolic leaf extract (200 and 300 mg/kg body weight) were used along with H-R-Z dosage, orally and once daily for 25 days and tried to ascertain their mechanistic action. For this, initially phytoconstituents of the extract were evaluated followed by extract standardization using RP-HPLC and FTIR methods. Furthermore, antioxidant activity of the extract was analyzed by DPPH assay. Finally, different treated groups were analyzed for hepatic oxidative stress markers, antioxidant markers, histopathological changes and gene expression study including CYP2E1, CYP7A1, NAT, NR1I2 and UGT1A1 genes involved in phase I and phase II xeno-metabolism. RESULTS: Estimated content of vasicine in RP-HPLC method and free-radical scavenging activity in DPPH assay was found to be 134.519 ± 0.00269µg/10mg of leaf extract and 47.81 µg/mL respectively. In H-R-Z treated group, a significant increase in the levels of thiobarbituric acid, significant reduction in the levels of GSH, and enzymatic markers and marked changes in hepatic histological architecture were observed. In addition, there was significance up-regulation of CYP7A and NAT genes, down-regulation of CYP2E1 gene and insignificant expression levels of NR1I2 and UGT1A1 genes were observed in H-R-Z group. Conversely, high dose of A. vasica extract effectively diminished these alterations by declining oxidative stress and boosting of antioxidant levels. In addition, it acted as bi-functional inducer of both phase I (CYP2E1) and phase II (NAT and UGT1A1) enzyme systems. CONCLUSION: Hence, we concluded that anti-TB drugs exposure has potential to generate reactive metabolites that eventually cause hepatotoxicity by altering oxidant-antioxidant levels and their own metabolism. This study not only emphasized on xeno-metabolism mediated hepatic alterations but also explore the benefit of A. vasica on these toxic insults.

Antitubercular polyhalogenated phenothiazines and phenoselenazine with reduced binding to CNS receptors.

Targeting energy metabolism in Mycobacterium tuberculosis (Mtb) is a new paradigm in the search for innovative anti-TB drugs. NADH:menaquinone oxidoreductase is a non-proton translocating type II NADH dehydrogenase (NDH-2) that is an essential enzyme in the respiratory chain of Mtb and is not found in mammalian mitochondria. Phenothiazines (PTZs) represent one of the most known class of NDH-2 inhibitors, but their use as anti-TB drugs is currently limited by the wide range of potentially serious off-target effects. In this work, we designed and synthesized a series of new PTZs by decorating the scaffold in an unconventional way, introducing various halogen atoms. By replacing the sulfur atom with selenium, a dibromophenoselenazine 20 was also synthesized. Among the synthesized poly-halogenated PTZs (HPTZs), dibromo and tetrachloro derivatives 9 and 11, along with the phenoselenazine 20, emerged with a better anti-TB profile than the therapeutic thioridazine (TZ). They targeted non-replicating Mtb, were bactericidal, and synergized with rifampin and bedaquiline. Moreover, their anti-TB activity was found to be related to the NDH-2 inhibition. Most important, they showed a markedly reduced affinity to dopaminergic and serotonergic receptors respect to the TZ. From this work emerged, for the first time, as the poly-halogenation of the PTZ core, while permitting to maintain good anti-TB profile could conceivably lead to fewer CNS side-effects risk, making more tangible the use of PTZs for this alternative therapeutic application.

Discovery of 1,2,3-triazole based quinoxaline-1,4-di-N-oxide derivatives as potential anti-tubercular agents.

A series of thirty one novel 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-3-methylquinoxaline-1,4-dioxide (7a-l), 3-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6-chloro-2-methylquinoxaline-1,4-dioxide (8a-l) and 2-(((1-(substituted phenyl)-1H-1,2,3-triazol-4-yl)methoxy)carbonyl)-6,7-dichloro-3-methylquinoxaline-1,4-dioxide (9a-g) analogues were synthesized, characterized using various analytical techniques and single crystal was developed for the compounds 8 g and 9f. Synthesized compounds were evaluated for in vitro anti-tubercular activity against Mycobacterium tuberculosis H37Rv strain and two clinical isolates Spec. 210 and Spec. 192. The titled compounds exhibited minimum inhibitory concentration (MIC) ranging from 30.35 to 252.00 µM. Among the tested compounds, 8e, 8 l, 9c and 9d exhibited moderate activity (MIC = 47.6 - 52.0 µM) and 8a exhibited significant anti-tubercular activity (MIC = 30.35 µM). Furthermore, 8e, 8 l, and 9d were found to be less toxic against human embryonic kidney, HEK 293 cell lines. Finally, a docking study was also performed using MTB DNA Gyrase (PDB ID: 5BS8) for the significantly active compound 8a to know the exact binding pattern within the active site of the target enzyme.

Molecular mechanism for the activation of the anti-tuberculosis drug isoniazid by Mn(III): First detection and unequivocal identification of the critical N-centered isoniazidyl radical and its exact location.

Isoniazid (INH), the most-widely used anti-tuberculosis drug, has been shown to be activated by Mn(III) to produce the reactive carbon-centered isonicotinic acyl radical, which was considered to be responsible for its anti-tuberculosis activity. However, it is still not clear whether the previously-proposed N-centered isoniazidyl radical intermediate can be initially produced or not; and if so, what is its exact location on the hydrazine group, distal- or proximal-nitrogen? Through complementary applications of ESR spin-trapping and HPLC/MS methods, here we show that the characteristic and transient N-centered isoniazidyl radical intermediate can be detected and identified from INH activation uniquely by Mn(III)Acetate not by Mn(III) pyrophosphate. The exact location of the radical was found to be at the distal-nitrogen of the hydrazine group by 15N-isotope-labeling techniques via using 15N-labeled INH. Diisonicotinyl hydrazine was identified as a new reaction product from INH/Mn(III). Analogous results were observed with other hydrazides. This study represents the first detection and unequivocal identification of the initial N-centered isoniazidyl radical and its exact location. These findings should provide a new perspective on the molecular mechanism of INH activation, which may have broad biomedical and toxicological significance for future research for more efficient hydrazide anti-tuberculosis drugs.

Theoretical insights on the binding of isoniazid to the active site residues of Mycobacterium tuberculosis catalase-peroxidase.

Isoniazid (INH) is known to cause the exclusive lethal action to Mycobacterium tuberculosis (M. tb.) cells because of the pathogen's own catalase-peroxidase (katG) enzyme that converts INH to a very reactive radical. Thus, in order to gain insights on the interaction of INH with the individual active site residues (Res) of katG, this study presents a computational approach via molecular docking and density functional theory (DFT) using augmented models to study the individual INH-Res interactions. Seven amino acid residues directly interacts with INH: Arg104, Asp137, His108, Ile228, Trp107, Tyr229, and Val230. The residues with the highest interaction energies are Arg104 (-39.64 kcal/mol) and Asp137 (-32.85 kcal/mol) mainly due to strong ion-dipole and H-bonding interactions present in the complexes, while the weakest interaction was observed for Ile228 (-13.78 kcal/mol). Molecular electrostatic potential surface revealed complementary regions for dipole interactions and charge distribution analysis further shows that INH generally donates electrons to the residues. The results in this study agrees with the previous experimental findings and provides new insights into the catalase dependent activation of INH and the methods presented may be valuable in the study of biological metabolism of molecules.

Docking- and pharmacophore-based virtual screening for the identification of novel Mycobacterium tuberculosis protein tyrosine phosphatase B (MptpB) inhibitor with a thiobarbiturate scaffold.

Mycobacterium tuberculosis (Mtb) protein tyrosine phosphatase B (MptpB) is an important virulence factor for Mtb that contributes to survival of the bacteria in macrophages. The absence of a human ortholog makes MptpB an attractive target for new therapeutics to treat tuberculosis. MptpB inhibitors could be an effective treatment to overcome emerging TB drug resistance. Adopting a structure-based virtual screening strategy, we successfully identified thiobarbiturate-based drug-like MptpB inhibitor 15 with an IC50 of 22.4 µM, and as a non-competitive inhibitor with a Ki of 24.7 µM. Importantly, not only did it exhibit moderate cell membrane permeability, compound 15 also displayed potent inhibition of intracellular TB growth in the macrophage, making it an excellent lead compound for anti-TB drug discovery. To the best of our knowledge, this novel thiobarbiturate is the first class of MptpB inhibitor reported so far that leveraged docking- and pharmacophore-based virtual screening approaches. The results of preliminary structure-activity relationship demonstrated that compound 15 identified herein was not a singleton and may inspire the design of novel selective and drug-like MptpB inhibitors.

Discovery of heterocyclic replacements for the coumarin core of anti-tubercular FadD32 inhibitors.

Previous work established a coumarin scaffold as a starting point for inhibition of Mycobacterium tuberculosis (Mtb) FadD32 enzymatic activity. After further profiling of the coumarin inhibitor 4 revealed chemical instability, we discovered that a quinoline ring circumvented this instability and had the advantage of offering additional substitution vectors to further optimize. Ensuing SAR studies gave rise to quinoline-2-carboxamides with potent anti-tubercular activity. Further optimization of ADME/PK properties culminated in 21b that exhibited compelling in vivo efficacy in a mouse model of Mtb infection.

Investigation of the anti-TB potential of selected propolis constituents using a molecular docking approach.

Human tuberculosis (TB), caused by Mycobacterium tuberculosis, is the leading bacterial killer disease worldwide and new anti-TB drugs are urgently needed. Natural remedies have long played an important role in medicine and continue to provide some inspiring templates for drug design. Propolis, a substance naturally-produced by bees upon collection of plant resins, is used in folk medicine for its beneficial anti-TB activity. In this study, we used a molecular docking approach to investigate the interactions between selected propolis constituents and four 'druggable' proteins involved in vital physiological functions in M. tuberculosis, namely MtPanK, MtDprE1, MtPknB and MtKasA. The docking score for ligands towards each protein was calculated to estimate the binding free energy, with the best docking score (lowest energy value) indicating the highest predicted ligand/protein affinity. Specific interactions were also explored to understand the nature of intermolecular bonds between the most active ligands and the protein binding site residues. The lignan (+)-sesamin displayed the best docking score towards MtDprE1 (-10.7 kcal/mol) while the prenylated flavonoid isonymphaeol D docked strongly with MtKasA (-9.7 kcal/mol). Both compounds showed docking scores superior to the control inhibitors and represent potentially interesting scaffolds for further in vitro biological evaluation and anti-TB drug design.

Iron-Based Metal-Organic Frameworks as a Theranostic Carrier for Local Tuberculosis Therapy.

PURPOSE: The purpose of the study was initial evaluation of applicability of metal organic framework (MOF) Fe-MIL-101-NH2 as a theranostic carrier of antituberculous drug in terms of its functionality, i.e. drug loading, drug dissolution, magnetic resonance imaging (MRI) contrast and cytotoxic safety. METHODS: Fe-MIL-101-NH2 was characterized using X-ray powder diffraction, FTIR spectrometry and scanning electron microscopy. The particle size analysis was determined using laser diffraction. Magnetic resonance relaxometry and MRI were carried out on phantoms of the MOF system suspended in polymer solution. Drug dissolution studies were conducted using Franz cells. For MOF cytotoxicity, commercially available fibroblasts L929 were cultured in Eagle's Minimum Essential Medium supplemented with 10% fetal bovine serum. RESULTS: MOF particles were loaded with 12% of isoniazid. The particle size (3.37-6.45 µm) depended on the micronization method used. The proposed drug delivery system can also serve as the MRI contrast agent. The drug dissolution showed extended release of isoniazid. MOF particles accumulated in the L929 fibroblast cytoplasmic area, suggesting MOF release the drug inside the cells. The cytotoxicity confirmed safety of MOF system. CONCLUSIONS: The application of MOF for extended release inhalable system proposes the novel strategy for delivery of standard antimycobacterial agents combined with monitoring of their distribution within the lung tissue.

Discovery of anti-microbial and anti-tubercular molecules from Fusarium solani: an endophyte of Glycyrrhiza glabra.

AIMS: Glycyrrhiza glabra is a high-value medicinal plant thriving in biodiversity rich Kashmir Himalaya. The present study was designed to explore the fungal endophytes from G. glabra as a source of bioactive molecules. METHODS AND RESULTS: The extracts prepared from the isolated endophytes were evaluated for anti-microbial activities using broth micro-dilution assay. The endophytic strain coded as A2 exhibiting promising anti-bacterial as well as anti-tuberculosis activity was identified as Fusarium solani by ITS-5.8S ribosomal gene sequencing technique. This strain was subjected to large-scale fermentation followed by isolation of its bioactive compounds using column chromatography. From the results of spectral data analysis and comparison with literature, the molecules were identified as 3,6,9-trihydroxy-7-methoxy-4,4-dimethyl-3,4-dihydro-1H-benzo[g]isochromene-5,10-dione (1), fusarubin (2), 3-O-methylfusarubin (3) and javanicin (4). Compound 1 is reported for the first time from this strain. All the four compounds inhibited the growth of various tested bacterial strains with MIC values in the range of <1 to 256 µg ml-1 . Fusarubin showed good activity against Mycobacterium tuberculosis strain H37Rv with MIC value of 8 µg ml-1 , whereas compounds 1, 3 and 4 exhibited moderate activity with MIC values of 256, 64, 32 µg ml-1 , respectively. CONCLUSIONS: To the best of our knowledge, this is the first study that reports significant anti-tuberculosis potential of bioactive molecules from endophytic F. solani evaluated against the virulent strain of M. tuberculosis. This study sets background towards their synthetic intervention for activity enhancement experiments in anti-microbial drug discovery programme. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the chemoprofile variation of same endophyte with respect to source plant and ecoregions, further studies are required to explore endophytes of medicinal plants of all unusual biodiversity rich ecoregions for important and or novel bioactive molecules.

Pyrrolidinone and pyrrolidine derivatives: Evaluation as inhibitors of InhA and Mycobacterium tuberculosis.

A series of GEQ analogues bearing pyrrolidinone or pyrrolidine cores were synthesized and evaluated against InhA, essential target for Mycobacterium tuberculosis (M.tb) survival. The compounds were also evaluated against M.tb H37Rv growth. Interestingly, some of the compounds, not efficient as InhA inhibitors, are active against M.tb with MICs up to 1.4 µM. In particular, compound 4b was screened with different M.tb mutated strains in order to identify the cellular target, but without success, suggesting a new possible mode of action.

A High Dose of Isoniazid Disturbs Endobiotic Homeostasis in Mouse Liver.

Overdose of isoniazid (INH), an antituberculosis drug, can be life-threatening because of neurotoxicity. In clinical practice for management of INH overdose and acute toxicity, the potential of INH-induced hepatotoxicity is also considered. However, the biochemical basis of acute INH toxicity in the liver remains elusive. In the current study, we used an untargeted metabolomic approach to explore the acute effects of INH on endobiotic homeostasis in mouse liver. We found that overdose of INH resulted in accumulation of oleoyl-l-carnitine and linoleoyl-l-carnitine in the liver, indicating mitochondrial dysfunction. We also revealed the interactions between INH and fatty acyl-CoAs by identifying INH-fatty acid amides. In addition, we found that overdose of INH led to the accumulation of heme and oxidized NAD in the liver. We also identified an INH and NAD adduct in the liver. In this adduct, the nicotinamide moiety in NAD was replaced by INH. Furthermore, we illustrated that overdose of INH depleted vitamin B6 in the liver and blocked vitamin B6-dependent cystathionine degradation. These data suggest that INH interacts with multiple biochemical pathways in the liver during acute poisoning caused by INH overdose.

A quantitative adaptation of the Wayne test for pyrazinamide resistance.

BACKGROUND: Pyrazinamide (PZA) is the most important drug against the latent stage of tuberculosis (TB) and is used in both first and second line treatment regimens. The continued increase in multi-drug resistant TB and the prevalence of PZA resistance makes the development of alternative assays for prompt identification of PZA resistance all the more important. METHODS: We standardized and evaluated a quantitative variant of the Wayne assay (QW) for determining PZA resistance in Mycobacterium tuberculosis strains. This assay quantifies M. tuberculosis metabolism of PZA and production of pyrazinoic acid (POA) using visible spectrophotometry. We evaluated this method using PZA concentrations of 400 µg/ml and 800 µg/ml at incubation periods of 3, 5 and 7 days. M. tuberculosis strains from 68 sputum samples were also tested with the standard Wayne assay, Tetrazolium Microplate Assay (TEMA), Bactec 460TB and pncA sequencing. We compared QW and standard Wayne assay against a dichotomous reference classification using concordant Bactec 460TB and pncA sequencing. Secondarily, we determined the quantitative correlation between both QW values and TEMA's minimum inhibitory concentration (MIC) against Bactec 460TB percentage growth. RESULTS: The standard Wayne showed sensitivity of 88% and specificity of 97.5%, giving a Youden Index (YI) of 0.855 against reference tests. The QW showed maximum YI of 0.934 on day 7 at 400 µg/ml PZA with 96% sensitivity and 97.4% specificity. Absorbance OD values for 400 µg/ml PZA were more accurate than 800 µg/ml PZA. Although QW showed high accuracy for PZA susceptibility, it did not correlate quantitatively with Bactec percentage growth. TEMA testing was unreliable and did not correlate with Bactec results. CONCLUSIONS: The proposed QW assay is an inexpensive method capable of providing standardization and automation of colorimetric PZA resistance testing, with better discriminatory than the standard Wayne assay.

Lauric acid and myristic acid from Allium sativum inhibit the growth of Mycobacterium tuberculosis H37Ra: in silico analysis reveals possible binding to protein kinase B.

CONTEXT: The bulb of Allium sativum Linn (Alliaceae) has numerous medicinal values. Though the petroleum ether extract of the bulb has shown to exhibit antimycobacterial activity, the phytochemical(s) responsible for this inhibitory activity is not known. OBJECTIVE: To characterize the bioactive compounds in the petroleum ether extract of Allium sativum (garlic) that inhibit the growth of Mycobacterium tuberculosis H37Ra. MATERIALS AND METHODS: Bioactivity-guided fractionation was employed to isolate the bioactive compounds. Antimycobacterial activity was evaluated by well-diffusion method and microplate alamar blue assay (MABA). Infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy were used to characterize the bioactive compounds. Autodock was used to obtain information on molecular recognition, and molecular dynamics simulation was performed using GROMACS. RESULTS: The bioactive compounds that inhibited the growth of M. tuberculosis H37Ra were found to be lauric acid (LA) and myristic acid (MA). The minimal inhibitory concentration of LA and MA was found to be 22.2 and 66.7 µg/mL, respectively. In silico analysis revealed that these fatty acids could bind at the cleft between the N-terminal and C-terminal lobes of the cytosolic domain of serine/threonine protein kinase B (PknB). DISCUSSION AND CONCLUSION: The inhibition activity was dependent on the alkyl chain length of the fatty acid, and the amino acid residues involved in binding to fatty acid was found to be conserved across the Pkn family of proteins. The study indicates the possibility of using fatty acid derivatives, involving Pkn family of proteins, to inhibit the signal transduction processes in M. tuberculosis.

Benzothiazole Derivative as a Novel Mycobacterium tuberculosis Shikimate Kinase Inhibitor: Identification and Elucidation of Its Allosteric Mode of Inhibition.

Mycobacterium tuberculosis shikimate kinase (Mtb-SK) is a key enzyme involved in the biosynthesis of aromatic amino acids through the shikimate pathway. Since it is proven to be essential for the survival of the microbe and is absent from mammals, it is a promising target for anti-TB drug discovery. In this study, a combined approach of in silico similarity search and pharmacophore building using already reported inhibitors was used to screen a procured library of 20,000 compounds of the commercially available ChemBridge database. From the in silico screening, 15 hits were identified, and these hits were evaluated in vitro for Mtb-SK enzyme inhibition. Two compounds presented significant enzyme inhibition with IC50 values of 10.69 ± 0.9 and 46.22 ± 1.2 µM. The best hit was then evaluated for the in vitro mode of inhibition where it came out to be an uncompetitive and noncompetitive inhibitor with respect to shikimate (SKM) and ATP, respectively, suggesting its binding at an allosteric site. Potential binding sites of Mtb-SK were identified which confirmed the presence of an allosteric binding pocket apart from the ATP and SKM binding sites. The docking simulations were performed at this pocket in order to find the mode of binding of the best hit in the presence of substrates and the products of the enzymatic reaction. Molecular dynamics (MD) simulations elucidated the probability of inhibitor binding at the allosteric site in the presence of ADP and shikimate-3-phosphate (S-3-P), that is, after the formation of products of the reaction. The inhibitor binding may prevent the release of the product from Mtb-SK, thereby inhibiting its activity. The binding stability and the key residue interactions of the inhibitor to this product complex were also revealed by the MD simulations. Residues ARG43, ILE45, and PHE57 were identified as crucial that were involved in interactions with the best hit. This is the first report of an allosteric binding site of Mtb-SK, which could largely address the selectivity issue associated with kinase inhibitors.