Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis.

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-ß-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis. Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

Mechanisms of Drug-Induced Tolerance in Mycobacterium tuberculosis.

Successful treatment of tuberculosis (TB) can be hampered by Mycobacterium tuberculosis populations that are temporarily able to survive antibiotic pressure in the absence of drug resistance-conferring mutations, a phenomenon termed drug tolerance. We summarize findings on M. tuberculosis tolerance published in the past 20 years. Key M. tuberculosis responses to drug pressure are reduced growth rates, metabolic shifting, and the promotion of efflux pump activity. Metabolic shifts upon drug pressure mainly occur in M. tuberculosis's lipid metabolism and redox homeostasis, with reduced tricarboxylic acid cycle activity in favor of lipid anabolism. Increased lipid anabolism plays a role in cell wall thickening, which reduces sensitivity to most TB drugs. In addition to these general mechanisms, drug-specific mechanisms have been described. Upon isoniazid exposure, M. tuberculosis reprograms several pathways associated with mycolic acid biosynthesis. Upon rifampicin exposure, M. tuberculosis upregulates the expression of its drug target rpoB Upon bedaquiline exposure, ATP synthesis is stimulated, and the transcription factors Rv0324 and Rv0880 are activated. A better understanding of M. tuberculosis's responses to drug pressure will be important for the development of novel agents that prevent the development of drug tolerance following treatment initiation. Such agents could then contribute to novel TB treatment-shortening strategies.

Differential Host Pro-Inflammatory Response to Mycobacterial Cell Wall Lipids Regulated by the Mce1 Operon.

The cell wall of wild-type (WT) Mycobacterium tuberculosis (Mtb), an etiologic agent of tuberculosis (TB) and a Mtb strain disrupted in a 13-gene operon mce1 (Δmce1) varies by more than 400 lipid species. Here, we examined Mtb lipid-induced response in murine macrophage, as well as in human T-cell subpopulations in order to gain an insight into how changes in cell wall lipid composition may modulate host immune response. Relative to WT Mtb cell wall lipids, the non-polar lipid extracts from Δmce1 enhanced the mRNA expression of lipid-sense nuclear receptors TR4 and PPAR-γ and dampened the macrophage expression of genes encoding TNF-α, IL-6, and IL-1ß. Relative to untreated control, WT lipid-pre-stimulated macrophages from healthy individuals induced a higher level of CD4-CD8- double negative T-cells (DN T-cells) producing TNF-α. Conversely, compared to WT, stimulation with Δmce1 lipids induced higher mean fluorescence intensity (MFI) in IL-10-producing DN T cells. Mononuclear cells from TB patients stimulated with WT Mtb lipids induced an increased production of TNF-α by CD8+ lymphocytes. Taken together, these observations suggest that changes in mce1 operon expression during a course of infection may serve as a strategy by Mtb to evade the host pro-inflammatory responses.

Evaluation of Locally Injected Mycobacterium Cell Wall Fraction in Horses with Sarcoids.

A reformulation of Mycobacterium cell wall fraction immunotherapeutic can be used to successfully treat sarcoids in horses. Sarcoids are reported to be the most common equine skin tumors with tumor type and location influencing the choice of treatment. Wide surgical excision is curative for many tumors, but may not always be feasible. Previous studies have reported sarcoid regression after injection with mycobacterial cell wall immunotherapeutics. A new formulation of the Mycobacterium phlei cell wall fraction immunostimulant (Immunocidin Equine) was used to treat cutaneous tumors in horses. Equids with skin tumors diagnosed as sarcoids were enrolled in the study. Sarcoids were injected at the initial visit with Immunocidin Equine and subsequently at approximately 2-week intervals. Of 17 cases, nine cases were completely resolved at the end of the study period evaluation or at the time of final follow-up (52.9%). Three cases were reported as improved (smaller), but not resolved (17.6%). Three cases were discontinued from the study as the respective masses were growing larger or not resolving (17.6%). One case (5.8%) with two masses had resolution of one mass, whereas the other tumor had a small regrowth 5 months after the last treatment. One case (5.8%) was lost to follow-up. All cases had mild to moderate swelling of the injection site, and some cases had discharge after the second, third, or fourth injections. No serious systemic side effects or complications were encountered during the study.

Structural and inhibition analysis of novel sulfur-rich 2-mercaptobenzothiazole and 1,2,3-triazole ligands against Mycobacterium tuberculosis DprE1 enzyme.

Mycobacterium tuberculosis decaprenylphosphoryl-ß-D-ribose oxidase (MtbDprE1) acts in concert with decaprenylphosphoryl-ß-D-ribose 2-epimerase (MtbDprE2) and catalyzes the epimerization of DPR into DPA. DPA is the sole precursor for synthesis of arabinogalactan and lipoarabinomannan in the mycobacterial cell wall. MtbDprE1 is a unique antimalarial drug target and many covalent and non-covalent inhibitors against MtbDprE1 have been studied for their antituberculosis activities. In the current study, we have purified MtbDprE1 enzyme and synthesized six sulfur-rich 2-mercaptobenzothiazole and 1, 2, 3-triazole conjugated ligands and performed binding analysis with MtbDprE1. All ligands have shown competitive binding, as observed for other covalently and noncovalently bound MtbDprE1 inhibitors. Molecular docking analysis of six ligands with MtbDprE1 shows that they occupy the substrate binding pocket of MtbDprE1 and are stabilized by hydrogen bonds and van der Waals interactions. Our study shows that sulfur-rich 2-mercaptobenzothiazole ligands act as specific inhibitors against MtbDprE1 and could be used as antituberculosis agents.

Establishment of an effective TLC bioautographic method for the detection of Mycobacterium tuberculosis H37Ra phosphoglucose isomerase inhibition by phosphoenolpyruvate.

A bioautographic assay based on thin layer chromatography was developed for phosphoenolpyruvate (PEP) detecting as a known but rarely studied inhibitor of phosphoglucose isomerase (PGI). The protocol with NADP(+)/NBT/PMS (ß-nicotinamide adenine dinucleotide phosphate/nitrotetrazolium blue chloride/phenazine methosulfate) staining was capable of detecting Mycobacterium tuberculosis H37Ra PGI inhibition using PEP. According to this method, visibly brighter spots (zones) against purple background are observed in the area of inhibition of the above-mentioned enzyme activity. The detection limit for PEP as an inhibitor of Mycobacterium tuberculosis H37Ra PGI was 226 µg per spot/zone. Noteworthy is that we are the first authors to have successfully used a bioautographic assay to detect Mycobacterium tuberculosis H37Ra PGI inhibition by PEP.