Discovery and Biosynthesis of Gladiolin: A Burkholderia gladioli Antibiotic with Promising Activity against Mycobacterium tuberculosis.

An antimicrobial activity screen of Burkholderia gladioli BCC0238, a clinical isolate from a cystic fibrosis patient, led to the discovery of gladiolin, a novel macrolide antibiotic with potent activity against Mycobacterium tuberculosis H37Rv. Gladiolin is structurally related to etnangien, a highly unstable antibiotic from Sorangium cellulosum that is also active against Mycobacteria. Like etnangien, gladiolin was found to inhibit RNA polymerase, a validated drug target in M. tuberculosis. However, gladiolin lacks the highly labile hexaene moiety of etnangien and was thus found to possess significantly increased chemical stability. Moreover, gladiolin displayed low mammalian cytotoxicity and good activity against several M. tuberculosis clinical isolates, including four that are resistant to isoniazid and one that is resistant to both isoniazid and rifampicin. Overall, these data suggest that gladiolin may represent a useful starting point for the development of novel drugs to tackle multidrug-resistant tuberculosis. The B. gladioli BCC0238 genome was sequenced using Single Molecule Real Time (SMRT) technology. This resulted in four contiguous sequences: two large circular chromosomes and two smaller putative plasmids. Analysis of the chromosome sequences identified 49 putative specialized metabolite biosynthetic gene clusters. One such gene cluster, located on the smaller of the two chromosomes, encodes a trans-acyltransferase (trans-AT) polyketide synthase (PKS) multienzyme that was hypothesized to assemble gladiolin. Insertional inactivation of a gene in this cluster encoding one of the PKS subunits abrogated gladiolin production, confirming that the gene cluster is responsible for biosynthesis of the antibiotic. Comparison of the PKSs responsible for the assembly of gladiolin and etnangien showed that they possess a remarkably similar architecture, obfuscating the biosynthetic mechanisms responsible for most of the structural differences between the two metabolites

Virulence Regulator EspR of Mycobacterium tuberculosis Is a Nucleoid-Associated Protein

The principal virulence determinant of Mycobacterium tuberculosis (Mtb), the ESX-1 protein secretion system, is positively controlled at the transcriptional level by EspR. Depletion of EspR reportedly affects a small number of genes, both positively or negatively, including a key ESX-1 component, the espACD operon. EspR is also thought to be an ESX-1 substrate. Using EspR-specific antibodies in ChIP-Seq experiments (chromatin immunoprecipitation followed by ultra-high throughput DNA sequencing) we show that EspR binds to at least 165 loci on the Mtb genome. Included in the EspR regulon are genes encoding not only EspA, but also EspR itself, the ESX-2 and ESX-5 systems, a host of diverse cell wall functions, such as production of the complex lipid PDIM (phenolthiocerol dimycocerosate) and the PE/PPE cell-surface proteins. EspR binding sites are not restricted to promoter regions and can be clustered. This suggests that rather than functioning as a classical regulatory protein EspR acts globally as a nucleoid-associated protein capable of long-range interactions consistent with a recently established structural model. EspR expression was shown to be growth phase-dependent, peaking in the stationary phase. Overexpression in Mtb strain H37Rv revealed that EspR influences target gene expression both positively or negatively leading to growth arrest. At no stage was EspR secreted into the culture filtrate. Thus, rather than serving as a specific activator of a virulence locus, EspR is a novel nucleoid-associated protein, with both architectural and regulatory roles, that impacts cell wall functions and pathogenesis through multiple genes

Towards a new tuberculosis drug: pyridomycin - nature's isoniazid

Tuberculosis, a global threat to public health, is becoming untreatable due to widespread drug resistance to frontline drugs such as the InhA-inhibitor isoniazid. Historically, by inhibiting highly vulnerable targets, natural products have been an important source of antibiotics including potent anti-tuberculosis agents. Here, we describe pyridomycin, a compound produced by Dactylosporangium fulvum with specific cidal activity against mycobacteria. By selecting pyridomycin-resistant mutants of Mycobacterium tuberculosis, whole-genome sequencing and genetic validation, we identified the NADH-dependent enoyl(Acyl-Carrier-Protein) reductase InhA as the principal target and demonstrate that pyridomycin inhibits mycolic acid synthesis in M. tuberculosis. Furthermore, biochemical and structural studies show that pyridomycin inhibits InhA directly as a competitive inhibitor of the NADH-binding site, thereby identifying a new, druggable pocket in InhA. Importantly, the most frequently encountered isoniazid-resistant clinical isolates remain fully susceptible to pyridomycin, thus opening new avenues for drug development

vitro synergy and enhanced murine brain penetration of saquinavir coadministered with mefloquine

ABSTRACT Highly active antiretroviral therapy has substantially improved prognosis in human immunodeficiency virus (HIV). However, the integration of proviral DNA, development of viral resistance, and lack of permeability of drugs into sanctuary sites (e.g., brain and lymphocyte) are major limitations to current regimens. Previous studies have indicated that the antimalarial drug chloroquine (CQ) has antiviral efficacy and a synergism with HIV protease inhibitors. We have screened a panel of antimalarial compounds for activity against HIV-1 in vitro. A limited efficacy was observed for CQ, mefloquine (MQ), and mepacrine (MC). However, marked synergy was observed between MQ and saquinavir (SQV), but not CQ in U937 cells. Furthermore, enhancement of the antiviral activity of SQV and four other protease inhibitors (PIs) by MQ was observed in MT4 cells, indicating a class specific rather than a drug-specific phenomenon. We demonstrate that these observations are a result of inhibition of multiple drug efflux proteins by MQ and that MQ also displaces SQV from orosomucoid in vitro. Finally, coadministration of MQ and SQV in CD-1 mice dramatically altered the tissue distribution of SQV, resulting in a Ͼ3-fold and Ͼ2-fold increase in the tissue/blood ratio for brain and testis, respectively. This pharmacological enhancement of in vitro antiviral activity of PIs by MQ now warrants further examination in vivo

Pharmacological determinants of anti-tuberculosis drug activity

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Reactivity of ClDaf and Pyr with different metalic salts

<p>Evaluation of the reactivity of ClDaf and Pyridomycin when mixed with 10 equivalents of different metalic salts. </p> <p>Samples were analyzed by analytical UPLC. </p> <p>Analytical (non-high resolution) spectrometry analysis (UHPLC-MS) were performed on an Ultimate 3000 UHPLC system, coupled with a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific). Chromatographic separation was achieved using an Acquity UPLC Peptide BEH C18 Column (300Å, 1.7 µm, 2.1 mm X 100 mm). Mobile phase system was composed of solvent A (H2O, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), typically run through a linear gradient from 0% to 100% of solvent B in 10 min. Elution of compounds was monitored by UV absorbance at 215 nm and 254 nm, and by mass spectrometry by electrospray ionization.</p> <p>Data published in: Caradec T, Anoz-Carbonell E, Petrov R, Billamboz M, Antraygues K, Cantrelle FX, Boll E, Beury D, Hot D, Drobecq H, Trivelli X, Hartkoorn RC. A Novel Natural Siderophore Antibiotic Conjugate Reveals a Chemical Approach to Macromolecule Coupling. ACS Cent Sci. 2023 Nov 10;9(11):2138-2149. doi: 10.1021/acscentsci.3c00965. PMID: 38033789; PMCID: PMC10683483.</p> <div> </div> <p> </p> <p> </p&gt

Tuberculosis drugs: new candidates and how to find more

The recent years have witnessed significant progress in the development of new drug candidates for the treatment of TB. While many of these are now in clinical trials, continued research is needed in order to sustain the drug discovery pipeline and meet the increasing needs of TB patients. These include shortening treatment, killing drug-resistant strains, and finding medications compatible with antiretroviral and diabetes therapy. Nowadays, TB drug discovery benefits from high-throughput screening methods, availability of conditional expression systems, and biophysical and biochemical techniques that enable target-based rational drug design. This article reviews the current state of TB drug development and discusses possible approaches to finding new leads

Analysis of metabolites produced by D.fulvum

<p>UPLC analysis of the metabolites produced by the soil bacterium D. fulvum when cultured in GYM media at different pH.</p> <p>Samples were analyzed by analytical UPLC. Analytical (non-high resolution) spectrometry analysis (UHPLC-MS) were performed on an Ultimate 3000 UHPLC system, coupled with a LCQ Fleet Ion Trap Mass Spectrometer (Thermo Scientific). Chromatographic separation was achieved using an Acquity UPLC Peptide BEH C18 Column (300Å, 1.7 µm, 2.1 mm X 100 mm). Mobile phase system was composed of solvent A (H2O, 0.1% formic acid) and B (acetonitrile, 0.1% formic acid), typically run through a linear gradient from 0% to 100% of solvent B in 10 min. Elution of compounds was monitored by UV absorbance at 215 nm and 254 nm, and by mass spectrometry by electrospray ionization.</p> <p> </p&gt

In Vitro Combination Studies of Benzothiazinone Lead Compound BTZ043 against Mycobacterium tuberculosis

Benzothiazinones (BTZ) are a new class of drug candidates to combat tuberculosis that inhibit decaprenyl-phosphoribose epimerase (DprE1), an essential enzyme involved in arabinan biosynthesis. Using the checkerboard method and cell viability assays, we have studied the interaction profiles of BTZ043, the current lead compound, with several antituberculosis drugs or drug candidates against Mycobacterium tuberculosis strain H37Rv, namely, rifampin, isoniazid, ethambutol, TMC207, PA-824, moxifloxacin, meropenem with or without clavulanate, and SQ-109. No antagonism was found between BTZ043 and the tested compounds, and most of the interactions were purely additive. Data from two different approaches clearly indicate that BTZ043 acts synergistically with TMC207, with a fractional inhibitory concentration index of 0.5. TMC207 at a quarter of the MIC (20 ng/ml) used in combination with BTZ043 (1/4 MIC, 0.375 ng/ml) had a stronger bactericidal effect on M. tuberculosis than TMC207 alone at a concentration of 80 ng/ml. This synergy was not observed when the combination was tested on a BTZ-resistant M. tuberculosis mutant, suggesting that DprE1 inhibition is the basis for the interaction. This finding excludes the possibility of synergy occurring through an off-target mechanism. We therefore hypothesize that sub-MICs of BTZ043 weaken the bacterial cell wall and allow improved penetration of TMC207 to its target. Synergy between two new antimycobacterial compounds, such as TMC207 and BTZ043, with novel targets, offers an attractive foundation for a new tuberculosis regimen

Cross-Resistance between Clofazimine and Bedaquiline through Upregulation of MmpL5 in Mycobacterium tuberculosis

The antileprosy drug clofazimine is also of interest for the treatment of multidrug-resistant tuberculosis. To understand possible resistance mechanisms, clofazimine-resistant Mycobacterium tuberculosis mutants were isolated in vitro, and, unexpectedly, found to be cross-resistant to bedaquiline. Mutations in the transcriptional regulator Rv0678, with concomitant upregulation of the multisubstrate efflux pump, MmpL5, accounted for this cross-resistance. Mutation in Rv0678 should therefore be considered a confounding factor for the treatment of tuberculosis with clofazimine or bedaquiline