scholarly journals 1H-Benzo[d]Imidazole Derivatives Affect MmpL3 in Mycobacterium tuberculosis

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Małgorzata Korycka-Machała ◽  
Albertus Viljoen ◽  
Jakub Pawełczyk ◽  
Paulina Borówka ◽  
Bożena Dziadek ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mycolic Acid ◽  
Cross Resistance ◽  
Metabolic Labeling ◽  
Drug Resistant ◽  
Content Type ◽  
Antitubercular Drugs ◽  
Imidazole Derivatives ◽  
Trehalose Dimycolate

ABSTRACT 1H-benzo[d]imidazole derivatives exhibit antitubercular activity in vitro at a nanomolar range of concentrations and are not toxic to human cells, but their mode of action remains unknown. Here, we showed that these compounds are active against intracellular Mycobacterium tuberculosis. To identify their target, we selected drug-resistant M. tuberculosis mutants and then used whole-genome sequencing to unravel mutations in the essential mmpL3 gene, which encodes the integral membrane protein that catalyzes the export of trehalose monomycolate, a precursor of the mycobacterial outer membrane component trehalose dimycolate (TDM), as well as mycolic acids bound to arabinogalactan. The drug-resistant phenotype was also observed in the parental strain overexpressing the mmpL3 alleles carrying the mutations identified in the resistors. However, no cross-resistance was observed between 1H-benzo[d]imidazole derivatives and SQ109, another MmpL3 inhibitor, or other first-line antitubercular drugs. Metabolic labeling and quantitative thin-layer chromatography (TLC) analysis of radiolabeled lipids from M. tuberculosis cultures treated with the benzoimidazoles indicated an inhibition of trehalose dimycolate (TDM) synthesis, as well as reduced levels of mycolylated arabinogalactan, in agreement with the inhibition of MmpL3 activity. Overall, this study emphasizes the pronounced activity of 1H-benzo[d]imidazole derivatives in interfering with mycolic acid metabolism and their potential for therapeutic application in the fight against tuberculosis.

scholarly journals Comparison of In Vitro Activity and MIC Distributions between the Novel Oxazolidinone Delpazolid and Linezolid against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis in China

2018 ◽  
Vol 62 (8) ◽  
Author(s):  
Zhaojing Zong ◽  
Wei Jing ◽  
Jin Shi ◽  
Shu'an Wen ◽  
Tingting Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Novel Mutation ◽  
Multidrug Resistant ◽  
23S Rrna ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Significant Difference ◽  
Mdr Tb

ABSTRACT Oxazolidinones are efficacious in treating mycobacterial infections, including tuberculosis (TB) caused by drug-resistant Mycobacterium tuberculosis. In this study, we compared the in vitro activities and MIC distributions of delpazolid, a novel oxazolidinone, and linezolid against multidrug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) in China. Additionally, genetic mutations in 23S rRNA, rplC, and rplD genes were analyzed to reveal potential mechanisms underlying the observed oxazolidinone resistance. A total of 240 M. tuberculosis isolates were included in this study, including 120 MDR-TB isolates and 120 XDR-TB isolates. Overall, linezolid and delpazolid MIC90 values for M. tuberculosis isolates were 0.25 mg/liter and 0.5 mg/liter, respectively. Based on visual inspection, we tentatively set epidemiological cutoff (ECOFF) values for MIC determinations for linezolid and delpazolid at 1.0 mg/liter and 2.0 mg/liter, respectively. Although no significant difference in resistance rates was observed between linezolid and delpazolid among XDR-TB isolates (P > 0.05), statistical analysis revealed a significantly greater proportion of linezolid-resistant isolates than delpazolid-resistant isolates within the MDR-TB group (P = 0.036). Seven (53.85%) of 13 linezolid-resistant isolates were found to harbor mutations within the three target genes. Additionally, 1 isolate exhibited an amino acid substitution (Arg126His) within the protein encoded by rplD that contributed to high-level resistance to linezolid (MIC of >16 mg/liter), compared to a delpazolid MIC of 0.25. In conclusion, in vitro susceptibility testing revealed that delpazolid antibacterial activity was comparable to that of linezolid. A novel mutation within rplD that endowed M. tuberculosis with linezolid, but not delpazolid, resistance was identified.

scholarly journals In Vitro Activity and MIC of Sitafloxacin against Multidrug-Resistant and Extensively Drug-Resistant Mycobacterium tuberculosis Isolated in Thailand

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Manoon Leechawengwongs ◽  
Therdsak Prammananan ◽  
Sarinya Jaitrong ◽  
Pamaree Billamas ◽  
Nampueng Makhao ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Critical Concentration ◽  
Multidrug Resistant ◽  
Quinolone Resistance ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Resistant Strains ◽  
Drug Resistant Strains

ABSTRACT New fluoroquinolones (FQs) have been shown to be more active against drug-resistant Mycobacterium tuberculosis strains than early FQs, such as ofloxacin. Sitafloxacin (STFX) is a new fluoroquinolone with in vitro activity against a broad range of bacteria, including M. tuberculosis. This study aimed to determine the in vitro activity of STFX against all groups of drug-resistant strains, including multidrug-resistant M. tuberculosis (MDR M. tuberculosis), MDR M. tuberculosis with quinolone resistance (pre-XDR), and extensively drug-resistant (XDR) strains. A total of 374 drug-resistant M. tuberculosis strains were tested for drug susceptibility by the conventional proportion method, and 95 strains were randomly submitted for MIC determination using the microplate alamarBlue assay (MABA). The results revealed that all the drug-resistant strains were susceptible to STFX at a critical concentration of 2 μg/ml. Determination of the MIC90s of the strains showed different MIC levels; MDR M. tuberculosis strains had a MIC90 of 0.0625 μg/ml, whereas pre-XDR and XDR M. tuberculosis strains had identical MIC90s of 0.5 μg/ml. Common mutations within the quinolone resistance-determining region (QRDR) of gyrA and/or gyrB did not confer resistance to STFX, except that double mutations of GyrA at Ala90Val and Asp94Ala were found in strains with a MIC of 1.0 μg/ml. The results indicated that STFX had potent in vitro activity against all the groups of drug-resistant M. tuberculosis strains and should be considered a new repurposed drug for treatment of multidrug-resistant and extensively drug-resistant TB.

scholarly journals Aminoglycoside Cross-Resistance in Mycobacterium tuberculosis Due to Mutations in the 5′ Untranslated Region ofwhiB7

2013 ◽  
Vol 57 (4) ◽  
pp. 1857-1865 ◽  
Author(s):  
Analise Z. Reeves ◽  
Patricia J. Campbell ◽  
Razvan Sultana ◽  
Seidu Malik ◽  
Megan Murray ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Mycobacterium Tuberculosis ◽  
Untranslated Region ◽  
Regulatory Region ◽  
Cross Resistance ◽  
Drug Resistant ◽  
Second Line ◽  
Content Type ◽  
Low Level ◽  
Level Resistance

ABSTRACTSince the discovery of streptomycin's bactericidal activity againstMycobacterium tuberculosis, aminoglycosides have been utilized to treat tuberculosis (TB). Today, the aminoglycosides kanamycin and amikacin are used to treat multidrug-resistant (MDR) TB, and resistance to any of the second-line injectable antibiotics, including kanamycin, amikacin, or capreomycin, is a defining characteristic of extensively drug-resistant (XDR) TB. Resistance to kanamycin and streptomycin is thought to be due to the acquisition of unlinked chromosomal mutations. However, we identified eight independent mutations in the 5′ untranslated region of the transcriptional activatorwhiB7that confer low-level resistance to both aminoglycosides. The mutations lead to 23- to 145-fold increases inwhiB7transcripts and subsequent increased expression of botheis(Rv2416c) andtap(Rv1258c). Increased expression ofeisconfers kanamycin resistance in these mutants, while increased expression oftap, which encodes an efflux pump, is a previously uncharacterized mechanism of low-level streptomycin resistance. Additionally, high-level resistance to streptomycin arose at a much higher frequency inwhiB7mutants than in a wild-type (WT) strain. AlthoughwhiB7is typically associated with intrinsic antibiotic resistance inM. tuberculosis, these data suggest that mutations in an uncharacterized regulatory region ofwhiB7contribute to cross-resistance against clinically used second-line antibiotics. As drug resistance continues to develop and spread, understanding the mechanisms and molecular basis of antibiotic resistance is critical for the development of rapid molecular tests to diagnose drug-resistant TB strains and ultimately for designing regimens to treat drug-resistant cases of TB.

scholarly journals In VitroActivity of AZD5847 against Geographically Diverse Clinical Isolates of Mycobacterium tuberculosis

2014 ◽  
Vol 58 (7) ◽  
pp. 4222-4223 ◽  
Author(s):  
Jim Werngren ◽  
Maria Wijkander ◽  
Nasrin Perskvist ◽  
V. Balasubramanian ◽  
Vasan K. Sambandamurthy ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lavage Fluid ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Cross Resistance ◽  
Drug Candidate ◽  
Content Type ◽  
Geographical Regions ◽  
Lung Biopsies

ABSTRACTThe MIC of the novel antituberculosis (anti-TB) drug AZD5847 was determined against 146 clinical isolates from diverse geographical regions, including eastern Europe, North America, Africa, and Asia, using the automated Bactec Mycobacterial Growth Indicator Tube (MGIT) 960 system. These isolates originated from specimen sources such as sputum, bronchial alveolar lavage fluid, pleural fluid, abscess material, lung biopsies, and feces. The overall MIC90was 1.0 mg/liter (range, 0.125 to 4 mg/liter). The MICs of AZD5847 for isolates ofMycobacterium tuberculosiswere similar among drug-sensitive strains, multidrug-resistant (MDR) strains, and extensively drug resistant (XDR) strains. The goodin vitroactivity of AZD5847 againstM. tuberculosisand the lack of cross-resistance make this agent a promising anti-TB drug candidate.

scholarly journals In VitroandIn VivoActivities of the Nitroimidazole TBA-354 against Mycobacterium tuberculosis

2014 ◽  
Vol 59 (1) ◽  
pp. 136-144 ◽  
Author(s):  
A. M. Upton ◽  
S. Cho ◽  
T. J. Yang ◽  
Y. Kim ◽  
Y. Wang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Pharmacokinetic Profile ◽  
Multidrug Resistant ◽  
Phase Iii ◽  
Drug Resistant ◽  
Next Generation ◽  
Content Type ◽  
Resistant Tuberculosis

ABSTRACTNitroimidazoles are a promising new class of antitubercular agents. The nitroimidazo-oxazole delamanid (OPC-67683, Deltyba) is in phase III trials for the treatment of multidrug-resistant tuberculosis, while the nitroimidazo-oxazine PA-824 is entering phase III for drug-sensitive and drug-resistant tuberculosis. TBA-354 (SN31354[(S)-2-nitro-6-((6-(4-trifluoromethoxy)phenyl)pyridine-3-yl)methoxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazine]) is a pyridine-containing biaryl compound with exceptional efficacy against chronic murine tuberculosis and favorable bioavailability in preliminary rodent studies. It was selected as a potential next-generation antituberculosis nitroimidazole following an extensive medicinal chemistry effort. Here, we further evaluate the pharmacokinetic properties and activity of TBA-354 againstMycobacterium tuberculosis. TBA-354 is narrow spectrum and bactericidalin vitroagainst replicating and nonreplicatingMycobacterium tuberculosis, with potency similar to that of delamanid and greater than that of PA-824. The addition of serum protein or albumin does not significantly alter this activity. TBA-354 maintains activity againstMycobacterium tuberculosisH37Rv isogenic monoresistant strains and clinical drug-sensitive and drug-resistant isolates. Spontaneous resistant mutants appear at a frequency of 3 × 10−7.In vitrostudies andin vivostudies in mice confirm that TBA-354 has high bioavailability and a long elimination half-life.In vitrostudies suggest a low risk of drug-drug interactions. Low-dose aerosol infection models of acute and chronic murine tuberculosis reveal time- and dose-dependentin vivobactericidal activity that is at least as potent as that of delamanid and more potent than that of PA-824. Its superior potency and pharmacokinetic profile that predicts suitability for once-daily oral dosing suggest that TBA-354 be studied further for its potential as a next-generation nitroimidazole.

scholarly journals Structure-Activity Relationships of Wollamide Cyclic Hexapeptides with Activity against Drug-Resistant and Intracellular Mycobacterium tuberculosis

2019 ◽  
Vol 63 (3) ◽  
Author(s):  
Zeinab G. Khalil ◽  
Timothy A. Hill ◽  
Luis M. De Leon Rodriguez ◽  
Rink-Jan Lohman ◽  
Huy N. Hoang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Mammalian Cells ◽  
Antimycobacterial Activity ◽  
Drug Resistant ◽  
Amino Acid Residues ◽  
Content Type ◽  
Cyclic Hexapeptides ◽  
Rats And Mice

ABSTRACT Wollamides are cyclic hexapeptides, recently isolated from an Australian soil Streptomyces isolate, that exhibit promising in vitro antimycobacterial activity against Mycobacterium bovis Bacille Calmette Guérin without displaying cytotoxicity against a panel of mammalian cells. Here, we report the synthesis and antimycobacterial activity of 36 new synthetic wollamides, collated with all known synthetic and natural wollamides, to reveal structure characteristics responsible for in vitro growth-inhibitory activity against Mycobacterium tuberculosis (H37Rv, H37Ra, CDC1551, HN878, and HN353). The most potent antimycobacterial wollamides were those where residue VI d-Orn (wollamide B) was replaced by d-Arg (wollamide B1) or d-Lys (wollamide B2), with all activity being lost when residue VI was replaced by Gly, l-Arg, or l-Lys (wollamide B3). Substitution of other amino acid residues mainly reduced or ablated antimycobacterial activity. Significantly, whereas wollamide B2 was the most potent in restricting M. tuberculosis in vitro, wollamide B1 restricted M. tuberculosis intracellular burden in infected macrophages. Wollamide B1 synergized with pretomanid (PA-824) in inhibiting M. tuberculosis in vitro growth but did not antagonize prominent first- and second-line tuberculosis antibiotics. Furthermore, wollamide B1 exerted bactericidal activity against nonreplicating M. tuberculosis and impaired growth of multidrug- and extensively drug-resistant clinical isolates. In vivo pharmacokinetic profiles for wollamide B1 in rats and mice encourage further optimization of the wollamide pharmacophore for in vivo bioavailability. Collectively, these observations highlight the potential of the wollamide antimycobacterial pharmacophore.

scholarly journals Can Inhibitor-Resistant Substitutions in the Mycobacterium tuberculosis β-Lactamase BlaC Lead to Clavulanate Resistance?: a Biochemical Rationale for the Use of β-Lactam–β-Lactamase Inhibitor Combinations

2013 ◽  
Vol 57 (12) ◽  
pp. 6085-6096 ◽  
Author(s):  
Sebastian G. Kurz ◽  
Kerstin A. Wolff ◽  
Saugata Hazra ◽  
Christopher R. Bethel ◽  
Andrea M. Hujer ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Clavulanic Acid ◽  
Treatment Strategies ◽  
Acid Resistance ◽  
Therapeutic Modality ◽  
Drug Resistant ◽  
Content Type ◽  
Extensively Drug Resistant ◽  
Lactamase Inhibitor

ABSTRACTThe current emergence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis calls for novel treatment strategies. Recently, BlaC, the principal β-lactamase ofMycobacterium tuberculosis, was recognized as a potential therapeutic target. The combination of meropenem and clavulanic acid, which inhibits BlaC, was found to be effective against even extensively drug-resistantM. tuberculosisstrains when testedin vitro. Yet there is significant concern that drug resistance against this combination will also emerge. To investigate the potential of BlaC to evolve variants resistant to clavulanic acid, we introduced substitutions at important amino acid residues ofM. tuberculosisBlaC (R220, A244, S130, and T237). Whereas the substitutions clearly led toin vitroclavulanic acid resistance in enzymatic assays but at the expense of catalytic activity, transformation of variant BlaCs into anM. tuberculosisH37Rv background revealed that impaired inhibition of BlaC did not affect inhibition of growth in the presence of ampicillin and clavulanate. From these data we propose that resistance to β-lactam–β-lactamase inhibitor combinations will likely not arise from structural alteration of BlaC, therefore establishing confidence that this therapeutic modality can be part of a successful treatment regimen againstM. tuberculosis.

scholarly journals A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis

2016 ◽  
Vol 84 (8) ◽  
pp. 2264-2273 ◽  
Author(s):  
Shinya Watanabe ◽  
Kazunori Matsumura ◽  
Hiroki Iwai ◽  
Keiji Funatogawa ◽  
Yuji Haishima ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
16S Rrna ◽  
Binding Sites ◽  
Mycolic Acid ◽  
Acid Biosynthesis ◽  
Mutation Site ◽  
Content Type ◽  
Antigen 85 ◽  
Decoding Region

Mycobacterium tuberculosiscontains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decreaseM. tuberculosisviability and virulence. Here, we describe an rRNA mutation, U1406A, which was generatedin vitroand confers resistance to kanamycin while highly attenuatingM. tuberculosisvirulence. The mutant showed decreased expression of 20% (n= 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuatedM. tuberculosisvirulence by affecting these processes.

scholarly journals The Cyclic Peptide Ecumicin Targeting ClpC1 Is Active against Mycobacterium tuberculosis In Vivo

2014 ◽  
Vol 59 (2) ◽  
pp. 880-889 ◽  
Author(s):  
Wei Gao ◽  
Jin-Yong Kim ◽  
Jeffrey R. Anderson ◽  
Tatos Akopian ◽  
Seungpyo Hong ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
High Throughput Screening ◽  
Cyclic Peptide ◽  
Genome Mining ◽  
Subcutaneous Administration ◽  
Multiple Drug ◽  
Drug Resistant ◽  
Content Type

ABSTRACTDrug-resistant tuberculosis (TB) has lent urgency to finding new drug leads with novel modes of action. A high-throughput screening campaign of >65,000 actinomycete extracts for inhibition ofMycobacterium tuberculosisviability identified ecumicin, a macrocyclic tridecapeptide that exerts potent, selective bactericidal activity againstM. tuberculosisin vitro, including nonreplicating cells. Ecumicin retains activity against isolated multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) strains ofM. tuberculosis. The subcutaneous administration to mice of ecumicin in a micellar formulation at 20 mg/kg body weight resulted in plasma and lung exposures exceeding the MIC. Complete inhibition ofM. tuberculosisgrowth in the lungs of mice was achieved following 12 doses at 20 or 32 mg/kg. Genome mining of lab-generated, spontaneous ecumicin-resistantM. tuberculosisstrains identified the ClpC1 ATPase complex as the putative target, and this was confirmed by a drug affinity response test. ClpC1 functions in protein breakdown with the ClpP1P2 protease complex. Ecumicin markedly enhanced the ATPase activity of wild-type (WT) ClpC1 but prevented activation of proteolysis by ClpC1. Less stimulation was observed with ClpC1 from ecumicin-resistant mutants. Thus, ClpC1 is a valid drug target againstM. tuberculosis, and ecumicin may serve as a lead compound for anti-TB drug development.

scholarly journals Mutations inpepQConfer Low-Level Resistance to Bedaquiline and Clofazimine in Mycobacterium tuberculosis

2016 ◽  
Vol 60 (8) ◽  
pp. 4590-4599 ◽  
Author(s):  
Deepak Almeida ◽  
Thomas Ioerger ◽  
Sandeep Tyagi ◽  
Si-Yang Li ◽  
Khisimuzi Mdluli ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Multidrug Resistant ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Content Type ◽  
Low Level ◽  
Resistant Tuberculosis ◽  
Multidrug Resistant Tuberculosis

ABSTRACTThe novel ATP synthase inhibitor bedaquiline recently received accelerated approval for treatment of multidrug-resistant tuberculosis and is currently being studied as a component of novel treatment-shortening regimens for drug-susceptible and multidrug-resistant tuberculosis. In a limited number of bedaquiline-treated patients reported to date, ≥4-fold upward shifts in bedaquiline MIC during treatment have been attributed to non-target-based mutations inRv0678that putatively increase bedaquiline efflux through the MmpS5-MmpL5 pump. These mutations also confer low-level clofazimine resistance, presumably by a similar mechanism. Here, we describe a new non-target-based determinant of low-level bedaquiline and clofazimine cross-resistance inMycobacterium tuberculosis: loss-of-function mutations inpepQ(Rv2535c), which corresponds to a putative Xaa-Pro aminopeptidase.pepQmutants were selected in mice by treatment with clinically relevant doses of bedaquiline, with or without clofazimine, and were shown to have bedaquiline and clofazimine MICs 4 times higher than those for the parental H37Rv strain. Coincubation with efflux inhibitors verapamil and reserpine lowered bedaquiline MICs against both mutant and parent strains to a level below the MIC against H37Rv in the absence of efflux pump inhibitors. However, quantitative PCR (qPCR) revealed no significant differences in expression ofRv0678,mmpS5, ormmpL5between mutant and parent strains. Complementation of apepQmutant with the wild-type gene restored susceptibility, indicating that loss of PepQ function is sufficient for reduced susceptibility bothin vitroand in mice. Although the mechanism by which mutations inpepQconfer bedaquiline and clofazimine cross-resistance remains unclear, these results may have clinical implications and warrant further evaluation of clinical isolates with reduced susceptibility to either drug for mutations in this gene.

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