scholarly journals Arylvinylpiperazine Amides, a New Class of Potent Inhibitors Targeting QcrB ofMycobacterium tuberculosis

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Caroline S. Foo ◽  
Andréanne Lupien ◽  
Maryline Kienle ◽  
Anthony Vocat ◽  
Andrej Benjak ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Parent Compound ◽  
New Drugs ◽  
Cross Resistance ◽  
Terminal Oxidase ◽  
Content Type ◽  
Allelic Exchange ◽  
Isolation And Characterization

ABSTRACTNew drugs are needed to control the current tuberculosis (TB) pandemic caused by infection withMycobacterium tuberculosis. We report here on our work with AX-35, an arylvinylpiperazine amide, and four related analogs, which are potent antitubercular agentsin vitro. All five compounds showed good activity againstM. tuberculosisin vitroand in infected THP-1 macrophages, while displaying only mild cytotoxicity. Isolation and characterization ofM. tuberculosis-resistant mutants to the arylvinylpiperazine amide derivative AX-35 revealed mutations in theqcrBgene encoding a subunit of cytochromebc1oxidase, one of two terminal oxidases of the electron transport chain. Cross-resistance studies, allelic exchange, transcriptomic analyses, and bioenergetic flux assays provided conclusive evidence that the cytochromebc1-aa3is the target of AX-35, although the compound appears to interact differently with the quinol binding pocket compared to previous QcrB inhibitors. The transcriptomic and bioenergetic profiles ofM. tuberculosistreated with AX-35 were similar to those generated by other cytochromebc1oxidase inhibitors, including the compensatory role of the alternate terminal oxidase cytochromebdin respiratory adaptation. In the absence of cytochromebdoxidase, AX-35 was bactericidal againstM. tuberculosis. Finally, AX-35 and its analogs were active in an acute mouse model of TB infection, with two analogs displaying improved activity over the parent compound. Our findings will guide future lead optimization to produce a drug candidate for the treatment of TB and other mycobacterial diseases, including Buruli ulcer and leprosy.IMPORTANCENew drugs againstMycobacterium tuberculosisare urgently needed to deal with the current global TB pandemic. We report here on the discovery of a series of arylvinylpiperazine amides (AX-35 to AX-39) that represent a promising new family of compounds with potentin vitroandin vivoactivities againstM. tuberculosis. AX compounds target the QcrB subunit of the cytochromebc1terminal oxidase with a different mode of interaction compared to those of known QcrB inhibitors. This study provides the first multifaceted validation of QcrB inhibition by recombineering-mediated allelic exchange, gene expression profiling, and bioenergetic flux studies. It also provides further evidence for the compensatory role of cytochromebdoxidase upon QcrB inhibition. In the absence of cytochromebdoxidase, AX compounds are bactericidal, an encouraging property for future antimycobacterial drug development.

scholarly journals Novel Pyrimidines as Antitubercular Agents

2018 ◽  
Vol 62 (3) ◽  
Author(s):  
Daigo Inoyama ◽  
Steven D. Paget ◽  
Riccardo Russo ◽  
Srinivasan Kandasamy ◽  
Pradeep Kumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Small Molecules ◽  
New Drugs ◽  
Cross Resistance ◽  
Antitubercular Agents ◽  
Front Line ◽  
Content Type ◽  
Pharmacokinetic Profiles ◽  
Global Pandemic

ABSTRACTMycobacterium tuberculosisinfection is responsible for a global pandemic. New drugs are needed that do not show cross-resistance with the existing front-line therapeutics. A triazine antitubercular hit led to the design of a related pyrimidine family. The synthesis of a focused series of these analogs facilitated exploration of theirin vitroactivity,in vitrocytotoxicity, and physiochemical and absorption-distribution-metabolism-excretion properties. Select pyrimidines were then evaluated for their pharmacokinetic profiles in mice. The findings suggest a rationale for the further evolution of this promising series of antitubercular small molecules, which appear to share some similarities with the clinical compound PA-824 in terms of activation, while highlighting more general guidelines for the optimization of small-molecule antitubercular agents.

scholarly journals Mutation ofRv2887, amarR-Like Gene, Confers Mycobacterium tuberculosis Resistance to an Imidazopyridine-Based Agent

2015 ◽  
Vol 59 (11) ◽  
pp. 6873-6881 ◽  
Author(s):  
Kathryn Winglee ◽  
Shichun Lun ◽  
Marco Pieroni ◽  
Alan Kozikowski ◽  
William Bishai
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Efflux Pump ◽  
Transcriptional Regulator ◽  
Cross Resistance ◽  
Loss Of Function ◽  
Strong Activity ◽  
Content Type ◽  
Novel Drug

ABSTRACTDrug resistance is a major problem inMycobacterium tuberculosiscontrol, and it is critical to identify novel drug targets and new antimycobacterial compounds. We have previously identified an imidazo[1,2-a]pyridine-4-carbonitrile-based agent, MP-III-71, with strong activity againstM. tuberculosis. In this study, we evaluated mechanisms of resistance to MP-III-71. We derived three independentM. tuberculosismutants resistant to MP-III-71 and conducted whole-genome sequencing of these mutants. Loss-of-function mutations inRv2887were common to all three MP-III-71-resistant mutants, and we confirmed the role ofRv2887as a gene required for MP-III-71 susceptibility using complementation. The Rv2887 protein was previously unannotated, but domain and homology analyses suggested it to be a transcriptional regulator in the MarR (multiple antibiotic resistance repressor) family, a group of proteins first identified inEscherichia colito negatively regulate efflux pumps and other mechanisms of multidrug resistance. We found that two efflux pump inhibitors, verapamil and chlorpromazine, potentiate the action of MP-III-71 and that mutation ofRv2887abrogates their activity. We also used transcriptome sequencing (RNA-seq) to identify genes which are differentially expressed in the presence and absence of a functional Rv2887 protein. We found that genes involved in benzoquinone and menaquinone biosynthesis were repressed by functional Rv2887. Thus, inactivating mutations ofRv2887, encoding a putative MarR-like transcriptional regulator, confer resistance to MP-III-71, an effective antimycobacterial compound that shows no cross-resistance to existing antituberculosis drugs. The mechanism of resistance ofM. tuberculosisRv2887mutants may involve efflux pump upregulation and also drug methylation.

scholarly journals A New NonpolarN-Hydroxy Imidazoline Lead Compound with Improved Activity in a Murine Model of Late-Stage Trypanosoma brucei brucei Infection Is Not Cross-Resistant with Diamidines

2014 ◽  
Vol 59 (2) ◽  
pp. 890-904 ◽  
Author(s):  
Carlos H. Ríos Martínez ◽  
Florence Miller ◽  
Kayathiri Ganeshamoorthy ◽  
Fabienne Glacial ◽  
Marcel Kaiser ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Late Stage ◽  
Parent Compound ◽  
Central Nervous System Infection ◽  
Sleeping Sickness ◽  
Intrinsic Activity ◽  
Cross Resistance ◽  
Content Type

ABSTRACTTreatment of late-stage sleeping sickness requires drugs that can cross the blood-brain barrier (BBB) to reach the parasites located in the brain. We report here the synthesis and evaluation of four newN-hydroxy and 12 newN-alkoxy derivatives of bisimidazoline leads as potential agents for the treatment of late-stage sleeping sickness. These compounds, which have reduced basicity compared to the parent leads (i.e., are less ionized at physiological pH), were evaluatedin vitroagainstTrypanosoma brucei rhodesienseandin vivoin murine models of first- and second-stage sleeping sickness. Resistance profile, physicochemical parameters,in vitroBBB permeability, and microsomal stability also were determined. TheN-hydroxy imidazoline analogues were the most effectivein vivo, with 4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)-N-(4-((1-hydroxy-4,5-dihydro-1H-imidazol-2-yl)amino)phenyl)benzamide (14d) showing 100% cures in the first-stage disease, while 15d, 16d, and 17d appeared to slightly improve survival. In addition, 14d showed weak activity in the chronic model of central nervous system infection in mice. No evidence of reduction of this compound with hepatic microsomes and mitochondria was foundin vitro, suggesting thatN-hydroxy imidazolines are metabolically stable and have intrinsic activity againstT. brucei. In contrast to its unsubstituted parent compound, the uptake of 14d inT. bruceiwas independent of known drug transporters (i.e.,T. bruceiAT1/P2 and HAPT), indicating a lower predisposition to cross-resistance with other diamidines and arsenical drugs. Hence, theN-hydroxy bisimidazolines (14d in particular) represent a new class of promising antitrypanosomal agents.

scholarly journals Fluorescence-Reported Allelic Exchange Mutagenesis Reveals a Role for Chlamydia trachomatis TmeA in Invasion That Is Independent of Host AHNAK

2017 ◽  
Vol 85 (12) ◽  
Author(s):  
M. J. McKuen ◽  
K. E. Mueller ◽  
Y. S. Bae ◽  
K. A. Fields
Keyword(s):  
Tissue Culture ◽  
Host Cells ◽  
Coding Sequences ◽  
Content Type ◽  
Allelic Exchange ◽  
Infection Models ◽  
Virulence Protein ◽  
Complete Deletion

ABSTRACT Development of approaches to genetically manipulate Chlamydia is fostering important advances in understanding pathogenesis. Fluorescence-reported allelic exchange mutagenesis (FRAEM) now enables the complete deletion of specific genes in C. trachomatis L2. We have leveraged this technology to delete the coding sequences for a known type III effector. The evidence provided here indicates that CT694/CTL0063 is a virulence protein involved in chlamydial invasion. Based on our findings, we designate the gene product corresponding to ct694-ctl0063 translocated membrane-associated effector A (TmeA). Deletion of tmeA did not impact development of intracellular chlamydiae. However, the absence of TmeA manifested as a decrease in infectivity in both tissue culture and murine infection models. The in vitro defect was reflected by impaired invasion of host cells. TmeA binds human AHNAK, and we demonstrate here that AHNAK is transiently recruited by invading chlamydiae. TmeA, however, is not required for endogenous AHNAK recruitment. TmeA also impairs AHNAK-dependent actin bundling activity. This TmeA-mediated effect likely does not explain impaired invasion displayed by the tmeA strain of Chlamydia, since AHNAK-deficient cells revealed no invasion phenotype. Overall, our data indicate the efficacy of FRAEM and reveal a role of TmeA during chlamydial invasion that manifests independently of effects on AHNAK.

scholarly journals In Vitro Isolation and Characterization of Oxazolidinone-Resistant Mycobacterium tuberculosis

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Matthew B. McNeil ◽  
Devon D. Dennison ◽  
Catherine D. Shelton ◽  
Tanya Parish
Keyword(s):  
Mycobacterium Tuberculosis ◽  
23S Rrna ◽  
Clinical Settings ◽  
Content Type ◽  
Isolation And Characterization ◽  
Mutant Strains ◽  
Ribosomal Protein L3 ◽  
Resistant Strains

ABSTRACT Oxazolidinones are promising candidates for the treatment of Mycobacterium tuberculosis infections. We isolated linezolid-resistant strains from H37Rv (Euro-American) and HN878 (East-Asian) strains; resistance frequencies were similar in the two strains. Mutations were identified in ribosomal protein L3 (RplC) and the 23S rRNA (rrl). All mutant strains were cross resistant to sutezolid; a subset was cross resistant to chloramphenicol. Mutations in rrl led to growth impairment and decreased fitness that may limit spread in clinical settings.

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 Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Matthew B. McNeil ◽  
Theresa O’Malley ◽  
Devon Dennison ◽  
Catherine D. Shelton ◽  
Bjorn Sunde ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
New Drugs ◽  
Content Type ◽  
Mechanism Of Resistance ◽  
Primary Mechanism ◽  
Resistant Mutants ◽  
Mycobacterial Cell Wall

ABSTRACT The Mycobacterium tuberculosis protein MmpL3 performs an essential role in cell wall synthesis, since it effects the transport of trehalose monomycolates across the inner membrane. Numerous structurally diverse pharmacophores have been identified as inhibitors of MmpL3 largely based on the identification of resistant isolates with mutations in MmpL3. For some compounds, it is possible there are different primary or secondary targets. Here, we have investigated resistance to the spiral amine class of compounds. Isolation and sequencing of resistant mutants demonstrated that all had mutations in MmpL3. We hypothesized that if additional targets of this pharmacophore existed, then successive rounds to generate resistant isolates might reveal mutations in other loci. Since compounds were still active against resistant isolates, albeit with reduced potency, we isolated resistant mutants in this background at higher concentrations. After a second round of isolation with the spiral amine, we found additional mutations in MmpL3. To increase our chance of finding alternative targets, we ran a third round of isolation using a different molecule scaffold (AU1235, an adamantyl urea). Surprisingly, we obtained further mutations in MmpL3. Multiple mutations in MmpL3 increased the level and spectrum of resistance to different pharmacophores but did not incur a fitness cost in vitro. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores. IMPORTANCE Mycobacterium tuberculosis is a major global human pathogen, and new drugs and new drug targets are urgently required. Cell wall biosynthesis is a major target of current tuberculosis drugs and of new agents under development. Several new classes of molecules appear to have the same target, MmpL3, which is involved in the export and synthesis of the mycobacterial cell wall. However, there is still debate over whether MmpL3 is the primary or only target for these classes. We wanted to confirm the mechanism of resistance for one series. We identified mutations in MmpL3 which led to resistance to the spiral amine series. High-level resistance to these compounds and two other series was conferred by multiple mutations in the same protein (MmpL3). These mutations did not reduce growth rate in culture. These results support the hypothesis that MmpL3 is the primary mechanism of resistance and likely target for these pharmacophores.

scholarly journals Peptidoglycan Hydrolases RipA and Ami1 Are Critical for Replication and Persistence of Mycobacterium tuberculosis in the Host

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Claire Healy ◽  
Alexandre Gouzy ◽  
Sabine Ehrt
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Cell Division ◽  
Drug Target ◽  
New Drugs ◽  
Chemotherapeutic Regimen ◽  
Content Type ◽  
Cell Wall Polymer ◽  
Redundant Functions

ABSTRACT Synthesis and cleavage of the cell wall polymer peptidoglycan (PG) are carefully orchestrated processes and are essential for the growth and survival of bacteria. Yet, the function and importance of many enzymes that act on PG in Mycobacterium tuberculosis remain to be elucidated. We demonstrate that the activity of the N-acetylmuramyl-l-alanine amidase Ami1 is dispensable for cell division in M. tuberculosis in vitro yet contributes to the bacterium’s ability to persist during chronic infection in mice. Furthermore, the d,l-endopeptidase RipA, a predicted essential enzyme, is dispensable for the viability of M. tuberculosis but required for efficient cell division in vitro and in vivo. Depletion of RipA sensitizes M. tuberculosis to rifampin and to cell envelope-targeting antibiotics. Ami1 helps sustain residual cell division in cells lacking RipA, but the partial redundancy provided by Ami1 is not sufficient during infection, as depletion of RipA prevents M. tuberculosis from replicating in macrophages and leads to dramatic killing of the bacteria in mice. Notably, RipA is essential for persistence of M. tuberculosis in mice, suggesting that cell division is required during chronic mouse infection. Despite the multiplicity of enzymes acting on PG with redundant functions, we have identified two PG hydrolases that are important for M. tuberculosis to replicate and persist in the host. IMPORTANCE Tuberculosis (TB) is a major global heath burden, with 1.6 million people succumbing to the disease every year. The search for new drugs to improve the current chemotherapeutic regimen is crucial to reducing this global health burden. The cell wall polymer peptidoglycan (PG) has emerged as a very successful drug target in bacterial pathogens, as many currently used antibiotics target the synthesis of this macromolecule. However, the multitude of genes encoding PG-synthesizing and PG-modifying enzymes with apparent redundant functions has hindered the identification of novel drug targets in PG synthesis in Mycobacterium tuberculosis. Here, we demonstrate that two PG-cleaving enzymes are important for virulence of M. tuberculosis. In particular, the d,l-endopeptidase RipA represents a potentially attractive drug target, as its depletion results in the clearance of M. tuberculosis from the host and renders the bacteria hypersusceptible to rifampin, a frontline TB drug, and to several cell wall-targeting antibiotics.

scholarly journals Activity of ketoconazole against Mycobacterium tuberculosis in vitro and in the mouse model

2007 ◽  
Vol 56 (8) ◽  
pp. 1047-1051 ◽  
Author(s):  
Sean T. Byrne ◽  
Steven M. Denkin ◽  
Peihua Gu ◽  
Eric Nuermberger ◽  
Ying Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Term Treatment ◽  
New Drugs ◽  
Bacteriostatic Effect ◽  
Short Term ◽  
Short Term Treatment ◽  
Neutral Conditions

There is an urgent need for the development of new drugs that are active against drug-resistant Mycobacterium tuberculosis strains and can shorten tuberculosis (TB) therapy. It has previously been reported that the azole class of antifungals has anti-TB activity in vitro. This study evaluated ketoconazole (KTC) for activity against M. tuberculosis. The MIC of KTC for different M. tuberculosis strains ranged from 8 to 16 μg ml−1 under both acidic and neutral conditions, with the minimum bactericidal concentration being about twofold higher than the MIC. KTC had enhanced activity against old, non-growing bacilli in vitro when combined with pyrazinamide (PZA) and rifampicin (RIF). A single oral dose of KTC at 75 mg kg−1 led to an inhibitory serum concentration 2 h after administration. The in vivo activity of KTC was evaluated in established pulmonary TB in the murine model, compared alone and in combination with isoniazid (INH), PZA and RIF. KTC alone exhibited little effect after short-term treatment, with a borderline bacteriostatic effect on spleen colony counts but not on lung counts. KTC, when added in combination with INH, PZA and RIF, significantly improved the treatment outcome in the lungs (compared with treatment with INH, PZA and RIF). The lowest numbers of bacilli in lungs were found in mice treated with KTC, PZA and RIF. Further investigation is necessary to determine the role of KTC in the treatment of TB.

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.

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