SAR and identification of 2-(quinolin-4-yloxy)acetamides as Mycobacterium tuberculosis cytochrome bc1 inhibitors

Gene Deletion ◽

Cytochrome Bc1 ◽

Resistant Mutants

New antimycobacterial 2-(quinoline-4-yloxy)acetamides were prepared, and using gene deletion and resistant mutants, we conclude that the compound class inhibits the mycobacterial cytochrome bc1 complex.

A Novel 6-Benzyl Ether Benzoxaborole Is Active against Mycobacterium tuberculosisIn Vitro

10.1128/aac.01205-17 ◽

2017 ◽

Vol 61 (9) ◽

Cited By ~ 1

Author(s):

Nipul Patel ◽

Theresa O'Malley ◽

Yong-Kang Zhang ◽

Yi Xia ◽

Bjorn Sunde ◽

...

Keyword(s):

Liquid Medium ◽

Inhibitory Concentration ◽

Solid Medium ◽

Eukaryotic Cells ◽

Intracellular Bacteria ◽

Benzyl Ether ◽

Content Type ◽

Resistant Mutants

ABSTRACT We identified a novel 6-benzyl ether benzoxaborole with potent activity against Mycobacterium tuberculosis. The compound had an MIC of 2 μM in liquid medium. The compound was also able to prevent growth on solid medium at 0.8 μM and was active against intracellular bacteria (50% inhibitory concentration [IC50] = 3.6 μM) without cytotoxicity against eukaryotic cells (IC50 > 100 μM). We isolated resistant mutants (MIC ≥ 100 μM), which had mutations in Rv1683, Rv3068c, and Rv0047c.

De Novo Emergence of Genetically Resistant Mutants of Mycobacterium tuberculosis from the Persistence Phase Cells Formed against Antituberculosis Drugs In Vitro

10.1128/aac.01343-16 ◽

2016 ◽

Vol 61 (2) ◽

Cited By ~ 29

Author(s):

Jees Sebastian ◽

Sharmada Swaminath ◽

Rashmi Ravindran Nair ◽

Kishor Jakkala ◽

Atul Pradhan ◽

...

Keyword(s):

Hydroxyl Radical ◽

Prolonged Exposure ◽

De Novo ◽

Random Mutagenesis ◽

Content Type ◽

Genome Wide ◽

Resistant Mutants ◽

Phase Population

ABSTRACT Bacterial persisters are a subpopulation of cells that can tolerate lethal concentrations of antibiotics. However, the possibility of the emergence of genetically resistant mutants from antibiotic persister cell populations, upon continued exposure to lethal concentrations of antibiotics, remained unexplored. In the present study, we found that Mycobacterium tuberculosis cells exposed continuously to lethal concentrations of rifampin (RIF) or moxifloxacin (MXF) for prolonged durations showed killing, RIF/MXF persistence, and regrowth phases. RIF-resistant or MXF-resistant mutants carrying clinically relevant mutations in the rpoB or gyrA gene, respectively, were found to emerge at high frequency from the RIF persistence phase population. A Luria-Delbruck fluctuation experiment using RIF-exposed M. tuberculosis cells showed that the rpoB mutants were not preexistent in the population but were formed de novo from the RIF persistence phase population. The RIF persistence phase M. tuberculosis cells carried elevated levels of hydroxyl radical that inflicted extensive genome-wide mutations, generating RIF-resistant mutants. Consistent with the elevated levels of hydroxyl radical-mediated genome-wide random mutagenesis, MXF-resistant M. tuberculosis gyrA de novo mutants could be selected from the RIF persistence phase cells. Thus, unlike previous studies, which showed emergence of genetically resistant mutants upon exposure of bacteria for short durations to sublethal concentrations of antibiotics, our study demonstrates that continuous prolonged exposure of M. tuberculosis cells to lethal concentrations of an antibiotic generates antibiotic persistence phase cells that form a reservoir for the generation of genetically resistant mutants to the same antibiotic or another antibiotic. These findings may have clinical significance in the emergence of drug-resistant tubercle bacilli.

Emergence of Fluoroquinolone Resistance in Mycobacterium tuberculosis during Continuously Dosed Moxifloxacin Monotherapy in a Mouse Model

10.1128/aac.49.9.3977-3979.2005 ◽

2005 ◽

Vol 49 (9) ◽

pp. 3977-3979 ◽

Cited By ~ 23

Author(s):

Amy Sarah Ginsburg ◽

Ronggai Sun ◽

Heather Calamita ◽

Cherise P. Scott ◽

William R. Bishai ◽

...

Keyword(s):

Mouse Model ◽

Fluoroquinolone Resistance ◽

Drug Concentrations ◽

Resistant Mutants ◽

Selection Of

ABSTRACT Fluoroquinolone resistance in tuberculosis may rapidly emerge. Mice infected with high titers of aerosolized Mycobacterium tuberculosis and treated for 8 weeks with four concentrations of moxifloxacin (0.125, 0.25, 0.50, and 1.0%) mixed into the diet had drug concentrations of 2.4, 4.1, 5.3, and 17.9 μg/ml, respectively, in blood. Selection of fluoroquinolone-resistant mutants occurred in all surviving mice.

Characteristic Resistance Mechanism ofMycobacterium tuberculosisto DC-159a, a New Respiratory Quinolone

10.1128/aac.00417-10 ◽

2011 ◽

Vol 55 (8) ◽

pp. 3958-3960 ◽

Cited By ~ 12

Author(s):

Jun-ichiro Sekiguchi ◽

Areeya Disratthakit ◽

Shinji Maeda ◽

Norio Doi

Keyword(s):

Resistance Mechanism ◽

Multidrug Resistant ◽

Double Mutation ◽

Resistant Mutants ◽

Characteristic Resistance

ABSTRACTA G88C mutation in GyrA is one of the key alterations by whichMycobacterium tuberculosismutants acquire DC-159a resistancein vitro. A novel double mutation in GyrA, G88C D94H, conferred high DC-159a resistance. Different mutation patterns in GyrA were demonstrated for DC-159a-resistant mutants and quinolone-resistant multidrug-resistant (QR-MDR)M. tuberculosisisolates, with a mutation either at position 90 or 94 and double mutations at 90 and 91 or at 90 and 94. DC-159a might be promising for QRM. tuberculosistreatment.

Rational Design of Biosafety Level 2-Approved, Multidrug-Resistant Strains ofMycobacterium tuberculosisthrough Nutrient Auxotrophy

mBio ◽

10.1128/mbio.00938-18 ◽

2018 ◽

Vol 9 (3) ◽

Cited By ~ 9

Author(s):

Catherine Vilchèze ◽

Jacqueline Copeland ◽

Tracy L. Keiser ◽

Torin Weisbrod ◽

Jacqueline Washington ◽

...

Keyword(s):

Rational Design ◽

Multidrug Resistant ◽

New Drugs ◽

Drug Resistant ◽

Content Type ◽

Auxotrophic Mutants ◽

Level 3 ◽

Resistant Mutants ◽

Biosafety Level

ABSTRACTMultidrug-resistant (MDR) tuberculosis, defined as tuberculosis resistant to the two first-line drugs isoniazid and rifampin, poses a serious problem for global tuberculosis control strategies. Lack of a safe and convenient model organism hampers progress in combating the spread of MDR strains ofMycobacterium tuberculosis. We reasoned that auxotrophic MDR mutants ofM. tuberculosiswould provide a safe means for studying MDRM. tuberculosiswithout the need for a biosafety level 3 (BSL3) laboratory. Two different sets of triple auxotrophic mutants ofM. tuberculosiswere generated, which were auxotrophic for the nutrients leucine, pantothenate, and arginine or for leucine, pantothenate, and methionine. These triple auxotrophic strains retained their acid-fastness, their ability to generate both a drug persistence phenotype and drug-resistant mutants, and their susceptibility to plaque-forming mycobacterial phages. MDR triple auxotrophic mutants were obtained in a two-step fashion, selecting first for solely isoniazid-resistant or rifampin-resistant mutants. Interestingly, selection for isoniazid-resistant mutants of the methionine auxotroph generated isolates with single point mutations inkatG, which encodes an isoniazid-activating enzyme, whereas similar selection using the arginine auxotroph yielded isoniazid-resistant mutants with large deletions in the chromosomal region containingkatG. TheseM. tuberculosisMDR strains were readily sterilized by second-line tuberculosis drugs and failed to kill immunocompromised mice. These strains provide attractive candidates forM. tuberculosisbiology studies and drug screening outside the BSL3 facility.IMPORTANCEElimination ofMycobacterium tuberculosis, the bacterium causing tuberculosis, requires enhanced understanding of its biology in order to identify new drugs against drug-susceptible and drug-resistantM. tuberculosisas well as uncovering novel pathways that lead toM. tuberculosisdeath. To circumvent the need for a biosafety level 3 (BSL3) laboratory when conducting research onM. tuberculosis, we have generated drug-susceptible and drug-resistant triple auxotrophic strains ofM. tuberculosissuitable for use in a BSL2 laboratory. These strains originate from a double auxotrophicM. tuberculosisstrain, H37Rv ΔpanCDΔleuCD, which was reclassified as a BSL2 strain based on its lack of lethality in immunocompromised and immunocompetent mice. A third auxotrophy (methionine or arginine) was introduced via deletion ofmetAorargB, respectively, sinceM. tuberculosisΔmetAandM. tuberculosisΔargBare unable to survive amino acid auxotrophy and infect their host. The resulting triple auxotrophicM. tuberculosisstrains retained characteristics ofM. tuberculosisrelevant for most types of investigations.

Multiple Mutations in Mycobacterium tuberculosis MmpL3 Increase Resistance to MmpL3 Inhibitors

mSphere ◽

10.1128/msphere.00985-20 ◽

2020 ◽

Vol 5 (5) ◽

Author(s):

Matthew B. McNeil ◽

Theresa O’Malley ◽

Devon Dennison ◽

Catherine D. Shelton ◽

Bjorn Sunde ◽

...

Keyword(s):

Cell Wall ◽

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.

Ursolic acid as a potential inhibitor of Mycobacterium tuberculosis cytochrome bc1 oxidase—a molecular modelling perspective

Journal of Molecular Modeling ◽

10.1007/s00894-021-04993-w ◽

2022 ◽

Vol 28 (2) ◽

Author(s):

Ntombikayise Tembe ◽

Kgothatso E. Machaba ◽

Umar Ndagi ◽

Hezekiel M. Kumalo ◽

Ndumiso N. Mhlongo

Keyword(s):

Ursolic Acid ◽

Molecular Modelling ◽

Cytochrome Bc1 ◽

Potential Inhibitor

Effect of rpoB Mutations Conferring Rifampin Resistance on Fitness of Mycobacterium tuberculosis

10.1128/aac.48.4.1289-1294.2004 ◽

2004 ◽

Vol 48 (4) ◽

pp. 1289-1294 ◽

Cited By ~ 155

Author(s):

Deneke H. Mariam ◽

Yohannes Mengistu ◽

Sven E. Hoffner ◽

Dan I. Andersson

Keyword(s):

Clinical Problem ◽

Relative Fitness ◽

Macrophage Cell ◽

Gene Encoding ◽

Laboratory Medium ◽

Rifampin Resistance ◽

Resistant Mutants ◽

Major Drug

ABSTRACT Rifampin is a major drug used in the treatment of tuberculosis infections, and increasing rifampin resistance represents a worldwide clinical problem. Resistance to rifampin is caused by mutations in the rpoB gene, encoding the β-subunit of RNA polymerase. We examined the effect of three different rpoB mutations on the fitness of Mycobacterium tuberculosis. Rifampin-resistant mutants were isolated from a virulent clinical isolate of M. tuberculosis (strain Harlingen) in vitro at a mutation frequency of 2.3 × 10−8. Mutations in the rpoB gene were identified, and the growth rates of three defined mutants were measured by competition with the susceptible parent strain in laboratory medium and by single cultures in a macrophage cell line and in laboratory medium. All of the mutants showed a decreased growth rate in the three assays. The relative fitness of the mutants varied between 0.29 and 0.96 (that of the susceptible strain was set to 1.0) depending on the specific mutant and assay system. Unexpectedly, the relative fitness ranking of the mutants differed between the different assays. In conclusion, rifampin resistance is associated with a cost that is conditional.

Gene Deletion Contributes to the Virulence of an Outbreak Strain of Mycobacterium Tuberculosis via Immune Subversion

Journal of Infection ◽

10.1016/j.jinf.2006.10.007 ◽

2007 ◽

Vol 54 (3) ◽

pp. e188-e189

Author(s):

S.M. Newton ◽

R.J. Smith ◽

K.A. Wilkinson ◽

M.P. Nicol ◽

N.J. Garton ◽

...

Keyword(s):

Gene Deletion ◽

Outbreak Strain

Mycobacterium tuberculosis stimulates IL-1β production by macrophages in an ESAT-6 dependent manner with the involvement of serum amyloid A3

10.1101/2020.04.04.025411 ◽

2020 ◽

Author(s):

Bock-Gie Jung ◽

Ramakrishna Vankayalapati ◽

Buka Samten

Keyword(s):

Gene Deletion ◽

Serum Amyloid A ◽

Serum Amyloid ◽

Mouse Bone Marrow ◽

Dependent Manner ◽

Rna Seq ◽

Amyloid A ◽

Highly Expressed Genes ◽

Mouse Lungs

ABSTRACTTo explore interleukin (IL)-1β production in tuberculosis, we infected mouse bone marrow-derived macrophages (BMDM) with Mycobacterium tuberculosis (Mtb) H37Rv, its early secreted antigenic target protein of 6 kDa (ESAT-6) gene deletion (H37Rv:Δ3875) or complemented strain (H37Rv:Δ3875C) and evaluated IL-1β production. H37Rv induced significantly increased IL-1β production by BMDMs compared to non-infected BMDMs. In contrast, H37Rv:Δ3875 induced significantly less mature IL-1β production despite eliciting comparable levels of pro-IL-1β and IL-8 from BMDMs compared to H37Rv and H37Rv:Δ3875C. Blocking either NLRP3 or K+ efflux diminished H37Rv-induced IL-1β production by BMDMs. Infection of mice intranasally with H37Rv:Δ3875 induced less IL-1β production in the lungs compared with H37Rv.Intranasal delivery of ESAT-6 but not CFP10 induced production of IL-1β in mouse lungs and RNA-Seq analysis identified serum amyloid A (SAA) 3 as one of the highly expressed genes in mouse lungs. Infection of mice with H37Rv but not H37Rv:Δ3875 induced expression of lung SAA3 mRNA and protein, consistent with the effect of intranasal delivery of ESAT-6. Silencing SAA3 reduced Mtb-induced IL-1β production by BMDMs. We conclude that the production of SAA3 is required for Mtb stimulated IL-1β production by macrophages in tuberculosis infection.