scholarly journals Pantothenate and Pantetheine Antagonize the Antitubercular Activity of Pyrazinamide

2014 ◽  
Vol 58 (12) ◽  
pp. 7258-7263 ◽  
Author(s):  
Nicholas A. Dillon ◽  
Nicholas D. Peterson ◽  
Brandon C. Rosen ◽  
Anthony D. Baughn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Nicotinic Acid ◽  
Active Form ◽  
Laboratory Strain ◽  
Antitubercular Activity ◽  
First Line ◽  
Auxotrophic Strain ◽  
Content Type ◽  
Related Compounds ◽  
Pyrazinoic Acid

ABSTRACTPyrazinamide (PZA) is a first-line tuberculosis drug that inhibits the growth ofMycobacterium tuberculosisvia an as yet undefined mechanism. AnM. tuberculosislaboratory strain that was auxotrophic for pantothenate was found to be insensitive to PZA and to the active form, pyrazinoic acid (POA). To determine whether this phenotype was strain or condition specific, the effect of pantothenate supplementation on PZA activity was assessed using prototrophic strains ofM. tuberculosis. It was found that pantothenate and other β-alanine-containing metabolites abolished PZA and POA susceptibility, suggesting that POA might selectively target pantothenate synthesis. However, when the pantothenate-auxotrophic strain was cultivated using a subantagonistic concentration of pantetheine in lieu of pantothenate, susceptibility to PZA and POA was restored. In addition, we found that β-alanine could not antagonize PZA and POA activity against the pantothenate-auxotrophic strain, indicating that the antagonism is specific to pantothenate. Moreover, pantothenate-mediated antagonism was observed for structurally related compounds, includingn-propyl pyrazinoate, 5-chloropyrazinamide, and nicotinamide, but not for nicotinic acid or isoniazid. Taken together, these data demonstrate that while pantothenate can interfere with the action of PZA, pantothenate synthesis is not directly targeted by PZA. Our findings suggest that targeting of pantothenate synthesis has the potential to enhance PZA efficacy and possibly to restore PZA susceptibility in isolates withpanD-linked resistance.

scholarly journals Pyrazinoic Acid Decreases the Proton Motive Force, Respiratory ATP Synthesis Activity, and Cellular ATP Levels

2011 ◽  
Vol 55 (11) ◽  
pp. 5354-5357 ◽  
Author(s):  
Ping Lu ◽  
Anna C. Haagsma ◽  
Hoang Pham ◽  
Janneke J. Maaskant ◽  
Selena Mol ◽  
...  
Keyword(s):  
Active Form ◽  
Atp Synthesis ◽  
Proton Motive Force ◽  
Motive Force ◽  
Antituberculosis Drug ◽  
First Line ◽  
Content Type ◽  
Atp Levels ◽  
Synthesis Activity ◽  
Pyrazinoic Acid

ABSTRACTPyrazinoic acid, the active form of the first-line antituberculosis drug pyrazinamide, decreased the proton motive force and respiratory ATP synthesis rates in subcellular mycobacterial membrane assays. Pyrazinoic acid also significantly lowered cellular ATP levels inMycobacterium bovisBCG. These results indicate that the predominant mechanism of killing by this drug may operate by depletion of cellular ATP reserves.

scholarly journals Pyrazinoic Acid Inhibits the Bifunctional Enzyme (Rv2783) in Mycobacterium tuberculosis by Competing with tmRNA

Pathogens ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 230 ◽  
Author(s):  
Lei He ◽  
Peng Cui ◽  
Wanliang Shi ◽  
Qiong Li ◽  
Wenhong Zhang ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Molecular Mechanism ◽  
Messenger Rna ◽  
Mutant Allele ◽  
Active Form ◽  
Antitubercular Activity ◽  
Bifunctional Enzyme ◽  
Catalytic Activities ◽  
Insight Into ◽  
Pyrazinoic Acid

Pyrazinamide (PZA) is a key drug for tuberculosis treatment. The active form of PZA, pyrazinoic acid (POA), appears to inhibit multiple targets in M. tuberculosis. Recently, the bifunctional enzyme Rv2783 was reported as a new target of POA. However, the mechanism by which POA inhibits Rv2783 is not yet clear. Here, we report how a new A2104C substitution in Rv2783c, identified in PZA-resistant clinical isolates, conferred resistance to PZA in M. tuberculosis. Expression of the mutant allele recapitulated the PZA resistance. All catalytic activities of Rv2783, but not the mutant, were inhibited by POA. Additionally, POA competed with transfer-messenger RNA (tmRNA) for binding to Rv2783, other than the mutant. These results provide new insight into the molecular mechanism of the antitubercular activity of PZA.

scholarly journals Structure-Based Drug Design and Characterization of Sulfonyl-Piperazine Benzothiazinone Inhibitors of DprE1 from Mycobacterium tuberculosis

2018 ◽  
Vol 62 (10) ◽  
Author(s):  
Jérémie Piton ◽  
Anthony Vocat ◽  
Andréanne Lupien ◽  
Caroline S. Foo ◽  
Olga Riabova ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Design ◽  
Antitubercular Activity ◽  
Cysteine Residue ◽  
Drug Candidate ◽  
Structure Based Drug Design ◽  
Content Type ◽  
Active Site Cysteine ◽  
Covalent Adducts

ABSTRACT Macozinone (MCZ) is a tuberculosis (TB) drug candidate that specifically targets the essential flavoenzyme DprE1, thereby blocking synthesis of the cell wall precursor decaprenyl phosphoarabinose (DPA) and provoking lysis of Mycobacterium tuberculosis. As part of the MCZ backup program, we exploited structure-guided drug design to produce a new series of sulfone-containing derivatives, 2-sulfonylpiperazin 8-nitro 6-trifluoromethyl 1,3-benzothiazin-4-one, or sPBTZ. These compounds are less active than MCZ but have a better solubility profile, and some derivatives display enhanced stability in microsomal assays. DprE1 was efficiently inhibited by sPBTZ, and covalent adducts with the active-site cysteine residue (C387) were formed. However, despite the H-bonding potential of the sulfone group, no additional bonds were seen in the crystal structure of the sPBTZ-DprE1 complex with compound 11326127 compared to MCZ. Compound 11626091, the most advanced sPBTZ, displayed good antitubercular activity in the murine model of chronic TB but was less effective than MCZ. Nonetheless, further testing of this MCZ backup compound is warranted as part of combination treatment with other TB drugs.

scholarly journals Identification of Novel Efflux Proteins Rv0191, Rv3756c, Rv3008, and Rv1667c Involved in Pyrazinamide Resistance in Mycobacterium tuberculosis

2017 ◽  
Vol 61 (8) ◽  
Author(s):  
Yumeng Zhang ◽  
Jia Zhang ◽  
Peng Cui ◽  
Ying Zhang ◽  
Wenhong Zhang
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Efflux Pump ◽  
Binding Studies ◽  
Content Type ◽  
Clinical Strains ◽  
Efflux Pump Inhibitors ◽  
Level Resistance ◽  
Pyrazinoic Acid ◽  
Increased Susceptibility ◽  
Efflux Proteins

ABSTRACT Pyrazinamide (PZA) is a critical drug used for the treatment of tuberculosis (TB). PZA is a prodrug that requires conversion to the active component pyrazinoic acid (POA) by pyrazinamidase (PZase) encoded by the pncA gene. Although resistance to PZA is mostly caused by pncA mutations and less commonly by rpsA, panD, and clpC1 mutations, clinical strains without these mutations are known to exist. While efflux of POA was demonstrated in Mycobacterium tuberculosis previously, the efflux proteins involved have not been identified. Here we performed POA binding studies with an M. tuberculosis proteome microarray and identified four efflux proteins (Rv0191, Rv3756c, Rv3008, and Rv1667c) that bind POA. Overexpression of the four efflux pump genes in M. tuberculosis caused low-level resistance to PZA and POA but not to other drugs. Furthermore, addition of efflux pump inhibitors such as reserpine, piperine, and verapamil caused increased susceptibility to PZA in M. tuberculosis strains overexpressing the efflux proteins Rv0191, Rv3756c, Rv3008, and Rv1667c. Our studies indicate that these four efflux proteins may be responsible for PZA/POA efflux and cause PZA resistance in M. tuberculosis. Future studies are needed to assess their roles in PZA resistance in clinical strains.

scholarly journals Acetylation of isoniazid - a novel mechanism of isoniazid resistance in Mycobacterium tuberculosis

2020 ◽  
Author(s):  
K. B. Arun ◽  
Aravind Madhavan ◽  
Billu Abraham ◽  
M. Balaji ◽  
K. C. Sivakumar ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Isonicotinic Acid ◽  
Active Form ◽  
Drug Susceptibility ◽  
Cell Wall Biosynthesis ◽  
First Line ◽  
Isoniazid Resistance ◽  
Acetyl Group ◽  
Resistant Strains ◽  
Susceptibility Assay

AbstractIsoniazid (INH), one of the first-line drugs used for the treatment of tuberculosis, is a pro-drug which is converted into its active form by the intracellular KatG enzyme of Mycobacterium tuberculosis. The activated drug hinders cell wall biosynthesis by inhibiting InhA protein. INH resistant strains of M. tuberculosis usually have mutations in katG, inhA, ahpC, kasA, and ndh genes. However, INH resistant strains which do not have mutations in any of these genes are reported, suggesting that these strains may adopt some other mechanism to become resistant to INH. In the present study we characterized Rv2170, a putative acetyltransferase in M. tuberculosis, to elucidate its role in inactivating isoniazid. The purified recombinant protein was able to catalyze transfer of acetyl group to INH from acetyl CoA. HPLC and LC-MS analyses showed that following acetylation by Rv2170, INH is broken down into isonicotinic acid and acetylhydrazine. Drug susceptibility assay and confocal analysis showed that M. smegmatis, which is susceptible to INH, is not inhibited by INH acetylated with Rv2170. Recombinant M. smegmatis and M. tuberculosis H37Ra overexpressing Rv2170 were found to be resistant to INH at minimum inhibitory concentrations that inhibited wildtype strains. In addition, intracellular M. tuberculosis H37Ra overexpressing Rv2170 survived better in macrophages when treated with INH. Our results strongly indicate that Rv2170 acetylates INH, and this could be one of the strategies adopted by at least some M. tuberculosis strains to overcome INH toxicity.

scholarly journals Oxadiazoles Have Butyrate-Specific Conditional Activity against Mycobacterium tuberculosis

2016 ◽  
Vol 60 (6) ◽  
pp. 3608-3616 ◽  
Author(s):  
Julie V. Early ◽  
Allen Casey ◽  
Maria Angeles Martinez-Grau ◽  
Isabel C. Gonzalez Valcarcel ◽  
Michal Vieth ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Carbon Source ◽  
Mammalian Cells ◽  
Sole Carbon Source ◽  
Specific Activity ◽  
Antitubercular Activity ◽  
Content Type ◽  
Phenotypic Screen

Mycobacterium tuberculosisis a global pathogen of huge importance which can adapt to several host niche environments in which carbon source availability is likely to vary. We developed and ran a phenotypic screen using butyrate as the sole carbon source to be more reflective of the host lung environment. We screened a library of ∼87,000 small compounds and identified compounds which demonstrated good antitubercular activity againstM. tuberculosisgrown with butyrate but not with glucose as the carbon source. Among the hits, we identified an oxadiazole series (six compounds) which had specific activity againstM. tuberculosisbut which lacked cytotoxicity against mammalian cells.

scholarly journals Identification of Novel Mutations in LprG (rv1411c),rv0521,rv3630,rv0010c,ppsC, andcyp128Associated with Pyrazinoic Acid/Pyrazinamide Resistance inMycobacterium tuberculosis

2018 ◽  
Vol 62 (7) ◽  
Author(s):  
Wanliang Shi ◽  
Jiazhen Chen ◽  
Shuo Zhang ◽  
Wenhong Zhang ◽  
Ying Zhang
Keyword(s):  
Cytochrome P450 ◽  
Active Form ◽  
Mechanisms Of Action ◽  
Novel Mutations ◽  
Content Type ◽  
Pyrazinoic Acid ◽  
New Mutations

ABSTRACTDespite progress, the mechanisms of action and resistance of pyrazinamide (PZA) are not well understood. We characterized 109 mutants resistant to pyrazinoic acid (POA), the active form of PZA, and found that while most (n= 101 [93%]) mutants hadpanDmutations and 4 (4%) hadclpC1mutations (S91G), new mutations inlprG(rv1411c) andrv0521(n= 4 [4%]),rv3630,rv0010c,ppsC, andcyp128(cytochrome P450 128) were identified, shedding new light on the mechanisms of action and resistance of PZA inM. tuberculosis.

scholarly journals Metallochaperones Are Needed for Mycobacterium tuberculosis and Escherichia coli Nicotinamidase-Pyrazinamidase Activity

2019 ◽  
Vol 202 (2) ◽  
Author(s):  
Patricia Sheen ◽  
Anuntxi Monsalve ◽  
Jhanina Campos ◽  
Rodolfo Huerta ◽  
Ricardo Antiparra ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Reference Strain ◽  
Mechanisms Of Action ◽  
Efflux Rate ◽  
Content Type ◽  
Causes Of Disease ◽  
Pyrazinoic Acid

ABSTRACT Mycobacterium tuberculosis nicotinamidase-pyrazinamidase (PZAse) is a metalloenzyme that catalyzes conversion of nicotinamide-pyrazinamide to nicotinic acid-pyrazinoic acid. This study investigated whether a metallochaperone is required for optimal PZAse activity. M. tuberculosis and Escherichia coli PZAses (PZAse-MT and PZAse-EC, respectively) were inactivated by metal depletion (giving PZAse-MT–Apo and PZAse-EC–Apo). Reactivation with the E. coli metallochaperone ZnuA or Rv2059 (the M. tuberculosis analog) was measured. This was repeated following proteolytic and thermal treatment of ZnuA and Rv2059. The CDC1551 M. tuberculosis reference strain had the Rv2059 coding gene knocked out, and PZA susceptibility and the pyrazinoic acid (POA) efflux rate were measured. ZnuA (200 μM) achieved 65% PZAse-EC–Apo reactivation. Rv2059 (1 μM) and ZnuA (1 μM) achieved 69% and 34.3% PZAse-MT–Apo reactivation, respectively. Proteolytic treatment of ZnuA and Rv2059 and application of three (but not one) thermal shocks to ZnuA significantly reduced the capacity to reactivate PZAse-MT–Apo. An M. tuberculosis Rv2059 knockout strain was Wayne positive and susceptible to PZA and did not have a significantly different POA efflux rate than the reference strain, although a trend toward a lower efflux rate was observed after knockout. The metallochaperone Rv2059 restored the activity of metal-depleted PZAse in vitro. Although Rv2059 is important in vitro, it seems to have a smaller effect on PZA susceptibility in vivo. It may be important to mechanisms of action and resistance to pyrazinamide in M. tuberculosis. Further studies are needed for confirmation. IMPORTANCE Tuberculosis is an infectious disease caused by the bacterium Mycobacterium tuberculosis and remains one of the major causes of disease and death worldwide. Pyrazinamide is a key drug used in the treatment of tuberculosis, yet its mechanism of action is not fully understood, and testing strains of M. tuberculosis for pyrazinamide resistance is not easy with the tools that are presently available. The significance of the present research is that a metallochaperone-like protein may be crucial to pyrazinamide’s mechanisms of action and of resistance. This may support the development of improved tools to detect pyrazinamide resistance, which would have significant implications for the clinical management of patients with tuberculosis: drug regimens that are appropriately tailored to the resistance profile of a patient’s individual strain lead to better clinical outcomes, reduced onward transmission of infection, and reduction of the development of resistant strains that are more challenging and expensive to treat.

scholarly journals Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Brandon C. Rosen ◽  
Nicholas A. Dillon ◽  
Nicholas D. Peterson ◽  
Yusuke Minato ◽  
Anthony D. Baughn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Coenzyme A ◽  
Fatty Acyl ◽  
Wild Type ◽  
Intrinsic Resistance ◽  
First Line ◽  
Minimum Concentration ◽  
Content Type ◽  
Acyl Coenzyme A ◽  
Long Chain

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

scholarly journals Phenotypically Adapted Mycobacterium tuberculosis Populations from Sputum Are Tolerant to First-Line Drugs

2016 ◽  
Vol 60 (4) ◽  
pp. 2476-2483 ◽  
Author(s):  
Obolbek Turapov ◽  
Benjamin D. O'Connor ◽  
Asel A. Sarybaeva ◽  
Caroline Williams ◽  
Hemu Patel ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Liquid Medium ◽  
Culture Supernatant ◽  
Ex Vivo ◽  
First Line ◽  
Content Type ◽  
Host Environment ◽  
Solid Media ◽  
Bactericidal Effects

ABSTRACTTuberculous sputum contains multipleMycobacterium tuberculosispopulations with different requirements for isolationin vitro. These include cells that form colonies on solid media (plateableM. tuberculosis), cells requiring standard liquid medium for growth (nonplateableM. tuberculosis), and cells requiring supplementation of liquid medium with culture supernatant (SN) for growth (SN-dependentM. tuberculosis). Here, we describe protocols for the cryopreservation and direct assessment of antimicrobial tolerance of theseM. tuberculosispopulations within sputum. Our results show that first-line drugs achieved only modest bactericidal effects on all three populations over 7 days (1 to 2.5 log10reductions), and SN-dependentM. tuberculosiswas more tolerant to streptomycin and isoniazid than the plateable and nonplateableM. tuberculosisstrains. Susceptibility of plateableM. tuberculosisto bactericidal drugs was significantly increased after passagein vitro; thus, tolerance observed in the sputum samples from the population groups was likely associated with mycobacterial adaptation to the host environment at some time prior to expectoration. Our findings support the use of a simpleex vivosystem for testing drug efficacies against mycobacteria that have phenotypically adapted during tuberculosis infection.

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