The Experts below are selected from a list of 270 Experts worldwide ranked by ideXlab platform
John T Welch - One of the best experts on this subject based on the ideXlab platform.
-
Efficacy of Pyrazinoic Acid dry powder aerosols in resolving necrotic and non-necrotic granulomas in a guinea pig model of tuberculosis.
PloS one, 2018Co-Authors: Stephanie A. Montgomery, John T Welch, Jennifer D. Hayden, Phillip Durham, Ellen F. Young, Katelyn E. Zulauf, Laura Rank, Brittany K. Miller, Feng-chang Lin, Anthony J. HickeyAbstract:New therapeutic strategies are needed to treat drug resistant tuberculosis (TB) and to improve treatment for drug sensitive TB. Pyrazinamide (PZA) is a critical component of current first-line TB therapy. However, the rise in PZA-resistant TB cases jeopardizes the future utility of PZA. To address this problem, we used the guinea pig model of TB and tested the efficacy of an inhaled dry powder combination, referred to as Pyrazinoic Acid/ester Dry Powder (PDP), which is comprised of Pyrazinoic Acid (POA), the active moiety of PZA, and Pyrazinoic Acid ester (PAE), which is a PZA analog. Both POA and PAE have the advantage of being able to act on PZA-resistant Mycobacterium tuberculosis. When used in combination with oral rifampicin (R), inhaled PDP had striking effects on tissue pathology. Effects were observed in lungs, the site of delivery, but also in the spleen and liver indicating both local and systemic effects of inhaled PDP. Tissue granulomas that harbor M. tuberculosis in a persistent state are a hallmark of TB and they pose a challenge for therapy. Compared to other treatments, which preferentially cleared non-necrotic granulomas, R+PDP reduced necrotic granulomas more effectively. The increased ability of R+PDP to act on more recalcitrant necrotic granulomas suggests a novel mechanism of action. The results presented in this report reveal the potential for developing therapies involving POA that are optimized to target necrotic as well as non-necrotic granulomas as a means of achieving more complete sterilization of M. tuberculosis bacilli and preventing disease relapse when therapy ends.
-
Inhaled Pyrazinoic Acid Esters for the Treatment of Tuberculosis.
Pharmaceutical research, 2016Co-Authors: Ellen Young, John T Welch, Ellen F. Perkowski, Seidu Malik, Jennifer D. Hayden, Phillip Durham, L. Zhong, Miriam Braunstein, Anthony J. HickeyAbstract:Purpose Analog development of existing drugs and direct drug delivery to the lungs by inhalation as treatments for multiple and extensively drug resistant (MDR and XDR) tuberculosis (TB) represent new therapeutic strategies. Pyrazinamide (PZA) is critical to drug sensitive TB therapy and is included in regimens for MDR TB. However, PZA-resistant Mycobacterium tuberculosis (Mtb) strains threaten its use. Pyrazinoic Acid esters (PAEs) are PZA analogs effective against Mtb in vitro, including against the most common PZA resistant strains. However, PAEs require testing for TB efficacy in animal models.
-
A dry powder combination of Pyrazinoic Acid and its n-propyl ester for aerosol administration to animals.
International journal of pharmaceutics, 2016Co-Authors: Phillip Durham, John T Welch, Ellen Young, Miriam Braunstein, Anthony J. HickeyAbstract:Combining the advantage of higher efficacy due to local pulmonary administration of Pyrazinoic Acid (POA) and potent effect of Pyrazinoic Acid ester (PAE) delivered as an aerosol would aid in tuberculosis therapy. A combination spray dried dry powder, composed of POA, PAE (n-propyl POA), maltodextrin and leucine, was prepared for aerosol delivery to animals. Solid-state characteristics of morphology (scanning electron microscopy) crystallinity (X-ray powder diffraction), thermal properties (thermogravimetric analysis and differential scanning calorimetry) and moisture content (Karl Fisher) were evaluated. Particle size distributions, by volume (laser diffraction) for the dispersed powder and by mass (inertial impaction) were determined. Efficient delivery of the powder to a nose only animal exposure chamber employed a novel rotating brush/micro-fan apparatus. Spherical, crystalline particles were prepared. The volume median diameter, ∼1.5μm, was smaller than the mass median aerodynamic diameter, ∼3.0μm, indicating modest aggregation. Drug content variations were observed across the particle size distribution and may be explained by PAE evaporative losses. Delivery to the nose-only exposure chamber indicated that boluses could be administered at approximately 3min intervals to avoid aerosol accumulation and effect uniform dose delivery with successive doses suitable for future pharmacokinetic and pharmacodynamic studies.
-
pyrazinamide but not Pyrazinoic Acid is a competitive inhibitor of nadph binding to mycobacterium tuberculosis fatty Acid synthase i
Bioorganic & Medicinal Chemistry Letters, 2011Co-Authors: Halimah Sayahi, Oren Zimhony, William R Jacobs, Alexander Shekhtman, John T WelchAbstract:Pyrazinamide (PZA), an essential component of short-course anti-tuberculosis chemotherapy, was shown by Saturation Transfer Difference (STD) NMR methods to act as a competitive inhibitor of NADPH binding to purified Mycobacterium tuberculosis fatty Acid synthase I (FAS I). Both PZA and Pyrazinoic Acid (POA) reversibly bind to FAS I but at different binding sites. The competitive binding of PZA and NADPH suggests potential FAS I binding sites. POA was not previously known to have any specific binding interactions. The STD NMR of NADPH bound to the mycobacterial FAS I was consistent with the orientation reported in published single crystal X-ray diffraction studies of fungal FAS I. Overall the differences in binding between PZA and POA are consistent with previous recognition of the importance of intracellular accumulation of POA for anti-mycobacterial activity.
-
Pyrazinoic Acid and its n propyl ester inhibit fatty Acid synthase type i in replicating tubercle bacilli
Antimicrobial Agents and Chemotherapy, 2007Co-Authors: Oren Zimhony, John T Welch, Catherine Vilcheze, Masayoshi Arai, William R JacobsAbstract:The activity of different analogs of pyrazinamide on Mycobacterium tuberculosis fatty Acid synthase type I (FASI) in replicating bacilli was studied. Palmitic Acid biosynthesis was diminished by 96% in bacilli treated with n-propyl pyrazinoate, 94% in bacilli treated with 5-chloro-pyrazinamide, and 97% in bacilli treated with Pyrazinoic Acid, the pharmacologically active agent of pyrazinamide. We conclude that the minimal structure of pyrazine ring with an acyl group is sufficient for FASI inhibition and antimycobacterial activity.
Thomas Dick - One of the best experts on this subject based on the ideXlab platform.
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequen...
-
Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Michelle Yee, Pooja Gopal, Thomas DickAbstract:ABSTRACT Previously, we showed that mutations in Mycobacterium tuberculosis panD , involved in coenzyme A biosynthesis, cause resistance against Pyrazinoic Acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against Pyrazinoic Acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined
Pooja Gopal - One of the best experts on this subject based on the ideXlab platform.
-
Pyrazinamide triggers degradation of its target aspartate decarboxylase
Nature communications, 2020Co-Authors: Pooja Gopal, Priya Ragunathan, Michelle Yee, Jickky Palmae Sarathy, Joon Shin, Shashi Bhushan, Junhao Zhu, Tatos Akopian, Olga Kandror, Teck Kwang LimAbstract:Pyrazinamide is a sterilizing first-line tuberculosis drug. Genetic, metabolomic and biophysical analyses previously demonstrated that Pyrazinoic Acid, the bioactive form of the prodrug pyrazinamide (PZA), interrupts biosynthesis of coenzyme A in Mycobacterium tuberculosis by binding to aspartate decarboxylase PanD. While most drugs act by inhibiting protein function upon target binding, we find here that Pyrazinoic Acid is only a weak enzyme inhibitor. We show that binding of Pyrazinoic Acid to PanD triggers degradation of the protein by the caseinolytic protease ClpC1-ClpP. Thus, the old tuberculosis drug pyrazinamide exerts antibacterial activity by acting as a target degrader, a mechanism of action that has recently emerged as a successful strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA. It has been shown that the bioactive component of pyrazinamide, Pyrazinoic Acid (POA), blocks coenzyme A biosynthesis in M. tuberculosis by binding to the aspartate decarboxylase PanD. Here the authors show that pyrazinamide triggers degradation of PanD by stimulating its degradation by the caseinolytic protease Clp.
-
Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jansy Sarathy, Veronique Dartois, Wilson Nartey, Priya Ragunathan, Firat Kaya, Michelle Yee, Claudia Setzer, Malathy Sony Subramanian Manimekalai, Gerhard GrüberAbstract:Previously, we showed that a major in vitro and in vivo mechanism of resistance to Pyrazinoic Acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Bi...
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequen...
-
Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Michelle Yee, Pooja Gopal, Thomas DickAbstract:ABSTRACT Previously, we showed that mutations in Mycobacterium tuberculosis panD , involved in coenzyme A biosynthesis, cause resistance against Pyrazinoic Acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against Pyrazinoic Acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.
-
Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD
2017Co-Authors: Pooja Gopal, Jansy Sarathy, Veronique Dartois, Wilson Nartey, Priya Ragunathan, Firat Kaya, Michelle Yee, Claudia Setzer, Malathy Sony Subramanian Manimekalai, Gerhard GrüberAbstract:Previously, we showed that a major in vitro and in vivo mechanism of resistance to Pyrazinoic Acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Binding was abolished for mutant PanD proteins. Taken together, these findings support a mechanism of action of POA in which the bioactive component of PZA inhibits coenzyme A biosynthesis via binding to aspartate decarboxylase PanD. Together with previous works, these results establish PanD as a genetically, metabolically, and biophysically validated target of PZA
Eric L Nuermberger - One of the best experts on this subject based on the ideXlab platform.
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequen...
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequencing revealed that 82 and 18% of the strains contained missense mutations in panD and clpC1, respectively. Consistent with their lower fitness and POA resistance level, independent mas/ppsA-E mutants were not found. In conclusion, we show that the POA/PZA resistance mechanisms due to panD and clpC1 missense mutations are recapitulated in vivo. Whereas the representative clpC1 mutant was attenuated for growth in the mouse infection model, providing a possible explanation for their absence among clinical isolates, the growth kinetics of the representative panD mutant was unaffected. Why POA/PZA resistance-conferring panD mutations are observed in POA-treated mice but not yet among clinical strains isolated from PZA-treated patients remains to be determined
-
high systemic exposure of Pyrazinoic Acid has limited antituberculosis activity in murine and rabbit models of tuberculosis
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Jean Philippe Lanoix, Rokeya Tasneen, Paul Obrien, Jansy Sarathy, Hassan Safi, Michael L Pinn, David Alland, Veronique Dartois, Eric L NuermbergerAbstract:: Pyrazinamide (PZA) is a prodrug requiring conversion to Pyrazinoic Acid (POA) by an amidase encoded by pncA for in vitro activity. Mutation of pncA is the most common cause of PZA resistance in clinical isolates. To determine whether the systemic delivery of POA or host-mediated conversion of PZA to POA could circumvent such resistance, we evaluated the efficacy of orally administered and host-derived POA in vivo Dose-ranging plasma and intrapulmonary POA pharmacokinetics and the efficacy of oral POA or PZA treatment against PZA-susceptible tuberculosis were determined in BALB/c and C3HeB/FeJ mice. The activity of host-derived POA was assessed in rabbits infected with a pncA-null mutant and treated with PZA. Median plasma POA values for the area under the concentration-time curve from 0 h to infinity (AUC0-∞) were 139 to 222 μg·h/ml and 178 to 287 μg·h/ml after doses of PZA and POA of 150 mg/kg of body weight, respectively, in mice. Epithelial lining fluid POA concentrations in infected mice were comparable after POA and PZA administration. In chronically infected BALB/c mice, PZA at 150 mg/kg reduced lung CFU counts by >2 log10 after 4 weeks. POA was effective only at 450 mg/kg, which reduced lung CFU counts by ∼0.7 log10 POA had no demonstrable bactericidal activity in C3HeB/FeJ mice, nor did PZA administered to rabbits infected with a PZA-resistant mutant. Oral POA administration and host-mediated conversion of PZA to POA producing plasma POA exposures comparable to PZA administration was significantly less effective than PZA. These results suggest that the intrabacillary delivery of POA and that producing higher POA concentrations at the site of infection will be more effective strategies for maximizing POA efficacy.
Michelle Yee - One of the best experts on this subject based on the ideXlab platform.
-
Pyrazinamide triggers degradation of its target aspartate decarboxylase
Nature communications, 2020Co-Authors: Pooja Gopal, Priya Ragunathan, Michelle Yee, Jickky Palmae Sarathy, Joon Shin, Shashi Bhushan, Junhao Zhu, Tatos Akopian, Olga Kandror, Teck Kwang LimAbstract:Pyrazinamide is a sterilizing first-line tuberculosis drug. Genetic, metabolomic and biophysical analyses previously demonstrated that Pyrazinoic Acid, the bioactive form of the prodrug pyrazinamide (PZA), interrupts biosynthesis of coenzyme A in Mycobacterium tuberculosis by binding to aspartate decarboxylase PanD. While most drugs act by inhibiting protein function upon target binding, we find here that Pyrazinoic Acid is only a weak enzyme inhibitor. We show that binding of Pyrazinoic Acid to PanD triggers degradation of the protein by the caseinolytic protease ClpC1-ClpP. Thus, the old tuberculosis drug pyrazinamide exerts antibacterial activity by acting as a target degrader, a mechanism of action that has recently emerged as a successful strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA. It has been shown that the bioactive component of pyrazinamide, Pyrazinoic Acid (POA), blocks coenzyme A biosynthesis in M. tuberculosis by binding to the aspartate decarboxylase PanD. Here the authors show that pyrazinamide triggers degradation of PanD by stimulating its degradation by the caseinolytic protease Clp.
-
Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jansy Sarathy, Veronique Dartois, Wilson Nartey, Priya Ragunathan, Firat Kaya, Michelle Yee, Claudia Setzer, Malathy Sony Subramanian Manimekalai, Gerhard GrüberAbstract:Previously, we showed that a major in vitro and in vivo mechanism of resistance to Pyrazinoic Acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Bi...
-
In Vivo-Selected Pyrazinoic Acid-Resistant Mycobacterium tuberculosis Strains Harbor Missense Mutations in the Aspartate Decarboxylase PanD and the Unfoldase ClpC1
ACS infectious diseases, 2017Co-Authors: Pooja Gopal, Jean Philippe Lanoix, Rokeya Tasneen, Jansy Sarathy, Veronique Dartois, Eric L Nuermberger, Michelle Yee, George Rasic, Thomas DickAbstract:Through mutant selection on agar containing Pyrazinoic Acid (POA), the bioactive form of the prodrug pyrazinamide (PZA), we recently showed that missense mutations in the aspartate decarboxylase PanD and the unfoldase ClpC1, and loss-of-function mutation of polyketide synthases Mas and PpsA-E involved in phthiocerol dimycocerosate synthesis, cause resistance to POA and PZA in Mycobacterium tuberculosis. Here we first asked whether these in vitro-selected POA/PZA-resistant mutants are attenuated in vivo, to potentially explain the lack of evidence of these mutations among PZA-resistant clinical isolates. Infection of mice with panD, clpC1, and mas/ppsA-E mutants showed that whereas growth of clpC1 and mas/ppsA-E mutants was attenuated, the panD mutant grew as well as the wild-type. To determine whether these resistance mechanisms can emerge within the host, mice infected with wild-type M. tuberculosis were treated with POA, and POA-resistant colonies were confirmed for PZA and POA resistance. Genome sequen...
-
Missense Mutations in the Unfoldase ClpC1 of the Caseinolytic Protease Complex Are Associated with Pyrazinamide Resistance in Mycobacterium tuberculosis.
Antimicrobial agents and chemotherapy, 2017Co-Authors: Michelle Yee, Pooja Gopal, Thomas DickAbstract:ABSTRACT Previously, we showed that mutations in Mycobacterium tuberculosis panD , involved in coenzyme A biosynthesis, cause resistance against Pyrazinoic Acid, the bioactive component of the prodrug pyrazinamide. To identify additional resistance mechanisms, we isolated mutants resistant against Pyrazinoic Acid and subjected panD wild-type strains to whole-genome sequencing. Eight of the nine resistant strains harbored missense mutations in the unfoldase ClpC1 associated with the caseinolytic protease complex.
-
Pyrazinoic Acid Inhibits Mycobacterial Coenzyme A Biosynthesis by Binding to Aspartate Decarboxylase PanD
2017Co-Authors: Pooja Gopal, Jansy Sarathy, Veronique Dartois, Wilson Nartey, Priya Ragunathan, Firat Kaya, Michelle Yee, Claudia Setzer, Malathy Sony Subramanian Manimekalai, Gerhard GrüberAbstract:Previously, we showed that a major in vitro and in vivo mechanism of resistance to Pyrazinoic Acid (POA), the bioactive component of the critical tuberculosis (TB) prodrug pyrazinamide (PZA), involves missense mutations in the aspartate decarboxylase PanD, an enzyme required for coenzyme A biosynthesis. What is the mechanism of action of POA? Upon demonstrating that treatment of M. bovis BCG with POA resulted in a depletion of intracellular coenzyme A and confirming that this POA-mediated depletion is prevented by either missense mutations in PanD or exogenous supplementation of pantothenate, we hypothesized that POA binds to PanD and that this binding blocks the biosynthetic pathway. Here, we confirm both hypotheses. First, metabolomic analyses showed that POA treatment resulted in a reduction of the concentrations of all coenzyme A precursors downstream of the PanD-mediated catalytic step. Second, using isothermal titration calorimetry, we established that POA, but not its prodrug PZA, binds to PanD. Binding was abolished for mutant PanD proteins. Taken together, these findings support a mechanism of action of POA in which the bioactive component of PZA inhibits coenzyme A biosynthesis via binding to aspartate decarboxylase PanD. Together with previous works, these results establish PanD as a genetically, metabolically, and biophysically validated target of PZA
