Validation of Drug-Like Inhibitors against Mycobacterium Tuberculosis L-Aspartate α-Decarboxylase Using Nuclear Magnetic Resonance (1H NMR)

Sharma, Reetu; Florea, Mara; Nau, Werner M.; Swaminathan, Kunchithapadam

doi:10.1371/journal.pone.0045947

Cited by 11 publications

(13 citation statements)

References 25 publications

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“…The structure was very similar to the wild-type protein structure with a root-mean-square difference in the cα positions of 0.32 Å. The crystallization condition contained tartrate, a known inhibitor of the enzyme 28 . A molecule of tartrate fit well into electron density in the active site that could not be accounted for by the protein.…”

Section: Resultsmentioning

confidence: 81%

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

Sun

Pérez

et al. 2020

Nat Commun

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Pyrazinamide has been a mainstay in the multidrug regimens used to treat tuberculosis. It is active against the persistent, non-replicating mycobacteria responsible for the protracted therapy required to cure tuberculosis. Pyrazinamide is a pro-drug that is converted into pyrazinoic acid (POA) by pyrazinamidase, however, the exact target of the drug has been difficult to determine. Here we show the enzyme PanD binds POA in its active site in a manner consistent with competitive inhibition. The active site is not directly accessible to the inhibitor, suggesting the protein must undergo a conformational change to bind the inhibitor. This is consistent with the slow binding kinetics we determined for POA. Drug-resistant mutations cluster near loops that lay on top of the active site. These resistant mutants show reduced affinity and residence time of POA consistent with a model where resistance occurs by destabilizing the closed conformation of the active site.

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Section: Resultsmentioning

confidence: 81%

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

Sun

Pérez

et al. 2020

Nat Commun

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“…400 MHz NMR spectrometer, equipped on a 5 mm BBI probe head at 298 K as described by Sharma et al 26 . Basic 1 H-1D NMR spectra were collected with presaturation of solvent water (relaxation delay 2 s; solvent presaturation applied during the relaxation delay).…”

Section: Sds-mentioning

confidence: 99%

Pyrazinamide triggers degradation of its target aspartate decarboxylase

et al. 2020

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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.

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“…NMR samples containing 2 mM L-Asp, 10 µM of PanDWT or PanD127TRASC131 were prepared in deuterated water (D2O) buffer. NMR experiments were carried out using a Bruker Avance 400 MHz NMR spectrometer, equipped on a 5 mm BBI probe head at 298 K as described by Sharma et al 26 . Basic 1 H-1D NMR spectra were collected with pre-saturation of solvent water (relaxation delay 2 s; solvent pre-saturation applied during the relaxation delay).…”

Section: H-1d Nmr Studies Of L-asp To β-Ala Conversionmentioning

confidence: 99%

Pyrazinamide triggers degradation of its target aspartate decarboxylase

Gopal

Sarathy

Yee

et al. 2019

Preprint

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AbstractThe introduction of pyrazinamide (PZA) in the tuberculosis drug regimen shortened treatment from 12 to 6 months 1. PZA is a prodrug that is activated by a Mycobacterium tuberculosis (Mtb) amidase to release its bioactive component pyrazinoic acid (POA) 2. Aspartate decarboxylase PanD, a proenzyme activated by autocatalytic cleavage (Supplementary Fig. 1A, 3) and required for Coenzyme A (CoA) biosynthesis, emerged as a target of POA 4-7. In vitro and in vivo screening to isolate spontaneous POA-resistant Mtb mutants identified missense mutations in either panD or the unfoldase clpC1, encoding a component of the caseinolytic protease ClpC1-ClpP 4,6-9. Overexpression and binding studies of PanD or ClpC1 pointed to PanD as the direct target of POA whereas clpC1 mutations appeared to indirectly cause resistance 4,5,7,9,10. Indeed, supplementing growth media with CoA precursors downstream of the PanD catalyzed step conferred POA resistance 4,7,11. Metabolomic analyses and biophysical studies using recombinant proteins confirmed targeting of PanD by POA 5. However, the exact molecular mechanism of PanD inhibition by POA remained unknown. While most drugs act by inhibiting protein function upon target binding, we show here that POA is not a bona fide enzyme inhibitor. Rather, POA binding to PanD triggers degradation of the protein by ClpC1-ClpP. Thus, the old tuberculosis drug PZA promotes degradation of its target. While novel for an antibacterial, drug-induced target degradation has recently emerged as a strategy in drug discovery across disease indications. Our findings provide the basis for the rational discovery of next generation PZA.

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Validation of Drug-Like Inhibitors against Mycobacterium Tuberculosis L-Aspartate α-Decarboxylase Using Nuclear Magnetic Resonance (1H NMR)

Cited by 11 publications

References 25 publications

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

The molecular basis of pyrazinamide activity on Mycobacterium tuberculosis PanD

Pyrazinamide triggers degradation of its target aspartate decarboxylase

Pyrazinamide triggers degradation of its target aspartate decarboxylase

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