Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis

Sarah M. Batt; Szilvi Toth; Beatriz Rodriguez; Katherine A. Abrahams; Natacha Veerapen; Giacomo Chiodarelli; Liam R. Cox; Patrick J. Moynihan; Joel Lelievre; Klaus Fütterer; Gurdyal S. Besra

doi:10.1099/mic.0.001288

Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis

Sarah M. Batt, Szilvi Toth, Beatriz Rodriguez, Katherine A. Abrahams, Natacha Veerapen, Giacomo Chiodarelli, Liam R. Cox, Patrick J. Moynihan, Joel Lelievre, Klaus Fütterer, Gurdyal S. Besra

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Abstract

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

Original language	English
Article number	001288
Journal	Microbiology
Volume	169
Issue number	1
DOIs	https://doi.org/10.1099/mic.0.001288
Publication status	Published - 23 Jan 2023

Keywords

decaprenylphosphoryl-D-arabinose
enzyme kinetics
Mt-DprE1
Mt-DprE2
mycobacterial cell wall
Mycobacterium tuberculosis and substrate inhibition

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1099/mic.0.001288Licence: Creative Commons: Attribution (CC BY)

BattSM2023AssayFinal published version, 3.09 MBLicence: Creative Commons: Attribution (CC BY)

https://www.microbiologyresearch.org/content/journal/micro/10.1099/mic.0.001288

1 Finished
1 Active

The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways
Besra, D., Bhatt, A., Futterer, K., Alderwick, L. & Zhang, J.
Medical Research Council
1/03/19 → 28/02/25
Project: Research Councils
MICA: Addressing the burgeoning problem of tuberculosis: Exploiting phenotypic hits to identify new protein targets for drug discovery
Besra, D., Cox, L. & Futterer, K.
Medical Research Council
1/04/18 → 31/03/22
Project: Research Councils

Cite this

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title = "Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis",

abstract = "DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.",

keywords = "decaprenylphosphoryl-D-arabinose, enzyme kinetics, Mt-DprE1, Mt-DprE2, mycobacterial cell wall, Mycobacterium tuberculosis and substrate inhibition",

author = "Batt, {Sarah M.} and Szilvi Toth and Beatriz Rodriguez and Abrahams, {Katherine A.} and Natacha Veerapen and Giacomo Chiodarelli and Cox, {Liam R.} and Moynihan, {Patrick J.} and Joel Lelievre and Klaus F{\"u}tterer and Besra, {Gurdyal S.}",

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AU - Batt, Sarah M.

AU - Toth, Szilvi

AU - Rodriguez, Beatriz

AU - Abrahams, Katherine A.

AU - Veerapen, Natacha

AU - Chiodarelli, Giacomo

AU - Cox, Liam R.

AU - Moynihan, Patrick J.

AU - Lelievre, Joel

AU - Fütterer, Klaus

AU - Besra, Gurdyal S.

PY - 2023/1/23

Y1 - 2023/1/23

N2 - DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

AB - DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis . Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

KW - decaprenylphosphoryl-D-arabinose

KW - enzyme kinetics

KW - Mt-DprE1

KW - Mt-DprE2

KW - mycobacterial cell wall

KW - Mycobacterium tuberculosis and substrate inhibition

U2 - 10.1099/mic.0.001288

DO - 10.1099/mic.0.001288

M3 - Article

SN - 1350-0872

VL - 169

JO - Microbiology

JF - Microbiology

IS - 1

M1 - 001288

ER -

Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis

Abstract

Keywords

UN SDGs

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Projects

The Mycobacterium tuberculosis Cell Envelope: unravelling complex cell wall assembly, degradation and re-cycling pathways

MICA: Addressing the burgeoning problem of tuberculosis: Exploiting phenotypic hits to identify new protein targets for drug discovery

Cite this