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Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid

Abstract

Isoniazid is a key drug used in the treatment of tuberculosis. Isoniazid is a pro-drug, which, after activation by the katG-encoded catalase peroxidase, reacts nonenzymatically with NAD+ and NADP+ to generate several isonicotinoyl adducts of these pyridine nucleotides. One of these, the acyclic 4S isomer of isoniazid-NAD, targets the inhA-encoded enoyl-ACP reductase, an enzyme essential for mycolic acid biosynthesis in Mycobacterium tuberculosis. Here we show that the acyclic 4R isomer of isoniazid-NADP inhibits the M. tuberculosis dihydrofolate reductase (DHFR), an enzyme essential for nucleic acid synthesis. This biologically relevant form of the isoniazid adduct is a subnanomolar bisubstrate inhibitor of M. tuberculosis DHFR. Expression of M. tuberculosis DHFR in Mycobacterium smegmatis mc2155 protects cells against growth inhibition by isoniazid by sequestering the drug. Thus, M. tuberculosis DHFR is the first new target for isoniazid identified in the last decade.

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Figure 1: Chemical structures of the mixture of isoniazid-NAD or isoniazid-NADP adducts.
Figure 2: Subnanomolar inhibition of M.tuberculosis DHFR by the bisubstrate analog, isoniazid-NADP.
Figure 3: Crystal structure of M.tuberculosis DHFR complexed with isoniazid-NADP.
Figure 4: Expression of M.tuberculosis DHFR in M. smegmatis mc2155 confers a two-fold increase in resistance to isoniazid.

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Acknowledgements

We thank W.R. Jacobs, Jr. (Albert Einstein College of Medicine) for providing plasmid pSD26 and valuable discussions, A. Bhatt for assistance with the MIC experiments and P.F. Cook for valuable discussions concerning bisubstrate inhibitors. This work was supported by a US National Institutes of Health grant to J.S.B. (AI33696).

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Correspondence to John S Blanchard.

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Supplementary Fig. 1

Crystal structures of dihydrofolate reductase from various organisms. (PDF 384 kb)

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Argyrou, A., Vetting, M., Aladegbami, B. et al. Mycobacterium tuberculosis dihydrofolate reductase is a target for isoniazid. Nat Struct Mol Biol 13, 408–413 (2006). https://doi.org/10.1038/nsmb1089

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