Substrate specificity of the deazaflavin‐dependent nitroreductase from Mycobacterium tuberculosis responsible for the bioreductive activation of bicyclic nitroimidazoles
Open Access
- 24 October 2011
- journal article
- Published by Wiley in The FEBS Journal
- Vol. 279 (1), 113-125
- https://doi.org/10.1111/j.1742-4658.2011.08404.x
Abstract
The bicyclic 4‐nitroimidazoles PA‐824 and OPC‐67683 represent a promising novel class of therapeutics for tuberculosis and are currently in phase II clinical development. Both compounds are pro‐drugs that are reductively activated by a deazaflavin (F420) dependent nitroreductase (Ddn). Herein we describe the biochemical properties of Ddn including the optimal enzymatic turnover conditions and substrate specificity. The preference of the enzyme for the (S) isomer of PA‐824 over the (R) isomer is directed by the presence of a long hydrophobic tail. Nitroimidazo‐oxazoles bearing only short alkyl substituents at the C‐7 position of the oxazole were reduced by Ddn without any stereochemical preference. However, with bulkier substitutions on the tail of the oxazole, Ddn displayed stereospecificity. Ddn mediated metabolism of PA‐824 results in the release of reactive nitrogen species. We have employed a direct chemiluminescence based nitric oxide (NO) detection assay to measure the kinetics of NO production by Ddn. Binding affinity of PA‐824 to Ddn was monitored through intrinsic fluorescence quenching of the protein facilitating a turnover‐independent assessment of affinity. Our results indicate that (R)‐PA‐824, despite not being turned over by Ddn, binds to the enzyme with the same affinity as the active (S) isomer. This result, in combination with docking studies in the active site, suggests that the (R) isomer probably has a different binding mode than the (S) with the C‐3 of the imidazole ring orienting in a non‐productive position with respect to the incoming hydride from F420. The results presented provide insight into the biochemical mechanism of reduction and elucidate structural features important for understanding substrate binding.Keywords
This publication has 42 references indexed in Scilit:
- Unexpected Abundance of Coenzyme F 420 -Dependent Enzymes in Mycobacterium tuberculosis and Other ActinobacteriaJournal of Bacteriology, 2010
- Identification and characterization of two families of F420H2‐dependent reductases from Mycobacteria that catalyse aflatoxin degradationMolecular Microbiology, 2010
- Nitroaromatic Compounds, from Synthesis to BiodegradationMicrobiology and Molecular Biology Reviews, 2010
- Conversion of NO 2 to NO by reduced coenzyme F 420 protects mycobacteria from nitrosative damageProceedings of the National Academy of Sciences, 2009
- Structure−Activity Relationships of Antitubercular Nitroimidazoles. 2. Determinants of Aerobic Activity and Quantitative Structure−Activity RelationshipsJournal of Medicinal Chemistry, 2009
- Structure−Activity Relationships of Antitubercular Nitroimidazoles. 1. Structural Features Associated with Aerobic and Anaerobic Activities of 4- and 5-NitroimidazolesJournal of Medicinal Chemistry, 2009
- PA-824 Kills Nonreplicating Mycobacterium tuberculosis by Intracellular NO ReleaseScience, 2008
- Tuberculous Granulomas Are Hypoxic in Guinea Pigs, Rabbits, and Nonhuman PrimatesInfection and Immunity, 2008
- Synthesis and antitubercular activity of 7-(R)- and 7-(S)-methyl-2-nitro-6-(S)-(4-(trifluoromethoxy)benzyloxy)-6,7-dihydro-5H-imidazo[2,1-b][1,3]oxazines, analogues of PA-824Bioorganic & Medicinal Chemistry Letters, 2008
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976