Novel insight into the reaction of nitro, nitroso and hydroxylamino benzothiazinones and of benzoxacinones with Mycobacterium tuberculosis DprE1

Robert Bates,Sarah Batt,Alfonso Mendoza-Losana,Gurdyal Besra,Lluís Ballell,Natacha Veerapen,Ute Möllmann,Michael Rees,Adrian Richter,Kerstin Voigt,Peter Imming,Chun-wa Chung,Ines Rudolph,Klaus Fütterer,Onkar M. P. Singh,Argyrides Argyrou

doi:10.1038/s41598-018-31316-6

Abstract

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. We prepared a number of oxidized and reduced forms of nitro-BTZs to probe the mechanism of inactivation of the enzyme and to identify opportunities for further chemistry. The kinetics of inactivation of DprE1 was examined using an enzymatic assay that monitored reaction progress up to 100 min, permitting compound ranking according to kinact/Ki values. The side-chain at the 2-position and heteroatom identity at the 1-position of the BTZs were found to be important for inhibitory activity. We obtained crystal structures with several compounds covalently bound. The data suggest that steps upstream from the covalent end-points are likely the key determinants of potency and reactivity. The results of protein mass spectrometry using a 7-chloro-nitro-BTZ suggest that nucleophilic reactions at the 7-position do not operate and support a previously proposed mechanism in which BTZ activation by a reduced flavin intermediate is required. Unexpectedly, a hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1. With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme.

Highlights

Nitro-substituted 1,3-benzothiazinones are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties
After BTZs have entered the catalytic pocket of DprE1 containing the reduced form of the flavin cofactor (FADH2; see Fig. 1), which was generated by oxidation of the substrate, the nitro group of the BTZs is reduced to nitroso which can form a covalent bond with a nearby cysteine residue (Cys[387], Mycobacterium tuberculosis (Mtb) enzyme numbering)[6,7]
This was accomplished by the use of asymmetrically substituted thiourea reagents

Summary

Introduction

Nitro-substituted 1,3-benzothiazinones (nitro-BTZs) are mechanism-based covalent inhibitors of Mycobacterium tuberculosis decaprenylphosphoryl-β-D-ribose-2′-oxidase (DprE1) with strong antimycobacterial properties. A hydroxylamino-BTZ showed time-dependent inhibition and mass spectrometry corroborated that this hydroxylamino-BTZ is a mechanism-based suicide inhibitor of DprE1 With this BTZ derivative, we propose a new covalent mechanism of inhibition of DprE1 that takes advantage of the oxidation cycle of the enzyme. BTZs can covalently inhibit DprE1 only in the presence of its natural substrate or close analogues, e.g. farnesylphosphoryl-β-D-ribofuranose (FPR) that is often used in enzymatic assays because reductive activation of the nitro to the nitroso form of the BTZs is mediated by enzyme-bound FADH29,10. We synthesized a novel BTZ, two nitroso- and one hydroxylamino-containing compound (see Results) in order to further probe the mechanism of inactivation of DprE1 through the combined used of enzyme inhibition kinetics, mass spectrometry and X-ray crystallography

Methods

Results

Conclusion