S-Adenosyl-N-decyl-aminoethyl, a Potent Bisubstrate Inhibitor of Mycobacterium tuberculosis Mycolic Acid Methyltransferases

Julien Vaubourgeix,Fabienne Bardou,Fanny Boissier,Sylviane Julien,Patricia Constant,Olivier Ploux,Mamadou Daffé,Annaïk Quémard,Lionel Mourey

doi:10.1074/jbc.m809599200

Abstract

S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC(50) values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.

Highlights

One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s
Mixtures were first incubated in the presence or absence of AdoMet analogs (i.e. SADAE (10 ␮M), dissolved in dimethyl sulfoxide (DMSO) such that the maximum final solvent concentration in the reaction medium was 5% (v/v); AdoHcy (1 mM) and sinefungin (3 mM), evaluated either with or without DMSO) for 15 min at 37 °C, and we added 50 ␮M of either [1-14C]acetate for mycolic acids (MAs) biosynthesis assays or [CH314C]AdoMet for mycolic acid methyltransferases (MA-MTs) activity assays
The neosynthesized and radiolabeled MAs were methylated and analyzed by TLC on Silica Gel 60 (Macherey-Nagel) run in dichloromethane for M. smegmatis strains and in petroleum ether/ diethyl ether (9:1) for M. tuberculosis [18], followed by phosphorimaging (Variable Mode Imager Typhoon TRIO, Amersham Biosciences). ␣-MAs were purified by preparative TLC on Silica Gel 60, and fractionated on AgNO3-impregnated silica gel TLC plates developed with dichloromethane [9, 19]

Summary

Introduction

One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s. The different types of MAs are defined by the presence of decorations introduced at proximal and distal positions of the meromycolic chain (Fig. 1A) by a family of paralogous S-adenosylmethionine-dependent methyltransferases (AdoMet-MTs), the mycolic acid methyltransferases (MA-MTs). These chemical modifications are known to be important for the pathogenicity, virulence, and persistence of M. tuberculosis. E. coli (Ki of 30 and 0.22 ␮M, respectively) [13, 14] They are active only against the isolated enzyme, whereas S-adenosyl-N-decyl-aminoethyl (SADAE), a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain (Fig. 2), is active against CFAS both in vitro (Ki,app ϭ 6 ␮M) and in vivo (complete inhibition at 150 ␮M)

Methods

Results

Conclusion