New drugs are required to counter the tuberculosis (TB) pandemic. Here, we describe the synthesis and characterization of 1,3-benzothiazin-4-ones (BTZs), a new class of antimycobacterial agents that kill Mycobacterium tuberculosis in vitro, ex vivo, and in mouse models of TB. Using genetics and biochemistry, we identified the enzyme decaprenylphosphoryl-beta-d-ribose 2'-epimerase as a major BTZ target. Inhibition of this enzymatic activity abolishes the formation of decaprenylphosphoryl arabinose, a key precursor that is required for the synthesis of the cell-wall arabinans, thus provoking cell lysis and bacterial death. The most advanced compound, BTZ043, is a candidate for inclusion in combination therapies for both drug-sensitive and extensively drug-resistant TB.

SummaryTuberculosis is still a leading cause of death in developing countries, for which there is an urgent need for new pharmacological agents. The synthesis of the novel antimycobacterial drug class of benzothiazinones (BTZs) and the identification of their cellular target as DprE1 (Rv3790), a component of the decaprenylphosphoryl-b-D-ribose 2Ј-epimerase complex, have been reported recently. Here, we describe the identification and characterization of a novel resistance mechanism to BTZ in Mycobacterium smegmatis. The overexpression of the nitroreductase NfnB leads to the inactivation of the drug by reduction of a critical nitro-group to an amino-group. The direct involvement of NfnB in the inactivation of the lead compound BTZ043 was demonstrated by enzymology, microbiological assays and gene knockout experiments. We also report the crystal structure of NfnB in complex with the essential cofactor flavin mononucleotide, and show that a common amino acid stretch between NfnB and DprE1 is likely to be essential for the interaction with BTZ. We performed docking analysis of NfnB-BTZ in order to understand their interaction and the mechanism of nitroreduction. Although Mycobacterium tuberculosis seems to lack nitroreductases able to inactivate these drugs, our findings are valuable for the design of new BTZ molecules, which may be more effective in vivo.

Two galactosyl transferases can apparently account for the full biosynthesis of the cell wall galactan of mycobacteria. Evidence is presented based on enzymatic incubations with purified natural and synthetic galactofuranose (Galf) acceptors that the recombinant galactofuranosyl transferase, GlfT1, from Mycobacterium smegmatis, the Mycobacterium tuberculosis Rv3782 ortholog known to be involved in the initial steps of galactan formation, harbors dual ␤-(134) and ␤-(135) Galf transferase activities and that the product of the enzyme, decaprenyl-P-P-GlcNAc-Rha-Galf-Galf, serves as a direct substrate for full polymerization catalyzed by another bifunctional Galf transferase, GlfT2, the Rv3808c enzyme.The mycobacterial cell wall-including the essential, covalently linked complex of peptidoglycan, heteropolymeric arabinogalactan, and highly hydrophobic mycolic acids-is responsible for many of the pathophysiological features of members of the Mycobacterium genus (9). Several antituberculosis drugs affect the formation of this complex. Isoniazid, ethionamide, thiocarlide, and thiacetazone inhibit mycolic acid synthesis (14,29,30,36,38); ethambutol specifically disrupts the synthesis of arabinan (20,24,35,39); and D-cycloserine, an inhibitor of peptidoglycan synthesis (11), has some clinical utility. However, drug resistance, particularly the multiple and extensive forms, is of pressing public health concern (15, 31), presaging the need for a broader array of targets and drugs affecting both cell wall synthesis and other aspects of mycobacterial metabolism. However, our understanding of the synthesis of the mycobacterial cell wall is elementary compared to that of other bacteria.The initiation point for arabinogalactan biogenesis is the mycobacterial version of the bacterial carrier lipid, bactoprenol, identified as decaprenyl phosphate (C 50 -P) (10), and the sequential addition of GlcNAc-P, rhamnose (Rha), and single galactofuranose (Galf) units, donated by the appropriate nucleotide sugars (23, 25) (Fig. 1). At some stage, the arabinofuranose (Araf) units are added, donated not by a nucleotide sugar but a lipid carrier, C 50 -P-Araf (20, 39). Several of the responsible glycosyl transferases taking part in this process have been identified (1,3,5,18,19,25,26,32,34).We recently described the galactofuranosyl transferase, Rv3782, responsible for attaching the first and, perhaps, the second Galf unit to the C 50 -P-P-GlcNAc-Rha acceptor (22). Due to its role in the initiation of galactan formation, we now name it galactofuranosyl transferase 1 (GlfT1). Previously, yet another galactofuranosyl transferase, Rv3808c (originally called GlfT but now named GlfT2), was recognized and proved to be bifunctional in that it was responsible for the formation of the bulk of the galactan, containing alternating 5-and 6-linked ␤-Galf units (19,25,32). In the present study, we examine the precise roles of GlfT1 and GlfT2 in mycobacterial cell wall galactan synthesis through the application of in vitro reactions with purified natural acc...

SUMMARY UDP-galactofuranose (UDP-Galf) is a substrate for two types of enzymes, UDP-galactopyranose mutase and galactofuranosyltransferases, which are present in many pathogenic organisms but absent from mammals. In particular, these enzymes are involved in the biosynthesis of cell wall galactan, a polymer essential for the survival of the causative agent of tuberculosis, Mycobacterium tuberculosis. We describe here the synthesis of derivatives of UDP-Galf modified at C-5 and C-6 using a chemoenzymatic route. In cell-free assays, these compounds prevented the formation of mycobacterial galactan, via the production of short “dead-end” intermediates resulting from their incorporation into the growing oligosaccharide chain. Modified UDP-furanoses thus constitute novel probes for the study of the two classes of enzymes involved in mycobacterial galactan assembly, and studies with these compounds may ultimately facilitate the future development of new therapeutic agents against tuberculosis.

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