These data showed the same pattern for both MMNAT and TBNAT. Open in a separate window Figure 3 Reversibility of the inhibition of TBNAT and MMNAT by compound 1.Each enzyme (MMNAT, TBNAT, 0.07 mM, 50 L) was preincubated either alone or with 15-fold molar excess 1 at 24C for 1 h. contributes to the pool of propionyl-CoA, a precursor that Rabbit Polyclonal to Lamin A is incorporated into cell-wall lipids. Arylamine (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the gene or inhibiting the NAT enzyme prevents intracellular survival and results in depletion of cell-wall lipids. TBNAT has been investigated as a potential target for TB therapies. From a previous high-throughput screen, 3-benzoyl-4-phenyl-1-methylpiperidinol was identified as a selective inhibitor of prokaryotic NAT that exhibited antimycobacterial activity. The compound resulted in time-dependent irreversible inhibition of the NAT activity when tested against NAT from (MMNAT). To further evaluate the antimycobacterial activity and the NAT inhibition of this compound, four piperidinol analogues were tested. All five compounds exert potent antimycobacterial activity against with MIC values of 2.3C16.9 M. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT WAY-362450 activity. Here we investigate the mechanism of NAT inhibition by studying protein-ligand interactions using mass spectrometry in combination with enzyme analysis and structure determination. We propose a covalent mechanism of NAT inhibition that involves the formation of a reactive intermediate and selective cysteine residue modification. These piperidinols present a unique class of antimycobacterial compounds that have a novel mode of action different from known anti-tubercular drugs. Introduction Tuberculosis (TB) remains the leading cause of death by bacterial infection [1]. According to WHO reports, latent infection represents the major pool of worldwide TB cases, making the treatment of latent TB an important strategy towards eradicating the disease [2]. Persistence of (is capable of using cholesterol as a carbon source inside the macrophage. The catabolism of cholesterol affects the propionate pool in mycobacteria and augments the production of virulence lipids [7]C[9]. Propionyl-CoA (Pr-CoA) is converted to methylmalonyl-CoA (Mm-CoA), which is considered to be the building block of multimethyl-branched mycolic acids such as Phthiocerol Dimycocerosate (PDIM) [8]. Several gene clusters that were shown to be involved in cholesterol degradation are also essential for mycobacterium survival inside the macrophage [10]C[12]. The catabolism of the sterol nucleus of cholesterol in involves the action of the products of a gene cluster which includes (Figure 1) [13], [14], the gene encoding for arylamine gene in and BCG and its relation to cholesterol catabolism.The accession numbers, detailed at http://genolist.pasteur.fr/TubercuList/, for these genes in WAY-362450 H37Rv are as follows: Rv3570c (and BCG. NAT is a cytosolic enzyme that is found in and many other organisms [20]. This enzyme catalyses the transfer of an acyl group, usually an acetyl, to an arylamine substrate using a conserved cysteine residue by a Ping-Pong bi-bi mechanism [21]. The genes from and Bacillus CalmetteCGurin (BCG) are identical and are encoded in virtually identical gene clusters in both organisms (Figure 1). Deleting the gene from BCG resulted in delayed growth and caused morphological changes of the BCG bacilli. Moreover, the mutant severely lacked mycolic acids and virulence-lipid content (PDIM and the cord factor). These effects were overcome when the mutant strain was complemented with the target gene [19]. Chemical inhibition of the NAT activity within mycobacteria resulted in similar changes in morphology, cell-wall lipids and intracellular survival to those WAY-362450 observed upon deleting the gene [22]. Furthermore, the chemically treated strains showed high sensitivity to gentamicin and hygromycin, which have weak activity against mycobacteria [19]. This enzyme is thus an attractive therapeutic target in the search.