Piperidinols that show anti-tubercular activity as inhibitors of arylamine N-acetyltransferase: an essential enzyme for mycobacterial survival inside macrophages

Latent M. tuberculosis infection presents one of the major obstacles in the global eradication of tuberculosis (TB). Cholesterol plays a critical role in the persistence of M. tuberculosis within the macrophage during latent infection. Catabolism of cholesterol contributes to the pool of propionyl-CoA, a precursor that is incorporated into cell-wall lipids. Arylamine N-acetyltransferase (NAT) is encoded within a gene cluster that is involved in the cholesterol sterol-ring degradation and is essential for intracellular survival. The ability of the NAT from M. tuberculosis (TBNAT) to utilise propionyl-CoA links it to the cholesterol-catabolism pathway. Deleting the nat 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 M. marinum (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 M. tuberculosis with MIC values of 2.3-16.9 µM. Treatment of the MMNAT enzyme with this set of inhibitors resulted in an irreversible time-dependent inhibition of NAT 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

Editorial : Competence in scientific agriculture

<div><p>Human arylamine <i>N</i>-acetyltransferase 1 (hNAT1) has become an attractive potential biomarker for estrogen-receptor-positive breast cancers. We describe here the mechanism of action of a selective non-covalent colorimetric biosensor for the recognition of hNAT1 and its murine homologue, mNat2, over their respective isoenzymes, leading to new opportunities in diagnosis. On interaction with the enzyme, the naphthoquinone probe undergoes an instantaneous and striking visible color change from red to blue. Spectroscopic, chemical, molecular modelling and biochemical studies reported here show that the color change is mediated by selective recognition between the conjugate base of the sulfonamide group within the probe and the conjugate acid of the arginine residue within the active site of both hNAT1 and mNat2. This represents a new mechanism for selective biomarker sensing and may be exploited as a general approach to the specific detection of biomarkers in disease.</p></div

Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine [Nu]-acetyltransferase 1

A naphthoquinone inhibitor of human arylamine N-acetyltransferase 1 (hNAT1), a potential cancer biomarker and therapeutic target, has been reported which undergoes a distinctive concomitant color change from red to blue upon binding to the enzyme. Here we describe the use of in silico modeling alongside structure-activity relationship studies to advance the hit compound towards a potential probe to quantify hNAT1 levels in tissues. Derivatives with both a fifty-fold higher potency against hNAT1 and a two-fold greater absorption coefficient compared to the initial hit have been synthesized; these compounds retain specificity for hNAT1 and its murine homologue mNat2 over the isoenzyme hNAT2. A relationship between pKa, inhibitor potency and colorimetric properties has also been uncovered. The high potency of representative examples against hNAT1 in ZR-75-1 cell extracts also paves the way for the development of inhibitors with improved intrinsic sensitivity which could enable detection of hNAT1 in tissue samples and potentially act as tools for elucidating the unknown role hNAT1 plays in ER+ breast cancer; this could in turn lead to a therapeutic use for such inhibitors

Competitive inhibition of 1 towards mNat2 and active site differences in mammalian NATs

<p>(<b>a</b>) <b>Left panel</b>: Structure of compound <b>1</b>; <b>Right panel</b>: Dixon plot shows competitive inhibition of mNat2 (9 ng) by <b>1</b> at different pABA concentrations (25 µM (circles), 50 µM (triangles), 100 µM (diamonds), and 250 µM (squares)). Initial rates of the mNat2 catalysed reaction were determined by monitoring the rate of hydrolysis of AcCoA (400 μM) (<b>b</b>) Summary table of active site differences of human and murine NATs and the effects of their interaction with <b>1</b>. Blue and red columns indicate the color of <b>1</b> on interaction with the protein.</p

Inhibitor binding pocket of hNAT1 and mNat2.

<p>(<b>a</b>) The active site of hNAT1 crystal structure (PDB:2PQT) in surface representation with <b>1</b> docked in stick representation. The hNAT1 residues involved in inhibitor binding and selectivity are shown in stick representation and labeled with carbon in green, nitrogen in blue, oxygen in red, and sulfur in yellow. <b>1</b> is labeled with carbon atoms in light orange, nitrogen in blue, oxygen in red, and sulfur in yellow. (<b>b</b>) The active site of mNat2 structural model with docked compound <b>1</b> is shown using the same representation as in (a).</p

Reagents and conditions: (i) MeNH₂.HCl, paraformaldehyde, MeCN, cat. HCl, Δ, 16 h.

<p>Reagents and conditions: (i) MeNH₂.HCl, paraformaldehyde, MeCN, cat. HCl, Δ, 16 h.</p

Reversibility of the inhibition of TBNAT and MMNAT by compound 1.

<p>Each enzyme (MMNAT, TBNAT, 0.07 mM, 50 µL) was preincubated either alone or with 15-fold molar excess 1 at 24°C for 1 h. Each sample was then dialysed against 1 L fresh assay buffer (20 mM Tris-HCl pH 8) at 4°C for 16 h. The enzyme activities of the samples were measured before dialysis and then measured after dialysis by measuring the rate of Ac-CoA hydrolysis in the presence of HLZ as described in Methods. The mean ± S.D. of three measurements of the activity is shown. Loss of enzyme activity upon dialysis is likely to be due to the oxidation of the active site sulfhydryl group, especially since dialysis was performed in the absence of dithiothreitol.</p

Specificity of compound 1 for prokaryotic NAT enzymes.

<p>Compound <b>1</b> was tested at 30 µM against pure recombinant NAT enzymes from <i>M. smegmatis</i> (MSNAT), <i>P. aeruginosa</i> (PANAT), <i>S. typhimurium</i> (STNAT), MMNAT and TBNAT, and also against two eukaryotic enzymes, hamster NAT2 (shNAT2) and human NAT1. The results are shown as the mean ± S.D. of triplicate determinations of the percentage inhibition of hydrolysis of Ac-CoA in the presence of 5-aminosalicylic acid (5ASA) and against TBNAT using hydralazine as a substrate. The inhibition is represented as a percentage compared to an uninhibited control from triplicate measurements. The structure of compound <b>1</b> is shown and the piperidinol nucleus is highlighted by the shaded area.</p