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

From arylamine N-acetyltransferase to folate-dependent acetyl CoA hydrolase : impact of folic acid on the activity of (HUMAN)NAT1 and its homologue (MOUSE)NAT2

Acetyl Coenzyme A-dependent N-, O- and N,O-acetylation of aromatic amines and hydrazines by arylamine N-acetyltransferases is well characterised. Here, we describe experiments demonstrating that human arylamine N-acetyltransferase Type 1 and its murine homologue (Type 2) can also catalyse the direct hydrolysis of acetyl Coenzyme A in the presence of folate. This folate-dependent activity is exclusive to these two isoforms; no acetyl Coenzyme A hydrolysis was found when murine arylamine N-acetyltransferase Type 1 or recombinant bacterial arylamine N-acetyltransferases were incubated with folate. Proton nuclear magnetic resonance spectroscopy allowed chemical modifications occurring during the catalytic reaction to be analysed in real time, revealing that the disappearance of acetyl CH3 from acetyl Coenzyme A occurred concomitantly with the appearance of a CH3 peak corresponding to that of free acetate and suggesting that folate is not acetylated during the reaction. We propose that folate is a cofactor for this reaction and suggest it as an endogenous function of this widespread enzyme. Furthermore, in silico docking of folate within the active site of human arylamine N-acetyltransferase Type 1 suggests that folate may bind at the enzyme's active site, and facilitate acetyl Coenzyme A hydrolysis. The evidence presented in this paper adds to our growing understanding of the endogenous roles of human arylamine N-acetyltransferase Type 1 and its mouse homologue and expands the catalytic repertoire of these enzymes, demonstrating that they are by no means just xenobiotic metabolising enzymes but probably also play an important role in cellular metabolism. These data, together with the characterisation of a naphthoquinone inhibitor of folate-dependent acetyl Coenzyme A hydrolysis by human arylamine N-acetyltransferase Type 1/murine arylamine N-acetyltransferase Type 2, open up a range of future avenues of exploration, both for elucidating the developmental role of these enzymes and for improving chemotherapeutic approaches to pathological conditions including estrogen receptor-positive breast cancer

Identification of NAD(P)H Quinone Oxidoreductase Activity in Azoreductases from P. aeruginosa: Azoreductases and NAD(P)H Quinone Oxidoreductases Belong to the Same FMN-Dependent Superfamily of Enzymes

Water soluble quinones are a group of cytotoxic anti-bacterial compounds that are secreted by many species of plants, invertebrates, fungi and bacteria. Studies in a number of species have shown the importance of quinones in response to pathogenic bacteria of the genus Pseudomonas. Two electron reduction is an important mechanism of quinone detoxification as it generates the less toxic quinol. In most organisms this reaction is carried out by a group of flavoenzymes known as NAD(P)H quinone oxidoreductases. Azoreductases have previously been separate from this group, however using azoreductases from Pseudomonas aeruginosa we show that they can rapidly reduce quinones. Azoreductases from the same organism are also shown to have distinct substrate specificity profiles allowing them to reduce a wide range of quinones. The azoreductase family is also shown to be more extensive than originally thought, due to the large sequence divergence amongst its members. As both NAD(P)H quinone oxidoreductases and azoreductases have related reaction mechanisms it is proposed that they form an enzyme superfamily. The ubiquitous and diverse nature of azoreductases alongside their broad substrate specificity, indicates they play a wide role in cellular survival under adverse conditions

Investigating the endogenous role of human n-acetyltransferase 1, as potential breast cancer biomarker, using chemical biology

Human N-acetyltransferase 1 (hNAT1) is one of the ten most highly overexpressed genes in oestrogen-receptor-positive (ER+ve) breast cancers and its overexpression is strongly related to tumour grade. N-acetyltransferases from prokaryotic and eukaryotic kingdoms catalyse the transfer of an acetyl group from acetyl coenzyme A (CoA) to a variety of arylamines and arylhydrazines. While the other human isoenzyme hNAT2 has widely been assessed as a phase-II xenobiotic metabolising enzyme, the exact endogenous role of hNAT1 is still unknown. The association of hNAT1 with ER levels in breast tumours may imply a role in cancer progression, making it an attractive potential biomarker for ER+ve breast cancers and/or a novel therapeutic target. Mice offer a good animal model for investigations on human NATs: hNAT1 and mouse NAT2 (mNat2) are orthologous genes and the corresponding proteins, hNAT1 and mNat2, are homologous on the basis of sequence identity (82&amp;percent;), substrate specificity and expression profile. Investigating selective inhibitors for hNAT1 and mNat2 is described in this thesis with the aim of using these inhibitors to aid in determining the in vivo function of hNAT1 and its mouse homologue. Naphthoquinone 1 was identified as a selective competitive inhibitor for hNAT1 and mNat2 (IC50,hNAT1=1.65 μM and IC50,mNat2=1.86μM) from a high-throughput screening of 5000 drug-like compounds against five distinct pure recombinant NATs. This compound also displays a distinctive colour change from red (λmax = 484nm) to blue (λmax = 610nm) in the presence of both hNAT1 and mNat2, but not the other human and murine isoenzymes. The colourimetric change was also observed by titration of compound 1 with an alkali. Physicochemical, biochemical and computational studies were conducted on naphthoquinone 7, an analogue of 1 with improved pharmacological properties (IC50,mNat2=0.99μM) and colour intensity, to support the hypothesis that the colour change event is related to deprotonation of the sulfonamide-NH of the ligand by the side-chain guanidine of Arg127 within the active site of both enzymes, hNAT1 and mNat2. Furthermore, the comparison of the arylamine substrate profiles of eight different mammalian NATs, alongside their preferences for inhibitor 7, provided substantial elements on the key role of Phe125, Arg127 and Tyr129 on isoenzyme selectivity for both substrate and inhibitor. This supports the development of this family of naphthoquinones as highly selective inhibitors of hNAT1 and mNat2 to elucidate the endogenous role of these proteins via Chemical Genetics, and as colourimetric biosensors to detect and quantify hNAT1 in breast cancer tissues. Selective recognition of hNAT1 by antibody allowed a preliminary estimation of the enzyme overex-pressed in the breast cancer cell line ZR-75-1: 1pg per cell, for which the binding affinity and the colourimetric properties of compound 7 were found to need further improvement. With the goal of improving both inhibitory potency and colourimetric properties of compound 7, a set of analogues varying at R1, R2 and R3 positions was chemically synthesised. The resulting substitutions al-tered inhibitory activity, range of colour change, molar extinction coefficient and acidity of the naphthoquinone derivatives, with compound 20 offering a tenfold increase in inhibitory potency towards both hNAT1 and mNat2 over 7, but less suitable colourimetric properties. Besides investigating the ability of different eukaryotic and prokaryotic NATs to use also n-propionylCoA as substrate, hNAT1 and mNat2 were exclusively identified to act as folate-dependent acetylCoA hydrolases compared to a panel of diverse eukaryotic and prokaryotic NATs, from which new hypotheses are proposed in the endogenous role of these enzymes in relation to folate, fat catabolism and cancer. </p

Selective small molecule inhibitors of the potential breast cancer marker, human arylamine N-acetyltransferase 1, and its murine homologue, mouse arylamine N-acetyltransferase 2.

The identification, synthesis and evaluation of a series of rhodanine and thiazolidin-2,4-dione derivatives as selective inhibitors of human arylamine N-acetyltransferase 1 and mouse arylamine N-acetyltransferase 2 is described. The most potent inhibitors identified have submicromolar activity and inhibit both the recombinant proteins and human NAT1 in ZR-75 cell lysates in a competitive manner. (1)H NMR studies on purified mouse Nat2 demonstrate that the inhibitors bind within the putative active site of the enzyme