Identification and characterization of potent, selective, and efficacious inhibitors of human arylamine N-acetyltransferase 1.

Arylamine N-acetyltransferase 1 (NAT1) plays a pivotal role in the metabolism of carcinogens and is a drug target for cancer prevention and/or treatment. A protein-ligand virtual screening of 2 million chemicals was ranked for predicted binding affinity towards the inhibition of human NAT1. Sixty of the five hundred top-ranked compounds were tested experimentally for inhibition of recombinant human NAT1 and N-acetyltransferase 2 (NAT2). The most promising compound 9,10-dihydro-9,10-dioxo-1,2-anthracenediyl diethyl ester (compound 10) was found to be a potent and selective NAT1 inhibitor with an in vitro IC50 of 0.75 µM. Two structural analogs of this compound were selective but less potent for inhibition of NAT1 whereas a third structural analog 1,2-dihydroxyanthraquinone (a compound 10 hydrolysis product also known as Alizarin) showed comparable potency and efficacy for human NAT1 inhibition. Compound 10 inhibited N-acetylation of the arylamine carcinogen 4-aminobiphenyl (ABP) both in vitro and in DNA repair-deficient Chinese hamster ovary (CHO) cells in situ stably expressing human NAT1 and CYP1A1. Compound 10 and Alizarin effectively inhibited NAT1 in cryopreserved human hepatocytes whereas inhibition of NAT2 was not observed. Compound 10 caused concentration-dependent reductions in DNA adduct formation and DNA double-strand breaks following metabolism of aromatic amine carcinogens beta-naphthylamine and/or ABP in CHO cells. Compound 10 inhibited proliferation and invasion in human breast cancer cells and showed selectivity towards tumorigenic versus non-tumorigenic cells. In conclusion, our study identifies potent, selective, and efficacious inhibitors of human NAT1. Alizarin's ability to inhibit NAT1 could reduce breast cancer metastasis particularly to bone.

Defining eligible patients for allele-selective chemotherapies targeting NAT2 in colorectal cancer.

Therapies targeting somatic bystander genetic events represent a new avenue for cancer treatment. We recently identified a subset of colorectal cancer (CRC) patients who are heterozygous for a wild-type and a low activity allele (NAT2*6) but lack the wild-type allele in their tumors due to loss of heterozygosity (LOH) at 8p22. These tumors were sensitive to treatment with a cytotoxic substrate of NAT2 (6-(4-aminophenyl)-N-(3,4,5-trimethoxyphenyl)pyrazin-2-amine, APA), and pointed to NAT2 loss being a therapeutically exploitable vulnerability of CRC tumors. To better estimate the total number of treatable CRC patients, we here determined whether tumor cells retaining also other NAT2 low activity variants after LOH respond to APA treatment. The prevalent low activity alleles NAT2*5 and NAT2*14, but not NAT2*7, were found to be low metabolizers with high sensitivity to APA. By analysis of two different CRC patient cohorts, we detected heterozygosity for NAT2 alleles targetable by APA, along with allelic imbalances pointing to LOH, in ~ 24% of tumors. Finally, to haplotype the NAT2 locus in tumor and patient-matched normal samples in a clinical setting, we develop and demonstrate a long-read sequencing based assay. In total, > 79.000 CRC patients per year fulfil genetic criteria for high sensitivity to a NAT2 LOH therapy and their eligibility can be assessed by clinical sequencing.

Exploration of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazoles as selective, multitargeted antimycobacterial agents.

We report the biological evaluation of 5-(5-nitrothiophen-2-yl)-4,5-dihydro-1H-pyrazole derivatives against bacteria, eukaryotic cell lines and the assessment of their mechanisms of action to determine their prospects of being developed into potent antituberculosis agents. The compounds were evaluated for their antibacterial property against Mycobacterium tuberculosis H37Rv, multidrug-resistant M. tuberculosis, Mycobacterium bovis BCG, Mycobacterium aurum, Escherichia coli, and Staphylococcus aureus using high-throughput spot-culture growth inhibition assay. They were found to be selective toward slow-growing mycobacteria and Gram-positive bacteria. In M. bovis BCG, they exhibited a bactericidal mode of action. Cytotoxicity was assessed in human THP-1 and murine RAW 264.7 cell lines, and the compounds showed a lower cytotoxicity potential when compared with their antibacterial activity. They were found to be excellent whole-cell efflux pump inhibitors of the mycobacterial surrogate M. aurum, performing better than known efflux pump inhibitor verapamil. The 5-nitrothiophene moiety was identified for the first time as a prospective inhibitor scaffold of mycobacterial arylamine N-acetyltransferase enzyme, which is the key enzyme in metabolizing isoniazid, a first-line antituberculosis drug. The two aforementioned findings make the compounds potential hits in the development of adjunctive tuberculosis therapy.

Allosteric regulation of arylamine N-acetyltransferase 1 by adenosine triphosphate.

In the present study, a screen of adenosine analogs as potential modulators of arylamine-N-acetyltransferase 1 activity identified ATP as an inhibitor within its range of physiological concentrations. Kinetically, ATP was a non-competitive inhibitor with respect to the acetyl acceptor but a competitive inhibitor with respect to the acetyl donor (acetyl-coenzyme A). In silico modelling predicted that ATP bound within the active site cleft arranged with the triphosphate group in close proximity to arginine 127. Since lysine 100 has previously been implicated in the binding of acetyl-coenzyme A to the enzyme, this amino acid was mutated to either an arginine or a glutamine. Both substitutions significantly changed the affinity of ATP for the enzyme, as well as the nature of the interaction to one with a large Hill coefficient (>3). Under these conditions, ATP was a strong allosteric modulator of arylamine-N-acetyltransferase 1 activity. Western blot analysis identified lysine 100 as a site of post-translational modification by acetylation. The results suggest that acetylation of lysine 100 converts arylamine-N-acetyltransferase 1 into a switch modulated by ATP. This observation provides important understanding of the molecular regulation of NAT1 activity and may reveal possible insight into the endogenous role of the enzyme.

Cytotoxic and N-Acetyltransferase Inhibitory Meroterpenoids from Ganoderma cochlear.

Seven compounds, including two pairs of new meroterpenoids, (+)- and (-)-gancochlearol C (1), (+)- and (-)-cochlearoid Q (3), and a new meroterpenoid gancochlearol D (2), together with four known meroterpenoids were isolated from the aqueous EtOH extract of the fruiting bodies of Ganoderma cochlear. Their structures were determined by spectroscopic data. The isolated compounds were evaluated for their cytotoxic activity against three human lung cancer cells (H1975, PC9, A549) and N-acetyltransferase inhibitory property. The results show that (+)-gancochlearol C could inhibit N-acetyltransferase with an IC50 value of 5.29 µM. In addition, ganomycin F was found to show moderate activity against the H1975 human lung cancer cell line, with an IC50 value of 19.47 µM.

Genetic and small molecule inhibition of arylamine N-acetyltransferase 1 reduces anchorage-independent growth in human breast cancer cell line MDA-MB-231.

Arylamine N-acetyltransferase 1 (NAT1) expression is reported to affect proliferation, invasiveness, and growth of cancer cells. MDA-MB-231 breast cancer cells were engineered such that NAT1 expression was elevated or suppressed, or treated with a small molecule inhibitor of NAT1. The MDA-MB-231 human breast cancer cell lines were engineered with a scrambled shRNA, a NAT1 specific shRNA or a NAT1 overexpression cassette stably integrated into a single flippase recognition target (FRT) site facilitating incorporation of these different genetic elements into the same genomic location. NAT1-specific shRNA reduced NAT1 activity in vitro by 39%, increased endogenous acetyl coenzyme A levels by 35%, and reduced anchorage-independent growth (sevenfold) without significant effects on cell morphology, growth rates, anchorage-dependent colony formation, or invasiveness compared to the scrambled shRNA cell line. Despite 12-fold overexpression of NAT1 activity in the NAT1 overexpression cassette transfected MDA-MB-231 cell line, doubling time, anchorage-dependent cell growth, anchorage-independent cell growth, and relative invasiveness were not changed significantly when compared to the scrambled shRNA cell line. A small molecule (5E)-[5-(4-hydroxy-3,5-diiodobenzylidene)-2-thioxo-1,3-thiazolidin-4-one (5-HDST) was 25-fold more selective towards the inhibition of recombinant human NAT1 than N-acetyltransferase 2. Incubation of MDA-MB-231 cell line with 5-HDST resulted in 60% reduction in NAT1 activity and significant decreases in cell growth, anchorage-dependent growth, and anchorage-independent growth. In summary, inhibition of NAT1 activity by either shRNA or 5-HDST reduced anchorage-independent growth in the MDA-MB-231 human breast cancer cell line. These findings suggest that human NAT1 could serve as a target for the prevention and/or treatment of breast cancer.

Human Arylamine N-Acetyltransferase 1 Is Inhibited by the Dithiocarbamate Pesticide Thiram.

Thiram (tetramethylthiuram disulfide) is a representative dithiocarbamate (DTC) pesticide used in both the field and as a seed protectant. The widespread use of Thiram and other DTC pesticides has raised concerns for health, because these compounds can exert neuropathic, endocrine disruptive, and carcinogenic effects. These toxic effects are thought to rely, at least in part, on the reaction of Thiram (and certain of its metabolites) with cellular protein thiols with subsequent loss of protein function. So far, a limited number of molecular targets of Thiram have been reported, including few enzymes such as dopamine ß-hydroxylase, 11ß-hydroxysteroid dehydrogenase, and brain glycogen phosphorylase. We provide evidence that Thiram is an inhibitor (KI = 23 µM; kinact = 0.085 second-1; kinact/KI = 3691 M-1⋅s-1) of human arylamine N-acetyltransferase 1 (NAT1), a phase II xenobiotic-metabolizing enzyme that plays a key role in the biotransformation of aromatic amine xenobiotics. Thiram was found to act as an irreversible inhibitor through the modification of NAT1 catalytic cysteine residue as also reported for other enzymes targeted by this pesticide. We also showed using purified NAT1 and human keratinocytes that Thiram impaired the N-acetylation of 3,4-dichloroaniline (3,4-DCA), a major toxic metabolite of aromatic amine pesticides (such as Diuron or Propanil). As coexposure to different classes of pesticides is common, our data suggest that pharmacokinetic drug-drug interactions between DTC pesticides such as Thiram and aromatic amine pesticides may occur through alteration of NAT1 enzymes functions.

Treatment of Rats with Apocynin Has Considerable Inhibitory Effects on Arylamine N-Acetyltransferase Activity in the Liver.

The effect of apocynin on the activity of arylamine N-acetyltransferases (NATs) in excised liver samples was examined using eighteen Sprague-Dawley rats. Three groups of six animals each were fed a normal diet alone or a treatment of 50 or 100 mg/kg/day of apocynin via gavages for eight (8) weeks. Chronic in vivo administration of apocynin led to significant (p < 0.001) reduction of in vitro liver NAT activity up to 93% as compared with untreated rats (18.80 ± 2.10 µmols p-anisidine/min/µg liver protein). In vitro exposure of untreated liver homogenates to apocynin led to a dose-dependent inhibition of NAT activity with IC50 = 0.69 ± 0.02 mM. In silico modelling of apocynin tautomers and radical species into human NAT crystal structures supported the hypothesis that thiol functionalities in NAT enzymes may be crucial in apocynin binding. The involvement of human NAT enzymes in different pathological conditions, such as cancer, has encouraged the research for selective NAT inhibitors in both humans and animal models with possible chemopreventive properties.

Identification of cancer chemopreventive isothiocyanates as direct inhibitors of the arylamine N-acetyltransferase-dependent acetylation and bioactivation of aromatic amine carcinogens.

Aromatic amines (AAs) are chemicals of industrial, pharmacological and environmental relevance. Certain AAs, such as 4-aminobiphenyl (4-ABP), are human carcinogens that require enzymatic metabolic activation to reactive chemicals to form genotoxic DNA adducts. Arylamine N-acetyltransferases (NAT) are xenobiotic metabolizing enzymes (XME) that play a major role in this carcinogenic bioactivation process. Isothiocyanates (ITCs), including benzyl-ITC (BITC) and phenethyl-ITC (PEITC), are phytochemicals known to have chemopreventive activity against several aromatic carcinogens. In particular, ITCs have been shown to modify the bioactivation and subsequent mutagenicity of carcinogenic AA chemicals such as 4-ABP. However, the molecular and biochemical mechanisms by which these phytochemicals may modulate AA carcinogens bioactivation and AA-DNA damage remains poorly understood. This manuscript provides evidence indicating that ITCs can decrease the metabolic activation of carcinogenic AAs via the irreversible inhibition of NAT enzymes and subsequent alteration of the acetylation of AAs. We demonstrate that BITC and PEITC react with NAT1 and inhibit readily its acetyltransferase activity (k(i) = 200 M(-1).s(-1) and 66 M(-1).s(-1) for BITC and PEITC, respectively). Chemical labeling, docking approaches and substrate protection assays indicated that inhibition of the acetylation of AAs by NAT1 was due to the chemical modification of the enzyme active site cysteine. Moreover, analyses of AAs acetylation and DNA adducts in cells showed that BITC was able to modulate the endogenous acetylation and bioactivation of 4-ABP. In conclusion, we show that direct inhibition of NAT enzymes may be an important mechanism by which ITCs exert their chemopreventive activity towards AA chemicals.

Effect of acetaminophen on sulfamethazine acetylation in male volunteers.

The effect of acetaminophen on sulfamethazine N-acetylation by human N-acetyltrasferase-2 (NAT2) was studied in 19 (n=19) healthy male volunteers in two different phases. In the first phase of the study the volunteers were given an oral dose of sulfamethazine 500 mg alone and blood and urine samples were collected. After the 10-day washout period the same selected volunteers were again administered sulfamethazine 500 mg along with 1000 mg acetaminophen. The acetylation of sulfamethazine by human NAT2 in both phases with and without acetaminophen was determined by HPLC to establish their respective phenotypes. In conclusion obtained statistics of present study revealed that acetaminophen significantly (P<0.0001) decreased sulfamethazine acetylation in plasma of both slow and fast acetylator male volunteers. A highly significant (P<0.0001) decrease in plasma-free and total sulfamethazine concentration was also observed when acetaminophen was co-administered. Urine acetylation status in both phases of the study was found not to be in complete concordance with that of plasma. Acetaminophen significantly (P<0.0001) increased the acetyl, free and total sulfamethazine concentration in urine of both slow and fast acetylators. Urine acetylation analysis has not been found to be a suitable approach for phenotypic studies.

Cantharidins induce ER stress and a terminal unfolded protein response in OSCC.

Mortality and morbidity associated with oral squamous cell carcinoma (OSCC) remain unacceptably high with disfiguring treatment options and a death rate of 1 per hour in the United States. The approval of cituximab for advanced OSCC has been the only new treatment for these patients since the 1970s, although it has not significantly increased overall survival. To address the paucity of effective new therapies, we undertook a high-throughput screen to discover small molecules and natural products that could induce endoplasmic reticulum (ER) stress and enforce a terminal unfolded protein response (UPR) in OSCC. The terpenoid cantharidin (CNT), previously used to treat various malignancies in culture-specific medical practices for over 2,000 y, emerged as a hit. CNT and its analog, cantharidic acid, potently induced protein and gene expression profiles consistent with the activation of ER stress, the UPR, and apoptosis in OSCC cells. Murine embryonic fibroblasts null for the UPR-associated transcription factors Atf4 or Chop were significantly protected from CNT, implicating a key role for the UPR in the death response. These data validate that our high-throughput screen can identify novel modulators of UPR signaling and that such compounds might provide a new therapeutic approach to treating patients with OSCC.

Differences between murine arylamine N-acetyltransferase type 1 and human arylamine N-acetyltransferase type 2 defined by substrate specificity and inhibitor binding.

BACKGROUND: The mouse has three arylamine N-acetyltransferase genes, (MOUSE)Nat1, (MOUSE)Nat2 and (MOUSE)Nat3. These are believed to correspond to (HUMAN)NAT1, (HUMAN)NAT2 and NATP in humans. (MOUSE)Nat3 encodes an enzyme with poor activity and human NATP is a pseudogene. (MOUSE)Nat2 is orthologous to (HUMAN)NAT1 and their corresponding proteins are functionally similar, but the relationship between (MOUSE)Nat1 and (HUMAN)NAT2 is less clear-cut. METHODS: To determine whether the (MOUSE)NAT1 and (HUMAN)NAT2 enzymes are functionally equivalent, we expressed and purified (MOUSE)NAT1*1 and analysed its substrate specificity using a panel of arylamines and hydrazines. To understand how specific residues contribute to substrate selectivity, three site-directed mutants of (MOUSE)NAT2*1 were prepared: these were (MOUSE)NAT2_F125S, (MOUSE)NAT2_R127G and (MOUSE)NAT2_R127L. All three exhibited diminished activity towards "(MOUSE)NAT2-specific" arylamines but were more active against hydrazines than (MOUSE)NAT1*1. The inhibitory and colorimetric properties of a selective naphthoquinone inhibitor of (HUMAN)NAT1 and (MOUSE)NAT2 were investigated. RESULTS: Comparing (MOUSE)NAT1*1 with other mammalian NAT enzymes demonstrated that the substrate profiles of (MOUSE)NAT1 and (HUMAN)NAT2 are less similar than previously believed. Three key residues (F125, R127 and Y129) in (HUMAN)NAT1*4 and (MOUSE)NAT2*1 were required for enzyme inhibition and the associated colour change on naphthoquinone binding. In silico modelling of selective ligands into the appropriate NAT active sites further implicated these residues in substrate and inhibitor specificity in mouse and human NAT isoenzymes. CONCLUSIONS: Three non-catalytic residues within (HUMAN)NAT1*4 (F125, R127 and Y129) contribute both to substrate recognition and inhibitor binding by participating in distinctive intermolecular interactions and maintaining the steric conformation of the catalytic pocket. These active site residues contribute to the definition of substrate and inhibitor selectivity, an understanding of which is essential for facilitating the design of second generation (HUMAN)NAT1-selective inhibitors for diagnostic, prognostic and therapeutic purposes. In particular, since the expression of (HUMAN)NAT1 is related to the development and progression of oestrogen-receptor-positive breast cancer, these structure-based tools will facilitate the ongoing design of candidate compounds for use in (HUMAN)NAT1-positive breast tumours.

Norcantharidin induces growth inhibition and apoptosis of glioma cells by blocking the Raf/MEK/ERK pathway.

BACKGROUND: Malignant gliomas represent the most common primary brain tumors. The prognosis of patients with malignant gliomas is poor in spite of current intensive therapy and novel therapeutic modalities are needed. Here we report that norcantharidin is effective in growth inhibition of glioma cell lines in vitro. METHODS: Glioma cell lines (U87 and C6) were treated with norcantharidin. The effects of norcantharidin on the proliferation and apoptosis of glioma cells were measured by 3-[4,5-dimethylthiazol-2-thiazolyl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and flow cytometry. Western blotting was employed to determine the signaling pathway changes. RESULTS: The results showed that norcantharidin effectively inhibited cell growth and induced apoptosis in glioma cells, which was concurrent with inhibition of the expression of phospho-MEK and phospho-ERK. Furthermore, the expression anti-apoptotic proteins Bcl-2 and Mcl-1 significantly reduced, but no changes in Bcl-xL and Bax. CONCLUSIONS: Our findings demonstrate that norcantharidin is effective for growth inhibition of glioma cell lines and suggest that norcantharidin may be a new therapeutic option for patients with glioma.

Structure-activity relationships and colorimetric properties of specific probes for the putative cancer biomarker human arylamine N-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.

Dimethocaine, a synthetic cocaine derivative: studies on its in vitro metabolism catalyzed by P450s and NAT2.

Dimethocaine (DMC), a synthetic derivative of cocaine, is distributed and consumed as "new psychoactive substance" (NPS) without any safety testing at the forefront. It is mainly metabolized by N-acetylation, N-deethylation or hydroxylation. Therefore, the aim of the presented study was to determine the human NAT and P450 isozymes involved in this major metabolic steps, to measure the kinetics of the reactions, and to estimate the contribution on in vivo hepatic clearance. For these studies, cDNA-expressed NATs and P450s were used and formation of metabolites after incubation was measured using LC-MS or LC-MS(n). For N-acetylation, NAT2 could be shown to be the only isoform catalyzing the reaction in vitro hence assuming to be the only relevant enzyme for in vivo acetylation. Kinetic profiles of all P450 catalyzed metabolite formations followed classic Michaelis-Menten behavior with enzyme affinities (Km values) between 3.6 and 220 µM. Using the relative activity factor approach, the net clearances for deethylation of DMC were calculated to be 3% for P450 1A2, 1% for 2C19, <1% for 2D6, and 96% for 3A4. The net clearances for hydroxylation of DMC were calculated to be 32% for P450 1A2, 5% for 2C19, 51% for 2D6, and 12% for 3A4. Furthermore, these data were confirmed by chemical inhibition tests in human liver microsomes. As DMC is metabolized via two main steps and different P450 isoforms were involved in the hepatic clearance of DMC, a clinically relevant interaction with single P450 inhibitors should not be expected. However, a slow acetylation phenotype or inhibition of NAT2 could lead to decreased N-acetylation and hence leading to an increased risk of side effects caused by this arylamine.

Interaction of human arylamine N-acetyltransferase 1 with different nanomaterials.

Humans are exposed to nanoparticles in the environment as well as those in nanomaterials developed for biomedical applications. However, the safety and biologic effects of many nanoparticles remain to be elucidated. Over the past decade, our understanding of the interaction of proteins with various nanomaterials has grown. The protein corona can determine not only how nanoparticles interact with cells but also their biologic effects and toxicity. In this study, we describe the effects that several different classes of nanoparticles exert on the enzymatic activity of the cytosolic protein human arylamine N-acetyltransferase 1 (NAT1), a drug-metabolizing enzyme widely distributed in the body that is also responsible for the activation and detoxification of known carcinogens. We investigated three metal oxides (zinc oxide, titanium dioxide, and silicon dioxide), two synthetic clay nanoparticles (layered double hydroxide and layered silicate nanoparticles), and a self-assembling thermo-responsive polymeric nanoparticle that differ in size and surface characteristics. We found that the different nanoparticles induced very different responses, ranging from inhibition to marked enhancement of enzyme activity. The layered silicates did not directly inactivate NAT1, but was found to enhance substrate-dependent inhibition. These differing effects demonstrate the multiplicity of nanoparticle-protein interactions and suggest that enzyme activity may be compromised in organs exposed to nanoparticles, such as the lungs or reticulo-endothelial system.

High throughput screening of 7-methylpicene-1,2-diol as arylamine N-acetyltransferase (NAT) inhibitor to establish a isoniazid supplement in anti-tubercular therapy.

Mycobacterium tuberculosis (Mtb), due to its unusual organization crosses different immune barriers and causes tuberculosis. The advent of multidrug resistance tuberculosis (MDR-TB) has attained alarming situation. Hence, computational drug design has been performed in this work to find potent molecules for this purpose. Isoniazid is a widely used frontline drug against tuberculosis. But reports justified the inactivity of isoniazid on acetylation by Arylamine N-acetyltransferase (NAT). 35 countries were highlighted to have isoniazid resistance from survey in 1998. Hence, Mtb NAT has been selected as the target in the present case and hundred compounds were screened in order to find potent NAT inhibitor to raise the efficacy of isoniazid. Molecular docking with Biosolveit LeadIT and Autodock 4.2 simulation was performed. The result showed 7- methylpicene-1, 2-diol to have -26.77 and -8.26 kcal/mol score in LeadIT and Autodock 4.2. The work validated 7- methylpicene-1, 2-diol to be a potent NAT inhibitor to supplement isoniazid.

Acrolein, an α,ß-unsaturated aldehyde, irreversibly inhibits the acetylation of aromatic amine xenobiotics by human arylamine N-acetyltransferase 1.

Acrolein is an electrophilic α,ß-unsaturated aldehyde of industrial, pharmaceutic, and toxicologic importance to which we are exposed in environmental, occupational, and therapeutic situations. Acrolein is known to exert different biologic effects through reactions with cellular macromolecules such as DNA, certain proteins, or glutathione. In many situations (such as in tobacco smoke or other fumes), exposure to acrolein occurs concomitantly with other compounds such as aromatic amine chemicals. Interestingly, it has been shown that acrolein could impact the cellular metabolism of aromatic xenobiotics through an indirect mechanism based on the transcriptional induction of phase II xenobiotic-metabolizing enzymes. Here we report a novel mechanism by which acrolein acts on the metabolism of aromatic foreign chemicals. We provide molecular, kinetic, and cellular evidence that acrolein can react directly and irreversibly with arylamine N-acetyltransferases, a major family of xenobiotic-metabolizing enzymes involved in the metabolization of aromatic amine chemicals. Formation of an acrolein adduct with a catalytic cysteine residue in the active site is responsible for the impairment of aromatic amine acetylation by the enzyme. This biochemical process may represent an additional mechanism by which acrolein impacts the metabolism and fate of aromatic amine drugs and pollutants.

Design, synthesis and structure-activity relationships of 3,5-diaryl-1H-pyrazoles as inhibitors of arylamine N-acetyltransferase.

The synthesis and inhibitory potencies of a novel series of 3,5-diaryl-1H-pyrazoles as specific inhibitors of prokaryotic arylamine N-acetyltransferase enzymes is described. The series is based on hit compound 1 3,5-diaryl-1H-pyrazole identified from a high-throughout screen that has been carried out previously and found to inhibit the growth of Mycobacterium tuberculosis.

Arylamine N-acetyltransferases: a structural perspective.

Arylamine N-acetyltransferase (NAT) plays an important role in metabolism and detoxification of many compounds including drugs and environmental carcinogens through chemical modification of the amine group with an acetyl group. Recent studies have suggested that NATs are also involved in cancer cell growth and inhibition of the enzymes may be a potential target for cancer chemotherapy. Three-dimensional (3D) structures are available for NATs from both prokaryotes and eukaryotes. These structures provide valuable insights into the acetylation mechanism, features of the active site and the structural determinants that govern substrate/inhibitor-binding specificity. Such insights allow a more precise understanding of the structure-activity relationships for NAT substrates and inhibitors. Furthermore, the structural elucidation of NATs has generated powerful tools in the design of small molecule inhibitors that should alleviate cancer, based on the important role of the enzyme in cancer biology.