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.

Acetylation of putative arylamine and alkylaniline carcinogens in immortalized human fibroblasts transfected with rapid and slow acetylator N-acetyltransferase 2 haplotypes.

Exposure to alkylanilines found in tobacco smoke and indoor air is associated with risk of bladder cancer. Genetic factors significantly influence the metabolism of arylamine carcinogens and the toxicological outcomes that result from exposure. We utilized nucleotide excision repair (NER)-deficient immortalized human fibroblasts to examine the effects of human N-acetyltransferase 1 (NAT1), CYP1A2, and common rapid (NAT2*4) and slow (NAT2*5B or NAT2*7B) acetylator human N-acetyltransferase 2 (NAT2) haplotypes on environmental arylamine and alkylaniline metabolism. We constructed SV40-transformed human fibroblast cells that stably express human NAT2 alleles (NAT2*4, NAT2*5B, or NAT2*7B) and human CYP1A2. Human NAT1 and NAT2 apparent kinetic constants were determined following recombinant expression of human NAT1 and NAT2 in yeast for the arylamines benzidine, 4-aminobiphenyl (ABP), and 2-aminofluorene (2-AF), and the alkylanilines 2,5-dimethylaniline (DMA), 3,4-DMA, 3,5-DMA, 2-6-DMA, and 3-ethylaniline (EA) compared with those of the prototype NAT1-selective substrate p-aminobenzoic acid and NAT2-selective substrate sulfamethazine. Benzidine, 3,4-DMA, and 2-AF were preferential human NAT1 substrates, while 3,5-DMA, 2,5-DMA, 3-EA, and ABP were preferential human NAT2 substrates. Neither recombinant human NAT1 or NAT2 catalyzed the N-acetylation of 2,6-DMA. Among the alkylanilines, N-acetylation of 3,5-DMA was substantially higher in human fibroblasts stably expressing NAT2*4 versus NAT2*5B and NAT2*7B. The results provide important insight into the role of the NAT2 acetylator polymorphism (in the presence of competing NAT1 and CYP1A2-catalyzed N-acetylation and N-hydroxylation) on the metabolism of putative alkyaniline carcinogens. The N-acetylation of two alkylanilines associated with urinary bladder cancer (3-EA and 3,5-DMA) was modified by NAT2 acetylator polymorphism.

A Preliminary Assessment of the Structure-Activity Relationship of Benzimidazole-Based Anti-Proliferative Agents.

PDK1 is pivotal in the development and progression of several cancers. A 3D pharmacophore was developed for pyrazole derivatives displaying anti-proliferative activity and PDK1 inhibition. The pharmacophore was utilized in the design of benzimidazole analogs. Our preliminary results indicate the pharmacophore should be useful in designing PDK1 inhibitors and anti-proliferative agents.

High-Throughput Patch Clamp Screening in Human α6-Containing Nicotinic Acetylcholine Receptors.

Nicotine, the addictive component of tobacco products, is an agonist at nicotinic acetylcholine receptors (nAChRs) in the brain. The subtypes of nAChR are defined by their α- and ß-subunit composition. The α6ß2ß3 nAChR subtype is expressed in terminals of dopaminergic neurons that project to the nucleus accumbens and striatum and modulate dopamine release in brain regions involved in nicotine addiction. Although subtype-dependent selectivity of nicotine is well documented, subtype-selective profiles of other tobacco product constituents are largely unknown and could be essential for understanding the addiction-related neurological effects of tobacco products. We describe the development and validation of a recombinant cell line expressing human α6/3ß2ß3V273S nAChR for screening and profiling assays in an automated patch clamp platform (IonWorks Barracuda). The cell line was pharmacologically characterized by subtype-selective and nonselective reference agonists, pore blockers, and competitive antagonists. Agonist and antagonist effects detected by the automated patch clamp approach were comparable to those obtained by conventional electrophysiological assays. A pilot screen of a library of Food and Drug Administration-approved drugs identified compounds, previously not known to modulate nAChRs, which selectively inhibited the α6/3ß2ß3V273S subtype. These assays provide new tools for screening and subtype-selective profiling of compounds that act at α6ß2ß3 nicotinic receptors.