Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery

Abstract

Arylamine N-acetyltransferases (NATs) are polymorphic drug-metabolizing enzymes, acetylating arylamine carcinogens and drugs including hydralazine and sulphonamides. The slow NAT phenotype increases susceptibility to hydralazine and isoniazid toxicity and to occupational bladder cancer. The two polymorphic human NAT loci show linkage disequilibrium. All mammalian Nat genes have an intronless open reading frame and non-coding exons. The human gene products NAT1 and NAT2 have distinct substrate specificities: NAT2 acetylates hydralazine and human NAT1 acetylates p-aminosalicylate (p-AS) and the folate catabolite para-aminobenzoylglutamate (p-abaglu). Human NAT2 is mainly in liver and gut. Human NAT1 and its murine homologue are in many adult tissues and in early embryos. Human NAT1 is strongly expressed in oestrogen receptor-positive breast cancer and may contribute to folate and acetyl CoA homeostasis. NAT enzymes act through a catalytic triad of Cys, His and Asp with the architecture of the active site-modulating specificity. Polymorphisms may cause unfolded protein. The C-terminus helps bind acetyl CoA and differs among NATs including prokaryotic homologues. NAT in Salmonella typhimurium supports carcinogen activation and NAT in mycobacteria metabolizes isoniazid with polymorphism a minor factor in isoniazid resistance. Importantly, nat is in a gene cluster essential for Mycobacterium tuberculosis survival inside macrophages. NAT inhibitors are a starting point for novel anti-tuberculosis drugs. Human NAT1-specific inhibitors may act in biomarker detection in breast cancer and in cancer therapy. NAT inhibitors for co-administration with 5-aminosalicylate (5-AS) in inflammatory bowel disease has prompted ongoing investigations of azoreductases in gut bacteria which release 5-AS from prodrugs including balsalazide.

Keywords: acetyl CoA; arylamine N-acetyltransferase; breast cancer; catalytic triad; hydralazine; isoniazid; pharmacogenetics; tuberculosis.

Figure 1

Metabolic reactions catalysed by arylamine N-acetyltransferase. Acetylation reactions from the top show N-acetylation of an arylamine, p-aminobenzoic acid; N-acetylation of isoniazid; O-acetylation of an arylhydroxylamine and the bottom reaction shows N,O acetyltransfer in a hydroxamate. The top three reactions use acetyl CoA as a cofactor while the bottom reaction does not. The bottom two reactions are associated with carcinogenesis. From Sim et al., .

Figure 2

JR Vane at the opening of the Pharmacology Department, Oxford 1991. John Vane is on the right, David Smith, Head of Department, is in the middle and the head of research of Squibb Pharmaceuticals, who gave the endowment before the Bristol-Meyers takeover is on the left.

Figure 3

Hydralazine inhibits binding of complement component C4. Radiolabelled C4 was activated by a model for immune complexes (C1s bound to Sepharose) and the inhibition of binding was compared using hydralazine or its acetylated metabolite, methyltriazolophthalazine synthesized by EW Gill. From Sim et al. (1984).

Figure 4

Two possible routes of acetylation of hydralazine. From the crystal structure and the different tautommers and ionization states it is proposed that hydralazine may also be acetylated initially at the N in the heterocyclic ring. In either case, cyclization is proposed to generate methyltriazolophthalazine. From Abuhammad et al. (2010).

Figure 5

Isoniazid pharmacokinetics in a population. After Evans et al. (1960).

Figure 6

Specificity profile of NATs. A range of different NAT enzymes were prepared and their specificity was determined with the same panel of substrates in each case The specific activity profiles of three eukaryotic NATs and three prokaryotic NATs have been reported. Specific activities are presented as percent compared with the most active substrate for each enzyme. STNAT, NAT from S. typhimurium; PANAT, NAT from P. aeruginosa; MSNAT, NAT from M. smegmatis; SMZ, sulfamethazine; PRO, procainamide; 5-AS, 5-aminosalicylic acid; PABA, 4-aminobenzoic acid; 2-AF, 2-aminofluorene; 4-CA, 4-chloroaniline; 4-BA, 4-bromoaniline; 4-IA, 4-iodoaniline; ANS, 4-anisidine; EOA, 4-ethoxyaniline; BOA, 4-butoxyaniline; HOA, 4-hexyloxyaniline; POA, 4-phenoxyaniline; AMV, 4-aminoveratrole; INH, isoniazid; CBZ, 4-chlorobenzoic hydrazide; HDZ, hydralazine (Westwood et al., 2006).

Figure 7

Generation of knockout mice. The Nat2 gene has been interrupted by a cassette in frame, which includes the β-galactosidase gene. Embryos can be stained with X-gal, which is converted to a blue colour in the knockout mice. Ashows 12.5 day embryos with wild type, heterozygote (Nat2+/−), a homozygote null (Nat2−/−). B shows 8.5 day whole mount embryo with wild type on the right and homozygote Nat2−/− on the left. Staining in the developing neural tube is marked. Panel A is from Cornish et al., . Panel B was prepared by L. Wakefield.

Figure 8

Active site of S. typhimurium NAT. The residues forming the catalytic triad are shown in pale yellow Cys⁶⁹, His¹⁰⁷ and Asp¹²². The active site Cys⁶⁹ has been labelled with bromacetanilide (bright green). The His residue is behind the labelled Cys, and the Asp residue is in front. The pale pink, green and blue residues correspond to the three domains of the NAT structure.

Figure 9

Inhibition of mycobacterial NAT by piperidinol. (A) The piperidinol inhibitor shows specificity for prokaryotic enzymes. (B) The mechanism of the reaction for the piperidinol inhibitor to NAT is through a chemical transformation to the corresponding phenyl vinyl ketone (PVK), which then binds to the active site Cys residue. C shows the inhibitor bound to the active site Cys residue. Electron density is shown around another Cys, which has no such modification.

Figure 10

Binding of substrates to azoreductase paAzoR1 from Pseudomonas aeruginosa. (A) Balsalazide (from Ryan et al., 2010a) structure was solved to 2.3 Å. Balsalazide is shown in purple and FMN cofactor is shown in yellow. (B) Nitrofurazone (Ryan et al., 2011). Structure was solved at 2.08 Å. Nitrofurazone is in brown while stably bound water molecule involved in reduction is a black ball. C shows the tautomerization and charge relay leading to reduction of the nitro group.