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Case Study 10: A Case to Investigate Acetyl Transferase Kinetics

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Enzyme Kinetics in Drug Metabolism

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2342))

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Abstract

Major routes of metabolism for marketed drugs are predominately driven by enzyme families such as cytochromes P450 and UDP-glucuronosyltransferases. Less studied conjugative enzymes, like N-acetyltransferases (NATs), are commonly associated with detoxification pathways. However, in the clinic, the high occurrence of NAT polymorphism that leads to slow and fast acetylator phenotypes in patient populations has been linked to toxicity for a multitude of drugs. A key example of this is the observed clinical toxicity in patients who exhibit the slow acetylator phenotype and were treated with isoniazid. Toxicity in patients has led to detailed characterization of the two NAT isoforms and their polymorphic genotypes. Investigation in recombinant enzymes, genotyped hepatocytes, and in vivo transgenic models coupled with acetylator status-driven clinical studies have helped understand the role of NATs in drug development, clinical study design and outcomes, and potential roles in human disease models. The selected case studies herein document NAT enzyme kinetics to explore substrate overlap from two human isoforms, preclinical species considerations, and clinical genotype population concerns.

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References

  1. Cerny MA (2016) Prevalence of non-cytochrome P450-mediated metabolism in Food and Drug Administration-approved oral and intravenous drugs: 2006–2015. Drug Metab Dispos 44(8):1246–1252. https://doi.org/10.1124/dmd.116.070763

    Article  CAS  PubMed  Google Scholar 

  2. Yamada S, Arikawa S (2014) An ectotherm homologue of human predicted gene NAT16 encodes histidine N acetyltransferase responsible for Nα-acetylhistidine synthesis. Biochim Biophys Acta 1840(1):434–442

    Article  CAS  PubMed  Google Scholar 

  3. Westwood IM, Sim E (2007) Kinetic characterisation of arylamine N-acetyltransferase from Pseudomonas aeruginosa. BMC Biochem 20(8):3

    Article  CAS  Google Scholar 

  4. Vatsis KP, Weber WW, Bell DA, Dupret JM, Evans DM, Grant DM, Hein DW, Lin HJ, Meyer UA, Relling MV (1995) Nomenclature for N-acetyltransferases. Pharmacogenetics 5:1–17

    Article  CAS  PubMed  Google Scholar 

  5. Sim E, Westwood I, Fullam E (2007) Arylamine N-acetyltransferases. Expert Opin Drug Metab Toxicol 3(2):169–184

    Article  CAS  PubMed  Google Scholar 

  6. Blanc A, Vivien-Roels B, Pevet P, Buisson B (2003) Melatonin and 5-methoxytryptophol (5-ML) in nervous and/or neurosensory structures of a gastropod Mollusc (Helix aspersa maxima): synthesis and diurnal rhythms. Gen Comp Endocrinol 131(2):168–175

    Article  CAS  PubMed  Google Scholar 

  7. Trepanier LA, Ray K, Winand NJ, Spielberg SP, Cribb AE (1997) Cytosolic arylamine N-acetyltransferase (NAT) deficiency in the dog and other canids due to an absence of NAT genes. Biochem Pharmacol 54(1):73–80

    Article  CAS  PubMed  Google Scholar 

  8. Sim E, Abuhammad A, Ryan A (2014) Arylamine N-acetyltransferases: from drug metabolism and pharmacogenetics to drug discovery. Br J Pharmacol 171:2705–2725

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Minchin RF (1995) Acetylation of P-aminobenzoylglutamate, a folic acid catabolite, by recombinant human arylamine N-acetyltransferase and U937 cells. Biochem J 307:1–3

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Adam PJ, Berry J, Loader JA, Tyson K, Craggs G, Smith P, De Belin J, Steers G, Pezzella F, Sachsenmeir KF, Stamps AC, Herath A, Sim E, O’Hare MJ, Harris A, Terret JA (2003) Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro. Mol Cancer Res 1(11):826–835

    CAS  PubMed  Google Scholar 

  11. Sim E, Payton M, Noble M, Minchin R (2000) An update on genetic, structural and functional studies of arylamine N-acetyltransferases in eucaryotes and procaryotes. Hum Mol Genet 9(16):2435–2441

    Article  CAS  PubMed  Google Scholar 

  12. Ohtani T, Hiroi A, Sakurana M, Furukawa F (2003) Slow acetylator genotypes as possible risk factor for infectious mononucleosis-like syndrome induced by salazosulfapyridine. Br J Dermatol 148(5):1035–1039

    Article  CAS  PubMed  Google Scholar 

  13. Romano A (2000) Recognising antibacterial hypersensitivity in children. Paediatr Drugs 2(2):101–112

    Article  CAS  PubMed  Google Scholar 

  14. Zielinska E, Niewiarowski W, Bodalski J, Stanczyk A, Bolanowski W, Rebowski G (1997) Arylamine N-acetyltransferase (NAT2) gene mutations in children with allergic diseases. Clin Pharmacol Ther 62(6):635–642

    Article  CAS  PubMed  Google Scholar 

  15. Huang Y-S, Chern H-D, Su W-J, Wu J-C, Liang-Shinn YS-Y, Chang F-Y, Lee S-D (2002) Polymorphism of the N-acetyltransferase 2 gene as a susceptibility risk factor for antituberculosis drug-induced hepatitis. Hepatology 35(4):883–889

    Article  CAS  PubMed  Google Scholar 

  16. Butcher NJ, Minchin RF (2012) Arylamine N-acetyltransferase 1: a novel drug target in cancer development. Pharmacol Rev 64(1):147–165

    Article  CAS  PubMed  Google Scholar 

  17. Rodrigues-Lima F, Dairou J, Busi F, Dupret JM (2010) Human arylamine N-acetyltransferase 1: a drug-metabolizing enzyme and a drug target? Curr Drug Targets 11:759–766

    Article  CAS  PubMed  Google Scholar 

  18. Sim E, Pinter K, Upton A, Sandy J, Bhakta S, Noble M (2003) Arylamine N-acetyltransferases: a pharmacogenomic approach to drug metabolism and endogenous function. Biochem Soc Trans 31:615–619

    Article  CAS  PubMed  Google Scholar 

  19. Riddle B, Jencks W (1971) Acetyl-coenzyme A: arylamine N-acetyltransferase. Role of the acetyl-enzyme intermediate and the effects of substituents on the rate. J Biol Chem 246(10):3250–3258

    Article  CAS  PubMed  Google Scholar 

  20. Hickman D, Palamanda JR, Unadkat JD, Sim E (1995) Enzyme kinetic properties of human recombinant arylamine N-acetyltransferase 2 allotypic variants expressed in Escherichia coli. Biochem Pharmacol 50(5):697–703

    Article  CAS  PubMed  Google Scholar 

  21. Weber WW, Cohen SN (1967) N-acetylation of drugs: isolation and properties of an N-acetyltransferase from rabbit liver. Mol Pharmacol 3(3):266–273

    CAS  PubMed  Google Scholar 

  22. Minchin RF, Butcher NJ (2015) The role of lysine (100) in the binding of acetylcoenzyme A to human arylamine N-acetyltransferase 1: implications for other acetyltransferases. Biochem Pharmacol 94(3):195–202

    Article  CAS  PubMed  Google Scholar 

  23. Sandy J, Mustaq A, Holton SJ, Schartau P, Noble MEM, Sim E (2005) Investigation of the catalytic triad of arylamine N-acetyltransferases: essential residues required for acetyl transfer to arylamines. Biochem J 15:115–123

    Article  CAS  Google Scholar 

  24. Wang H, Liu L, Hanna PE, Wagner CR (2005) Catalytic mechanism of hamster arylamine N-acetyltransferase 2. Biochemistry 44(33):11295–11306

    Article  CAS  PubMed  Google Scholar 

  25. Wang H, Vath GM, Gleason KJ, Hanna PE, Wagner CR (2004) Probing the mechanism of hamster arylamine N-acetyltransferase 2 acetylation by active site modification, site-directed mutagenesis, and pre-steady state and steady state kinetic studies. Biochemistry 43(35):8234–8246

    Article  CAS  PubMed  Google Scholar 

  26. Sinclair JC, Sandy J, Delgoda R, Sim E, Noble ME (2000) Structure of arylamine N-acetyltransferase reveals a catalytic triad. Nat Struct Biol 7(7):560–564

    Article  CAS  PubMed  Google Scholar 

  27. Ma MK, Woo MH, McLeod HL (2002) Genetic basis of drug metabolism. Am J Health Syst Pharm 59(21):2061–2069

    Article  CAS  PubMed  Google Scholar 

  28. Grant DM, Hughes NC, Janezic SA, Goodfellow GH, Chen HJ, Gaedigk A, Yu VL, Grewal R (1997) Human acetyltransferase polymorphisms. Mutat Res 376

    Google Scholar 

  29. Hein DW, Grant DM, Sim E (2000) Update on consensus arylamine N-acetyltransferase gene nomenclature. Pharmacogenetics 10:291–292

    Article  CAS  PubMed  Google Scholar 

  30. Hughes HB (1953) On the metabolic fate of isoniazid. J Pharmacol Exp Ther 109:444–452

    CAS  PubMed  Google Scholar 

  31. Ellard GA, Gammon PT (1977) Acetylator phenotyping of tuberculosis patients using matrix isoniazid or sulphadimidine and its prognostic significance for treatment with several intermittent isoniazid-containing regimens. Br J Clin Pharmacol 4:5–14

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Evans DA, Manley KA, McKusick VA (1960) Genetic control of isoniazid metabolism in man. Br Med J 13(2):485–491

    Article  Google Scholar 

  33. Grant DM, Morike K, Eichelbaum M, Meyer UA (1990) Acetylation pharmacogenetics. The slow acetylator phenotype is caused by decreased or absent arylamine N-acetyltransferase in human liver. J Clin Investig 85:968–972

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Peters JH, Miller KS, Brown P (1965) Studies on the metabolic basis for the genetically determined capacities for isoniazid inactivation in man. J Pharmacol Exp Ther 150(2):298–304

    CAS  PubMed  Google Scholar 

  35. Weber WW, Hein DW (1985) N-acetylation pharmacogenetics. Pharmacol Rev 37(1):26–79

    Google Scholar 

  36. Ramappa V, Aithal GP (2013) Hepatotoxicity related to anti-tuberculosis drugs: mechanisms and management. J Clin Exp Hepatol 3:37–49

    Article  PubMed  Google Scholar 

  37. Hughes HB, Biehl JP, Jones AP, Schmit LH (1954) Metabolism of isoniazid in man as related to the occurrence of peripheral neuritis. Am Rev Tuberc 70(2):266–273

    CAS  PubMed  Google Scholar 

  38. Cai Y, Yi J, Zhou C, Shen X (2012) Pharmacogenetic study of drug-metabolising enzyme polymorphisms on the risk of anti-tuberculosis drug-induced liver injury: a meta-analysis. PLoS One 7(10):e47769

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Klein DJ, Boukouvala S, McDonagh EM, Shuildiner SR, Laurieri N, Thorn CF, Altman RB, Klein TE (2016) PharmGKB summary: isoniazid pathway, pharmacokinetics (PK). Pharmacogenet Genomics 26(9):436–444

    Article  PubMed  PubMed Central  Google Scholar 

  40. Boukouvala S, Fakis G (2005) Arylamine N-acetyltransferases: what we learn from genes and genomes. Drug Metab Rev 3:511–564

    Article  CAS  Google Scholar 

  41. Butcher NJ, Boukouvala S, Sim E, Minchin RF (2002) Pharmacogenetics of the arylamine N-acetyltransferases. Pharmacogenomics J 2:30–42

    Article  CAS  PubMed  Google Scholar 

  42. Laurieri N, Sim E (eds) (2018) Arylamine N-acetyltransferases in health and disease: from pharmacogenetics to drug discovery and diagnostics, 1st edn. World Scientific Publishing Company, NJ

    Google Scholar 

  43. Ratain MJ, Mick R, Berezin F, Janisch L, Schilsky RL, Vogelzang NJ, Lane LB (1993) Phase I study of amonafide dosing based on Acetylator phenotype. Cancer Res 53:2304–2308

    CAS  PubMed  Google Scholar 

  44. Campbell W, Tilstone WJ, Lawson DH, Hutton I, Lawrie TDV (1976) Acetylator phenotype and the clinical pharmacology of slow-release procainamide. Br J Clin Pharmacol 3:1023–1026

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Haroldsen PE, Sisic Z, Datt J, Musson DG, Ingenito G (2017) Acetylator status impacts amifampridine phosphate (Firdapse™) pharmacokinetics and exposure to a greater extent than renal function. Clin Ther 39(7):1360–1370

    Article  CAS  PubMed  Google Scholar 

  46. Abouraya M, Sacco JC, Hayes K, Thomas S, Kitchens CS, Trepanier LA (2012) Dapsone-associated methemoglobinemia in a patient with slow NAT2*5B haplotype and impaired cytochrome b5 reductase activity. J Clin Pharmacol 52:272–278

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bluhm R, Adedoyin A, McCarver DG, Branch RA (1999) Development of dapsone toxicity in patients with inflammatory dermatoses: activity of acetylation and hydroxylation of dapsone as risk factors. Clin Pharmacol Ther 65:598–605

    Article  CAS  PubMed  Google Scholar 

  48. May DG, Porter JA, Uetrecht JP, Wilkinson GR, Branch RA (1990) The contribution of N-hydroxylation and acetylation to dapsone pharmacokinetics in normal subjects. Clin Pharmacol Ther 48:619–627

    Article  CAS  PubMed  Google Scholar 

  49. Sacco JC, Abouraya M, Motsinger-Reif A, Yale SH, McCarty CA, Trepanier LA (2012) Evaluation of polymorphisms in the sulfonamide detoxification genes NAT2, CYB5A, and CYB5R3 in patients with sulfonamide hypersensitivity. Pharmacogenet Genomics 22:733–740

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Tam CM, Chan SL, Kam KM, Sim E, Staples D, Sole KM, Al-Ghusein H, Mitchison DA (2000) Rifapentine and isoniazid in the continuation phase of a 6-month regimen. Interim report: no activity of isoniazid in the continuation phase. Int J Tuber Lung Dis 4:262–267

    CAS  Google Scholar 

  51. Tahir IM, Iqbal T, Saleem S, Mehboob H, Akhter N, Riaz M (2016) Effect of acetaminophen on sulfamethazine acetylation in male volunteers. Int J Immunopathol Pharmacol 39:17–22

    Article  CAS  Google Scholar 

  52. Toth K, Csukly G, Sirok D, Belic A, Kiss A, Hafra E, Deri M, Menus A, Bitter I, Monostory K (2016) Optimization of clonazepam therapy adjusted to patient’s CYP3A status and NAT2 genotype. Int J Neuripsyschopharmacol 19:1–9

    CAS  Google Scholar 

  53. Ramirez-Alcantara V, Montrose MH (2014) Acute murine colitis reduces colonic 5-aminosalicylic acid metabolism by regulation of N-acetyltransferase-2. Am J Physiol Gastrointest Liver Physiol 306:G1002–G1010

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Committee TAN-aGN human NAT1 alleles (haplotypes). http://nat.mbg.duth.gr/Human%20NAT1%20alleles_2013.htm. Accessed 2 May 2020

  55. Committee TAN-aGN human NAT2 alleles (haplotypes). http://nat.mbg.duth.gr/Human%20NAT2%20alleles_2013.htm. Accessed 2 May 2020

  56. Blum M, Grant DM, McBride W, Heim M, Meyer UA (1990) Human arylamine N-acetyltransferase genes: isolation, chromosomal localization, and functional expression. DNA Cell Biol 9:193–203

    Article  CAS  PubMed  Google Scholar 

  57. Husain A, Zhang Z, Doll MA, States JC, Barker DF, Hein DW (2007) Functional analysis of the human N-acetyltransferase 1 major promoter: quantitation of tissue expression and identification of critical sequence elements. Drug Metab Dispos 35:1649–1656

    Article  CAS  PubMed  Google Scholar 

  58. Hickman D, Pope J, Patil SD, Fakis G, Smelt V, Stanley LA, Payton M, Unadkat JD, Sim E (1998) Expression of arylamine N-acetyltransferase in human intestine. Gut 42:402–409

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Argikar UA, Dumouchel JL, Dunne CE, Bushee AJ (2017) Ocular non-P450 oxidative, reductive, hydrolytic, and conjugative drug metabolizing enzymes. Drug Metab Rev 49(3):372–394

    Article  CAS  PubMed  Google Scholar 

  60. Dupret JM, Goodfellow GH, Janezic SA, Grant DM (1994) Structure-function studies of human arylamine N-acetyltransferases NAT1 and NAT2. J Biol Chem 269(43):26830–26835

    Article  CAS  PubMed  Google Scholar 

  61. Goodfellow GH, Dupret J-M, Grant DM (2000) Identification of amino acids imparting acceptor substrate selectivity to human arylamine acetyltransferases NAT1 and NAT2. Biochem J 348:159–166

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Lui L, Wagner CR, Hanna PE (2008) Human arylamine N-acetyltransferase 1: in vitro and intracellular inactivation by nitrosoarene metabolites of toxic and carcinogenic arylamines. Chem Res Toxicol 21:2005–2016

    Article  CAS  Google Scholar 

  63. Deng ZJ, Butcher NJ, Mortimer GM, Jia Z, Monteiro MJ, Martin DJ, Minchin RF (2014) Interaction of human arylamine N-acetyltransferase 1 with different nanomaterials. Drug Metab Dispos 42:377–383

    Article  PubMed  CAS  Google Scholar 

  64. Butcher NJ, Arulpragasam A, Minchin RF (2004) Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine: a mechanism for the slow acetylator phenotype and substrate-dependent down-regulation. J Biol Chem 279(21):22131–22137

    Article  CAS  PubMed  Google Scholar 

  65. Zhu Y, Hein D (2008) Functional effects of single nucleotide polymorphisms in the coding region of human N-acetyltransferase 1. Pharmacogenomics J 8:339–348

    Article  CAS  PubMed  Google Scholar 

  66. Butcher NJ, Arulpragasam A, Minchin R (2004) Proteasomal degradation of N-acetyltransferase 1 is prevented by acetylation of the active site cysteine. J Biol Chem 279(21):22131–22137

    Article  CAS  PubMed  Google Scholar 

  67. Millner LM, Doll MA, Cai J, States JC, Hein DW (2012) Phenotype of the most common “slow Acetylator” arylamine N-acetyltransferase 1 genetic variant (NAT1*14B) is substrate-dependent. Drug Metab Dispos 40:198–204

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Ishibe N, Sinha R, Hein DW, Kulldorff M, Strickland P, Fretland AJ, Chow W-H, Kadlubar FF, Lang NP, Rothman N (2002) Genetic polymorphisms in heterocyclic amine metabolism and risk of colorectal adenomas. Pharmacogenetics 12:145–150

    Article  CAS  PubMed  Google Scholar 

  69. Johnson N, Bell P, Jonovska V, Budge M, Sim E (2004) NAT gene polymorphisms and susceptibility to Alzheimer’s disease: identification of a novel NAT1 allelic variant. BMC Med Genet 5:6

    Article  PubMed  PubMed Central  Google Scholar 

  70. Carlisle SM, Trainor PJ, Hong KU, Doll MA, Hein DW (2020) CRISPR/Cas9 knockout of human arylamine N-acetyltransferase 1 in MDA-MB-231 breast cancer cells suggests a role in cellular metabolism. Sci Rep 10

    Google Scholar 

  71. Wakefield L, Robinson J, Long H, Ibbitt JC, Cooke S, Hurst HC, Sim E (2008) Arylamine N-acetyltransferase 1 expression in breast cancer cell lines: a potential marker in estrogen receptor-positive tumors. Genes Chromosomes Cancer 47:118–126

    Article  CAS  PubMed  Google Scholar 

  72. Husain A, Zhang Z, Doll MA, States JC, Barker DF, Hein DW (2007) Identification of N-acetyltransferase 2 (NAT2) transcription start sites and quantitation of NAT2-specific mRNA in human tissues. Drug Metab Dispos 35:721–727

    Article  CAS  PubMed  Google Scholar 

  73. Sim E, Walters K, Boukouvala S (2008) Arylamine N-acetyltransferases: from structure to function. Drug Metab Rev 40:479–510

    Article  CAS  PubMed  Google Scholar 

  74. Tsirka T, Boukouvala S, Agianian B, Fakis G (2014) Polymorphism p.Val231Ile alters substrate selectivity of drug-metabolizing arylamine N-acetyltransferase 2 (NAT2) isoenzyme of rhesus macaque and human. Gene 536:65–73

    Article  CAS  PubMed  Google Scholar 

  75. Allen CE, Doll MA, Hein DW (2017) N-acetyltransferase 2 genotype-dependent N-acetylation of hydralazine in human hepatocytes. Drug Metab Dispos 45:1276–1281

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Doll MA, Hein DW (2017) Genetic heterogeneity among slow acetylator N-acetyltransferase 2 phenotypes in cryopreserved human hepatocytes. Arch Toxicol 91:2655–2661

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Mthiyane T, Millard J, Adamson J, Balakrishna Y, Connolly C, Owen A, Rustomjee R, Dheda K, McIlleron H, Pym AS (2020) N-acetyltransferase 2 genotypes among Zulu-speaking South Africans and isoniazid and N acetyl-isoniazid pharmacokinetics during antituberculosis treatment. Antimicrob Agents Chemother 64(4):e02376–e02319

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Sabbagh A, Langaney A, Darlu P, Gerard N, Krishnamoorthy R, Poloni ES (2008) Worldwide distribution of NAT2 diversity: implications for NAT2 evolutionary history. BMC Genet 9:21. https://doi.org/10.1186/1471-2156-9-21

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  79. Soejima M, Sugiura T, Kawaguchi Y, Kawamoto M, Katsumata Y, Takagi K, Nakajima A, Mitamura T, Mimori A, Hara M, Kamatani N (2007) Association of the diplotype configuration at the N-acetyltransferase 2 gene with adverse events with co-trimoxazole in Japanese patients with systemic lupus erythematosus. Arthritis Res Ther 9:R23. https://doi.org/10.1186/ar2134

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  80. Cooper GS, Treadwell EL, Dooley MA, St Clair EW, Gilkeson GS, Taylor JA (2004) N-acetyl transferase genotypes in relation to risk of developing systemic lupus erythematosus. J Rheumatol 31:76–80

    CAS  PubMed  Google Scholar 

  81. Zschieschang P, Hiepe F, Gromnica-Ihle E, Roots I, Cascorbi I (2002) Lack of association between arylamine N-acetyltransferase 2 (NAT2) polymorphism and systemic lupus erythematosus. Pharmacogenetics 12:559–563

    Article  CAS  PubMed  Google Scholar 

  82. Rocha L, Garcia C, de Mendonca A, Gil J, Bishop D, Lechner M (1999) N-acetyltransferase (NAT2) genotype and susceptibility of sporadic Alzheimer’s disease. Pharmacogenetics 9(1):9–15

    Article  CAS  PubMed  Google Scholar 

  83. Meyer D, Parkin DP, Seifart HI, Maritz JS, Englebrecht AH, Werely CJ, van Helden PD (2003) NAT2 slow acetylator function as a risk indicator for age-related cataract formation. Pharmacogenetics 13:285–289

    Article  CAS  PubMed  Google Scholar 

  84. Cartwright RA, Rogers HJ, Barham-Hall D, Glashan RW, Ahmad RA, Higgins E, Kahn MA (1982) Role of n-acetyltransferase phenotypes in bladder carcinogenesis: a pharmacogenetic epidemiological approach to bladder cancer. Lancet 320(8303):842–846

    Article  Google Scholar 

  85. Hein DW (2006) N-acetyltransferase 2 genetic polymorphism: effects of carcinogen and haplotype on urinary bladder cancer risk. Oncogene 25:1649–1658

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  86. Golka K, Prior V, Blaszkewicz M, Bolt HM (2002) The enhanced bladder cancer susceptibility of NAT2 slow acetylators towards aromatic amines: a review considering ethnic differences. Toxicol Lett 128:229–241

    Article  CAS  PubMed  Google Scholar 

  87. Quan L, Chattopadhyay K, Nelson HH, Chan KK, Xiang Y-B, Zhang W, Wang R, Gao Y-T, Yuan J-M (2016) Differential association for N-acetyltransferase 2 genotype and phenotype with bladder cancer risk in Chinese population. Oncotarget 7:40012–40024

    Article  PubMed  PubMed Central  Google Scholar 

  88. Nunes T, Rocha JF, Vaz-da-Silva M, Igreja B, Wright LC, Falcao A, Almeida L, Soares-da-Silva P (2010) Safety, tolerability, and pharmacokinetics of Etamicastat, a novel dopamine-β-hydroxylase inhibitor, in a rising multiple-dose study in young healthy subjects. Drugs R D 10(4):225–242

    Article  PubMed  PubMed Central  Google Scholar 

  89. Loureiro AI, Rocha JF, Fernandes-Lopes C, Nunes T, Wright LC, Almeida L, Soares-da-Silva P (2014) Human disposition, metabolism and excretion of Etamicastat, a reversible, peripherally selective dopamine β-hydroxylase inhibitor. Br J Clin Pharmacol 77(6):1017–1026

    Article  CAS  PubMed  Google Scholar 

  90. Nunes T, Rocha JF, Vaz-da-Silva M, Falcao A, Almeida L, Soares-da-Silva P (2011) Pharmacokinetics and tolerability of Etamicastat following single and repeated administration in elderly versus young healthy male subjects: an open-label, single-center, parallel-group study. Clin Ther 33(6):776–791

    Article  CAS  PubMed  Google Scholar 

  91. Rocha JF, Vaz-da-Silva M, Nunes T, Igreja B, Loureiro AI, Bonifacio MJ, Wright LC, Falcao A, Almeida L, Soares-da-Silva P (2012) Single-dose tolerability, pharmacokinetics, and pharmacodynamics of Etamicastat (BIA 5-453), a new dopamine β-hydroxylase inhibitor, in healthy subjects. J Clin Pharmacol 52(2):156–170

    Article  CAS  PubMed  Google Scholar 

  92. Loureiro AI, Fernandes-Lopes C, Bonifacio MJ, Wright LC, Soares-da-Silva P (2013) N-acetylation of Etamicastat, a reversible dopamine-b-hydroxylase inhibitor. Drug Metab Dispos 41:2081–2086

    Article  CAS  PubMed  Google Scholar 

  93. Rioux N, Mitchell LH, Tiller P, Plant K, Shaw J, Frost K, Ribich S, Moyer MP, Copeland RA, Chesworth C, Waters NJ (2015) Structural and kinetic characterization of a novel N-acetylated aliphatic amine metabolite of the PRMT inhibitor, EPZ011652. Drug Metab Dispos 43:936–943

    Article  CAS  PubMed  Google Scholar 

  94. Madeo F, Eisenberg T, Pietrocola F, Kroemer G (2018) Spermidine in health and disease. Science 359:eaan2788

    Article  PubMed  CAS  Google Scholar 

  95. Coleman CS, Stanley BA, Jones AD, Pegg AE (2004) Spermidine/spermine-N1-acetyltransferase-2 (SSAT2) acetylates thialysine and is not involved in polyamine metabolism. Biochem J 384:139–148

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  96. Hyovnen MT, Weisell J, Khomutov AR, Alhonen L, Vepsalainen J, Keinanen TA (2013) Metabolism of triethylenetetramine and 1,12-diamino-3,6,9-triazadodecane by the spermidine/spermine-N1-acetyltransferase and thialysine acetyltransferase. Drug Metab Dispos 41:30–32

    Article  CAS  Google Scholar 

  97. Tampaki M, Gatselis NK, Savvanis S, Koullias E, Saitis A, Gabeta S, Deutsch M, Manesis E, Dalekos GN, Koskinas J (2019) Wilson disease: 30-year data on epidemiology, clinical presentation, treatment modalities and disease outcomes from two tertiary Greek centers. Eur J Gastroenterol Hepatol 32(12):1545–1552. https://doi.org/10.1097/meg.0000000000001670

    Article  Google Scholar 

  98. Kodama H, Murata Y, Iitsuka T, Abe T (1997) Metabolism of administered triethylenetetramine dihydrochloride in humans. Life Sci 61(9):899–907. https://doi.org/10.1016/S0024-3205(97)00592-4

    Article  CAS  PubMed  Google Scholar 

  99. Lu J, Poppitt SD, Othman AA, Sunderland T, Ruggiero K, Willett MS, Diamond LE, Garcia WD, Roesch BG, Cooper GJS (2010) Pharmacokinetics, pharmacodynamics, and metabolism of triethylenetetramine in healthy human participants: an open-label trial. J Clin Pharmacol 50:647–658. https://doi.org/10.1177/0091270009349379

    Article  CAS  PubMed  Google Scholar 

  100. Cerrada-Gimenez M, Weisell J, Hyvonen MT, Park MH, Alhonen L, Vepsalainen J, Keinanen TA (2011) Complex N-acetylation of triethylenetetramine. Drug Metab Dispos 39:2242–2249

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Bras APM, Hoff HR, Aoki FY, Sitar DS (1998) Amantadine acetylation may be effected by acetyltransferases other than NAT1 or NAT2. Can J Physiol Pharmacol 76:701–706

    Article  CAS  PubMed  Google Scholar 

  102. Bras APM, Janne J, Porter CW, Sitar DS (2001) Spermidine/spermine N1-acetyltransferase catalyzes amantadine acetylation. Drug Metab Dispos 29:676–680

    CAS  PubMed  Google Scholar 

  103. Battaglia V, DeStano Shields C, Murray-Stewart T, Casero RA Jr (2014) Polyamine catabolism in carcinogenesis: potential targets for chemotherapy and chemoprevention. Amino Acids 46:511–519. https://doi.org/10.1007/s00726-013-1529-6

    Article  CAS  PubMed  Google Scholar 

  104. Bewley MC, Graziano V, Jiang J, Matz E, Studier FW, Pegg AE, Coleman CS, Flanagan JM (2006) Structures of wild-type and mutant human spermidine/spermine N1-acetyltransferase, a potential therapeutic drug target. Proc Natl Acad Sci U S A 103:2063–2068

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Di Paolo ML, Cervelli M, Mariottini P, Leonetti A, Polticelli F, Rosini M, Milelli A, Basagni F, Venerando R, Agostinelli E, Minarini A (2019) Exploring the activity of polyamine analogues on polyamine and spermine oxidase: methoctramine, a potent and selective inhibitor of polyamine oxidase. J Enzyme Inhib Med Chem 34(1):740–752

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  106. Thomas T, Thomas TJ (2003) Polyamine metabolism and cancer. J Cell Mol Med 7(2):113–126

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Thomas TJ, Thomas T, John S, Hsu H-C, Yang P, Keinanen TA, Hyovnen MT (2016) Tamoxifen metabolite endoxifen interferes with the polyamine pathway in breast cancer. Amino Acids 48:2293–2302

    Article  CAS  PubMed  Google Scholar 

  108. Klotz U (1985) Clinical pharmacokinetics of sulphasalazine, its metabolites and other prodrugs of 5-aminosalicylic acid. Clin Pharmacokinet 10(4):285–302

    Article  CAS  PubMed  Google Scholar 

  109. Azad Khan AK, Nurazzaman M, Truelove SC (1983) The effect of the acetylator phenotype on the metabolism of sulphasalazine in man. J Med Genet 20(1):30–36

    Article  CAS  PubMed  Google Scholar 

  110. Adam AM, Rogers HJ, Amiel SA, Rubens RD (1984) The effect of acetylator phenotype on the disposition of aminoglutethimide. Br J Clin Pharmacol 18:495–505

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Sanders GL, Rawlins MD (1979) Phenelzine: acetylator status and clinical response. Br J Clin Pharmacol 7:451–452

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. Grant DM, Blum M, Beer M, Meyer UA (1991) Monomorphic and polymorphic human arylamine N-acetyltransferases: a comparison of liver isozymes and expressed products of two cloned genes. Mol Pharmacol 39:184–191

    CAS  PubMed  Google Scholar 

  113. Palamanda JR, Hickman D, Ward A, Sim E, Romkes-Sparks M, Unadkat JD (1995) Dapsone acetylation by human liver arylamine N-acetyltransferase and interaction with antiopportunistic infection drugs. Drug Metab Dispos 23:473–477

    CAS  PubMed  Google Scholar 

  114. Weisell J, Hyvenen MT, Hakkinen MR, Grigorenko NA, Pietila M, Lampinen A, Kochetkov SN, Alhonen L, Vepsalainen J, Keinanen TA, Khomutov AR (2010) Synthesis and biological characterization of novel charge-deficient spermine analogues. J Med Chem 53:5738–5748

    Article  CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Molecular Pharmacology and Physiology Graduate Training Program, Brown University, Providence, RI, USA

    Jennifer L. Dumouchel

  2. Translational Medicine, Novartis Institutes for BioMedical Research, Inc., Cambridge, MA, USA

    Valerie M. Kramlinger

Authors
  1. Jennifer L. Dumouchel
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  2. Valerie M. Kramlinger
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Corresponding author

Correspondence to Jennifer L. Dumouchel .

Editor information

Editors and Affiliations

  1. Department of Pharmaceutical Sciences, Temple University School of Pharmacy, Philadelphia, PA, USA

    Swati Nagar

  2. Translational Medicine, Novartis Institutes for Biomedical Research, Inc., Cambridge, MA, USA

    Upendra A. Argikar

  3. Independent, Harleysville, PA, USA

    Donald Tweedie

Appendix 1

Appendix 1

The following represents a general NAT enzyme kinetics protocol that assesses the metabolism of p-aminobenzoic acid (PABA) to N-acetyl p-aminobenzoic acid (N-acetyl-PABA) by human liver and kidney cytosolic fractions. Alternative in vitro systems that have been reported in the literature include liver S9 fractions and recombinant human NAT [63, 64, 93]. Before using the outlined protocol, protein linearity and time studies should be assessed for each substrate as described earlier in this book (see Chapter 22. Case Study 2). For this 30-minute incubation example, the final protein concentration was 0.5 mg/mL.

1.1 Reagents

  1. (a)

    Cytosolic fractions, pooled human liver and kidney, 5 mg/mL protein

  2. (b)

    Potassium phosphate, monobasic K2PO4

  3. (c)

    Potassium phosphate dibasic, KH2PO4

  4. (d)

    Ethylenediaminetetraacetic acid, EDTA

  5. (e)

    Acetyl coenzyme A sodium salt, acetyl CoA

  6. (f)

    4-Aminobenzoic acid, PABA

  7. (g)

    4-Acetamidobenzoic acid, N-acetyl-PABA

  8. (h)

    4-Aminobenzoic acid-d4

  9. (i)

    Tris (2-carboxyethyl) phosphine hydrochloride, TCEP

  10. (j)

    1,4-Dithiothreitol, DTT

1.2 Protocol

  1. 1.

    Prepare the 0.1 M phosphate buffer and store at 4 °C until use:

    1. (a)

      Prepare 1 L 100 mM K2HPO4 stock solution and 0.5 L 100 mM KH2PO4 stock solution in deionized water

    2. (b)

      Add 802 mL dibasic phosphate stock solution (0.1 M K2HPO4) and 198 mL monobasic phosphate stock (0.1 M KH2PO4) and mix well

    3. (c)

      Stir and titrate buffer to pH 7.4 with either mono or dibasic phosphate stock solution, as appropriate

  2. 2.

    Prepare the following stock solutions on the day of the experiment and store on ice until use:

    1. (a)

      100 mM EDTA in 0.1 M phosphate buffer

    2. (b)

      20 mM acetyl-CoA in phosphate buffer

    3. (c)

      100 mM DTT in phosphate buffer

  3. 3.

    Combine all incubation constituents according to Table 5, except cofactor, and incubate in a 37 °C water bath for 2 min

  4. 4.

    Add cofactor, acetyl-CoA, to start the reaction

  5. 5.

    Incubate for 30 min and stop the reaction by adding an equal volume of acetonitrile or methanol with 100 ng/mL of internal standard (4-aminobenzoic acid-d4)

  6. 6.

    Centrifuge quenched reaction mixture at 13,000 × g for 10 min

  7. 7.

    Analyze samples by LC-MS/MS. Based on instrument sensitivity, samples can be run with or without a 1–100× water dilution alongside an authentic product (N-acetyl-PABA) reference standard curve. (See Chapter 22. Case Study 2 for a discussion on analytical sensitivity and impact detectors’ dynamic ranges)

Table 5 Summarized incubation conditions, added volumes, and final reagent concentrations for 0.5 mL incubation volume
Full size table

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Dumouchel, J.L., Kramlinger, V.M. (2021). Case Study 10: A Case to Investigate Acetyl Transferase Kinetics. In: Nagar, S., Argikar, U.A., Tweedie, D. (eds) Enzyme Kinetics in Drug Metabolism. Methods in Molecular Biology, vol 2342. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1554-6_29

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  • DOI: https://doi.org/10.1007/978-1-0716-1554-6_29

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1553-9

  • Online ISBN: 978-1-0716-1554-6

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