Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.711649
Title: Investigating the endogenous role of human N-acetyltransferase 1, as potential breast cancer biomarker, using chemical biology
Author: Laurieri, Nicola
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2012
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Abstract:
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&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.
Supervisor: Sim, Edith Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.711649  DOI: Not available
Keywords: Pharmacology ; Medical sciences
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