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Mechanisms of Action, Repair and Resistance

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Mechanisms of Action, Repair and Resistance 102 Current Molecular Pharmacology, 2012, 5, 102-114 Temozolomide: Mechanisms of Action, Repair and Resistance Jihong Zhang1, Malcolm F.G. Stevens2 and Tracey D. Bradshaw*,2 1Faculty of Life Science, Kunming University of Science and Technology, Kunming, Yunnan, 650093, China 2Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK Abstract: Glioblastoma multiforme is the most common aggressive adult primary tumour of the central nervous system. Treatment includes surgery, radiotherapy and adjuvant temozolomide (TMZ) chemotherapy. TMZ is an alkylating agent prodrug, delivering a methyl group to purine bases of DNA (O6-guanine; N7-guanine and N3-adenine). The primary cytotoxic lesion, O6-methylguanine (O6-MeG) can be removed by methylguanine methyltransferase (MGMT; direct repair) in tumours expressing this protein, or tolerated in mismatch repair-deficient (MMR-) tumours. Thus MGMT or MMR deficiency confers resistance to TMZ. Inherent- and acquired resistance to TMZ present major obstacles to successful treatment. Strategies devised to thwart resistance and enhance response to TMZ, including inhibition of DNA repair mechanisms which contribute to TMZ resistance, are under clinical evaluation. Depletion of MGMT prior to alkylating agent chemotherapy prevents O6-MeG repair; thus, MGMT pseudosubstrates O6-benzylguanine and lomeguatrib are able to sensitise tumours to TMZ. Disruption of base excision repair (BER) results in persistence of potentially lethal N7- and N3- purine lesions contributing significantly to TMZ cytoxicity particularly when O6-MeG adducts are repaired or tolerated. Several small molecule inhibitors of poly(ADP-ribose)polymerase-1 (PARP-1), a critical BER protein are yielding promising results clinically, both in combination with TMZ and as single agent chemotherapy in patients whose tumours possess homologous recombination DNA repair defects. Another validated, but as yet preclinical protein target, mandatory to BER is abasic (AP) endonuclease-1 (APE-1); in preclinical tests, APE-1 inhibition potentiates TMZ activity. An alternative strategy is synthesis of a molecule, evoking an irrepairable cytotoxic O6-G lesion. Preliminary efforts to achieve this goal are described. Keywords: Base excision repair, glioblastoma multiforme, methyltransferase, mismatch repair, O6-methylguanine methylguanine, temozolomide. INTRODUCTION administration [6], but labile above pH 7, with a plasma half- life of 1.8 hours at pH 7.4 [7]. Thus, TMZ is rapidly Malignant glioma is the most common adult primary absorbed intact, but then undergoes spontaneous breakdown tumour of the central nervous system (CNS). Median to form monomethyl triazene 5-(3-methyltriazen-1-yl)- survival from time of diagnosis is approximately 12 – 15 imidazole-4-carboxamide (MTIC). MTIC further reacts with months [1-2]. Glioblastoma multiforme (GBM; grade IV water to liberate 5-aminoimidazole-4-carboxamide (AIC) astrocytoma) is the most prevalent and aggressive adult and the highly reactive methyldiazonium cation (Fig. 1). The primary brain tumour whose hallmark features include active species methyldiazonium cation preferentially uncontrolled cellular proliferation, diffuse infiltration, methylates DNA at N7 positions of guanine in guanine rich resistance to apoptosis, robust angiogenesis and rampant regions (N7-MeG; 70%), but also methylates N3 adenine genomic instability [3]. The current standard of care for (N3-MeA; 9%) and O6 guanine residues (O6-MeG; 6%) [8- newly diagnosed GBM patients includes surgery, 9]. radiotherapy and adjuvant temozolomide (TMZ) treatment, There is a narrow pH window close to physiological pH conferring a median survival time of 14.6 months compared at which the whole process of TMZ prodrug activation to with 12.2 months for patients receiving radiotherapy alone. methyl group transfer can occur. Brain tumours possess a Although TMZ (Temodar; Schering-Plough Corporation) more alkaline pH compared with surrounding healthy tissue, offers some hope to GBM patients, a best 5-year survival a situation which favours prodrug activiation preferentially rate of only 9.8% is achieved [4-5]. within tumour tissue [10]. TEMOZOLOMIDE PRODRUG ACTIVATION Thus TMZ is used to treat (but not exclusively) brain tumours, imparting significant therapeutic benefit to GBM TMZ, a small (194 Da) lipophilic molecule (Fig. 1), is an patients [4]. orally available monofunctional DNA alkylating agent of the imidazotetrazine class. TMZ acts as a prodrug, stable at TEMOZOLOMIDE CYTOTOXICITY acidic pH values, a property which permits oral Temozolomide cytotoxicity is primarily mediated through O6-MeG, a carcinogenic, mutagenic and toxic lesion *Address correspondence to this author at the Centre for Biomolecular [11-14]. Direct repair of O6-MeG by the suicide enzyme Sciences, School of Pharmacy, University of Nottingham, NG7 2RD, UK; methylguanine-DNA methyltransferase (MGMT) removes Tel: +44(0)115 9513419; Fax: +44(0)115 9515102; E-mail: [email protected] the methyl adduct, restoring guanine (Fig. 2). Unrepaired 1874-4672/12 $58.00+.00 © 2012 Bentham Science Publishers Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 103 H2NOC H2NOC H N N N CH3 N H O N N 2 - CO N H+ N N 2 NH CH3 O H2NOC NH2 NuH + N + NuCH3 + N2 + H NH CH3N2 (Nu = nucleophilic centre on DNA) Fig. (1). Structure and activation route of prodrug temozolomide. O6-MeG mispairs with thymine (not cytosine) during DNA DNA REPAIR MECHANISMS CONTRIBUTING TO replication, alerting DNA mismatch repair (MMR) [15-16]. TEMOZOLOMIDE RESISTANCE MMR exclusively recognises the mispaired thymine on the daughter strand and excises it, yet O6-MeG persists in the Direct Repair template strand. Therefore, futile cycles of thymine re- insertion and excision result in persistent DNA strand MGMT (O6-Alkylguanine-DNA alkyltransferase; AGT) repairs O6-alkylguanine adducts in a single step, breaks, causing replication fork collapse [17]. G2/M cell cycle arrest is triggered, occurring in the second cell cycle independently of any other protein or cofactors (Fig. 2). It is following treatment [18-20] via ATR/CHK1-dependent a small protein (22 kDa) present in both the cytoplasm and signalling [21]; ultimately, apoptosis ensues [22] (Figs. 3 and nucleus. Upon DNA alkylation, a shift towards more nuclear 4). A good response to TMZ therefore requires functional localisation may facilitate the repair process [24]. MGMT is MMR and low levels of MGMT. able to repair not only O6-MeG, but also guanine residues with longer O6-alkyl adducts such as ethyl, chloroethyl, The quantitatively more abundant N7-MeG and N3-MeA hydroxyethyl, n-propyl, n-butyl, and more bulky cyclic lesions are rapidly repaired by DNA base excision repair lesions conferred by benzyl or pyridyloxobutyl groups, but (BER; Figs. 4 and 5). N7-MeG appears not to be markedly with diminishing efficiency as adduct size increases [25-27]. cytotoxic: in contrast, N3-MeA lesions are lethal if not The O6-alkyl group is transferred from guanine to the active intercepted [23]. site cysteine residue (Cys 145) of MGMT in a Therefore, the most important DNA repair systems stoichiometric, auto-inactivating reaction, thereby repairing impacting the mechanism of action and cytotoxicty of TMZ DNA and inactivating MGMT [28]. MGMT binds damaged are MGMT (direct repair), MMR and BER (Figs. 4 and 5). substrate DNA in the minor groove, the target base is then MGMT MGMT Cysteine 145 Cysteine 145 CH3 H NOC 2 N N CH3 N NN O O CH3 66 N N O N HN H H TMZ N N H2N N H2N N d Ribose d Ribose O6-methylguanine guanine Fig. (2). Repair of O6-methylguanine adducts by O6-methylguanine-DNA methyltransferase. 104 Current Molecular Pharmacology, 2012, Vol. 5, No. 1 Zhang et al. A B 600 500 MGMT U373V U373M SNB19M SNB19V 400 m) m ( 300 50 I G 200 100 0 SNB19V SNB19M U373V U373M Cell Line C Control 24 h 48 h 72 h 120 h Fig. (3). A) Effect of temozolomide on glioblastoma multiforme cell growth. Cells were seeded at a density of 650 per well. After 24 h, temozolomide was introduced. At the time of drug addition and following 7 days incubation, MTT assays were performed to determine cell growth. B) Expression of methylguaninemethyltransferase protein in SNB19M and U373M cells. Western blot assays were performed following separation of protein from whole cell lysates. C) Effect of temozolomide on SNB19V cell cycle. MGMT+ G survival O6-Me-G MMR+ cytotoxicity MGMT- TMZ MMR- N7-,N3-Me-Purine mutations tolerated BER survival Fig. (4). Key DNA repair mechanisms influencing cellular response to temozolomide. flipped out of the helix and bound to MGMT, altering the alkylating agents such as TMZ. Tissue expression is conformation of the DNA binding domain allowing variable, with high protein expression in liver and lower alkylated MGMT to be detached from DNA and degraded expression in haematopoietic tissues and brain [30-31]. through the ubiquitin/proteasomal system [15, 29]. Tumour MGMT expression is immensely variable, highest levels are found in breast, ovarian and lung tumours, MGMT protects cells from carcinogens; however, it is whereas lowest activity is observed in gliomas, pancreatic also able to protect cancer cells from chemotherapeutic Temozolomide: Mechanisms of Action, Repair and Resistance Current Molecular Pharmacology, 2012, Vol. 5, No. 1 105 TMZ O6-MeG N7-MeG N3-MeA DR MMR BER MGMT MSH2-MSH6 Glycosylase MSH2-MSH3 (Mutsα and β) APE-1 MLH1-PMS2 PARP MLH1-PMS1 (MutlLα and β) short patch long patch
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