6 Mechanism of action of methotrexate Mechanism of action of methotrexate. Methotrexate (MTX) enters the cell through the reduced folate carrier (a) using an endocytic pathway activated by a folate receptor (b). After entering the cell, methotrexate is polyglutamated (Glu) by the the enzyme folylpolyglutamate synthase (c). Methotrexate and its polyglutamates inhibit the enzyme dihydrofolate reductase (d), thereby blocking the conversion of dihydrofolate (FH2) to tetrahydrofolate (FH4). As tetrahydrofolate stores are depleted, thymidylate (TMP) synthesis (e) is reduced,which ultimately inhibits DNA synthesis (f). Long-chain polyglutamates of MTX have the same affinity as MTX for the target enzyme dihydrofolate reductase, but have markedly increased inhibitory effects on both thymidylate synthesis (e) and purine biosynthesis (f), which is required for RNA production. Adapted from figure 1 of Ref. 91.
8 BIOSINTESI DELLE BASI PURINICHE METENILTETRAIDROFOLATON10-FORMILTETRAIDROFOLATO
9 EFFETTI COLLATERALI DEGLI ANTIFOLATI mielosoppressione, trombocitopeniapolmonitefibrosi epatica e cirrosiembriotossicità
10 Folate metabolism and related pathways Folate metabolism and related pathways. This simplified figure illustrates the interconnectedness of folate metabolism and proteins for which functional polymorphisms have been identified. Polymorphisms have been found that are associated with pharmacogenetic outcomes in three key proteins in these pathways: the drug transporter protein reduced folate carrier (RFC); the regulatory enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR); and the drug target thymidylate synthase. Key enzymes are denoted as ovals, substrates as rectangles. Red ovals denote enzymes with genetic polymorphisms that have been investigated in pharmacogenetic studies. Orange ovals denote enzymes for which functional genetic polymorphisms have been described. 5-FU, 5-fluorouracil; AICAR, 5-aminoimidazole-4-carboxamine ribonucleotide; AICARFT, AICAR formyltransferase; CBS, cystathionine- -synthase; DHF, dihydrofolate; DHFR, DHF reductase; dTMP, deoxythymidine monophosphate; dUMP, deoxyuridine monophosphate; GAR, glycinamide ribonucleotide; GART, phosphoribosylglycinamide formyltransferase; hFR, human folate receptor; MTX, methotrexate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; SHMT, serine hydroxymethyltransferase; THF, tetrahydrofolate; X, various substrates for methylation.
11 ASPETTI FARMACOGENETICI SNP a livello del gene che codifica per MTHFRDiverso numero di sequenze ripetute a livello di TSERSNP a livello del gene che codifica per RCF
12 MECCANISMI DI RESISTENZA AGLI ANTIFOLATI impaired transport into cellsincreased efflux by ABC transporters
19 ANALOGHI DELLE NUCLEOBASI E DEI NUCLEOSIDI Figure 1. Common characteristics in metabolism and drug–target interactions of nucleoside analogues. Most of these agents are hydrophilic molecules and therefore require specialised transporter proteins to enter cells. Once inside, they are activated by intracellular metabolic steps to triphosphate derivatives. Active derivatives of nucleoside analogues can exert cytotoxic activity by being incorporated into and altering the DNA and RNA macromolecules or by interfering with various enzymes involved in synthesis of nucleic acids, such as DNA polymerases and ribonucleotide reductase. These actions result in inhibition of DNA synthesis and apoptotic cell death.
21 5-Fluorouracil metabolism 5-Fluorouracil metabolism. 5-Fluorouracil (5-FU; see structure) is converted to three main active metabolites: fluorodeoxyuridine monophosphate (FdUMP), fluorodeoxyuridine triphosphate (FdUTP) and fluorouridine triphosphate (FUTP). The main mechanism of 5-FU activation is conversion to fluorouridine monophosphate (FUMP), either directly by orotate phosphoribosyltransferase (OPRT) with phosphoribosyl pyrophosphate (PRPP) as the cofactor, or indirectly via fluorouridine (FUR) through the sequential action of uridine phosphorylase (UP) and uridine kinase (UK). FUMP is then phosphorylated to fluorouridine diphosphate (FUDP), which can be either further phosphorylated to the active metabolite fluorouridine triphosphate (FUTP), or converted to fluorodeoxyuridine diphosphate (FdUDP) by ribonucleotide reductase (RR). In turn, FdUDP can either be phosphorylated or dephosphorylated to generate the active metabolites FdUTP and FdUMP, respectively. An alternative activation pathway involves the thymidine phosphorylase catalysed conversion of 5-FU to fluorodeoxyuridine (FUDR), which is then phosphorylated by thymidine kinase (TK) to FdUMP. Dihydropyrimidine dehydrogenase (DPD)-mediated conversion of 5-FU to dihydrofluorouracil (DHFU) is the rate-limiting step of 5-FU catabolism in normal and tumour cells. Up to 80% of administered 5-FU is broken down by DPD in the liver.
22 MECCANISMI D’AZIONE DEL 5-FLUOROURACILE Inibizione della timidilato sintasi
24 MECCANISMI D’AZIONE DEL 5-FLUOROURACILE Inibizione della timidilato sintasiIncorporazione di FUTP nel RNA
25 Mechanism of thymidylate synthase inhibition by 5-fluorouracil Mechanism of thymidylate synthase inhibition by 5-fluorouracil. Thymidylate synthase (TS) catalyses the conversion of deoxyuridine monophosphate (dUMP) to deoxythymidine monophosphate (dTMP) with 5,10-methylene tetrahydrofolate (CH2THF) as the methyl donor. The 5-fluorouracil (5-FU) active metabolite fluorodeoxyuridine monophosphate (FdUMP) binds to the nucleotide-binding site of TS and forms a stable ternary complex with TS and CH2THF, blocking access of dUMP to the nucleotide-binding site and inhibiting dTMP synthesis. This results in deoxynucleotide (dNTP) pool imbalances and increased levels of deoxyuridine triphosphate (dUTP), both of which cause DNA damage. The extent of DNA damage caused by dUTP is dependent on the levels of the pyrophosphatase dUTPase and uracil-DNA glycosylase (UDG). dTMP can be salvaged from thymidine through the action of thymidine kinase (TK).
26 MECCANISMI D’AZIONE DEL 5-FLUOROURACILE Inibizione della timidilato sintasiIncorporazione di FUTP nel RNAIncorporazione di FdUTP nel DNA
27 Modulation of 5-fluorouracil activity Modulation of 5-fluorouracil activity. Summary of some of the strategies that have been investigated for increasing the anticancer activity of 5-fluorouracil (5-FU). Leucovorin (LV) increases the intracellular pool of 5,10-methylene tetrahydrofolate (CH2THF), thereby enhancing thymidylate synthase (TS) inhibition by fluorodeoxyuridine monophosphate (FdUMP). Eniluracil and uracil inhibit DPD-mediated degradation of 5-FU. Methotrexate (MTX) is thought to increase 5-FU activation by increasing phosphoribosyl pyrophosphate (PRPP) levels. Interferons (IFNs) have been reported to enhance thymidine phosphorylase (TP) activity, abrogate acute TS induction caused by 5-FU treatment and enhance 5-FU-mediated DNA damage. Capecitabine is a 5-FU pro-drug that is converted to 5'-deoxy-5-fluorouridine (5'DFUR) in the liver by the sequential action of carboxylesterase and cytidine deaminase. 5'DFUR is converted to 5-FU by TP.
28 Activation of p53 by 5-fluorouracil Activation of p53 by 5-fluorouracil. 5-Fluorouracil (5-FU) can activate p53 by more than one mechanism: incorporation of fluorouridine triphosphate (FUTP) into RNA, incorporation of fluorodeoxyuridine triphosphate (FdUTP) into DNA and inhibition of thymidylate synthase (TS) by fluorodeoxyuridine monophosphate (FdUMP) with resultant DNA damage. TS-directed cytotoxicity is abrogated by increased TS expression, whereas RNA-directed cytoxicity can be abrogated by increasing the intracellular levels of uridine.
29 EFFETTI TOSSICI DEL 5FUEFFETTI GI: anoressia e nausea; stomatite e diarreaMIELOSOPPRESSIONEMANIFESTAZIONI NEUROLOGICHETOSSICITÀ CARDIACAMANIFESTAZIONI CUTANEE
31 MECCANISMI DI RESISTENZA AL 5FU PERDITA O ATTIVITÀ DEGLI ENZIMI ATTIVATORI PIRIMIDINA MONOFOSFATO CHINASI LIVELLI DELL’ENZIMA DEGRADATIVO DPDPRODUZIONE DI FORME ALTERATE DI TS
32 a | Mutations in dihydropyrimidine dehydrogenase (DPD) affect 5-FU metabolism46, 49. The wild-type DPD gene contains a GT nucleotide sequence at exon 14 (wt), required for normal catabolism of 5-FU to dihydro-5-FU. A G-to-A transition at the exon 14 splice site leads to deletion of the entire exon during transcription (mut), and synthesis of a nonfunctional protein. Patients with low hepatic DPD activity cannot efficiently metabolize 5-FU (which is used to treat colon and breast cancer) and its accumulation causes toxicity. b | Polymorphisms in thymidylate synthase (TS) affect 5-FU efficacy. In tumour tissue, the active metabolite of 5-FU, 5-fluorodeoxyuridine monophosphate (5-FdUMP), inhibits TS. The presence of triple tandem repeats of 28 base pairs in the enhancer region of the TS gene is associated with higher TS activity and lower probability of response to 5-FU when compared with patients that have only two tandem repeats.
33 Modulation of 5-fluorouracil activity Modulation of 5-fluorouracil activity. Summary of some of the strategies that have been investigated for increasing the anticancer activity of 5-fluorouracil (5-FU). Leucovorin (LV) increases the intracellular pool of 5,10-methylene tetrahydrofolate (CH2THF), thereby enhancing thymidylate synthase (TS) inhibition by fluorodeoxyuridine monophosphate (FdUMP). Eniluracil and uracil inhibit DPD-mediated degradation of 5-FU. Methotrexate (MTX) is thought to increase 5-FU activation by increasing phosphoribosyl pyrophosphate (PRPP) levels. Interferons (IFNs) have been reported to enhance thymidine phosphorylase (TP) activity, abrogate acute TS induction caused by 5-FU treatment and enhance 5-FU-mediated DNA damage. Capecitabine is a 5-FU pro-drug that is converted to 5'-deoxy-5-fluorouridine (5'DFUR) in the liver by the sequential action of carboxylesterase and cytidine deaminase. 5'DFUR is converted to 5-FU by TP.
34 TRATTAMENTI COMBINATI CONTENENTI FLUOROPIRIMIDiNE UFT: ftorafur (profarmaco del 5FU)uracile (inibitore della DPD)S1: ftorafur (profarmaco del 5FU)5-cloro-2,4-diidrossipiridina (inibitore della DPD)potassio oxonato (inibitore della fosforibosil-pirofosfato transferasi, riduce i sintomiintestinali)
37 Modulation of 5-fluorouracil activity Modulation of 5-fluorouracil activity. Summary of some of the strategies that have been investigated for increasing the anticancer activity of 5-fluorouracil (5-FU). Leucovorin (LV) increases the intracellular pool of 5,10-methylene tetrahydrofolate (CH2THF), thereby enhancing thymidylate synthase (TS) inhibition by fluorodeoxyuridine monophosphate (FdUMP). Eniluracil and uracil inhibit DPD-mediated degradation of 5-FU. Methotrexate (MTX) is thought to increase 5-FU activation by increasing phosphoribosyl pyrophosphate (PRPP) levels. Interferons (IFNs) have been reported to enhance thymidine phosphorylase (TP) activity, abrogate acute TS induction caused by 5-FU treatment and enhance 5-FU-mediated DNA damage. Capecitabine is a 5-FU pro-drug that is converted to 5'-deoxy-5-fluorouridine (5'DFUR) in the liver by the sequential action of carboxylesterase and cytidine deaminase. 5'DFUR is converted to 5-FU by TP.
39 PATHWAYS PER L’ATTIVAZIONE/DEGRADAZIONE DI AraC
40 MECCANISMO D’AZIONE di AraC competizione con dCTP per l’incorporazione nel DNAinibizione della DNA polimerasi:blocco dell’allungamento della catena di DNAblocco della sintesi riparativa di DNAripetizione di segmenti di DNAaumentata possibilità di ricombinazione, crossover e amplificazione genicacompetizione con CDP-colinainibizione della sintesi di glicoproteine e glicolipidi di membranainibizione del trasferimento di galattosio, N-acetilglucosamina e acido sialico alle glicoproteine di membranainibizione della sintesi di acido CMP-neuraminico
41 MECCANISMI DI RESISTENZA AGLI ANALOGHI DELLA CITOSINA ALTERAZIONE DELLE ATTIVITÀ RELATIVE DEGLI ENZIMI ATTIVATORI O DEGRADATIVEALTERAZIONI A CARICO DEI TRASPORTATORI DI MEMBRANAESPANSIONE DEL POOL CELLULARE DI dCTPSOVRAESPRESSIONE DEL GENE ANTIAPOPTOTICO bcl-2FOSFORILAZIONE DI FATTORI DI TRASCRIZIONE E/O FATTORI DI RISPOSTA A DANNI A CARICO DEL DNA
45 ANALOGHI DELLA CITOSINA Gore et al. Nature Reviews Drug Discovery 5, 891–892 (November 2006)inibitori della metilazione del DNAapprovati per la terapia delle sindromi mielodisplastiche
46 Modificazioni Epigenetiche e Cancro degli istoniMetilazionedel DNARegione promotrice del DNADNAXMetilazione del DNABlocco della trascrizione del RNASilenziamento epigenetico di geni oncosoppressoriEpigenetics and Cancer (Slide 2 of 2)Alterations in epigenetic processes involved in the regulation of gene expression are associated with various types of cancer.1–3Histone deacetylation and other modifications can cause tightening of chromatin and can block transcriptional activation. Histone modification can also attract DNA methyltransferases to initiate cytosine methylation, which in turn can reinforce histone modification patterns conducive to silencing.3Transcriptional repression of tumor suppressor genes may allow cancer cell proliferation.2,4To explain how the aberrations in this process can lead to cancer, we will describe chromatin structure and how it plays a role in DNA transcription and gene expression, the role of HDAC in chromatin remodeling, and the role of abnormal or sustained HDAC in cancer development.References:1. Johnstone RW. Histone-deacetylase inhibitors: novel drugs for the treatment of cancer. Nat Rev Drug Discov. 2002;1:287–299.2. Arts J, de Schepper S, Van Emelen K. Histone deacetylase inhibitors: from chromatin remodeling to experimental cancer therapeutics. Curr Med Chem. 2003;10:2343–2350.3. Egger G, Liang G, Aparicio A, Jones PA. Epigenetics in human disease and prospects for epigenetic therapy. Nature. 2004;429:457–463.4. Marks PA, Richon VM, Miller TA, Kelly WK. Histone deacetylase inhibitors: new targeted anticancer drugs. In: DeVita VT Jr, Hellman S, Rosenberg SA, eds. Cancer: Principles & Practice of Oncology. 7th ed. Philadelphia, Pa: Lippincott Williams & Wilkins; 2005:439–445.
47 METILAZIONE DEL DNADNA methylation. b | A family of DNA methyltransferases (DNMTs) catalyse the methylation of the 5 position of the cytosine ring. After intracellular conversion to 5-aza-2'-deoxycytidine (decitabine), azacitidine (Z) is incorporated in place of cytidine into DNA, where it acts as a direct and irreversible inhibitor of DNMTs. Cells then divide in the absence of DNMTs, which results in progressive DNA hypomethylation and reactivation of previously silenced genes. Azacitidine also incorporates into RNA, but very little is known about the effects of this. Pink circles, methylated CpG; yellow circles, unmethylated CpG.
48 ANALOGHI DELLA CITOSINA competizione tra dFdCTP e dCTPinibizione DNA polimerasiinibizione della ribonuclotide reduttasiincorporazione di dFdCTP nel DNABlocco dell’allungamento della catena dopo l’incorporazione di un ulteriore nucleotide elusione dei sistemi di riparazione del DNAattiva anche su cellule in fase non-S
49 Gemcitabine is a prodrug that requires cellular uptake and intracellular phosphorylation in order to exert its action. Once administered, gemcitabine istransported into cells by nucleoside transporters. Gemcitabine is then phosphorylated into gemcitabine monophosphate (dFdCMP) by deoxycytidine kinase (DCK), and dFdCMP is subsequently phosphorylated to gemcitabine diphosphate (dFdCDP) and gemcitabine triphosphate (dFdCTP) by nucleoside monophosphate (UMP/CMP) and diphosphate kinase.Gemcitabine exerts its cytotoxic effect mainly through inhibition of DNA synthesis by being incorporated into the DNA strand as the active dFdCTP. It is known that gemcitabine has a unique mechanism of action known as ‘self-potentiation’. For example, dFdCDP potently inhibits ribonucleotide reductase, resulting in a decrease of competing deoxyribonucleotide pools necessary for DNA synthesis. Again, dFdCTP suppresses inactivation of dFdCMP by inhibiting deoxycytidine monophosphate deaminase (DCTD). On the other hand, more than 90% of administered gemcitabine is converted, and thus inactivated, by cytidine deaminase (CDA) into 2’-deoxy-2’,2’-difluorouridine (dFdU). Phosphorylated metabolites of gemcitabine are reduced by cellular 5’-nucleotidase (5’-NT), and dFdCMP is also converted, and inactivated, by DCTD into 2’-deoxy-2’,2’-difluorouridine monophosphate (dFdUMP).
52 The thiopurine pathway The thiopurine pathway. After uptake, mercaptopurine is converted through the salvage pathway into thioinosine monophosphate (TIMP) by hypoxanthine guanine phosphoribosyl transferase (HPRT1), with 5-phospho-D-ribose-1-pyrophosphate (PRPP), a component of the de novo purine-synthesis (DNPS) pathway, as the phosphoribosyl donor. TIMP is converted first into thioxanthosine monophosphate (TXMP), then to thioguanosine monophosphate (TGMP). Subsequently, TGMP can be converted into thioguanosine diphosphate (TGDP) and triphosphate (TGTP). Cytotoxic effects occur when deoxyTGTP or TGTP are incorporated into DNA or RNA, respectively, and when DNPS is inhibited, specifically when phosphoribosyl pyrophosphate amidotransferase (PPAT) is inhibited by methyl thionosine monophosphate (meTIMP). DNPS results in the production of the precursor purines inosine monophosphate (IMP) and adenosine monophosphate (AMP) and, finally, inosine and adenosine. The inactivation of mercaptopurine, which is catalysed by xanthine oxidase (XDH) or the polymorphic thiopurine methyltransferase (TPMT), competes with the synthesis of the active metabolites of mercaptopurine (the thioguanine nucleotides TGDP and TGTP). Drugs and drug metabolites are indicated in yellow, transporters in pink, enzymes in green and cellular metabolites in blueMeyling H. Cheok and William E. Evans Nature Reviews Cancer 6, (February 2006)
54 When mercaptopurine (MP) treatment is given to a cohort of patients, dosing can be uniform (a) (that is, conventional doses of 200 mg m-2 week-1 are given to all patients) or individualized according the patient's genotype (b). Given conventional doses of mercaptopurine (a, left panel), thiopurine methyltransferase (TPMT)-deficient patients (v/v; pink) show a markedly (tenfold) higher systemic exposure (a, middle panel) to active thioguanine nucleotides (TGN) than do wild-type patients (wt/wt; green), and heterozygous patients (wt/v; blue) show about twofold higher TGN concentrations. The higher concentrations of TGN translate into a significantly higher frequency of haematopoietic toxicity (a, right panel). When genotype-specifically adjusted doses are given in an individualized therapeutic regimen (b, left panel), similar cellular TGN concentrations are achieved in all patients (b, middle panel), and all three TPMT phenotypes can be treated without acute toxicity (b, right panel).
56 An enzymatic method of purine arabinonucleoside synthesis led to the development of nelarabine, the 6-methoxy derivative of ara-G, which is 10 times more soluble than ara-G4, 5. Nelarabine is demethoxylated intracellularly by adenosine deaminase to generate ara-G, which is then phosphorylated to give the active species, ara-GTP.
57 MECCANISMI DI RESISTENZA AGLI ANALOGHI DELLA GUANINA PERDITA O ATTIVITÀ DELL’ ENZIMA HGPRT TRASPORTO ATTRAVERSO LA MEMBRANA PLASMATICA DELLA VELOCITÀ DI DEGRADAZIONEALTERATA EFFICIENZA DEGLI ENZIMI CHE RIPARANO IL DNA DELL’ATTIVITÀ DELLA MRP5
59 Deficit o inibizione dell’enzima adenosina deaminasi (ADA) Accumulo di dAdenosina e dATPInibizione a feedback della ribonucleotide reduttasi+Inibizione della S-adenosil-omocisteina idrolasiInibizione sintesi e riparazione del DNAInibizione delle reazioni di metilazione
62 Clinically used purine nucleoside analogues Clinically used purine nucleoside analogues. a | Chemical structures of cladribine and fludarabine. b | Clofarabine, a hybrid of cladribine and fludarabine, is also substituted with a halogen at position 2 of the purine ring, which makes it resistant to deamination by adenosine deaminase2. Substitution of a fluorine moiety at the 2' carbon in the arabino configuration in clofarabine increases its stability in acid compared with cladribine and its resistance to cleavage of the glycosidic bond by bacterial purine nucleoside phosphorylases2, 3, which for fludarabine leads to the formation of 2-F adenine, a toxic compound with no antitumour selectivity.
63 FIGURE 2 | Schematic of clofarabine mechanism of action FIGURE 2 | Schematic of clofarabine mechanism of action. Clofarabine is transported into cells by both diffusion and facilitated diffusion. Clofarabine is monophosphorylated by deoxycytidine kinase (dCK) and then serially phosphorylated by other kinases to form clofarabine triphosphate, the active moiety. The triphosphate acts to terminate DNA chain elongation and inhibit repair through incorporation into the DNA chain by competitive inhibition of DNA polymerases27. It also inhibits ribonucleotide reductase with reduction of dNTP pools28, and induces apoptosis through direct and indirect action on mitochondria by releasing cytochrome c and other pro-apoptotic factors29.Bonate et al. Nature Reviews Drug Discovery 5, 855–863 (October 2006)
65 CARATTERISTICHE DEI DERIVATI DELLA DESOSSIADENOSINAResistenza alla adenosina deaminasiAttività anche su cellule non proliferantiinibizione della riparazione del DNAalterazioni mitocondrialiattivazione diretta del programma apoptoticoMielosoppressione ed effetto immunosoppressivo
67 Figure 6. Currently applied strategies to increase the cytotoxicity of nucleoside analogues. One approach is to use high-dose administration of nucleoside analogues. Another is the use of compounds that are insensitive to the effect of degradative enzymes or specific inhibitors of degradative enzymes. Fludarabine, cladribine, and troxacitabine are resistant to degradation by adenosine deaminase and cytidine deaminase, respectively, whereas eniluracil inhibits fluorouracil degradation by dihydropyrimidine dehydrogenase. Different combinations of nucleoside analogues or other cytotoxic drugs potentiate activity of nucleoside analogues by increasing drug metabolism. Finally, combination of nucleoside analogues with DNA-damaging agents agents inhibits DNA-repair induction.
69 MECCANISMI D’AZIONE DELLA IDROSSIUREA effetto scavenger sul radicale libero tirosinico presente nel sito attivoliberazione di NO
70 MECCANISMI DI RESISTENZA ALLA IDROSSIUREA DEI LIVELLI DI RIBONUCLEOTIDE REDUTTASIPRODUZIONE DI FORME ALTERATE DELL’ENZIMA
71 IMPIEGO DELLA IDROSSIUREA IN PATOLOGIE NON NEOPLASTICHE ANEMIA FALCIFORMEINFEZIONE DA HIVTROMBOCITEMIAPOLICITEMIA VERA
72 EFFETTI TOSSICI DEGLI ANALOGHI DELLA IDROSSIUREAMIELODEPRESSIONEEFFETTI GI E DERMATOLOGICI RISCHIO DI INSORGENZA DI LEUCEMIE SECONDARIEEFFETTO TERATOGENO
73 Azathioprine is converted to 6-mercaptopurine (6-MP) by non-enzymatic activation in red blood cells. Both 6-MP and 6-thioguanine (6-TG) are salvaged by the hypoxanthine–guanine phosphoribosyltransferase (HPRT) of immune effector cells and converted into their respective nucleoside monophosphates (TIMP and TGMP). In competing catabolic reactions, thiopurine S-methyltransferase (TPMT) inactivates 6-MP and 6-TG by S-methylation and xanthine oxidase (XO) converts 6-MP to 6-thiouric acid. TIMP and TGMP are also TPMT substrates. Methylated TIMP (meTIMP), but not meTGMP, is an effective inhibitor of de novo purine biosynthesis. TIMP that escapes catabolism is further metabolized by inosine monophosphate dehydrogenase (IMPDH) and guanine monophosphate synthetase (GMPS) to TGMP. Sequential action of deoxynucleoside kinases and reductase generates the thioGTP and thio-dGTP that are the substrates for incorporation of 6-TG into RNA and DNA.
74 a | Correction of replication errors a | Correction of replication errors. Erroneous incorporation of a non-complementary base by the replication apparatus (DNA polymerase plus the polymerase-associated proliferating cell nuclear antigen (PCNA) sliding clamp triggers recruitment of the mismatch recognition factor MutS (an MSH2 and MSH6 dimer). A second dedicated MMR factor MutL (a dimer of MLH1 and PMS2) is then recruited. The endonuclease activity of MutL introduces nicks that promote the loading of EXO1 on the 5' side of the misincorporated nucleotide. The 5'-to-3' exonucleolytic activity of EXO1 excises a stretch of daughter strand of DNA including the mismatch. The extensive gap left by EXO1 is filled by DNA polymerase –PCNA, which now incorporates the correct complementary nucleotide, and mismatch repair is completed by DNA ligase sealing the remaining nick. The key to successful correction is that the incorrect nucleotide resides, by definition, in the newly synthesized daughter DNA strand. b | The presence of a miscoding base analogue (X, for example, methyl 6-thioguanine (me6-TG) or O6-meG) in the template strand can confuse the MMR system. Here me6-TG derived by chemical S-methylation of 6-TG incorporated in an earlier round of replication, or O6-meG produced by a methylating agent, directs the incorporation of T. Me6-TG:T or O6-meG:T base pairs do not escape surveillance by MutS –MutL , which triggers mismatch correction attempts that remain incomplete because of the impossibility of incorporating a perfectly paired base opposite the lesion28, 31. The anomalous DNA structures generated by the incomplete repair attempts can be observed as DNA strand interruptions34. During the S phase of the next cell cycle, these are converted into potentially lethal aberrant DNA structures that are substrates for recombination. These trigger activation of ATR (ataxia telangiectasia and Rad3-related)–ATRIP (ATR-interacting protein) DNA-damage signalling, phosphorylation of CHEK1 and G2 cell-cycle arrest.