Inibitori delle DNA topoisomerasi

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1 Inibitori delle DNA topoisomerasi

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5 STRUTTURA DELLE DNA TOPOISOMERASI II
Regione ATPasica (omologa a GyrB) Regione catalitica (omologa a GyrA) Regione C-terminale (variabile)  a.a.660  a.a.1200 COOH NH2 ATP Y 804 NLS PO4

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7 MECCANISMO D’AZIONE DEI FARMACI ATTIVI SULLA DNA TOPOISOMERASI II

8 MECCANISMO D’AZIONE DEI FARMACI ATTIVI SULLA DNA TOPOISOMERASI II
The catalytic cycle of DNA topoisomerase II. The ATPase domains of topoisomerase II are shown in light blue, the core domain in dark blue, and the active site tyrosine residue in red. The C-terminal domain of the enzyme is not included in the diagram since its orientation, with respect to the rest of the molecule, is not known. The catalytic cycle is initiated by enzyme binding to two double-stranded DNA segments called the G segment (in red) and the T segment (in green) (Step 1). Next, two ATP molecules are bound, which is associated with dimerization of the ATPase domains (Step 2). The G segment is cleaved (Step 3) and the T segment is transported through the break in the G segment, which is accompanied by the hydrolysis of one ATP molecule (Step 4). The G segment is then religated and the remaining ATP molecule is hydrolyzed (Step 5). Upon dissociation of the two ADP molecules, the T segment is transported through the opening in the C-terminal part of the enzyme (Step 6) followed by closing of this gate. Finally, the N-terminal ATPase domains reopen, allowing the enzyme to dissociate from DNA (Step 7).

9 Effects of topoisomerase II-DNA cleavage complexes in the cell
Effects of topoisomerase II-DNA cleavage complexes in the cell. Topoisomerase II-DNA cleavage complexes normally are transient intermediates in the catalytic cycle of the enzyme and are present at low cellular concentrations. However, when levels of cleavage complexes rise, they can be converted to permanent DNA strand breaks that trigger DNA recombination/repair pathways or cell death pathways. Alternatively, chromosomal translocations can be generated that lead to specific types of leukemia. E.L. Baldwin1 and N. Osheroff1,2,* Etoposide, Topoisomerase II and Cancer Curr. Med. Chem. - Anti-Cancer Agents, 2005, 5,

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13 ANTRACICLINE

14 DAUNORUBICINA

15 EFFETTI TOSSICI DELLE ANTRACICLINE
mielotossicità (neutropenia, trombocitopenia) mucosite alopecia cardiotossicità gravi lesioni tessutali da stravaso

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20 ALTRI AGENTI INTERCALANTI
Actinomicina D

21 EPIPODOFILLOTOSSINE

22 DELLE EPIPODOFILLOTOSSINE
EFFETTI TOSSICI DELLE EPIPODOFILLOTOSSINE mielotossicità nausea, vomito (VM26 > VP16) alopecia reazioni di ipersensibilità mucosite (ad alte dosi)

23 ALTRI “VELENI” NON INTERCALANTI

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25 MECCANISMI DI RESISTENZA AI
VELENI DELLA TOPO II

26 MECCANISMI DI RESISTENZA AI
VELENI DELLA TOPO II  dei livelli di attività enzimatica produzione di forme alterate dell’enzima (i) a livello del sito di riconoscimento per il DNA (iI) a livello del sito di riconoscimento per il farmaco (iii) a livello della sequenza di localizzazione nucleare shift da isoforma  a isoforma 

27 ALTRI MECCANISMI DI RESISTENZA A ANTRACICLINE E EPIPODOFILLOTOSSINE
 espressione della P-gp  espressione della MRP-1  espressione di LRP limitatamente alle antracicline:  dei livelli intracellulari di glutatione e/o dell’attivita di enzimi glutatione-dipendenti (glutatione transferasi; glutatione perossidasi)

28 MECCANISMI DI RIPARAZIONE DEI DANNI INDOTTI DAI VELENI DELLA TOPO II

29 The catalytic cycle of DNA topoisomerase II
The catalytic cycle of DNA topoisomerase II. The ATPase domains of topoisomerase II are shown in light blue, the core domain in dark blue, and the active site tyrosine residue in red. The C-terminal domain of the enzyme is not included in the diagram since its orientation, with respect to the rest of the molecule, is not known. The catalytic cycle is initiated by enzyme binding to two double-stranded DNA segments called the G segment (in red) and the T segment (in green) (Step 1). Next, two ATP molecules are bound, which is associated with dimerization of the ATPase domains (Step 2). The G segment is cleaved (Step 3) and the T segment is transported through the break in the G segment, which is accompanied by the hydrolysis of one ATP molecule (Step 4). The G segment is then religated and the remaining ATP molecule is hydrolyzed (Step 5). Upon dissociation of the two ADP molecules, the T segment is transported through the opening in the C-terminal part of the enzyme (Step 6) followed by closing of this gate. Finally, the N-terminal ATPase domains reopen, allowing the enzyme to dissociate from DNA (Step 7).

30 INIBITORI CATALITICI DELLA DNA TOPOISOMERASI II

31 INIBITORI CATALITICI DELLA DNA TOPOISOMERASI II

32 INIBITORI CATALITICI DELLA DNA TOPOISOMERASI II

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34 Treatment of interphase cells with topoisomerase inhibitors leads to a concentration-dependent G2 arrest due to activation of the DNA damage or the catenation checkpoint. The G2 block can be overcome by checkpoint abrogators, such as caffeine, fostriecin, or okadaic acid. The chromosomes of cells treated with etoposide or other cleavable complex inducers are extensively fragmented, whereas chromosomes from cells treated with the catalytic topoisomerase II inhibitors merbarone, ICRF-187, and aclarubicin are intact but elongated and/or entangled. Data from [Downes et al], [Ishida et al], [Anderson ], and unpublished results.

35 STRUTTURA DELLA DNA TOPOISOMERASI I
1 197 651 696 765 NH2 COOH NLS Y 723 Lys

36 Figure 1 | Relaxation of DNA supercoiling by TOP1-mediated DNA cleavage complexes, and the trapping of TOP1 cleavage complexes by drugs, DNA modifications and during apoptosis. a | The generation of DNA supercoiling by DNA replication, transcription and chromatin remodelling. The unwinding of duplex DNA by macromolecular complexes tracking along the DNA (arrow) without rotating freely around the DNA double helix, which is also unable to rotate freely owing to its length or attachment to nuclear matrix regions, generates positive supercoiling ahead of the unwound segment and negative supercoiling behind (negative supercoiling not shown). b | The introduction of DNA single-strand breaks (nicks) by TOP1 provides swivel points that enable the rotation of the intact DNA strand around the break and facilitate DNA relaxation. The cleavage intermediate is referred to as a cleavage complex because TOP1 cleaves DNA by forming a covalent bond to the 3′ DNA terminus that it generates. The covalently linked catalytic tyrosine of TOP1 (Y723 for human TOP1) is shown as the yellow circle.

37 Figure 1 | Relaxation of DNA supercoiling by TOP1-mediated DNA cleavage complexes, and the trapping of TOP1 cleavage complexes by drugs, DNA modifications and during apoptosis. c | An expanded view of DNA relaxation by a TOP1 cleavage complex (TOP1cc). The first step (left) is a transesterification reaction whereby the catalytic tyrosine (Y) becomes linked to the 3′ DNA end (nicking step). In the second step (middle), the torsional strain that results from DNA supercoiling drives the rotation of the 5′ end of the nicked DNA strand around the intact strand. TOP1 encircles the rotating nicked DNA and slows its rotation (FIG. 3a). This process is referred to as ‘controlled rotation’. In the last step (right), the 5′ end of the nicked DNA is realigned with the corresponding 3′ end, which enables DNA religation (the closing step of the ‘nicking–closing reaction’). TOP1ccs are normally transient because the closing step is much faster than the nicking step. Drugs and DNA lesions inhibit religation by misaligning the ends of the broken DNA. d | TOP1ccs can be stabilized under three conditions: by drugs such as camptothecin (left), by DNA lesions (damage) that misalign the 5′ end of the nicked DNA, and by DNA and TOP1 modifications that occur during programmed cell death (apoptosis).

38 Figure 4 | Conversion of TOP1 cleavage complexes into DNA damage by replication-fork collision and transcription. a | When a TOP1 cleavage complex (TOP1cc) is on the leading strand (dark blue) for DNA synthesis, DNA polymerase elongates the nascent leading strand (light blue) up to the last base flanking the TOP1 cleavage site, thereby generating a replication double-strand break (RepDSB). Religation of the TOP1cc is blocked by the pairing of the template and leading strand immediately downstream of the TOP1cc. Because of the discontinuous replication on the lagging strand, a single-stranded DNA segment (light blue) probably exists immediately downstream of the TOP1 covalent complex.

39 Figure 4 | Conversion of TOP1 cleavage complexes into DNA damage by replication-fork collision and transcription. b | When a trapped TOP1cc is on the transcribed strand (dark blue), the RNA polymerase can reach the cleavage complex and arrest RNA elongation (dark purple). The RNA–DNA duplex prevents the religation of the TOP1cc, and TOP1 inhibition leads to an accumulation of negative supercoiling that could promote the formation of an R-loop. Inhibition of TOP1 SRkinase activity would also inactivate splicing because of ASF hypophosphorylation. In cancer cells treated with camptothecins, replication-mediated double-strand breaks (a) are probably the main mechanism that generates DNA damage.

40 Camptotheca acuminata

41 Figure 2 | The chemical structure of camptothecin and
its derivatives. a | Camptothecin is a 5-ring heterocyclic alkaloid that contains an α-hydroxylactone within its E-ring that is unstable at physiological pH. For all camptothecin derivatives, the carboxylate form is inactive as a TOP1 inhibitor and is sequested by tight binding to serum albumin. As a result, shortly after administration only a small fraction of camptothecin or its derivatives are in the active lactone form.

42 Figure 2 | The chemical structure of camptothecin and
its derivatives. a | Camptothecin is a 5-ring heterocyclic alkaloid that contains an α-hydroxylactone within its E-ring that is unstable at physiological pH. For all camptothecin derivatives, the carboxylate form is inactive as a TOP1 inhibitor and is sequested by tight binding to serum albumin. As a result, shortly after administration only a small fraction of camptothecin or its derivatives are in the active lactone form.

43 Figure 2 | Possible pathways of irinotecan metabolism
Figure 2 | Possible pathways of irinotecan metabolism. Irinotecan (CPT-11) can be converted into the active metabolite SN-38 by carboxylesterases (CES) outside or inside the cell. CPT-11 and SN-38 are both substrates for the ATP-binding cassette (ABC) transport proteins — P-glycoprotein (ABCB), ABCC and ABCG — which transport the drug out of the cell. Alternatively, CPT-11 and SN-38 can be inactivated by cytochrome P450 enzymes (CYP) or uridine diphosphate glycosyltransferase (UGT), respectively. If SN-38 persists, it binds to its target topoisomerase I (TOPI), interfering with DNA synthesis and repair processes, culminating in cell death. ADPRT, ADP-ribosyltransferase; APC, inactive metabolite of SN-38; CDC45L, cell-division cycle 45L; NPC, inactive metabolite of SN-38; SN-38G, SN-38 glucuronide; TDP, tyrosyl-DNA phosphodiesterase; XRCC1, X-ray-repair cross-complementing defective-1.

44 EFFETTI TOSSICI DELLE CAMPTOTECINE
mielotossicità (prev. neutropenia) nausea, vomito; diarrea (non gravi) debolezza alopecia febbre orticaria mucosite

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46 MECCANISMI DI RESISTENZA
ALLE CAMPTOTECINE  dei livelli di attività enzimatica produzione di forme alterate dell’enzima (i) a livello del sito di riconoscimento per il DNA (iI) a livello del sito di riconoscimento per il farmaco (iii) a livello della sequenza di localizzazione nucleare  dei livelli di attività della topo II  dei livelli di CPT11-converting enzyme (solo per irinotecano)