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Agenti Alchilanti

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Agenti Alchilanti

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    2. MOSTARDE AZOTATE

    3. The figure shows the historic development of the mustard family of agents, from mustard gas through to molecules that have progressively decreasing toxicity to normal cells, to a molecule designed to target the oestrogen receptor in tumour cells. It was realized that if the electrophilicity of mustard gas could be reduced, then less-toxic drugs might be obtained that could be administered orally. This led to the development of the subsequent compounds chlormethine, chlorambucil, melphalan, cyclophosphamide and estramustine, which are all still in use today. Melphalan is composed of a phenylalanine attached to the mustard, enhancing the selective uptake by tumour cells. Estramustine is the combination of a mustard and an oestrogen.The figure shows the historic development of the mustard family of agents, from mustard gas through to molecules that have progressively decreasing toxicity to normal cells, to a molecule designed to target the oestrogen receptor in tumour cells. It was realized that if the electrophilicity of mustard gas could be reduced, then less-toxic drugs might be obtained that could be administered orally. This led to the development of the subsequent compounds chlormethine, chlorambucil, melphalan, cyclophosphamide and estramustine, which are all still in use today. Melphalan is composed of a phenylalanine attached to the mustard, enhancing the selective uptake by tumour cells. Estramustine is the combination of a mustard and an oestrogen.

    25. a | The O6-alkylguanine DNA alkyltransferase (AGT) protein scans double-stranded DNA for alkylation at the O6 position of guanine. Covalent transfer of the alkyl group (in this figure shown as a methyl, CH3, group) to the conserved active-site cysteine inactivates the AGT protein and restores the guanine to normal. However, if the lesion consists of an N1, O6-ethanoguanine residue formed after chloroethylguanine alkylation, an AGT–DNA crosslink results (not shown).a | The O6-alkylguanine DNA alkyltransferase (AGT) protein scans double-stranded DNA for alkylation at the O6 position of guanine. Covalent transfer of the alkyl group (in this figure shown as a methyl, CH3, group) to the conserved active-site cysteine inactivates the AGT protein and restores the guanine to normal. However, if the lesion consists of an N1, O6-ethanoguanine residue formed after chloroethylguanine alkylation, an AGT–DNA crosslink results (not shown).

    26. b | If repair of the CH3-G lesion does not occur, a G A transition mutation or a strand break can result b | If repair of the CH3-G lesion does not occur, a G A transition mutation or a strand break can result

    28. Temozolomide acts as a prodrug, transporting a methylating agent (the methyldiazonium ion) to guanine bases within the major groove of DNA. The mechanism of activation involves chemical (as opposed to enzymatic) hydrolytic cleavage of the tetrazinone ring at physiological pH to give the unstable monomethyl triazene, which then undergoes further cleavage to liberate the stable 5-aminoimidazole-4-carboxamide and the highly reactive methyldiazonium methylating species. After methylating DNA (or otherwise decomposing by reacting with water to give methanol), the latter forms N2. The generation of the small stable molecules 5-aminoimidazole-4-carboxamide, CO2 and N2 provides the driving force for the mechanism of action of temozolomide. The antitumour activity of temozolomide correlates with its accumulation in tumours, where it methylates guanine O6 and N7 positions in DNA. Cellular selectivity might be attributable to the slightly different pH environments of normal versus malignant tissues in the brain, coupled with differential capacities to repair the methylated lesions by O6-alkyl-DNA alkyltransferase or other repair processes. A fragment of duplex DNA is shown with a methylated guanine base.Temozolomide acts as a prodrug, transporting a methylating agent (the methyldiazonium ion) to guanine bases within the major groove of DNA. The mechanism of activation involves chemical (as opposed to enzymatic) hydrolytic cleavage of the tetrazinone ring at physiological pH to give the unstable monomethyl triazene, which then undergoes further cleavage to liberate the stable 5-aminoimidazole-4-carboxamide and the highly reactive methyldiazonium methylating species. After methylating DNA (or otherwise decomposing by reacting with water to give methanol), the latter forms N2. The generation of the small stable molecules 5-aminoimidazole-4-carboxamide, CO2 and N2 provides the driving force for the mechanism of action of temozolomide. The antitumour activity of temozolomide correlates with its accumulation in tumours, where it methylates guanine O6 and N7 positions in DNA. Cellular selectivity might be attributable to the slightly different pH environments of normal versus malignant tissues in the brain, coupled with differential capacities to repair the methylated lesions by O6-alkyl-DNA alkyltransferase or other repair processes. A fragment of duplex DNA is shown with a methylated guanine base.

    30. Fig. 1. Molecular mechanisms underlying cytotoxicity induced by wide spectrum or N3-adenine selective methylating compounds, as single agents or combined with PARP inhibitors. Left panel: Temozolomode (TMZ) generates a variety of DNA adducts such as O6-methylguanine (O6-MeG) which is repaired by high levels of O6-alkylguanine DNA alkyltransferase (AGT) and N7-methylguanine (N7-MeG) or N3-methyladenine (N3-MeA) which are both removed by base excision repair (BER). In AGT-deficient cells, unrepaired O6-MeG triggers apoptosis as long as the Mismatch Repair (MR) system is functional. Inhibition of PARP avoids recruitments of BER components involved in the repair process of N-methylpurines; this results in generation of strand breaks and induction of apoptosis. Right panel: The selective N3-A methylating agent Me-Lex provokes PARP activation and ultimately leads to necrosis; addition of PARP inhibitor switches the modality of cell death from necrosis to apoptosis (see text for further details). Fig. 1. Molecular mechanisms underlying cytotoxicity induced by wide spectrum or N3-adenine selective methylating compounds, as single agents or combined with PARP inhibitors. Left panel: Temozolomode (TMZ) generates a variety of DNA adducts such as O6-methylguanine (O6-MeG) which is repaired by high levels of O6-alkylguanine DNA alkyltransferase (AGT) and N7-methylguanine (N7-MeG) or N3-methyladenine (N3-MeA) which are both removed by base excision repair (BER). In AGT-deficient cells, unrepaired O6-MeG triggers apoptosis as long as the Mismatch Repair (MR) system is functional. Inhibition of PARP avoids recruitments of BER components involved in the repair process of N-methylpurines; this results in generation of strand breaks and induction of apoptosis. Right panel: The selective N3-A methylating agent Me-Lex provokes PARP activation and ultimately leads to necrosis; addition of PARP inhibitor switches the modality of cell death from necrosis to apoptosis (see text for further details).

    40. Formation and effects of cisplatin adducts.   Cisplatin undergoes aquation to form [Pt(NH3)2Cl(OH2)]+ and [Pt(NH3)2(OH2)2]2+ once inside the cell. The platinum atom of cisplatin binds covalently to the N7 position of purines to form 1,2- or 1,3-intrastrand crosslinks, and interstrand crosslinks. Cisplatin–DNA adducts cause various cellular responses, such as replication arrest, transcription inhibition, cell-cycle arrest, DNA repair and apoptosis. Formation and effects of cisplatin adducts.   Cisplatin undergoes aquation to form [Pt(NH3)2Cl(OH2)]+ and [Pt(NH3)2(OH2)2]2+ once inside the cell. The platinum atom of cisplatin binds covalently to the N7 position of purines to form 1,2- or 1,3-intrastrand crosslinks, and interstrand crosslinks. Cisplatin–DNA adducts cause various cellular responses, such as replication arrest, transcription inhibition, cell-cycle arrest, DNA repair and apoptosis.

    41. Transduction of DNA-damage signals: p53 and cisplatin.   The p53 pathway can partially mediate cisplatin cytotoxicity. p53 is linked to DNA repair, cell-cycle arrest and apoptosis. Following DNA damage, p53 is activated and subsequently trans-activates different sets of downstream target genes, which in turn induce various cellular responses. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; BRCA1, breast cancer 1, early onset; CSB, Cockayne syndrome B; GADD45, growth arrest and DNA damage 45; MDM2, mouse double minute 2 homologue; PCNA, proliferating cell nuclear antigen; RPA, replication A; TFIIH, transcription factor IIH; XPC, xeroderma pigmentosum, complementation group C. Transduction of DNA-damage signals: p53 and cisplatin.   The p53 pathway can partially mediate cisplatin cytotoxicity. p53 is linked to DNA repair, cell-cycle arrest and apoptosis. Following DNA damage, p53 is activated and subsequently trans-activates different sets of downstream target genes, which in turn induce various cellular responses. ATM, ataxia telangiectasia mutated; ATR, ataxia telangiectasia and Rad3 related; BRCA1, breast cancer 1, early onset; CSB, Cockayne syndrome B; GADD45, growth arrest and DNA damage 45; MDM2, mouse double minute 2 homologue; PCNA, proliferating cell nuclear antigen; RPA, replication A; TFIIH, transcription factor IIH; XPC, xeroderma pigmentosum, complementation group C.

    42. Figure 3. The possible fates of a cell damaged by a platinum chemotherapy drug. The dashed arrows represent the specific, modulated signals elicited by lesion-recognition proteins, which subsequently engage effector pathways (repair, growth arrest, apoptosis, lesion-tolerant replication/transcription). Apparently opposed mechanisms, DNA repair and apoptosis, may be simultaneously activated so that cell death can be swiftly executed if the necessary steps for repair are not fully completed. However, apoptotic pathways are commonly aberrant in cancer cells, enabling cells to survive despite extensive damage. Figure 3. The possible fates of a cell damaged by a platinum chemotherapy drug. The dashed arrows represent the specific, modulated signals elicited by lesion-recognition proteins, which subsequently engage effector pathways (repair, growth arrest, apoptosis, lesion-tolerant replication/transcription). Apparently opposed mechanisms, DNA repair and apoptosis, may be simultaneously activated so that cell death can be swiftly executed if the necessary steps for repair are not fully completed. However, apoptotic pathways are commonly aberrant in cancer cells, enabling cells to survive despite extensive damage.

    45. Cisplatin and cell-death pathways.   Cisplatin induces necrosis and apoptosis — two different modes of cell death. DNA damage arrests the cell cycle, inhibits transcription and initiates apoptosis. Excessive DNA damage induces hyper-activation of poly(ADP-ribose)polymerase (PARP). PARP cleaves NAD+ and transfers ADP-ribose moieties (ADPR) to carboxyl groups of nuclear proteins. It thereby causes NAD+/ATP depletion, resulting in necrotic cell death if ATP depletion reaches lethal-inducing levels. TCR, transcription-coupled repair. Cisplatin and cell-death pathways.   Cisplatin induces necrosis and apoptosis — two different modes of cell death. DNA damage arrests the cell cycle, inhibits transcription and initiates apoptosis. Excessive DNA damage induces hyper-activation of poly(ADP-ribose)polymerase (PARP). PARP cleaves NAD+ and transfers ADP-ribose moieties (ADPR) to carboxyl groups of nuclear proteins. It thereby causes NAD+/ATP depletion, resulting in necrotic cell death if ATP depletion reaches lethal-inducing levels. TCR, transcription-coupled repair.

    46. Recognition of platinum–DNA adducts: the roles of HMGB1.   HMGB1 recognizes cisplatin-damaged DNA and modulates the efficiency of nucleotide excision repair (NER) in vitro. Moreover, HMGB1 interacts with RAG1/2, p53, and MutS . HMGB1 has been connected to the mismatch repair (MMR), V(D)J recombination, p53 and MAPK pathways. It also facilitates nucleosome remodelling and serves as a cytokine, being secreted by necrotic and immune cells. ACF, ATP-utilizing chromatin assembly and remodelling factor; HMGB1, high-mobility group box 1; MAPK, mitogen-activated protein kinase; NF- B, nuclear factor- B; RAG, recombination activating genes. Recognition of platinum–DNA adducts: the roles of HMGB1.   HMGB1 recognizes cisplatin-damaged DNA and modulates the efficiency of nucleotide excision repair (NER) in vitro. Moreover, HMGB1 interacts with RAG1/2, p53, and MutS . HMGB1 has been connected to the mismatch repair (MMR), V(D)J recombination, p53 and MAPK pathways. It also facilitates nucleosome remodelling and serves as a cytokine, being secreted by necrotic and immune cells. ACF, ATP-utilizing chromatin assembly and remodelling factor; HMGB1, high-mobility group box 1; MAPK, mitogen-activated protein kinase; NF- B, nuclear factor- B; RAG, recombination activating genes.

    50. Platinum might enter cells using either transporters — a significant one being the copper transporter CTR1 — or by passive diffusion. Loss of CTR1 results in less platinum entering cells and, consequently, drug resistance. Once inside cells, cisplatin is activated by the addition of water molecules to form a chemically reactive aqua species. This is facilitated by the relatively low chloride concentrations that are found within cells. In the cytoplasm, the activated aqua species preferentially reacts with species containing high sulphur levels by virtue of their containing many cysteine or methionine amino acids. These species include the tripeptide glutathione or metallothioneins. In some platinum-resistant cancer cells, glutathione and metallothionein levels are relatively high, so activated platinum is effectively 'mopped up' in the cytoplasm before DNA binding can occur, thereby causing resistance. Finally, active export of platinum from the cells through the copper exporters ATP7A and ATP7B as well as through the glutathione S-conjugate export GS-X pump (also known as MRP2 or ABCC2) can contribute to platinum drug resistance. GSTs, glutathione S-transferases Platinum might enter cells using either transporters — a significant one being the copper transporter CTR1 — or by passive diffusion. Loss of CTR1 results in less platinum entering cells and, consequently, drug resistance. Once inside cells, cisplatin is activated by the addition of water molecules to form a chemically reactive aqua species. This is facilitated by the relatively low chloride concentrations that are found within cells. In the cytoplasm, the activated aqua species preferentially reacts with species containing high sulphur levels by virtue of their containing many cysteine or methionine amino acids. These species include the tripeptide glutathione or metallothioneins. In some platinum-resistant cancer cells, glutathione and metallothionein levels are relatively high, so activated platinum is effectively 'mopped up' in the cytoplasm before DNA binding can occur, thereby causing resistance. Finally, active export of platinum from the cells through the copper exporters ATP7A and ATP7B as well as through the glutathione S-conjugate export GS-X pump (also known as MRP2 or ABCC2) can contribute to platinum drug resistance. GSTs, glutathione S-transferases

    53. Mechanisms of cisplatin uptake and efflux.   In addition to passive diffusion, cisplatin is also actively imported by the copper transporter CTR1. Copper-transporting P-type adenosine triphosphate (ATP7B) has a role in cisplatin efflux. Both copper and cisplatin have the ability to reduce the uptake of the other and can trigger the degradation and delocalization of CTR1. Moreover, copper and cisplatin show directional cross-resistance. MRP2, multidrug resistance protein. Mechanisms of cisplatin uptake and efflux.   In addition to passive diffusion, cisplatin is also actively imported by the copper transporter CTR1. Copper-transporting P-type adenosine triphosphate (ATP7B) has a role in cisplatin efflux. Both copper and cisplatin have the ability to reduce the uptake of the other and can trigger the degradation and delocalization of CTR1. Moreover, copper and cisplatin show directional cross-resistance. MRP2, multidrug resistance protein.

    54. Expression of MRP2 in ovarian cancer. Immunohistochemical staining of ovarian carcinoma for the putative cisplatin export pump shown by brown membrane staining; counterstained with haematoxylin. Expression of MRP2 in ovarian cancer. Immunohistochemical staining of ovarian carcinoma for the putative cisplatin export pump shown by brown membrane staining; counterstained with haematoxylin.

    55. Once the activated aqua platinum species (see Fig. 1 and note that this is the same for cisplatin and carboplatin) has entered the nucleus, preferential covalent binding to the nitrogen on position 7 of guanine occurs. The major covalent bis-adduct that is formed involves adjacent guanines on the same strand of DNA (the intrastrand crosslink); a minor adduct involves binding to guanines on opposite DNA strands (the interstrand crosslink). The main removal pathway for these DNA adducts is that of nucleotide-excision repair (NER); increased NER, especially through increased activity of the endonuclease protein ERCC1 (excision repair cross-complementing-1) can occur in tumours, and can lead to platinum drug resistance (as adducts are removed before apoptotic signalling pathways are triggered). In addition, resistance can occur through increased tolerance to platinum–DNA adducts — even though the DNA adducts are formed — either through loss of DNA mismatch repair, bypassing of adducts by polymerase and , or through downregulation of apoptotic pathways. BAX, BCL2-associated X protein; BID, BH3 interacting domain death agonist; HR23B, human Rad23B; MLH1, MutL homologue 1; MSH2/3/6, MutS homologue 2/3/6; PCNA, proliferating cell nuclear antigen; PMS2, postmeiotic segregation increased-2; RPA, replication protein A; TFB5, tenth subunit of TFIIH; TFIIH, general transcription factor IIH; XPA/B/C/D/F/G, xeroderma pigmentosum (XP), complementation group A/B/C/D/F/G.Once the activated aqua platinum species (see Fig. 1 and note that this is the same for cisplatin and carboplatin) has entered the nucleus, preferential covalent binding to the nitrogen on position 7 of guanine occurs. The major covalent bis-adduct that is formed involves adjacent guanines on the same strand of DNA (the intrastrand crosslink); a minor adduct involves binding to guanines on opposite DNA strands (the interstrand crosslink). The main removal pathway for these DNA adducts is that of nucleotide-excision repair (NER); increased NER, especially through increased activity of the endonuclease protein ERCC1 (excision repair cross-complementing-1) can occur in tumours, and can lead to platinum drug resistance (as adducts are removed before apoptotic signalling pathways are triggered). In addition, resistance can occur through increased tolerance to platinum–DNA adducts — even though the DNA adducts are formed — either through loss of DNA mismatch repair, bypassing of adducts by polymerase and , or through downregulation of apoptotic pathways. BAX, BCL2-associated X protein; BID, BH3 interacting domain death agonist; HR23B, human Rad23B; MLH1, MutL homologue 1; MSH2/3/6, MutS homologue 2/3/6; PCNA, proliferating cell nuclear antigen; PMS2, postmeiotic segregation increased-2; RPA, replication protein A; TFB5, tenth subunit of TFIIH; TFIIH, general transcription factor IIH; XPA/B/C/D/F/G, xeroderma pigmentosum (XP), complementation group A/B/C/D/F/G.

    57. Resistance can be tackled by: increasing the levels of platinum reaching tumours (for example, liposomal platinum products) thereby resulting in greater killing; combining existing platinum drugs with molecularly targeted drugs (for example, bevacizumab); using novel platinum drugs such as oxaliplatin that are capable of circumventing cisplatin-mediated resistance mechanisms; and using other drugs either alone (for example, TLK286) or in combination (for example, decitabine), which exploit particular cisplatin-mediated resistance mechanisms. GSH, reduced glutathione; GST, glutathione S-transferase.Resistance can be tackled by: increasing the levels of platinum reaching tumours (for example, liposomal platinum products) thereby resulting in greater killing; combining existing platinum drugs with molecularly targeted drugs (for example, bevacizumab); using novel platinum drugs such as oxaliplatin that are capable of circumventing cisplatin-mediated resistance mechanisms; and using other drugs either alone (for example, TLK286) or in combination (for example, decitabine), which exploit particular cisplatin-mediated resistance mechanisms. GSH, reduced glutathione; GST, glutathione S-transferase.

    61. Figure 5. Molecular differences between oxaliplatin and cisplatin. Schematic comparative view of the major sites of interactions between cisplatin- and oxaliplatin-DNA adducts and damage recognition processes that are thought to contribute to discriminating between the two drugs. The arrows indicate the sites of interaction with oxaliplatin (dotted line) and cisplatin (continuous line). Arrows length is linearly correlated with the relative intensity of the interaction. Translesion synthesis (TS; error prone, potentially mutagenic). Its efficacy depends on the specificity of DNA polymerase, damage recognition proteins and/or MMR-mediated processes. TS may be inhibited by the selective recognition of platinated DNA by either MMR proteins or HMG-1 which will shield the adducts from the replication machinery. In MMR-proficient cells, TS is usually more efficient for DACH-Pt over cisplatin adducts, whereas in MMR-defective cells, it is enhanced for cisplatin and lower for oxaliplatin adducts. NER. This is the major repair process involved in the removal of platinated DNA. No differences have been observed in the repair processes of oxaliplatin and cisplatin adducts. MMR (hMutL aand hMutS a) recognises cisplatin-DNA. Poor recognition has been shown for oxaliplatin adducts. Selective recognition of cisplatin over oxaliplatin adducts by HGM-1 proteins that may result in a specific block of TS and/or other repair pathways. Figure 5. Molecular differences between oxaliplatin and cisplatin. Schematic comparative view of the major sites of interactions between cisplatin- and oxaliplatin-DNA adducts and damage recognition processes that are thought to contribute to discriminating between the two drugs. The arrows indicate the sites of interaction with oxaliplatin (dotted line) and cisplatin (continuous line). Arrows length is linearly correlated with the relative intensity of the interaction. Translesion synthesis (TS; error prone, potentially mutagenic). Its efficacy depends on the specificity of DNA polymerase, damage recognition proteins and/or MMR-mediated processes. TS may be inhibited by the selective recognition of platinated DNA by either MMR proteins or HMG-1 which will shield the adducts from the replication machinery. In MMR-proficient cells, TS is usually more efficient for DACH-Pt over cisplatin adducts, whereas in MMR-defective cells, it is enhanced for cisplatin and lower for oxaliplatin adducts. NER. This is the major repair process involved in the removal of platinated DNA. No differences have been observed in the repair processes of oxaliplatin and cisplatin adducts. MMR (hMutL aand hMutS a) recognises cisplatin-DNA. Poor recognition has been shown for oxaliplatin adducts. Selective recognition of cisplatin over oxaliplatin adducts by HGM-1 proteins that may result in a specific block of TS and/or other repair pathways.

    63. BBR3464: DNA-Drug Adducts

    64. Structure of metallobleomycins and their domain organization.   a | Structure of bleomycins. The metal-binding domain is in green and the nitrogen atoms that coordinate the metal are black. The linker region is in red and the bithiazole tail is in blue. The disaccharide (R) and examples of the positively charged tail (R') are shown. b | Understanding the organization of the ligands of bleomycin around its metal is essential to understanding binding to its target — a left hand cannot fit into the right glove. This is the first atomic-resolution structure determined crystallographically of an intact bleomycin. The three-dimensional structure of Cu(II)–bleomycin A2 determined by X-ray crystallography bound to the bleomycin-resistance protein from Streptomyces verticillus105 is shown. The atoms are coloured as follows: N, blue; O, red; C, green; Cu(II) ion, magenta sphere; chloride ion, cyan sphere. The bithiazole tail is omitted for clarity. c | The model for double-stranded DNA cleavage by bleomycin is dependent on the structural model derived from bleomycin–Co(III)–OOH106. It is essential that the organization of the ligands around the metal faithfully replicate the organization of the ligands around bleomycin–Fe(III)–OOH, the structure of which cannot be examined due to chemical instability. The superimposition of the Cu(II) structure shown in b and the Co(III) structure shown in c demonstrates that ligand organization is the same, suggesting that bleomycin-Fe(III)-OOH might also have a similar structure. G, guanine. Structure of metallobleomycins and their domain organization.   a | Structure of bleomycins. The metal-binding domain is in green and the nitrogen atoms that coordinate the metal are black. The linker region is in red and the bithiazole tail is in blue. The disaccharide (R) and examples of the positively charged tail (R') are shown. b | Understanding the organization of the ligands of bleomycin around its metal is essential to understanding binding to its target — a left hand cannot fit into the right glove. This is the first atomic-resolution structure determined crystallographically of an intact bleomycin. The three-dimensional structure of Cu(II)–bleomycin A2 determined by X-ray crystallography bound to the bleomycin-resistance protein from Streptomyces verticillus105 is shown. The atoms are coloured as follows: N, blue; O, red; C, green; Cu(II) ion, magenta sphere; chloride ion, cyan sphere. The bithiazole tail is omitted for clarity. c | The model for double-stranded DNA cleavage by bleomycin is dependent on the structural model derived from bleomycin–Co(III)–OOH106. It is essential that the organization of the ligands around the metal faithfully replicate the organization of the ligands around bleomycin–Fe(III)–OOH, the structure of which cannot be examined due to chemical instability. The superimposition of the Cu(II) structure shown in b and the Co(III) structure shown in c demonstrates that ligand organization is the same, suggesting that bleomycin-Fe(III)-OOH might also have a similar structure. G, guanine.

    65. Formation of 'activated' bleomycin and cleavage of DNA.   a | The activated bleomycin can be formed by bleomycin binding to Fe(II), followed by oxygen binding and reduction by a reductant. This intermediate (green box) has a half-life of several minutes at 4°C (Ref. 9) and is responsible for initiating DNA damage. b | Both single-stranded and double-stranded DNA (dsDNA) cleavage is initiated by the activated bleomycin, which, directly or indirectly, removes the 4'-hydrogen atom from C4' of the deoxyribose moiety of a pyrimidine 3' to a guanine107. Depending on the availability of O2, this 4'-radical intermediate (blue box) can partition between two pathways. In the pathway on the left, the 4'-radical intermediate is oxidized to a 4'-carbocation to which H2O is added, generating the 4'-oxidized abasic site108. In the pathway on the right, the 4'-radical intermediate reacts with O2 to form a 4'-peroxy radical. The resulting 4'-peroxy radical is reduced to the 4'-hydroperoxide. The 4'-hydroperoxide then undergoes a complex series of chemical transformations, ultimately generating a gapped DNA with 3'-phosphoglycolate/5'-phosphate (3'-PG/5'-P) ends and a pyrimidine propenal109, 110. Ds-cleavage of DNA is effected by a single bleomycin molecule and consequently requires reactivation of bleomycin. This occurs only through the pathway on the right, and is proposed to result from reduction of the peroxy radical27, 111. Py, pyrimidine. Formation of 'activated' bleomycin and cleavage of DNA.   a | The activated bleomycin can be formed by bleomycin binding to Fe(II), followed by oxygen binding and reduction by a reductant. This intermediate (green box) has a half-life of several minutes at 4°C (Ref. 9) and is responsible for initiating DNA damage. b | Both single-stranded and double-stranded DNA (dsDNA) cleavage is initiated by the activated bleomycin, which, directly or indirectly, removes the 4'-hydrogen atom from C4' of the deoxyribose moiety of a pyrimidine 3' to a guanine107. Depending on the availability of O2, this 4'-radical intermediate (blue box) can partition between two pathways. In the pathway on the left, the 4'-radical intermediate is oxidized to a 4'-carbocation to which H2O is added, generating the 4'-oxidized abasic site108. In the pathway on the right, the 4'-radical intermediate reacts with O2 to form a 4'-peroxy radical. The resulting 4'-peroxy radical is reduced to the 4'-hydroperoxide. The 4'-hydroperoxide then undergoes a complex series of chemical transformations, ultimately generating a gapped DNA with 3'-phosphoglycolate/5'-phosphate (3'-PG/5'-P) ends and a pyrimidine propenal109, 110. Ds-cleavage of DNA is effected by a single bleomycin molecule and consequently requires reactivation of bleomycin. This occurs only through the pathway on the right, and is proposed to result from reduction of the peroxy radical27, 111. Py, pyrimidine.

    66. Formation of 'activated' bleomycin and cleavage of DNA.   a | The activated bleomycin can be formed by bleomycin binding to Fe(II), followed by oxygen binding and reduction by a reductant. This intermediate (green box) has a half-life of several minutes at 4°C (Ref. 9) and is responsible for initiating DNA damage. b | Both single-stranded and double-stranded DNA (dsDNA) cleavage is initiated by the activated bleomycin, which, directly or indirectly, removes the 4'-hydrogen atom from C4' of the deoxyribose moiety of a pyrimidine 3' to a guanine107. Depending on the availability of O2, this 4'-radical intermediate (blue box) can partition between two pathways. In the pathway on the left, the 4'-radical intermediate is oxidized to a 4'-carbocation to which H2O is added, generating the 4'-oxidized abasic site108. In the pathway on the right, the 4'-radical intermediate reacts with O2 to form a 4'-peroxy radical. The resulting 4'-peroxy radical is reduced to the 4'-hydroperoxide. The 4'-hydroperoxide then undergoes a complex series of chemical transformations, ultimately generating a gapped DNA with 3'-phosphoglycolate/5'-phosphate (3'-PG/5'-P) ends and a pyrimidine propenal109, 110. Ds-cleavage of DNA is effected by a single bleomycin molecule and consequently requires reactivation of bleomycin. This occurs only through the pathway on the right, and is proposed to result from reduction of the peroxy radical27, 111. Py, pyrimidine. Formation of 'activated' bleomycin and cleavage of DNA.   a | The activated bleomycin can be formed by bleomycin binding to Fe(II), followed by oxygen binding and reduction by a reductant. This intermediate (green box) has a half-life of several minutes at 4°C (Ref. 9) and is responsible for initiating DNA damage. b | Both single-stranded and double-stranded DNA (dsDNA) cleavage is initiated by the activated bleomycin, which, directly or indirectly, removes the 4'-hydrogen atom from C4' of the deoxyribose moiety of a pyrimidine 3' to a guanine107. Depending on the availability of O2, this 4'-radical intermediate (blue box) can partition between two pathways. In the pathway on the left, the 4'-radical intermediate is oxidized to a 4'-carbocation to which H2O is added, generating the 4'-oxidized abasic site108. In the pathway on the right, the 4'-radical intermediate reacts with O2 to form a 4'-peroxy radical. The resulting 4'-peroxy radical is reduced to the 4'-hydroperoxide. The 4'-hydroperoxide then undergoes a complex series of chemical transformations, ultimately generating a gapped DNA with 3'-phosphoglycolate/5'-phosphate (3'-PG/5'-P) ends and a pyrimidine propenal109, 110. Ds-cleavage of DNA is effected by a single bleomycin molecule and consequently requires reactivation of bleomycin. This occurs only through the pathway on the right, and is proposed to result from reduction of the peroxy radical27, 111. Py, pyrimidine.

    68. Figure 3 | Products of bleomycin-induced double-stranded DNA cleavage and a proposed model for this process.   a | Bleomycins initiate single-stranded DNA (ssDNA) cleavage at pyrimidines (Py, that is, T or C) 3' to a guanine (5'-G-Py-3', _ denotes the cleavage site) in a sequence-specific fashion. Rules for the specificity of double-stranded DNA (dsDNA) cleavage indicate that the primary cleavage site is always a good ssDNA cleavage site. The secondary site of cleavage depends on the residue 3' to the primary cleavage site. For sequences of 5'-G-Py-Pu-3' (Pu, purine), bleomycins generate 5'-staggered ends, while for 5'-G-Py-Py-3', bleomycins generate blunt ends. b | A model for dsDNA cleavage by a single bleomycin molecule requires bleomycin reactivation (Fig. 2) and bleomycin reorganization during or after the cleavage of the first strand of DNA. Based on structural information from bleomycin–Co(III)–OOH bound to a hot spot for dsDNA cleavage and to the same sequence in which a gapped 3'-phosphoglycolate/5'-phosphate end has been created at the primary cleavage site, a model for reorganization of bleomycin from one strand to the cleavage site in the second strand has been proposed14. The key to the reorganization is proposed to be the linker and the flexibility of the bithiazole tail that is bound by partial intercalation. The rotation around the bond between the two thiazole rings in the tail of bleomycin, together with other motions, make the peroxide of the activated drug available for interaction with the second DNA strand. Figure 3 | Products of bleomycin-induced double-stranded DNA cleavage and a proposed model for this process.   a | Bleomycins initiate single-stranded DNA (ssDNA) cleavage at pyrimidines (Py, that is, T or C) 3' to a guanine (5'-G-Py-3', _ denotes the cleavage site) in a sequence-specific fashion. Rules for the specificity of double-stranded DNA (dsDNA) cleavage indicate that the primary cleavage site is always a good ssDNA cleavage site. The secondary site of cleavage depends on the residue 3' to the primary cleavage site. For sequences of 5'-G-Py-Pu-3' (Pu, purine), bleomycins generate 5'-staggered ends, while for 5'-G-Py-Py-3', bleomycins generate blunt ends. b | A model for dsDNA cleavage by a single bleomycin molecule requires bleomycin reactivation (Fig. 2) and bleomycin reorganization during or after the cleavage of the first strand of DNA. Based on structural information from bleomycin–Co(III)–OOH bound to a hot spot for dsDNA cleavage and to the same sequence in which a gapped 3'-phosphoglycolate/5'-phosphate end has been created at the primary cleavage site, a model for reorganization of bleomycin from one strand to the cleavage site in the second strand has been proposed14. The key to the reorganization is proposed to be the linker and the flexibility of the bithiazole tail that is bound by partial intercalation. The rotation around the bond between the two thiazole rings in the tail of bleomycin, together with other motions, make the peroxide of the activated drug available for interaction with the second DNA strand.

    69. Proposed mechanisms for generation of 'activated bleomycin' in vivo.   Bleomycin binds to Cu(II) tightly and the ligands do not exchange113. Intracellularly, bleomycin–Cu(II) can be reduced to bleomycin–Cu(I)113, which can react with O2 to initiate radical-mediated transformations and DNA cleavage114. In addition, the ligands in the bleomycin–Cu(I) complex are now able to exchange115 and only this form can lose metal and bind Fe(II)113. Therefore, for the 'activated bleomycin' (bleomycin–Fe(III)–OOH) to be generated, bleomycin–Cu(II) must be reduced to bleomycin–Cu(I) intracellularly. The Cu(I) must then dissociate from the bleomycin and an Fe(II) must bind. Once Fe(II) is bound, the chemistry to form the activated bleomycin ensues (Fig. 2a). Proposed mechanisms for generation of 'activated bleomycin' in vivo.   Bleomycin binds to Cu(II) tightly and the ligands do not exchange113. Intracellularly, bleomycin–Cu(II) can be reduced to bleomycin–Cu(I)113, which can react with O2 to initiate radical-mediated transformations and DNA cleavage114. In addition, the ligands in the bleomycin–Cu(I) complex are now able to exchange115 and only this form can lose metal and bind Fe(II)113. Therefore, for the 'activated bleomycin' (bleomycin–Fe(III)–OOH) to be generated, bleomycin–Cu(II) must be reduced to bleomycin–Cu(I) intracellularly. The Cu(I) must then dissociate from the bleomycin and an Fe(II) must bind. Once Fe(II) is bound, the chemistry to form the activated bleomycin ensues (Fig. 2a).

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