MAY / JUNE 1992 VOLUME 5, NUMBER 3 @Copyright 1992 by the American Chemical Society
Invited Review Free-Radical Mechanisms Involved in the Formation of Sequence-Dependent Bistranded DNA Lesions by the Antitumor Antibiotics Bleomycin, Neocarzinostatin, and Calicheamicin Peter C. Dedon*Tt and Irving H. Goldberg' Division of Toxicology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 Received March 13, 1992
I ntroductlon The relationship between free radical-mediated DNA damage and the biologic effects of numerous toxic and therapeutic agents has been the focus of intense research activity in the fields of chemistry and biology. Historically, ionizing radiation has dominated this area of study, but the advent of cancer chemotherapy has fostered a large research effort directed at isolating and characterizing radical-mediated DNA-damaging molecules, such as bleomycin (BLM)' ( I , 2) and the enediyne antitumor agents (3). Significant progress has been made in defining the principles that underlie the interaction of radicalmediated agents with DNA, as demonstrated by the studies of iron-EDTA systems ( 4 , 5 ) , copperphenanthroline complexes (6),chiral transition-metal complexes (7), and metalloporphyrins (8). Ionizing radiation and most metal complexes appear to cause DNA damage by virtue of their ability to generate activated oxygen species, with hydroxyl radicals implicated as the major DNA-damaging species (9-1 1 ) . While both single-strand (SS) and double-strand (DS) DNA lesions result from the radical-mediated oxidative chemistry, the latter are probably the more significant lesion biologically, given the correlation of DS breaks with the lethality of radical-mediated agents (12, 13), the mutagenicity of bistranded DNA lesions (14-20), and the effect of DS
*
Massachusetts Institute of Technology. Harvard Medical School.
breaks on recombinatorial activity that leads to DNA rearrangements involved in carcinogenesis (21-23). Recent studies have revealed that the radical-mediated antitumor antibiotics BLM and two members of the enediyne family, neocarzinostatin (NCS) (24)and calicheamicin (CAL) (25), produce sequence-specific bistranded DNA damage that cannot be accounted for by the random placement of closely opposed SS lesions (26-29). While the DS lesions produced by BLM result from an activated oxygen species and depend on two separate cleavage eventa, the bistranded lesions produced by NCS and CAL, the most thoroughly characterized enediynes, probably occur by the action of carbon-centered radicals of a single drug molecule. In this account, we will review recent findings related to the mechanisms of action of BLM, NCS, and CAL and then focus on proposed models for the drug-mediated bistranded lesions. Two other enediynes, esperamicin (ESP) (30)and dynemicin (DYN) (31),produce DS breaks that do not appear to be strongly sequencespecific, so their DNA-damaging activities will not be covered extensively in this review. For details of BLM and enediyne activity not considered in this review the reader is referred to several thorough reviews of the chemistry, biochemistry, and biology of BLM by Hecht (32),Stubbe and Kozarich ( I ) , McGall and Stubbe (2),Grollman (33),and Petering Abbreviations: AP, apurinic/apyrimidinic; BLM,bleomycin;CAL, calicheamicin; DS, double strand; DYN,dynemicin; ESP, esperamicin; ESR, electron spin resonance;NCS, neocarzinostatin;SS,single strand.
0893-228X/92/2~05-031~~~3.o0/0 1992 American Chemical Society
Dedon and Goldberg
312 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
B2: R = -NH-NHxNH;l Bleomycinic acid: R =-OH Deamido A*: R = O H 0
N
I.
B
A
3
;.w'
0-sugar
Figure 1. Structural features of bleomycin. (A) Structures of several bleomycin analogues. (B) A proposed structure of the oxygen.Fe(II).bleomycin complex (I),based on known structures of the Cu(I1) and Co(II1) complexes of bleomycin reviewed in ref 41.
et al. (34);of NCS by Goldberg (24,35) and Dedon and Goldberg (3);and of CAL by Lee et al. (25).The current status of synthetic enediynes and related compounds has been extensively reviewed by Nicoloau and Dai (36), in addition to Dedon and Goldberg (3).
A. Drug Structures and Mechanisms of Activation Bleomycin. The bleomycins are a family of glycopeptide antibiotics, produced by Streptomyces verticillw and discovered by Umezawa ( 3 8 , that have demonstrated clinical utility against a variety of neoplasms (38).BLM A2,the most thoroughly studied form, is the major component of the clinically used formulation, Blenoxane (39). Its structure, shown in Figure l A , has been divided into three loose functional domains (40): the metal-complexing pyrimidine, 8-aminoalanine, and j3-hydroxyimidazole moieties; the DNA-biiding bithiazole and its terminal amine, the latter distinguishing the various forms of BLM; and the gulose and carbamoylated mannose sugars thought to be important for drug uptake into cells. The DNA-damaging activity of BLM appears to lie in an activated oxygen species that results from the interaction of BLM with a metal ion and dioxygen. This activated complex is capable of abstracting hydrogen atoms from the deoxyribose of DNA. A proposed structure of Fe(II).BLM is shown in Figure 1B (41). A variety of metal ions [e.g., Fe(II)/Fe(III), Cu(I)/Cu(II), Co(III), Zn(II), and Mn(II)] can complex with BLM to cause DNA damage (1, 41), but the current debate focuses on the biologic signifcance of either Fe(I1) or Cu(II)/Cu(I). Demetallo-BLM forms a Cu(I1) complex in blood plasma (42),and this is the form that is thought to enter cells. However, the absence of DNA degradation with Cu(II).BLM (43) has led to a model in which the Cu(I1) is replaced by Fe(I1) in the cell (37). Hecht and co-workers have argued that the Cu(II).BLM can be reduced slowly to a Cu(1) complex
that causes DNA damage, albeit with a slightly different sequence selectivity and different DNA damage products (44).While this controversy remains to be resolved, this review will focus on the more thoroughly studied FeBLM system. A proposal for the mechanism of activation of FeeBLM is shown in Figure 2, which is based on numerous studies (see refs 1,2, 34, 40, 45, and 46 and references therein). Since the exact form of the iron-oxygen complex responsible for hydrogen atom abstraction remains a matter of controversy (a highly valent iron-oxygen species is a likely candidate given the similarity of FeBLM to P450 systems; 1,47),the iron-oxygen complexes are presented in Figure 2 in the starting oxidation state of Fe(I1) or the final state of Fe(II1) for simplicity. In this model, molecular oxygen combines with Fe(II) and BLM to form a ternary complex A that has the properties of ferric iron-superoxide B (47, 48). This complex undergoes a one-electron reduction by another Fe(II)-BLM complex (47,49) or possibly by a thiol (43,49,50),to produce the so-called "activated BLM" C, the ESR-active form whose decay coincides with the production of DNA damage (47). The activated BLM complex has been shown to have features of an iron-peroxide complex (45,47,51),a conclusion supported by the observation that a mixture of Fe(III), BLM, and H202 also produces a complex identical to activated BLM in both physical properties (47) and DNA damage (52). By analogy to the well-characterized heme systems (53), the generation of the "FeO" species thought to be responsible for DNA damage requires cleavage of the peroxide 0-0 bond, either heterolytically or homolytically. With respect to the latter mechanism, evidence from studies performed by Padbury et al. (45) involving the acid interaction of 10-hydroperoxy-8,12-octadecadienoic with FeBLM suggested that the peroxide intermediate of activated BLM underwent homolytic 0-0bond cleavage to produce the equivalent of a hydroxyl radical and ('OH)Fe3+.BLM (D in Figure 2), the latter proposed to be
Invited Review
Chem. Res. Toxicol., Vol. 5, No. 3, 1992 313 RS'
R0,PO
''3RA\\ I 0N
(02)Fe"BLM
R03p:v
(peroxide bypass)
A
.1
1
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B
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E
(RO-)(0)Fe+3BLM (RO)('OH)Fe'3BLM (O=Fe+')BLM ???
/
'I2
(O=Fe4)BLM ???
Fe+'BLM or RSH Fe+3BLMor RS'
l
Hete olytic
Ho olytic
R=H
R = alkyl
(RO0.)Fef3BLM
€
"Activated BLM"
Figure 2. Proposed mechanism for the activation of bleomycin. R = H or alkyl. the DNA-damaging species (45). This model has been criticized on several grounds. First, the mechanism proposed by Padbury et al. (45)requires the production of an equivalent of hydroxyl radical, but only substoichiometric quantities of hydroxyl radical have been detected by ESR spin trapping (54,55). However, the fact that hydroxyl radical can be detected even at low levels suggests that homolytic 0-0 bond cleavage may be occurring to some extent. It is generally agreed that freely diffusible species such as hydroxyl radical, hydrogen peroxide, or superoxide are not responsible for the bulk of BLM-mediated DNA damage given the exquisite selectivity of BLM for the C4'-hydrogen of deoxyribose (56-60), the minor quantities of hydroxyl radical-mediated base damage (e.g., hydroxyguanosine formation) (61,62), and the lack of significant inhibition of DNA damage by hydroxyl radical scavengers (46, 63, 64). Perhaps the strongest evidence against homolytic 0-0 bond cleavage in activated BLM has been presented by Natrajan et al., who recently demonstrated that the 10hydroperoxy-8,12-octadecadienoicacid-activated FeBLM, while it resulted in minor quantities of DNA damage after prolonged incubation, did not result in DNA damage that correlated with consumption of the peroxide (46). Thus, the homolytic 0-0 bond cleavage in alkyl peroxide-activated BLM may play a role in the mechanism of BLMs oxygen-transfer activity, but evidence for its involvement in the production of DNA lesions by BLM is weak. Heterolytic 0-0 cleavage should produce the equivalent of (0)Fe3+(E in Figure 2), perhaps as a perferryl species (1,46,65), that may be responsible for C4'-hydrogen abstraction (46). Enediynes. In spite of what appears to be significant structural diversity (Figure 3), the enediynes share several common features in their DNA-damaging activities. Like BLM, the enediyne structures can be divided into domains (Figure 3): the naphthoate of NCS (66-681, the carbohydrate side chains of CAL and ESP (30, 69-71), and the anthraquinone of DYN (31, 72, 73) all serve as DNAbinding domains, while the enediyne core of each drug constitutes the DNA-damaging machinery. Additionally, the amino sugar of NCS and the ethylamino group of the
carbohydrate chain of CAL and ESP appear to be involved in catalyzing the thiol activation step (74, 75), while the cyclic carbonate of NCS appears to be involved with drug uptake into cells (76, 77). The DNA-damagingelement present in all the enediynes consists of a highly strained ring system with a pair of triply unsaturated C-C bonds and a C-C double bond. The DNA-damaging form arises from a spontaneous electronic rearrangement of this ring system (Bergman rearrangement; 78) that results in a diradical species, as shown in Figures 4 and 5 (3,24). In the naturally occurring enediynes, the cyclization reaction is held in check by an energy barrier imposed by the C12-C1 unsaturation of NCS, the bridgehead unsaturation of CAL and ESP, and the epoxide of DYN (79-84). Removal of this barrier is accomplished by nucleophilic attack by exogenous thiol at the C12 of NCS (Figure 4) (85-87) and by a reductively-generated intramolecular thiolate in CAL and ESP (Figure 5) (27,30, 74), with thiolate anion constituting the form earlier hypothesized (88) and recently demonstrated to be responsible for activation of NCS (89). Rehybridization of these carbons from sp2to sp3 with subsequent release of strain energy is hypothesized to drive the electronic rearrangement of the closely approximated acetylene groups to form the diradica12 (Figures 4 and 5) (79-84). Situated in the minor groove (20,27,30,31, 71,9&92), the carbon-centered radicals of the activated species abstract hydrogen atoms from the deoxyribose sugar, an activity unequivocally demonstrated for NCS and CAL by deuterium labeling studies (93-96). The DNA-damaging radical is part of the enediyne carbon skeleton rather than a form of activated oxygen, as in the case of BLM and most other radical-mediated DNA-damaging agents. The remainder of this review will focus on BLM, NCS, and CAL, given the well-characterized and highly sequence-specific nature of their bistranded lesions.
* Two numbering systems have been used to designate the radical centers of calicheamicin: the carbons can be numbered 3 and 6 with reference to the structure of the parent compound (see Figure 5) (25);or numbered 1 and 4 (equivalent to 3 and 6 in the parent structure, respectively) if only the dihydrobenzene ring is considered (28,94,95). The 3,6 numbering is used throughout this review.
Dedon and Goldberg
314 Chem. Res. Toxicol., Vol. 5, No. 3, 1992 4eocarzinostatin
CH,NH
OH
1)) nciiii c i n
Esperamicin A,
0
Figure 3. Structures of enediyne antitumor antibiotics.
6. Interactions with DNA Bleomycin. Considerable experimental evidence identifies a minor groove location for BLM binding to DNA. The most obvious evidence stems from the C4'hydrogen specificity of the drug (vide infra), which suggests that at least the metal-binding domain is positioned in the minor groove. Further support is derived from the effects of known minor groove-binding agents on BLM activity. Both anthramycin, a minor groove-specificDNA-alkylating agent that bonds to the 2-amino group of guanine, and distamycin A, an A-T-specific equilibrium minor groovebinding agent, interfere with BLM-mediated DNA damage
P
r
(97,98). On the other hand, agents that block the major groove, such as bulky glucose residues bound to cytosine in the major groove of T4 DNA (99,100) and guanine N7 adducts with aflatoxin B1 (99), do not interfere with this activity. However, it should be noted that methylation of cytosine a t C5 and adenine at N6, both major groove modifications, reduced BLM activity (100). The reason for this appears to lie in DNA structural changes induced by these modifications (i.e., B-to-Z transitions), and not direct blockage of drug binding (100). Identifying the specific mechanisms of binding appears to be more complicated than the question of groove location in DNA. The major contribution to DNA binding
fl
L
D
C
AR =
R.
Figure 4. Proposed mechanism of activation of neocarzinostatin. Based on Myers (86).
Figure 5. Proposed mechanism of activation of calicheamicin.
Invited Review
Chem. Res. Toxicol., Vol.5, No. 3, 1992 315
2 3'-phosphoglycolate
w1
base propenal
H20
Fe+3BLM
4'
OP0,R'
I
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il
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I
'ZN t
+
e
R 0,PO
Yo
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z'
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4-hydroxylated abasic site
La
u
Figure 6. Proposed mechanism for bleomycin-induced DNA damage at C4'.
arises from the bithiazole moiety and its associated amine and/or sulfonium residue (Figure 1) (see ref 1). The exact nature of the bithiazole-DNA interaction remains controversial, however, since evidence exists for intercalative (from partial to full) (101-105)and nonintercalative modes (98,106), the latter involving hydrogen bonding between the right-handed curved bithiazole and the matching curvature of the base substituents on the floor of the minor groove (106).The positive charge of the amine/sulfonium residue appears to play a crucial role in binding, since a dialkyl sulfide analog and bleomycinic acid, a BLM analog missing the terminal amide group altogether, both show evidence of reduced interaction with DNA (101,107,108). Hecht and co-workers have presented evidence that the metal-binding domain also contributes to both DNAbinding and sequence specificity (107-109). Cu(I1)-BLM was shown to unwind supercoiled DNA 100 times more efficiently than demetallo-BLM, perhaps as a result of allosteric alteration of the drug conformation and/or an ionic interaction of the positively-charged metal-binding domain with DNA (108).This is consistent with the observation that the mobility of the metal-binding domain is restricted when metallo-BLM is bound to DNA (110-112).Furthermore, BLM analogs with the bithiazole separated from the metal-binding region by varying lengths exhibited somewhat similar sequence-specific DNA damage (109).On the basis of these findings, Hecht and coworkers developed a model in which the bithiazole is primarily responsible for sequence-independent binding activity, while the metal-binding head determines the sequence specificity (109).However, Kuwahara and Sugiura argue on the basis of experimental evidence and computer modeling that the 2-amino group of guanine, which is always found 5' to the damaged residue (C or T), hydrogen bonds to the bithiazole and that this interaction is re-
sponsible for the sequence specificity (98).Resolution of this issue awaits more rigorous crystallographic or NMR structural studies. BLM-mediated DNA damage was found to be sequence-specific, involving mainly pyrimidine residues in a 3'-position to G G-C and G-T (113,114).The structures and mechanisms of formation of these lesions have been studied by characterization of damage products and by isotope labeling experiments, with the end result being a hypothesized unified mechanism for BLM-mediated DNA damage, as shown in Figure 6. It was originally observed that (1)BLM treatment of DNA resulted in the release of two products: free DNA bases and a malondialdehyde-like material (63,115,116)and (2)the ratio of these products could be varied by changing the oxygen tension (52). Early in the study of BLM activity, Takeshita et al. had proposed that C4'-hydrogen abstraction could account for the production of malondialdehyde-like material (113),but firm proof that BLM removed the C4'hydrogen atoms came from later isotope labeling studies (vide infra) (57). The malondialdehyde-like material was demonstrated by Giloni et al. to be base propenal (56),and they and other groups established that stoichiometric quantities of 3'-phosphoglycolate-ended DNA fragments accompanied the base propenals (56,117,118).The observed oxygen dependence for the generation of these produck, led Giloni et al. to propose a reaction mechanism involving peroxide formation at the C4'-radical(2 and 3) that eventually leads to a C3'-C4' bond scission (branch A of Figure 6) (56). Numerous mechanistic models for the formation of 3'phosphoglycolate and base propenal residues from the C4'-peroxide have been proposed (I,56,65,119).However, any proposed model must account for the following observations:
316 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
(1) Strand breakage and C2’-hydrogen removal, the latter specific for the C2’-pro-R-hydrogen(120,121),occur an order of magnitude faster than base propenal release (65, 121).
(2) One oxygen of the glycolate carboxyl group is derived from molecular oxygen, apparently the oxygen forming the C4’-peroxide,and the other from the original deoxyribose ring oxygen (121,122). (3) The oxygen of the aldehyde of the base propenal appears to be derived from solvent (H,O) (121). (4) The C3’ carbon-oxygen bond is broken during release of the 5‘-phosphate-ended fragment (120). None of the models proposed in refs 1,56,65, and 119 fulfill all these criteria. To account for these observations, McGall et al. (121) have very recently proposed a model, shown in Figure 6, branch A, for BLM-mediated formation of 3’-phosphoglycolate and base propenal. Instead of an initial C3’44’ bond cleavage, the Criegee-rearranged sugar 4 opens by cleavage of the 041’ bond induced by a rapid C2’-hydrogen removal. Subsequent slow release of base propenal can then occur by either of two pathways (C and D in Figure 6). Verification of this proposed mechanism awaits further study. The other major DNA damage observed with BLM is shown in branch B of Figure 6. It was observed that BLM caused the release of free base (1151, shown to be dependent on activated BLM (49), and the formation of alkali-labile sites (26, 123). Both events were suspected of being related to each other (50,52,124), and Wu et al. eventually demonstrated a direct relationship between free base release and the formation of an alkali-labile C4’ketone-containing abasic site (58). The structure of the sugar residue forming the abasic site was accomplished by Stubbe, Kozarich, and co-workers (125) and Hecht and co-workers (126), both of whom presented results consistent with the production of intermediates 10 and 11, the so-called 4’-hydroxylated abasic site (Figure 6). It was demonstrated by Rabow et al. that 10/11 is produced stoichiometrically with free base release (127). Given the oxygen independence of this pathway, two mechanistic models were proposed (128). The C4’-radical could be oxidized, perhaps by the putative (-OH)Fe4+-BLMgenerated by C4’-hydrogen abstraction into (0)Fe3+.BLM(E in Figure 2) (128), to yield a C4’-carbonium ion, which would produce the 4‘-hydroxylation product upon addition of H,O (Figure 6). Alternatively, in a manner similar to the oxygen rebound of P450 and heme systems, hydroxyl radical could be added to the C4’-radical to generate the same final product (45,53,129). Experimental support for the carbonium ion mechanism has come from Rabow et al. with the demonstration that solvent serves as the source of oxygen at the C4’-position in 10/ 11, and not O2 (128). The oxygen rebound pathway would necessitate exchange of the C4’-ketone oxygen with solvent, which did not occur on the time scale of their experiments (128). Similarly, the addition of H,O to a carbonium ion intermediate has been proposed to account for the C4’hydroxylation product observed in the oxidation of the C4‘-carbon of 2’-deoxyaristeromycin,a nucleoside analog with a carbon replacing the deoxyribose ring oxygen (130). The primary evidence for C4’-hydrogen abstraction by BLM has come from isotope labeling experiments performed in a collaborative effort by the Stubbe and Kozarich laboratories (57-60). In initial experiments, large and constant tritium selection effects were demonstrated for the production of both the free uracil release and uracilpropenal in poly(dA-[4’-3H]dU) treated with Fe-BLM under varying conditions of oxygen concentration (58,59).
Dedon and Goldberg
Using a [4’-2H]thymidine-labeled DNA restriction fragment, they recently demonstrated a primary kinetic isotope effect kH/kD = 2-4.5 for both types of product (60). Thus, it was demonstrated that C4’-hydrogen abstraction was the initial step in the DNA damage produced by BLM. These experiments also led to the conclusion that both the base release pathway and the glycolate/propenal pathway were the result of the partitioning of a common intermediate arising from the C4’-hydrogen abstraction by BLM. Neocarzinostatin. The modes of DNA binding by NCS have been more readily characterized than those of BLM. The drug binds to DNA (KD lo4 M; 131)in what appears to be a two-step process, with either external and intercalative modes or two types of intercalative binding (68). The naphthoate appears to behave like a classical intercalator, orientated parallel to the DNA bases (electric dichroism studies), and causing a helix unwinding and lengthening of 21’ and 3.3 A, respectively (66). Evidence for a minor groove binding site comes from two sources. First, major groove modifications (glucosyl moieties of T4 DNA and halogenated dU in synthetic DNA) did not alter the NCS binding constant, while netropsin and distamycin were able to compete with NCS for binding to DNA (90). As will be discussed in more detail later, NCS produces bistranded lesions with cleavage sites on opposite strands staggered 2 bp in a 3‘-direction (20,92), which suggests that the drug is oriented in the minor groove with its radicals abstracting hydrogen atoms from deoxyribose residues directly across the minor groove from each other (4). The DNA damage produced by NCS, shown in Figure 7, presents a more complicated picture than the exclusive C4’-hydrogen abstraction mediated by BLM. NCS was originally observed to cause mainly thiol- and oxygen-dependent (132-135) SS breaks with a base preference of T > A >> C > G (136, 137), but little sequence specificity (138). The major lesion involved a SS break with 3’phosphate and 5’-thymidine 5’-aldehyde ends (139, 140), which was subsequently shown, using [EI’-~H] thymidinelabeled DNA (141,142) and (143),to arise by drugmediated abstraction of the C5’-hydrogen followed by incorporation of O2 into the aldehyde (Figure 7). It has now been established that all NCS-mediated lesions require oxygen or its substitute for expression (144-146), except for covalent drug-DNA deoxyribose adducts (147, 148). The finding of a 1:l thiol-NCS adduct (85) and the fact that drug-mediated hydrogen abstraction occurred under anaerobic conditions (141) led to what is the currently accepted model in which nucleophilic addition of a thiol to NCS resulted in the formation of a diradical species that abstracted hydrogens from DNA (141). Later structural studies (see ref 24) led Myers (86) to propose the thiol activation pathway shown in Figure 4. While nucleoside 5‘-aldehydes accounted for a majority of the damage produced by C5’-hydrogen abstraction, the remaining lesions consisted of phosphate-ended fragments (240). The mechanism of the formation of this minor lesion was solved with the aid of the nitroaromatic radiation sensitizer and oxygen substitute, misonidazole (structure in Figure 8), which was found to increase the proportion of phosphate-ended fragments in NCS-treated DNA under anaerobic conditions (149). [5’-3H]Thymidine-labeled DNA was used to show that the 3’-phosphate-ended fragment in these minor lesions arose by hydrolysis of a labile 3’-formyl phosphate residue (150) (Figure 7 and structure 3 in Figure 8), which, under anaerobic conditions with misonidazole, accounted for virtually all the damage arising from C5’-hydrogen abstraction (149, 150). Subsequent experiments with
-
Invited Review
Chem. Res. Toricol., Vol. 5,No. 3, 1992 317 0 H
0' I H
6#
0
03p0'
-
'
I
N
nucleoside 5'-aldehy de
3'-phoiphate
P - d1 n
+
00,PO
I (RSH)
H
OPO".,
"op,o GJH0C = GOH/PC > GOH/HoC.The phosphate-ckarged gap present in both models was proposed to resemble the 3’-phosphoglycolate and 5’-phosphate ends present at BLM-induced SS breaks. Clearly, BLM cleavage is enhanced by a strand break on the opposite strand, especially one that contains charged fragment ends.
Again, however, the formation of a 3’-overhang is not consistent with the DS cleavage site structure found in larger DNA fragments without gaps (165,166). Steighner et al. also suggest that the Keller and Oppenheimer model is flawed from the standpoint of the kinetics of DNA damage formation (166). They argue that, since BLMmediated DS breaks are formed faster than base propenal is released (65),attack by a second molecule of BLM must occur before the phosphate-ended fragment is formed (166). However, while Burger et al. have demonstrated that strand breaks form faster than base propenals are released (65), it has not been established unequivocally which products form first to cause the strand break: the 3’-phosphoglycolate or the 5’-phosphate-ended fragment (I). Either way, a strand break with a negative charge results before base propenal is released, a situation that Keller and Oppenheimer suggest is attractive to the binding of a second molecule of BLM (167). It is possible that the presence of the base propenal serves to block access of the second BLM to the cleavage site, as suggested by the hairpin model with ddA filling the gap (Figure 9A) (167),which would support the arguments of Steighner et al. (166). Clearly, further study is required before this dilemma can be resolved. The model for BLM-induced DS lesions proposed by Steighner et al. is based on a survey of the chemical structures formed in BLM-mediated bistranded lesions (165,166). DS breaks produced by BLM in end-labeled restriction fragments were resolved in nondenaturing gels, and purified DNA from excised bands was resolved on a sequencing gel. The sequence identity and the nature of the chemical structure at individual cleavage sites were determined by comparing the mobilities of the fragments relative to Maxam-Gilbert chemical cleavage standards. They found that every DS break occurred with cleavage at the pyrimidine of a G-Py sequence on one strand accompanied by a cleavage directly opposite the pyrimidine or staggered one nucleotide in a 5’-direction on the opposite strand. There were no breaks occurring with a 3’-stagger to the cleavage sites, as were proposed to occur at GQGC sequences by Mirabelli et al. (163). Interestingly, the breaks in the strand opposite the G-PJ occurred at sites not normally associated with SS lesions, and they rarely had the predicted G-Py sequence (165). Further refinement of these findings was accomplished by examining BLM-induced damage a t several G-C steps known to be mutational hot spots for BLM (167) (see below). As illustrated in Figure 10, the major site of damage was determined to be the C of the expected GC sequence, and the damage at this “primary site” consisted of either a 3’-phosphoglycolate-ended fragment or an apyrimidinic (AP) site. Less frequently, damage was also noted to occur at the directly opposed G in the complementary strand (the secondary site), with lesions consisting again of 3’-phosphoglycolateresidues and AP sites (Figure 10). The crucial observation in these studies was that secondary site damage only occurred when there was a strand break at the primary site, i.e., damage at the secondary site was always part of a bistranded lesion. Using an elegant combination of amines to cleave AP sites and T4 polynucleotide kinase to remove 3‘-phosphates, they determined that bistranded lesions always occurred with a 3’-phosphoglycolate present at the primary site, while the secondary site in a bistranded lesion could be either an AP site or a strand break. An AP site as the primary damage did not occur in bistranded lesions. To account for these observations, they proposed that BLM abstracted a C4’-hydrogen at the primary site and,
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320 Chem. Res. Toxicol., Vol. 5, No. 3, 1992
$.Secondary A t t a c k coo-
NO Further Reaction
???’??? T C G C C A
poo-
coo-
???f??l??lP’Tlpl T C G C C A A G C G G T
T C G C C A A G C G T
-006
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coo-
??+’ p? T C G
C A
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-006
Figure 10. Mechanism proposed by Steighner and Povirk (166) for the production of bistranded lesions by bleomycin. Attack by BLM at the C4’-position occurs first at the primary site, with partitioning to form either an abasic site (no C4’-peroxy radical) or a 3’-phosphoglycolate-~ontaining strand break (peroxy radical present at C4’). The presence of a peroxy radical or peroxide at C4‘ of the primary site is proposed to reactivate BLM toward
attack at the secondary site on the opposite strand, which can again partition to form either an abasic site or a strand break. See text for details. Reprinted with permission from Steighner, R. J., and Povirk, L. F. (1990) Bleomycin-induced DNA lesions at mutational hot spots: Implications for the mechanism of double-strand cleavage. Proc. Natl. Acad. Sci. U.S.A. 87, 8350-8354. if the primary site was involved in a glycolate-forming lesion, the peroxy radical or peroxide intermediate served to regenerate activated BLM in either of two ways. First, the peroxide could interact with the spent Fe3+.BLM to form an alkyl peroxide-Fe3+.BLMcomplex, with subsequent homolytic 0-0 bond cleavage generating two products: the (‘OH)Fe3+-BLMequivalent of activated BLM suggested by Padbury et al. (Figure 2) (45) and a glycolate-forming intermediate, for which an oxygen radical is the logical candidate if the putative peroxide 0-0bond cleaves homolytically (45). The “activated BLM” would proceed to abstract the C4’-hydrogen atom from the secondary site on the opposite strand. However, such a mechanism seems improbable given the apparent incompatibility of the homolytically produced (‘OH)Fe3+.BLM with DNA damage (46). Furthermore, the mechanism of
Dedon and Goldberg
product formation from an oxygen radical-mediated /3scission of the C3’-C4’ bond would not be consistent with the proposed mechanism of BLM-mediated C4’-chemistry (121). Alternatively, the C4’-peroxy radical could reabstract the hydrogen atom from the equivalent of (OH-)Fe4+.BLM (128) to again regenerate activated BLM, (0)Fe3+.BLM, and the C4’-peroxide (166). If the primary site lesion evolved along the abasic site pathway, no peroxy radical would be formed and no reactivation of the BLM would occur. The findings of Povirk and co-workers provide a more complete model for BLM-mediated bistranded lesions than other proposals, but the proposed mechanism requires verification. To distinguish between two separate BLM complexes’ binding and abstracting hydrogens from opposite strands, as in the Keller and Oppenheimer model, and one BLM complex remaining bound to cause both lesions will require that the kinetics and mechanism of the formation of both 3’-phosphoglycolate and base propenal be firmly established. Neocarzinostatin. Like BLM, NCS- and CAL-induced DS lesions can result from random, independent cleavage at T and A residues at closely opposed sites, or by the preferred cleavage by a second drug molecule opposite an existing SS break site. Unlike BLM, however, the proposed diradical structure of the active NCS and CAL intermediates (Figures 4 and 5) suggests a third possibility: a concerted reaction involving simultaneous bistranded cleavage at a single site by a single drug molecule. Evidence supporting the last model has come from several types of DNA damage analyses, including supercoiled plasmid studies, sequencing gel analysis of cleavage fragments, isotope labeling studies, and molecular modeling and dynamics calculations. The finding of high ratios (30:l to 50:l) of SS to DS breaks under in vitro conditions with NCS using 2mercaptoethanol as the thiol activator (133,168) had led to the conjecture that DS breaks result from the nearrandom placement of SS breaks a t closely opposed sites. However, in vivo experiments in mammalian cells, where glutathione probably serves as the thiol activator (169-171), have given a ratio of -51 (13,172). This apparent discrepancy was resolved using a supercoiled plasmid model system to quantitate DS and SS lesions produced by NCS (89,92). In an initial set of experiments, it was determined that glutathione resulted in a ratio of SS to DS breaks of 6:1, while with 2-mercaptoethanol the ratio increased to 42:l (92). This indicated that the thiol had significant effect on the relative quantities of SS and DS lesions. Additionally, the low SS:DS ratio for glutathione and the observed linearity of the variation of the quantity of DS and SS breaks with drug concentration suggested that DS cleavages with glutathione as activator are caused by a single event or interdependent events, and not by the coincidence of random SS breaks on opposite strands (26). Subsequent studies revealed even lower SS:DS ratios when abasic sites were expressed as strand breaks and drug-DNA incubation time was extended to allow complete formation of glycolate-containing strand breaks (89), which were determined to form with a t l l z = 12 min (29). The increased reaction time resulted in a decrease in the SS:DS from 6:l to 5:l for glutathione, and from 41:l to 22:l for 2-mercaptoethanol(29). Expression of putative 4’-hydroxylated abasic sites as 3’-phosphopyridazine-ended strand breaks by hydrazine treatment resulted in a SS:DS of -4:1, while the additional cleavage of putative 2’-deoxyribonolactone-containingabasic sites
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Invited Review
A
3’
I
‘Y B
3‘
Figure 11. Stylized models of neocarzinostatin-induced bistranded lesions. (A) AGCeGCT site. (B) AGT-ACT site. by putrescine lowered the ratio to -2:l. This latter value is comparable to the ratio of -2:l for CAL determined by Drak et al. for direct strand breaks (28).Conclusive evidence for the presence of these two types of abasic sites in bistranded lesions produced by NCS came from DNA sequencing studies (20,29,89, 144,154). The trinucleotide sequence specificity of NCS-induced DS lesions also supports the hypothesis that bistranded lesions result from the action of a single drug molecule. NCS has been shown to produce two types of bistranded lesion. The first described lesion occurs at the AGC-GCT sequence (20,144) and results from C1’-hydrogen abstraction at the C of AGC (144,154), accompanied by a C5’-hydrogen abstraction at the T of the complementary GCT (20)(Figures 7 and 11A). Nearly every abasic site at the C of AGC is accompanied by a strand break at the T on the complementary strand (201,while strand breaks at the T of GCT occur frequently as isolated lesions (144). Direct evidence for C1’-hydrogen abstraction comes from deuterium labeling studies, in which a deuterium isotope selection effect kH/kD 4 was observed when deuterium replaced hydrogen at the C1’-position (158).Interestingly, substitution of an I residue, which lacks a 2-amino group on the base, for the G of AGC markedly reduces abasic site formation, whereas placement of an I residue opposite the C enhances abasic site formation 4- to 5-fold (144).The latter substitution also eliminates the deuterium isotope effect on abasic site formation at the C residue, which is expected if enhanced abasic site formation is due to an increase in the relative rate of hydrogen abstraction by the activated drug versus its dissociation from DNA. Molecular modeling and dynamics studies have been performed with the postactivated form of NCS (Figure 4) and an AGC-GCT-containing oligomer: 5’GAGCG. 5’CGCTC (173).Stable, low-energy structures can be rationalized that intercalate the naphthoate moiety between the A-T and G C base pairs with the diradical core oriented toward the 3’-end of the AGC-containing strand (Figure 12). This orientation laces the C6 radical close to the C5’ of thymidine (3.31 ) and the C2 radical close to the C1’ of cytidine (4.10 A). Examination of models of B-DNA
-
x
suggesta that the minor groove-oriented prochiral Hs of the C5’ position is the one abstracted by NCS, rather than which points toward the major groove. the HR, Chemical evidence to support this model comes from the recent studies of Meschwitz and Goldberg (96),in which deuterium was substituted for both C5’ hydrogen atoms of the T of AGC-GCT in a self-complementary oligonucleotide. Reaction of the oligonucleotide with NCS and subsequent ‘H-NMR analysis of the drug product revealed the presence of deuterium only at the C6-position in the drug (Figure 4), in agreement with the molecular modeling studies. Further confirmation of the model comes from recent experiments6 showing that deuterium from the C1’ of the C of AGC is abstracted exclusively by the C2-position of the activated drug. Isolated cleavage at the T of GCT, unaccompanied by abasic site formation at the C of AGC, can be rationalized by two different mechanisms: different drug binding modes, or the same binding mode as occurs in DS lesions but with abasic site formation being less efficient due to chemical or geometric reasons. Molecular modeling predicts a second stable binding mode in which the naphthoate intercalates between the GC/C-G step and the active core is oriented toward the 3‘-end of the GCT-containing strand (173).This alternative places the C2 of the drug close to the C5’-hydrogen of the T of GCT. However, in the deuterium labeling studies described previously, SS breaks at the T of GCT were the predominant lesion that occurred in the AGC-GCT-containingoligonucleotide, and yet deuterium was incorporated only at the C6-position of the drug (96). It is possible that the selection for SS breaks or bistranded lesions occurs as a result of minor groove geometry placing the C6-radical center closer to its target C5’-hydrogen on one strand more frequently than the C1’ on the other strand. Alternatively, the selectivity could result from differing chemical reactivities of the deoxyribose hydrogens, with the C1’-hydrogen of the C of AGC less readily removed or the primary damage more easily repaired by thiols, for example, than the C5’-hydrogen abstraction. Finally, two other possibilities that will be discussed shortly involve different reactivities of the C2- and C6-radicals of the drug, and selective quenching of the drug C2-radical and/or DNA radicals by intramolecular hydrogen transfer from the adducted thiol. SS and DS lesions at AGT-ACT may be similarly explained. A third NCS-DNA model involves the same intercalation site at the GC-GC step, but places the active core perpendicular to the helix axis so that the C6-radical now lies near the C5’-hydrogen of the T of GCT at a distance of 3.47 A (173). This could account for the deuterium incorporation noted previously. Another modeling study performed by Chen et al. (174),before the structure of the activated form of NCS was known, also places the enediyne core perpendicular to the minor groove in a way that could account for SS breaks. The second trinucleotide DS cleavage site occurs at AGTsACT (Figure 11B), which was discovered to be the major site of direct DS breaks produced by NCS (92). Studies of the chemistry of DNA damage at this site revealed an interesting discrepancy: bistranded lesions consisted of mainly C4’-hydrogen abstraction at the T of AGT, as suggested by the presence of 3’-phosphoglycolate residues and 4’-hydroxylated abasic sites, and C5’ chemistry at the T of ACT, while SS breaks appeared to be caused by a mixture of C4’- and C5’-hydrogen abstraction, S. M. Meschwitz, R. G. Schultz, G. Ashley, and I. H. Goldberg, unpublished observations.
322 Chem. Res. Toxicol., Vol.5,No.3, 1992
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G
Figure 12. A model of the interaction of postactivated neocarzinostatin with the AGCeGCT site for bistranded lesions. Dots represent putative radical centers of activated NCS. From Galat and Goldberg (173).
but were devoid of 3’-phosphoglycolate residues (Figures 7 and 11B) (29,92).This suggested that partitioning of C4’ chemistry was affected by the environment of the DS lesion. The thiol sensitivity of 3’-phosphoglycolate formation and the ability of misonidazole to substitute for dioxygen in the mechanism of glycolate production both suggested the presence of a reducihle intermediate in the glycolate/propenal pathway of C4’ chemistry. The misonidazole evidence strongly favors an oxygen radical, as discussed earlier for C5’ chemistry (29,89). The most significant finding, however, involved the appearance of 3’-phosphoglycolate residues in SS lesions when misonidazole was the oxygen source, which resulted in 3’phosphoglycolateaccounting for nearly 30% of the damage at the T of AGT in SS lesions (29).Two models for the partitioning of C4’ chemistry in bistranded lesions and SS lesions have been developed (29).In a SS lesion, C4’-hydrogen abstraction ultimately results in the formation of a C4’-peroxy radical (Figure 7) that can, in the majority of cases, partition to form the hydroxylated abasic site, or less frequently react to form an oxygen radical. In the presence of thiols and other reducing species, the oxygen radical is reduced to a hydroxyl group and the 4‘hydroxylated abasic site is produced at the expense of the glycolate/propenal products. In the presence of misonidazole, virtually all of the C4’ chemistry results from oxygen radical formation, a significant quantity of which survives long enough to undergo /3-scission to produce the 3’-phosphoglycolate. A second model explains the fact that significant quantities of 3’-phosphoglycolate are found only in bistranded lesions. An intramolecular tetroxide bridge may form between the peroxy radicals at the C5’-position of the T of ACT on one strand and peroxy radicals at the C4’or C5’-pition of the T of AGT on the other strand (Figure 13). A similar proposal, but involving tetroxide formation between separate strands of poly(uridy1ic acid), has been made to account for chain cleavage due to ionizing radiation (175). Preliminary molecular modeling experiments suggest that the tetroxide can readily form between deoxyribose residues staggered 3 bp in a 3‘-direction, as occu in NCS-mediated bistranded lesions, with little alteration of the minor groove dimension^.^ Breakdown of the tetroxide can then occur by several pathways, including the direct production of two oxygen radicals along with molecular oxygen and a concerted mechanism that results in glycolate production without an oxygen radical intermediate (Figure 13) (175,176).If the bridge were to involve C5’-positions on both strands, this might explain the dominance of the formyl phosphate pathway in bistranded lesions at AGT, where the C5’ chemistry is represented by
