Chemistry of drug-induced DNA lesions

Toxicology Letters, 61(1993) 0

3-l 5

1993 Elsevier Science Publishers B.V. All rights reserved 037%4274/93/$06.00

TOXLET 02847

Chemistry of drug-induced DNA lesions

Isa0 Saito Department

of Synthetic

Chemistry, Kyoto University, Kyoto (Japan)

Key words: Antitumor antibiotic neocarzinostatin;

DNA alkylation; Abasic-site structure

SUMMARY Recent results on the action mechanisms of naturally occurring DNA-damaging antitumor antibiotics have been described. These antibiotics include neocarzinostatin (NCS) and DNA alkylating, duocarmycin A and kapurimycin A3. A series of duplex hexanucleotides of modified bases were prepared and their selectivity for C5’ and C4’ oxidation in the NCS-mediated degradation was investigated. Based on the cleavage data, a new binding model that permits competitive hydrogen abstraction from C5’ and C4’ of the DNA deoxyribose moiety has been described. Chemistry of alkylation of self complementary octanucleotide d(CGTATACG), by antitumor antibiotic duocarmycin A was described. It was demonstrated that N3 of adenine, attacks the cyclopropane subunit of duocarmycin A to produce the covalently alkylated adduct. In contrast, antibiotic kapurimycin A3 alkylate N7 of guanine, of d(CGCG), to provide the corresponding covalent adduct. Heating at 90°C degraded the adduct to kapurimycin A3-guanine adduct and the respective abasic site-containing oligonucleotide. The structures of heat-induced abasic sites were unambiguously characterized.

INTRODUCTION

DNA is the biological target of a class of naturally occurring antitumor antibiotics, synthetic chemotherapeutic agents, as well as of many chemical carcinogens. DNA structure and function are altered by covalent modification by these DNA-damaging agents at their binding sites. Elucidation of the detail mechanisms of DNA lesions is extremely important not only for uncovering molecular mechanisms of chemical carcinogenesis but also for the design of sequence specific DNA-cleaving agents or potent chemotherapeutic agents. In recent years, there has been much interest in a class of naturally occurring DNA-damaging agents that interact at specific sites of DNA and modify or cleave the DNA strand at the site of binding [l]. Alkylation of DNA by naturally occurring

Correspondence to: Professor I. Saito, Department of Synthetic Chemistry, Faculty of Engineering, Kyoto University, Kyoto 606, Japan.

4

alkylating agents occurs at DNA purine base in a sequence-dependent manner to produce an alkaline-labile site which is followed by either chemical or enzyme-catalyzed hydrolysis of the N-glycoside bond to give a baseless site, called the ‘abasic’ (AP) site. A smaller number of DNA-damaging agents are also known to attack DNA deoxyribose moieties rather than DNA base to result in a formation of direct strand breaks and alkaline-labile breaks at abasic sites. Among the latter are bleomytins [2] and bicyclic enediyne families of antitumor antibiotics such as neocarzinostatin (NCS) [3], calicheamicins [4], esperamicins [5] and dynemicin A [6]. These enediyne antibiotics are converted by thiol activation under physiological conditions to highly reactive carbon-centered radicals which can then abstract hydrogen from the DNA deoxyribose moieties [3,7]. While the DNA damage resulting from hydrogen abstraction from Cl’, C4’ and C5’ positions of DNA deoxyribose has been demonstrated as illustrated below, the chemistry of the resulting radicals leading to oxidative cleavage of the DNA backbone has not yet fully been understood.

NCS,

Calicheamicin

Bleomycin (NCS)

In our laboratory, the action mechanism of naturally occurring DNA-damaging antitumor antibiotics has been studied extensively by using synthetic oligonucleotides of defined sequence as DNA substrate. These antibiotics include bleomycin, neocarzinostatin (NCS) and DNA alkylating, duocarmycin A and kapurimycin A3. Herein described are our own recent results on NCS and the DNA-alkylating antitumor antibiotics, duocarmycin A and kapurimycin A3. NEOCARZINOSTATIN

(NCS)

NCS is an antitumor antibiotic consisting of nonprotein chromophore (NCS-C, 1) and its carrier protein. NCS-C undergoes irreversible reaction with thiols to generate a biradical species which is capable of cleaving DNA with a high degree of base specificity (T > A >> C - G) upon aerobic incubation [3,7]. Based on the structural elucidation of the chromophore-thiol adduct, the activation mechanism shown in Scheme I (path A) has been proposed [8]; nucleophilic attack by thiol at Cl2 and epoxide ring opening generate an enyne[3]cumulene 2, which spontaneously cyclizes to form the indacene diradical3. While the intermediary formation of 2 at extremely

5

low temperature in organic solvent has been supported by *H-NMR [9], the exact nature of the thiol-I reaction under physiolo~cal conditions still remains to be clarified. We have recently observed an unprecedented cyclization of NCS chromophore 1 leading to the formation of a novel cyclization product 5 as a major product in the reaction with 2-mercaptoethanol in aqueous-buffered solution (path B) [IO].

Scheme I.

A soiution of NCS and 2-mercaptoethanol in Tris-HCl (pH 7.0) was incubated at 0°C under aerobic conditions. HPLC analysis of the reaction mixture after 12 h incubation revealed the formation of one major product. This product was isolated by HPLC, and its structure was assigned as 5 on the basis of spectroscopic data including NMR (‘H-‘H COSY, NOE, r3C--‘H COSY), FABMS, UV and FTIR. Quantitative analysis indicated that the yields of 5 and 4a are 59 and 9.5%, respectively. The ratio of 5 vs. 4a is highly dependent on the concentration of added isopropanol. In a solvent containing 80% isopropanol 4a (45%) was a major product together with 5 (5.1%). It is very likely that 5 is produced from 1 when complexed with its apoprotein in an aqueous solution (Scheme II).

1

nocx,cw?=4

WOH

+

da

Scheme II.

The fact that 5 is produced under anaerobic conditions indicates that the oxygen molecule may not be incorporated into 5. When the reaction was conducted in H,‘80, the labelled ‘$0 was incorporated into 5 as revealed by FABMS. When D,O was used as a solvent, deuterium was incorporated at the C7 position of 5 to the extent of 80%.

These results suggest that 5 is formed from zwitterionic Alternatively, the direct nucleophilic attack of water to also produce 5. Our results demonstrated that there are pathways in the reaction of NCS with 2-mercaptoethanol tions [lo].

precursor 6 (Scheme III). enyne[3]~umulene 7 would two distinct aromatization under physiological condi-

Scheme III.

We have recently demonstrated that the use of oligonucleotides as a sequenceselective substrate for NCS provides a very useful tool for understanding the chemistry of NCS-mediated DNA degradation. By using a self-complementary hexanucleotide d(GCATGC),, we were able to characterize the structure of the previously unidentified oxidized deoxy~bose moiety associated with spontaneous free base release [l 11.We also observed that previously unobserved C4’ hydroxylation of deoxyribose moiety does occur significantly at T3 of a self-complementary hexanucleotide d(C,G,T,A,C,G,), in competition with CS’ oxidation at A, (Scheme IV) [12]. OH OHCo T cs cGp0 0T -c@ + H’. H’ v 9 OPAcG OPAffi

Scheme IV.

A similar C4’ hydroxylation has also been observed in NCS-mediated degradation of calf thymus DNA. Specific detection methods of C4’ hydroxylated abasic sites recently developed in our laboratory have indicated that C4’ hydroxylation is estimated to be a minimum of 17% of the total event occurred by action of NCS on calf thymus DNA 1131. In order to get insight into the binding geometry of neocarzinostatin (NCS) that

permits competitive hydroxylation at CS and C4’ of the deoxyribose moiety of DNA, a series of hexanucleotides possessing A-T, G-C, inosine (1)-C and 2-aminoadenine (ANHZ)-T base pairs at the 5’ side of the target thymine were prepared and their selectivity for C5’ and C4’ oxidation in the NCS-mediated degradation was investigated. Quantitative product analysis indicated that preferential C5’ oxidation of the deoxyribose moiety of the target T occurs at -5’-AT- and 5’-IT- sites, whereas C5’ and C4’ oxidation occurs competitively at T of the -S-CTand -5’-ANH2T- sites as shown below.

96% 4%

(4' = 3%, 5' = 97%)

94%

(4' = 41%,

5' = 59%)

91%

I

I

I

GCiTGC CGT?CG t

GCGTGC

GCITGC

cGcPcG

CGCPCG 9%

6%

78%

37% Y% 1

GCATCG CGT?GC

(5' = 100%)

(4' = 62%,

5' = 38%)

1 4'=5% 5'=95%

I

56%

GCKTCG CG;rAGC 22%

(5' = 100%)

Based on the experimental results, an intercalation model that permits competitive hydrogen abstraction from C5’ and C4’ of the deoxyribose moiety has been proposed [14]. Molecular modeling studies using AMBER suggested that in the complex between d(GCATGCh and 4a, C6 carbon radical of 4a is close to the target C5’ (pro-S) hydrogen. In contrast, the tricyclic core of 4a is slightly raised so as to become closer to C4’ hydrogen in the complex between d(GCGTGC)/d(CGCACG) and 4a due to the steric repulsion between the guanine 2-amino group in the minor groove and the core moiety. The energy minimized complex between d(GCATGC)2 and 4a by using AMBER has indicated that the methylamino group of sugar moiety of the NCS chromophore is participating in the specific recognition of thymine residue by hydrogen bonding as illustrated in Figure 1. ALKYLATION

OF DNA

Various types of DNA-alkylating

agents are known in nature. These DNA-damag-

8

9

ing agents, including antitumor antibiotics and naturally occurring mutagens, possess a reactive functional group that behaves like a carbocation equivalent in order to facilitate covalent alkylation of the ring nitrogen of adenine or guanine base of DNA. Such reactive functional groups are in some cases conjugated cyclopropane, aziridine or epoxide. Representative DNA-alkylating antitumor antibiotics and their alkylating ring nitrogen of DNA purine base are shown below.

Mitomycin

C (Gunninc

N2)

Anthrarnycin

(Gunninc

N2)

Duocarmycin A

Duocarmycins are a new class of antitumor antibiotics produced by Streptomyces sp. and are effective against murine lymphocytec Leukemia ~388 and murine Sarcoma 180 in mice. The structural similarities between duocarmycin A and CC-1065 [lS] suggest that this antibiotic may be acting by a related mechanism involving alkylation of DNA.

Duocarmycin

A

CC-1065

10

According to the sequence specificity reported for the DNA alkylation with duoca~ycin A, we selected seIf-compIementa~ octanucleotide d(CGTATACG), as a substrate to investigate the chemistry of the DNA damage. A typical reaction mixture containing duocarmycin A (0.01 mM) and d(CGTATACG), (1 mM base cont.) in sodium cacodylate buffer (pH 7.0) was incubated at 0°C for 1 h. HPLC analysis indicated that approximately half of the octamer was consumed with formation of one major product 8. Upon heating at 90°C for 5 min, 8 was immediately converted to 9 and 10. The structures of 9 and 10 were confirmed by their spectroscopic data as well as chemical transformations shown in Scheme V [16]. These results clearly indicated that N, of adenine, attacks the cyclopropane subunit of duocarmycin A to produce 8 which upon heating is converted to abasic site-containing oligomer 9 with concomitant release of adenine adduct 10, Alkylation of A6 of d(CGTATACG) with duocarmycin A has a first-order kinetic with the rate constant of k = 6.8 x low5s-’ at 0°C and k = 2.8 x 10W4s-’ at 37°C. Covalent alkyiated adduct 8 was fairly stable at 0°C and no decomposition was observed after 2 days. At higher temperatures 8 was converted to 10 with a tyl of 134 h at 37°C and of 1.l h at 60°C.

NH

OH

8

OpCG

9 piperidine

CGTAT (Strand

+

pCG

Cleavage)

Scheme V.

Of particular interest is the clean formation of 9 from 8 upon brief heating (90°C, 5 min) at neutral pH. By contrast, under the prolonged heating (lOO’C, 30 mint

11

conditions, 9 was no more stable and completely decomposed to d(pCG) and d(CGTATp) bearing multiple modified sugar ends as revealed by HPLC. Therefore, it is suggested that the thermal cleavage of alkylated DNA previously reported [17] may result from the further degradation of the abasic sites such as 9. It is also noteworthy that incubation of duocarmycin A with calf thymus DNA followed by heating (90°C, 20 min) produces duocarmycin A-guanine adduct 11 together with 10 in a

1) Duocarmycin A

calf thymus DNA

2) 9O”C,20min

Duw Adcnine Adduci

10

Duo- GuanineAdducl

11

Scheme VI.

ratio of 1:1.3 as shown in Scheme VI, in sharp contrast to CC-1065 which produces only adenine adduct specifically upon incubation with calf thymus DNA [ 151. Kapurimycin A3 Kapurimycin A3 (kap A3,12), isolated from Streptomyces sp. DO-l 15, was found

Kapurimycin A3

(12)

12

to exhibit cytotoxic activity, and its epoxide ring has been suggested to alkylate guanine residue of DNA [18]. We have demonstrated the detailed chemistry of kap A3induced DNA damage by reacting this antibiotic with a self-complementary deoxytereanucleotide d(CGCG), [ 191. The reaction mixture containing 12 and d(CGCG)* in neutral pH was incubated at 0°C. HPLC analysis indicated that major (13) and minor (14) products were formed after 5 h reaction. Upon heating at 90°C (5 min), 13 was completely converted to abasic site 15 and kap-A3-guanine adduct 16. Quantitative analysis indicated that alkylation of the tetramer is maximum after 40 h with the formation of 13 (64%) and 14 (7%). Approximately 91% of 12 had been consumed with the efficiency of oligomer alkylation being 88%. Alkylation at G, of d(CGCG), by 12 has a first-order kinetic with a rate constant of k = 9.4 x 1O-6s-’ at 0°C whereas that at G, was 1.0 x 10e6 s-‘, Adduct 13 was fairly stable at 0°C with a ty2of 244 h. These studies provided evidence that 12 alkylate DNA at N7 of guanine to produce a thermolabile adduct which could then undergo depurination to produce a more stable kap A3-guanine adduct 16, together with the formation of its abasic site-containing oligomer 15. It should be noted here that these DNA-alkylating antibiotics, duocarmycin A and kapurimycin A3, react only with duplex oligonucleotides with a high site selectivity and efficiency. No reaction was observed with single-stranded DNA even after prolonged incubation as illustrated by Scheme VII.

90 “C

C

5 min

12 R2= 0

OH

i

+

OpCG

We have already shown that abasic (AP) sites such as 9 or 15 are only produced in high yields following heating (90°C, 3 min) of the alkylated DNA base. It is also known that a small portion of AP sites can be generated spontaneously under physiological conditions as well by hydrolysis of the N-glycosydic bond [20]. Despite the

13

Duocarmycin

A

d (CGTATACG) d (GCATATGC)

Knpurimycin

cxn

substrate

“p

d (CGTATACG) d (GCATATGC)

alkylation

d (CGAAAACG)

iW

k = 6.6 x 1O”sec” (al 0°C)

d (CGAAAACG)

yEs

Efficiency - 100%

d (GCTTTTGC)

-

A3 substrate

alkylation

d (ATCGTA)

No

d (TAGCAT)

Kl

d (ATCGTA) d (TAGCAT)

YES

“;” d (CGCG) d(GCGC)

KaP

-

d (CGCA) d (GCGC)

k = 9.4 x 10~6sec“ (at0°C) Efficiency - 88%

Scheme VII.

importance of AP sites in molecular biology, such as the intermediacy of AP sites in the repair of damaged DNA, or their reported mutagenicity during transcription [21], no detailed information is available about their structures and the chemical reactivity dGCATAT0

90°C. 40 mh

OH

OH

+pc

pH 7.0

4

-

OPC

dW’

o

b OPT

OH

9O”C, 40 min

dCp0

....OH

H i

-

CHO

dCp0

o OH

t ti

CHO

OH

NaBH4 i

“‘;qoH

Scheme VIII.

I

dcpo~OH

+

PT

14

of AP sites. We found that prolonged heating (90°C 40 min) of AP sites produces the 3’ end possessing tram a$ -unsaturated aldehyde residue via p-elimination as shown in Scheme VIII. These terminie are quantitated by means of HPLC following conversion to more stable alcohols by NaBH, reduction. In summary, duocarmycin A alkylates N3 of adenine and guanine, indicating that this antibiotic binds to the minor groove. In contrast, kapurimycin A3 specifically alkylate N7 of guanine, indicating its specific binding to the major groove. In addition, the structures of heat-induced abasic sites were unambiguously characterized.

Kapurimycin

A3

ma@groove H

‘H miwr

T-A

groove

base palr

minor groove

Duocarmycin

Duocarmycin

A

A C-G base palr

ACKNOWLEDGEMENTS

This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture, Japan. I am grateful to the Pola Kasei Co., Ltd., for providing NCS and to the Kyowa Hakko Kogyo Co., Ltd., for duocarmycin A and kapurimycin A3. REFERENCES Nielsen, P. (1991) Sequence-selective DNA recognition by synthetic ligands. Bioconjugate Chem. 2, l-12. Stubbe, J. and Kozarich, J.W. (1987) Mechanisms of bleomycin-induced DNA degradation. Chem. Rev. 87, 1107-1136. Goldberg, I.H. (1987) Free radical mechanisms in neocarzinostatin-induced DNA damage. Free Radical Med. 3,41-54. Lee, M.D., Ellestad, G.A. and Borders, D.B. (1991) Calicheamicins: Discovery, structure, chemistry, and interaction with DNA. Accounts Chem. Res. 24,235-243.

5 Golik, J., Clardy, J., Dubay, G., Groenewold, G., Kawaguchi, H., Konishi, M., Kirshna, B., Ohkuma, H., Saitoh, K.-i. and Doyle, T.W. (1987) Esperamicins, a novel class ofpotent antitumor antibiotics. 2. Structure of esperamicin X. J. Am. Chem. Sot. 109.3461-3462. 6 Snyder, J.P. and Tipsword, G.E. (1990) Proposal for blending classical and biradical me~h~isms in antitumor antibiotics: Dynemicin A. J. Am. Chem. Sot. 112,4040-4042. 7 Goldberg, I.H. (1991) Mechanism of neocarzinostatin action: Role of DNA microstructure in determination of chemistry of bistranded oxidative damage. Accounts Chem. Res. 24. 191-198. 8 Myers, A.G., Proteau, P.J. and Handel, T.M. (1988) Stereochemical assignment of neocarzinostatin chromophore. Structure of NCS chromophore-methyl thioglycolate adducts. J. Am. Chem. Sot. 110, 7212-7214. 9 Myers, A.G. and Proteau, P.J. (1989) Evidence for spontaneous, low-tem~rature biradical fo~ation from a highly reactive neocarzinostatin chromophore-thiol conjugate. J. Am. Chem. Sot. 11I, 1146 1147. 10 Sugiyama, H., Yamashita, K., Nishi, M. and Saito, I. (1992) A novel cyclization pathway in activation of neocarzinostatin chromophore by thiol under physiological conditions. Tetrahedron Lett. 33, 515518. 11 Kawabata, H., Takeshita, H., Fujiwara, T., Sugiyama, H., Matsuura, T. and Saito, I. (1989) Chemistry of neoca~inostatin-mediated degradation of d(GCATGC): M~hanism of spontaneous thymine release. Tetrahedron Lett. 32,42634266. 12 Saito, I., Kawabata, H., Fujiwara, T., Sugiyama, .H. and Matsuura, T. (1989) A novel ribose C-4 hydroxylation pathway in neocarzinostatin-mediated degradation of oligonucleotides. J. Am. Chem. Sot. 111.8302-8303. 13 Sugiyama, H., Kawabata, H., Fujiwara, T., Dannoue, Y. and Saito, 1. (1990) Specific detection of C-4’ hydroxylated abasic sites generated by bleomycin and ne~arzinostatin in DNA. J. Am. Chem. Sot. 112,5252-5257. 14 Sugiyama, H., Fujiwara, T., Kawabata, H., Yoda, N., Hirayama, N. and Saito, I. (1992) Chemistry of neocarzinostatin-mediated cleavage of oligonucleotides: Competitive ribose C5’ and C4’ hydroxylation. J. Am. Chem. Sot. 114,5573-5578. 15 Lin, C.-H. and Hurley, L.H. (1990) Determination of the major tautomeric form of the covalently modified adenine in the (t)-CC-1065-DNA adduct by ‘H- and lSN-NMR studies. Biochemistry 29, 9503-9507. 16 Sugiyama, H., Hosoda, M. and Saito, I. (1990) Covalent alkylation of DNA with duocarmycin A: Identification of abasic site structure. Tetrahedron Lett. 31, 7197-7200. 17 Boger, D.L., Ishizaki, T., Zarrinmayeh, H., Kitos, P.E. and Suntornwat, 0. (1990) Synthesis and preliminary evaluation of agents incorporated in the pharmacophore of the duocarmycin/pyridamycin alkylation subunit: Identification of the CC-106Slduocarmycin common pharmacophore. J. Org. Chem. 55,4499-4502. 18 Hara, M., Yoshida, M. and Nakano, H. (1990) Covalent modification and single-stranded scission of DNA by a new antitumor antibiotic kapurimycin A3. Biochemistry 29, 10449-10453. 19 Chan, K.L., Sugiyama, H. and Saito, I. (1991) DNA cleavage reaction of antitumor antibiotic, kapurimycin A3, with deoxytetranucleotide d(CGCG),. Tetrahedron Lett. 32, 7719-7722. 20 Kubo, K., Ide, H., Wallace, S.S. and Know, Y.W. (1992) A novel, sensitive, and specific assay for abasic sites, the most commonly produced DNA lesion. Biochemistry 31, 370333708. 21 Manoharan, M., Ransom, S.C., Mazumder, A., Gerlt, J.A., Wilde, J.E., Withka, J.A. and Bolton, P.H. (I 988) The characterization of abasic sites in DNA heteroduplexes by site specific labeling with 13C.J. Am. Chem. Sot. 110, 1620-1622.