Nuclear magnetic resonance solution structures of inter- and intrastrand adducts of DNA cross-linker SJG-136

SJG-136 (1) is a sequence-selective DNA-interactive agent that is about to enter phase II clinical trials for the treatment of malignant disease. Previous studies on the pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers, typified by SJG-136 and DSB-120 (2), have shown that these planar ligands react with the exocyclic NH(2) groups of two guanine bases in the base of the minor groove of DNA to form an irreversible interstrand cross-linked sequence-specific adduct. Using high-field NMR, we have characterized and modeled the previously predicted interstrand duplex adduct formed by SJG-136 with the self-complementary 5'-d(CICGATCICG)(2) duplex (4). This first SJG-136 NMR-refined adduct structure has been compared with previous high-field NMR studies of the adducts of the closely related PBD dimer DSB-120 with the same duplex and of the adduct of tomaymycin (3) formed with 5'-d(ATGCAT)(2). Surprisingly, the SJG-136 duplex adduct appears to be more closely related to the tomaymycin adduct than to the DSB-120 adduct with respect of the orientation and depth of insertion of the ligand within the minor groove. The intrastrand duplex adduct formed in the reaction of SJG-136 with the noncomplementary 5'-d(CTCATCAC)·(GTGATGAG) duplex (5) has also been synthesized and modeled. In this duplex adduct, the nature of the cross-link was confirmed, the central guanines were identified as the sites of alkylation, and the stereochemical configuration at C11 at both ends of the SJG-136 molecule was determined to be S. The NMR-refined solution structures produced for the intrastrand adduct confirm the previously proposed structure (which was based solely on mass spectroscopy). Both the inter- and intrastrand SJG-136 duplex adducts form with minimal distortion of the DNA duplex. These observations have an impact on the proposal for the mechanism of action of SJG-136 both in vitro and in vivo, on the repair of its adducts and mechanism of resistance in cells, and, potentially, on the type of pharmacodynamic assay to be used in clinical trials. SGJ-136 is currently in phase II clinical trials with several groups working on both dimeric cross-linking agents and monoalkylating ligands based on the PBD alkylating moiety. This study suggests subtle differences between the DNA binding of SJG-136 and the C2 unsubstituted analogue DSB-120 that are likely to be the origins of the differences in potency. Confirmation of the stereochemical configuration at the C11 position (particularly in the intrastrand adduct) provides confirmation of binding orientation that was previously only speculation in the HPLC MS study. Together, these observations are likely to be of value in the development of third-generation PBD-based cross-linkers and monoalkylating analogues.     

Effect of base sequence on the DNA cross-linking properties of pyrrolobenzodiazepine (PBD) dimers

Pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimers are synthetic sequence-selective DNA minor-groove cross-linking agents that possess two electrophilic imine moieties (or their equivalent) capable of forming covalent aminal linkages with guanine C2-NH₂ functionalities. The PBD dimer SJG-136, which has a C8–O–(CH₂)₃–O–C8′′ central linker joining the two PBD moieties, is currently undergoing phase II clinical trials and current research is focused on developing analogues of SJG-136 with different linker lengths and substitution patterns. Using a reversed-phase ion pair HPLC/MS method to evaluate interaction with oligonucleotides of varying length and sequence, we recently reported (JACS, 2009, 131, 13 756) that SJG-136 can form three different types of adducts: inter- and intrastrand cross-linked adducts, and mono-alkylated adducts. These studies have now been extended to include PBD dimers with a longer central linker (C8–O–(CH₂)₅–O–C8′), demonstrating that the type and distribution of adducts appear to depend on (i) the length of the C8/C8′-linker connecting the two PBD units, (ii) the positioning of the two reactive guanine bases on the same or opposite strands, and (iii) their separation (i.e. the number of base pairs, usually ATs, between them). Based on these data, a set of rules are emerging that can be used to predict the DNA–interaction behaviour of a PBD dimer of particular C8–C8′ linker length towards a given DNA sequence. These observations suggest that it may be possible to design PBD dimers to target specific DNA sequences.     

Interstrand cross-links and sequence specificity in the reaction of cis-dichloro(ethylenediamine)platinum(II) with DNA

Characterization of the adducts produced in DNA by the cancer chemotherapeutic drug cis-diamminedichloroplatinum(II) and a radiolabeled analogue, [3H]-cis-dichloro(ethylenediamine)platinum(II) ([3H]-cis-DEP) was recently reported [Eastman, A. (1983) Biochemistry 22, 3927]. Both drugs reacted at identical sites in DNA, most of which produced intrastrand cross-links. DNA-interstrand cross-links, which represent less than 1% of total platination, have now been characterized. DNA containing interstrand cross-links was enriched for on the basis of its renaturability after boiling. This DNA was digested to deoxyribonucleosides, and the adducts were separated by high-pressure liquid chromatography. A cross-link between two deoxyguanosines was observed to be the most prominent adduct. It is proposed that the major sequence in which this cross-link occurs is 5'-CG-3'. DNA that was incubated with [3H]-cis-DEP for 1 h showed low levels of interstrand cross-links. After removal of unreacted drug, their frequency increased significantly over 6 h with a maximum occurring at about 12 h. A similar phenomenon was seen in the case of intrastrand cross-links that contained adenine, in particular when the cross-link was between the terminal bases in an ANG trinucleotide sequence (N is any nucleotide). The primary site of reaction is at guanine, with a slow subsequent cross-link to the adenine. A model is presented that is consistent with the observation that adenine is always at the 5' terminus of these adducts. The proportion of adducts at ANG sequences also increased at elevated temperatures. This is discussed with regard to potential significance during hyperthermia treatment of patients.(ABSTRACT TRUNCATED AT 250 WORDS)     

The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5′-GATC-3′ sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.     

Construction of DNA substrates modified with psoralen at a unique site and study of the action mechanism of ABC excinuclease on these uniformly modified substrates

Psoralens bind to DNA noncovalently and upon exposure to near UV (320-400 nm) light produce covalent adducts. Thymidine residues in DNA, especially those at 5'-TpA-3' sequences, are most susceptible to the photochemical reaction. This property of the reaction and the recent advances in oligonucleotide synthesis and separation has enabled us to construct DNA fragments containing psoralen adducts at a specific site. The octanucleotide 5'-TCGTAGCT-3' was photoreacted (in the presence of the complementary strand) with the synthetic psoralen 4'-hydroxymethyl-4,5',8-trimethylpsoralen to obtain oligonucleotides adducted via the furan or pyrone ring at the internal thymine. These modified octanucleotides were ligated to nonmodified oligonucleotides to obtain a 40-base pair DNA fragment containing a psoralen adduct at a central location. The modified fragment having the thymine-furan side 4'-hydroxymethyl-4,5',8-trimethylpsoralen adduct was irradiated with 360 nm of light to produce an interstrand cross-link, and this cross-linked DNA was purified to homogeneity. These uniquely modified DNAs were used as substrates for Escherichia coli ABC excinuclease to determine its incision mechanism unambiguously and to determine the contact sites of the enzyme. ABC excinuclease mediates the cleavage of the 8th and 5th phosphodiester bonds 5' and 3', respectively, to psoralen monoadducts, and the 9th (5') and 3rd (3') phosphodiester bonds to the furan-side thymine of the cross-link. Preliminary DNaseI footprinting studies show that ABC excinuclease protects the whole 40-base pair fragment from DNaseI, and binding of the A and B subunits to the furan side-monoadducted substrate produces two hypersensitive phosphodiester bonds in the vicinity of the 5' incision site of ABC excinuclease.     

Stereochemistry modulates the stability of reduced interstrand cross-links arising from R- and S-alpha-CH3-gamma-OH-1,N2-propano-2'-deoxyguanosine in the 5'-CpG-3' DNA sequence

The solution structures of 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' interstrand DNA cross-links in the 5'-CpG-3' sequence were determined by NMR spectroscopy. These were utilized as chemically stable surrogates for the corresponding carbinolamine interstrand cross-links arising from the crotonaldehyde- and acetaldehyde-derived R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts. The results provide an explanation for the observation that interstrand cross-link formation in the 5'-CpG-3' sequence by the R- and S-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adducts is dependent upon stereochemistry, favoring the R-alpha-CH3-gamma-OH-1,N2-propanodeoxyguanosine adduct [Kozekov, I. D., Nechev, L. V., Moseley, M. S., Harris, C. M., Rizzo, C. J., Stone, M. P., and Harris, T. M. (2003) J. Am. Chem. Soc. 125, 50-61]. Molecular dynamics calculations, restrained by NOE-based distances and empirical restraints, revealed that both the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' and 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-links were located in the minor groove and retained Watson-Crick hydrogen bonds at the tandem cross-linked C.G base pairs. However, for the 5'-Cp-N2-dG-3'-R-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the center of the minor groove, whereas for the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link, the (alpha)-CH3 group was positioned in the 3' direction, showing steric interference with the DNA helix. The 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link exhibited a lower thermal stability as evidenced by NMR spectroscopy as a function of temperature. The two cross-links also exhibited apparent differences in the conformation of the interstrand three-carbon cross-link, which may also contribute to the lower apparent thermodynamic stability of the 5'-Cp-N2-dG-3'-S-(alpha)-CH3-propyl-5'-Cp-N2-dG-3' cross-link.     

Enantioselective recognition of 2,3-benzodiazepin-4-one derivatives with anticonvulsant activity on several polysaccharide chiral stationary phases

The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501) has potent in vitro cytotoxicity and in vivo antitumor activity. SJG-136 binds in the minor groove of DNA and produces G-G interstrand cross-links via reactive N(10)-C(11)/N(10')-C(ll') imine/carbinolamine moieties. We have developed a sensitive, specific liquid chromatography tandem mass spectrometry (LC/MS/MS) method for the quantitative determination of SJG-136 in plasma. SJG-136 was isolated by solid phase extraction through a C8 column, reverse-phase HPLC separation was accomplished on a C18 column with isocratic elution and MS/MS detection, monitoring the m/z 557-m/z 476 transition after electrospray ionization. The linear range and lower limit of quantitation from plasma standard curves were 2.8-1800 nM, and 5 nM, respectively. SJG-136 plasma protein binding was species-dependent. Values of the unbound fraction in human, rat and mouse were 25%, 16.2% and <1%, respectively. Protein binding was saturable in dog plasma where the unbound fraction increased from 10.8% to 22.3% over a 22-720 nM concentration range. SJG-136 pharmacokinetics after a single intravenous dose were best fit to a two-compartment open model with elimination half-life and plasma clearance values of 97 min and 6.1 mL/min/kg, respectively. SJG-136 did not accumulate in plasma following intravenous administration of 1.0 microg/kg doses for five consecutive days.     

DNA base composition determines the specificity of UvrABC endonuclease incision of a psoralen cross-link

The sequences flanking a psoralen interstrand cross-link may determine how it is repaired. Our comparison of the Escherichia coli UvrABC endonuclease incision of a variety of specific cross-link sequences in a single natural DNA fragment showed that DNA base composition determines which of two cross-linked DNA strands will be incised. G/C enrichment of the region 6-12 bases 5' of the modified T on the furan-side strand results in preferential incision of the furan-side strand. When the G/C-rich region is on the 3' side, or on neither side, incisions occur on either strand. These effects of DNA base composition suggest that UvrAB can bind in two ways to a psoralen cross-link.     

Repair of an interstrand DNA cross-link initiated by ERCC1-XPF repair/recombination nuclease

Interstrand DNA cross-link damage is a severe challenge to genomic integrity. Nucleotide excision repair plays some role in the repair of DNA cross-links caused by psoralens and other agents. However, in mammalian cells there is evidence that the ERCC1-XPF nuclease has a specialized additional function during interstrand DNA cross-link repair, beyond its role in nucleotide excision repair. We placed a psoralen monoadduct or interstrand cross-link in a duplex, 4-6 bases from a junction with unpaired DNA. ERCC1-XPF endonucleolytically cleaved within the duplex on either side of the adduct, on the strand having an unpaired 3' tail. Cross-links that were cleaved only on the 5' side were purified and reincubated with ERCC1-XPF. A second cleavage was then observed on the 3' side. Relevant partially unwound structures near a cross-link may be expected to arise frequently, for example at stalled DNA replication forks. The results show that the single enzyme ERCC1-XPF can release one arm of a cross-link and suggest a novel mechanism for interstrand cross-link repair.     

Covalent Bonding of Pyrrolobenzodiazepines (PBDs) to Terminal Guanine Residues within Duplex and Hairpin DNA Fragments

Pyrrolobenzodiazepines (PBDs) are covalent-binding DNA-interactive agents with growing importance as payloads in Antibody Drug Conjugates (ADCs). Until now, PBDs were thought to covalently bond to C2-NH₂ groups of guanines in the DNA-minor groove across a three-base-pair recognition sequence. Using HPLC/MS methodology with designed hairpin and duplex oligonucleotides, we have now demonstrated that the PBD Dimer SJG-136 and the C8-conjugated PBD Monomer GWL-78 can covalently bond to a terminal guanine of DNA, with the PBD skeleton spanning only two base pairs. Control experiments with the non-C8-conjugated anthramycin along with molecular dynamics simulations suggest that the C8-substituent of a PBD Monomer, or one-half of a PBD Dimer, may provide stability for the adduct. This observation highlights the importance of PBD C8-substituents, and also suggests that PBDs may bind to terminal guanines within stretches of DNA in cells, thus representing a potentially novel mechanism of action at the end of DNA strand breaks.     

Syntheses of DNA duplexes containing a C-C interstrand cross-link

Short DNA duplexes that contain a N4C-ethyl-N4C interstrand cross-link were prepared on controlled pore glass supports using a DNA synthesizer. The C-C cross-link was introduced via a convertible nucleoside on the support or by using a protected C-C cross-link phosphoramidite. An orthogonal protection scheme allowed selective chain growth in either a 3'-->5' or 5'-->3' direction. The cross-linked duplexes were purified by HPLC and characterized by MALDI-TOF mass spectrometry and/or by enzymatic digestion.     

An assay combining high-performance liquid chromatography and mass spectrometry to measure DNA interstrand cross-linking efficiency in oligonucleotides of varying sequences

The main method of evaluating the DNA interstrand cross-linking ability of cancer chemotherapeutic agents in naked DNA currently involves the electrophoresis of relatively long radiolabeled duplex DNA fragments (typically approximately 2000 bp) on neutral gels after incubation with the agent of interest. Denaturation by heating is carried out prior to loading, and a neutral gel allows reannealing of cross-linked DNA. To avoid the use of radioactivity we have developed a new method based on ion pair reversed phase liquid chromatography (RPLC) and mass spectrometry (MS) that allows characterization and quantitation of drug-DNA interstrand cross-links formed within short oligonucleotide duplexes (i.e., 12 bp). Advantages of this assay include rapid throughput, as compared to electrophoretic methods, and the use of readily available short nonradiolabeled oligonucleotides of any sequence, thereby facilitating investigation of sequence selectivity. A further advantage is that all species separated by the chromatographic process can be positively identified by MS. Using this new method, we have investigated the rate of DNA cross-linking and sequence selectivity of the interstrand cross-linking agent SJG-136, a pyrrolobenzodiazepine (PBD) dimer currently in phase I clinical trials. The assay was found to be sufficiently sensitive and selective to allow separation of the unbound and drug-bound oligonucleotide species by high-performance liquid chromatography (HPLC) and to allow positive identification of these individual species by MS. A further benefit, as compared with electrophoretic methods, is that kinetic information can be obtained and compared for different binding sequences.     

An asymmetric C8/C8'-tripyrrole-linked sequence-selective pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer DNA interstrand cross-linking agent spanning 11 DNA base pairs

A novel sequence-selective extended PBD dimer 4 has been synthesized that binds with high affinity to an interstrand cross-linking site spanning 11 DNA base pairs. Despite its molecular weight (984.07) and length, the molecule has significant DNA interstrand cross-linking potency (approximately 100-fold greater than the clinically used agent melphalan) and sub-micromolar cytotoxicity in a number of tumour cell lines, suggesting that it readily penetrates cellular and nuclear membranes to reach its DNA target.     

Contribution of XPF functional domains to the 5' and 3' incisions produced at the site of a psoralen interstrand cross-link.

XPF forms a heterodimeric complex with ERCC1 and is required for the repair of DNA interstrand cross-links. In association with ERCC1, it is involved in production of the 5' incision at the site of a psoralen interstrand cross-link as well as the 3' incision. The present study was carried out to determine the functional domains of XPF that are important in the production of the 5' and 3' incisions that occur at a site of a psoralen interstrand cross-link. Monoclonal antibodies (mAbs) were utilized that had been generated against polypeptide fragments of XPF and affinity-mapped to specific regions of XPF. These mAbs were examined for their ability to differentially inhibit production of dual incisions in DNA by normal human chromatin-associated protein extracts that contain XPF and ERCC1. These studies show that two regions of XPF, one N-terminal region from amino acids 12-166 and one C-terminal region from amino acids 702-854, are the most important in the production of the 5' incision. The same N-terminal region and the C-terminal region from amino acids 702-916 are also involved in the 3' incision, though to a much lesser extent. Since this C-terminal region corresponds to the proposed site of interaction of ERCC1 with XPF, these results suggest that binding of ERCC1 to XPF is critical for its ability to produce the 5' and 3' incisions at the site of an interstrand cross-link, possibly through activation or regulation of the endonucleolytic activity of the N-terminal domain of XPF.     

Interstrand cross-linking reaction in triplexes containing a monofunctional transplatin-adduct.

Our aim was to determine whether a single transplatin monofunctional adduct, either trans-[Pt(NH3)2(dC)Cl]+ or trans-[Pt(NH3)2(dG)Cl]+ within a homopyrimidine oligonucleotide, could further react and form an interstrand cross-link once the platinated oligonucleotide was bound to the complementary duplex. The single monofunctional adduct was located at either the 5' end or in the middle of the platinated oligonucleotide. In all the triplexes, specific interstrand cross-links were formed between the platinated Hoogsteen strand and the complementary purine-rich strand. No interstrand cross-links were detected between the platinated oligonucleotides and non-complementary DNA. The yield and the rate of the cross-linking reaction depend upon the nature and location of the monofunctional adducts. Half-lives of the monofunctional adducts within the triplexes were in the range 2-6 h. The potential use of the platinated oligonucleotides to modulate gene expression is discussed.     

Induction of AT-specific DNA-interstrand crosslinks by bizelesin in genomic and simian virus 40 DNA

Bizelesin is a bifunctional AT-specific DNA alkylating drug. Our study characterized the ability of bizelesin to induce interstrand crosslinks, a potential lethal lesion. In genomic DNA of BSC-1 cells, bizelesin formed from approx. 0.3 to 6.03+/-0.85 interstrand crosslinks per 106 base pairs, at 5-100 nM drug concentration, respectively, comparable to the number of total adducts previously determined in the same system (J.M. Woynarowski, M.M. McHugh, L.S. Gawron, T.A. Beerman, Biochemistry 34 (1995) 13042-13050). Bizelesin did not induce DNA-protein crosslinks or strand breaks. A model defined target, intracellular simian virus 40 (SV40) DNA, was employed to map at the nucleotide level sites of bizelesin adducts, including potential interstrand crosslinks. Preferential adduct formation was observed at AT tracts which are abundant in the SV40 matrix associated region and the origin of replication. Many sites, including each occurrence of 5'-T(A/T)4A-3', co-mapped on both DNA strands suggesting interstrand crosslinks, although monoadducts were also formed. Bizelesin adducts in naked SV40 DNA were found at similar sites. The localization of bizelesin-induced crosslinks in AT-rich tracts of replication-related regions is consistent with the potent anti-replicative properties of bizelesin. Given the apparent lack of other types of lesions in genomic DNA, interstrand crosslinks localized in AT-rich tracts, and to some extent perhaps also monoadducts, are likely to be lethal effects of bizelesin.     

Alteration in DNA cross-linking and sequence selectivity of a series of aziridinylbenzoquinones after enzymatic reduction by DT-diaphorase.

DT-diaphorase (DTD) mediated reduction of a series of 2,5-bis-substituted-3,6-diaziridinyl-1,4-benzoquinones was found to increase the level of DNA interstrand cross-linking (ISC) formed at neutral pH with an enhancement observed as the pH was decreased to 5.8. The analogues used were symmetrically alkyl-substituted carbamoyl ester analogues of AZQ (D1-D7), 3,6-diaziridinyl-1,4-benzoquinone (DZQ), the 2,5-dimethyl derivative (MeDZQ), and a 2,5-bis[(2-hydroxyethyl)amino] analogue (BZQ). At pH 5.8, the level of DNA ISC induced by enzymatic reduction was as follows: DZQ greater than MeDZQ much greater than D1 (methyl) greater than D3 (n-propyl) greater than D2 (AZQ; ethyl) greater than D5 (n-butyl) greater than D7 (sec-butyl) greater than D4 (isopropyl) D6 greater than (isobutyl). A similar trend was observed at pH 7.2. The level of DNA ISC induced by BZQ, which is not a substrate for DTD, was not increased by enzymatic reduction. Dicumarol, a known inhibitor of DTD, was capable of inhibiting the DNA ISC induced by these quinones upon enzymatic reduction. MeDZQ and DZQ reacted with guanines, as measured by Maxam and Gilbert sequencing, with a sequence selectivity similar to that of the nitrogen mustard class of antitumor agents. Enzymatic reduction of DZQ and MeDZQ by DTD was found to alter their sequence-selective alkylation. Reduced DZQ showed enhanced guanine alkylation in 5'-GC-3' sequences and new sites of adenine alkylation in 5'-(A/T)AA-3' sequences. Reduced MeDZQ only showed new sites of adenine alkylation at 5'-(A/T)AA-3' sequences but no enhancement of guanine alkylation. The new sites of adenine alkylation were found to be inhibited in the presence of magnesium and rapidly converted into apurinic sites.(ABSTRACT TRUNCATED AT 250 WORDS)