DNA ligands as radiomodifiers: studies with minor-groove binding bibenzimidazoles.

An iodinated bibenzimidazole, iodoHoechst 33258, was previously reported to markedly sensitize DNA and cells to UV-A, exemplifying the potential of iodinated DNA ligands as radiosensitizers, a rational extension of sensitization by halogenated pyrimidines. However, unlike the latter sensitizers, iodoHoechst 33258 is not a sensitizer of ionizing radiation, presumably due to the innate radioprotective properties of the uniodinated ligand. Experiments with purified DNA show that both Hoechst 33258 and Hoechst 33342 decrease the yield the radiation-induced DNA strand breakage. The ligands bind at discrete sites in the minor groove of DNA, and analysis on DNA sequencing gels show pronounced protection at the ligand binding sites, as well as more generalized protection. The extent of protection of strand breakage on plasmid DNA and the fact that it persists in the presence of 0.5 M NaCl (which prevents low affinity ionic binding between the high affinity sites) suggests that the protective effects of bound ligand are not confined to the high affinity binding sites in the minor groove. The mechanisms of this generalized protection is unknown, but there is some evidence indicating that the H-atom donation from the ligand may account for the site-specific protection. The extent of protection is much diminished, but still evident, in the presence of 100 mM mannitol, a known hydroxyl radical scavenger, indicating that some of the protective effects might relate to DNA damage mediated by direct action. Further evaluation of the mechanisms of protection should enable development of both more active radioprotectors and, by elimination of the radioprotective features from halogenated DNA ligands, more effective radiosensitizers.     

Mutagenicity of the antitumor antibiotic CC-1065 and its analogues in mammalian (V79) cells and bacteria

CC-1065 is a very potent antitumor antibiotic which binds in the minor groove of DNA with alkylation at N-3 of adenine. Since CC-1065 caused delayed deaths in mice at therapeutic doses, analogues were prepared whose antitumor and biochemical activities have been reported. In this study, the mutagenicity for V79 cells (6-thioguanine resistance) and Salmonella (histidine auxotrophy or azaguanine resistance) of selected analogues was compared to DNA-binding activity and the structure-activity relationship was determined. CC-1065, U-62,736, U-66,866, U-66,694, U-67,786, and U-68,415 all have an A segment with an intact cyclopropyl group and different B segments. The cyclopropyl group is absent from U-66,226 and U-63,360. Elimination of the cyclopropyl ring diminished the cytotoxic and mutagenic potency of the compounds such that U-63,360 was nearly three orders of magnitude less potent than CC-1065 in V79 cells. For the compounds with an intact cyclopropyl group, the order of cytotoxic and mutagenic potency (molar basis) in V79 cells generally correlated with binding to calf thymus DNA, and increased with the length of the B segment. Thus, the order of cytotoxicity was CC-1065 greater than U-68,415 greater than U-66,694 greater than U-66,866 greater than U-62,736. U-67,786 fell outside this pattern since it was more cytotoxic and mutagenic than U-66,694, although it was of a similar size and had similar DNA-binding activity. These results show that an electrophilic carbon afforded by an intact cyclopropyl group of this type is necessary but not sufficient to account for the high cytotoxic and mutagenic potency of CC-1065 and U-68,415. The size and characteristics of the B segment also affect the potency. At an equitoxic (10 or 50% lethal dose) dose, an inverse relationship exists between cytotoxic and mutagenic potency such that at the 50% lethal dose, the least cytotoxic compound (U-62,736) was more mutagenic than the most cytotoxic compound (CC-1065). We speculate that the more cytotoxic analogues are less mutagenic (at an equitoxic dose) because they may have greater structure-directed binding to less mutable DNA sites in the minor groove.     

Interaction of CC-1065 and its analogues with mouse DNA and chromatin

CC-1065 is a potent antitumor antibiotic which is cytotoxic to P388 and L1210 leukemia cells in vitro and in vivo. CC-1065 covalently binds to calf thymus DNA preferentially to adenine-thymine regions at N3 of adenine. Here, we compare CC-1065 interaction with P388-derived chromatin, DNA, and histones as measured by electronic absorption and circular dichroism. Two CC-1065 analogues (U-71,184 and its enantiomer, U-71,185) which show different biological activities from CC-1065 were also studied. The shape and temporal behavior of the induced circular dichroism curves generated by CC-1065 or its analogues bound to chromatin were similar to CC-1065 plus DNA. This suggested that CC-1065 and its analogues bind to the minor groove of chromatin DNA in a manner similar to calf thymus DNA. However, the binding of CC-1065 and its analogues to DNA induced a more intense circular dichroism band than binding to chromatin. The order of interaction for both chromatin and DNA was CC-1065 greater than U-71,184 greater than U-71,185. In contrast to the essentially irreversible binding to DNA after 24-h incubation, binding to chromatin was primarily a reversible interaction, the degree of reversibility being U-71,185 greater than U-71,184 = CC-1065. CC-1065 binds weakly and nonspecifically to histones.     

Involvement of DNA topoisomerase II in the selective resistance of a mammalian cell mutant to DNA minor groove ligands: ligand-induced DNA-protein crosslinking and responses to topoisomerase poisons

A mutant murine cell line has previously been reported to be resistant to the AT-specific DNA minor groove ligand 2',5'-bi-1H-benzimidazole, 2',(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl), trichloride (Ho33342), due to an enhanced capacity to remove ligand molecules from cellular DNA via a pathway which can be blocked by DNA topoisomerase poisons. We have studied the relationship between ligand resistance and DNA topoisomerase II activity. The cross-sensitivity patterns of the mutant were examined for covalently (anthramycin) and non-covalently (distamycin A) binding minor groove ligands, and DNA intercalating [adriamycin, mitoxantrone and 4'-(9-acridinylamino)methanesulphon-m-anisidide (mAMSA)] and non-intercalating (VP16-213) topoisomerase II poisons. The mutant was cross-resistant to distamycin A alone. The mutant showed no abnormality in: (i) the in vitro decatenation activity of topoisomerase II, (ii) VP16-213 or mAMSA induced protein-DNA cross-linking activities in nuclear extracts, (iii) 'cleavable complex' generation (or DNA strand scisson) in intact cells exposed to topoisomerase poisons. Ho33342 and the topoisomerase II inhibitor novobiocin were found to disrupt both the in vitro binding of nuclear extracted proteins, from mutant and parental cells, to plasmid DNA and the formation of drug-induced cleavable complexes in vitro. Unexpectedly, Ho33342 induced significant levels of DNA-protein crosslinking in both parental and mutant cells. We conclude that: (i) resistance of the mutant is limited to non-covalently binding minor groove ligands, (ii) Ho33342 can block the trapping of DNA topoisomerase II by enzyme poisons in vitro, (iii) Ho33342 can induce a novel form of DNA-protein cross-link in intact cells, and (iv) the resistance of the mutant is not dependent upon some abnormality in topoisomerase II function.     

Diaminobenzene schiff base, a novel class of DNA minor groove binder

Molecules that target the deoxyribonucleic acid (DNA) minor groove are relatively sequence specific and they can be excellent carrier structures for cytotoxic chemotherapeutic compounds which can help to minimize side effects. Two novel isomeric derivatives of diaminobenzene Schiff base [N,N'-bis (2-hydroxy-3-methoxybenzylidene)-1,2-diaminobenzene (2MJ) and N,N'-bis(2-hydroxy-3-methoxybenzylidene)-1,3-diaminobenzene (2MH)] were analyzed for their DNA minor groove binding (MGB) ability using viscometry, UV and fluorescence spectroscopy, computational modeling and clonogenic assay. The result shows that 2MJ and 2MH are strong DNA MGBs with the latter being more potent. 2MH can form interstrand hydrogen bond linkages at its oxygens with N3 of adenines. Changing the 2-hydroxy-3-methoxybenzylidene binding position to the 1,3 location on the diaminobenzene structure (2MJ) completely removed any viable hydrogen bond formation with the DNA and caused significant decrease in binding strength and minor groove binding potency. Neither compound showed any significant cytotoxicity towards human breast, colon or liver cancer cell lines.     

Control over the sequence specificity of DNA alkylation: syntheses and reactions with 32P-end-labelled DNA of N-alkyl-N-nitrosoureas linked to minor groove binding lexitropsins.

The syntheses of N-2-chloroethyl-N-nitrosoureas (Cl-ENU) that are covalently linked to a series of minor groove binding lexitropsins related to distamycin A are reported. The lexitropsins of 2-Cl-ENU show a sequence specificity for alkylating an adenine toward the ends of its DNA affinity binding domains. The reaction of DNA with 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea does not yield these products. Therefore, the linking of the 2-Cl-ENU to the minor groove binder qualitatively and quantitatively alters the DNA observed.     

Characterization of a duocarmycin-DNA adduct-recognizing protein in cancer cells.

Duocarmycins have been reported to derive their potent antitumor activity through a sequence-selective minor groove alkylation of N3 adenine in double-stranded DNA. We have used gel mobility shift assays to detect proteins that bind to DNA treated in vitro with duocarmycin SA and identified a protein, named duocarmycin-DNA adduct recognizing protein (DARP), which binds with increased affinity to duocarmycin-damaged DNA. Examination with partially purified DARP revealed that the protein recognized not only the DNA adduct of structurally related drug, CC-1065, but unexpectedly, the protein also recognized the DNA adduct of another chemotype of minor groove binder, anthramycin. These results demonstrate that DARP recognizes the structural alteration of DNA induced by these potent DNA-alkylating drugs, suggesting the possibility that the protein might modulate the antitumor activity of these drugs.     

A mammalian cell mutant with enhanced capacity to dissociate a bis-benzimidazole dye-DNA complex

The bis-benzimidazole dyes (specifically Hoechst 33258 and themore lipophilic derivative Hoechst 33342) are non-intercalatingAT base pair-specific ligands which bind to cellular DNA bynon-covalent association with the minor groove. The interactionof dye with cellular DNA is thought to be the principal pathwayfor the cytotoxic, mutagenic and DNA-damaging properties ofsuch agents. Upon binding and near UV light excitation, dyemolecules exhibit fluorescence enhancement such that dye/DNAassociation and dissociation in individual cells can be monitoredby flow cytometry. We have studied dye uptake and the DNA -dye dissociation characteristics of a Hoechst 33258-resistantmouse cell line (Hoe^R4l5) compared to the response of the parentalcell line Ltk^–. Hoe^R4l5 was found to show similar levelsof cross resistance ({small tilde} 10-fold) to Hoechst 33258and Hoechst 33342 comparedto parental responses except thatthe more lipophilic ligand was {small tilde}30-fold more toxic.Estimates of Hoechst 33342 uptake using flow cytometry or radiolabellingmethods indicated that resistance could not be attributed toreduced cellular uptake, low initial levels or different modesof DNA binding. Both cell lines showed similar initial levelsof dye-induced DNA strand-breakage. However, Hoechst 33342 resistancedid correlate with an enhanced capacity (10-fold) ofHoe^R4l5to remove dye from cellular DNA compared with the relativelylong retention (T_1/2 300 min) of ligand by the parental cellline. Our results are consistentwith the view that ligand persistencerather than indirect DNA damage is a more important factor inthe cytotoxicity of non-intercalating DNA-binding ligands. Amodel is presented of the cellular processes of DNA damage recognitionand surveillance for ligands which interact with the minor grooveof DNA.     

Influence of spermidine on sister chromatid exchanges induced by alkylating agents in mammalian cells in vitro

Sister chromatid exchanges (SCEs) are a very sensitive genetic end-point for in vitro identification of presumed carcinogenic and mutagenic agents, although the mechanism of their formation is still to be elucidated. The present work shows the influence of spermidine on SCE induction by two different DNA damaging agents: Mitomycin-C (MMC) and N-Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG). The SCE level induced by MMC was significantly decreased by spermidine. On the contrary, MNNG-induced SCEs were not affected. It has recently been suggested that MMC, via its reduced metabolite mitosene, produces bulky mono-and bi-adducts in DNA, mainly located in the minor groove of the double helix. MNNG, instead, directly methylates several electrophilic sites of DNA bases, such as the N7 and the O6 of guanines and the N3 of adenines. Both MMC and MNNG, despite their different mechanism of action, are potent SCE inducers. Spermidine, similarly to its structural analogue Spermine, is known to interact with DNA phosphate groups and to bind reversibly to the minor groove, thus stabilizing the double helix structure. Spermidine, being therefore ineffective on the MNNG-mediated DNA methylation, might affect DNA, making it structurally unavailable for MMC binding.     

Pharmacokinetics, pharmacodynamics and metabolism of the dimeric pyrrolobenzodiazepine SJG-136 in rats

Purpose  

The dimeric pyrrolobenzodiazepine SJG-136 (NSC 694501, SG2000) has potent in vitro antiproliferative activity and in vivo antitumor activity associated with binding in the minor groove of DNA and formation of covalent interstrand DNA cross-links. The pharmacokinetics and in vitro metabolism of SJG-136 and as well as the feasibility of using the Comet assay to measure in vivo interstrand DNA cross-links, was assessed in the rat.     

Mismatch repair deficiency is associated with resistance to DNA minor groove alkylating agents.

Mismatch DNA repair deficiency is associated with resistance to certain major groove alkylating agents including methylating agents and cisplatin. We have now studied the relevance of mismatch repair alterations to the cytotoxicity induced by drugs which alkylate N3 adenines in the minor groove of DNA. We have used the mismatch repair defective human colocarcinoma cell line HCT-116 which has a mutation in the hMLH1 gene, and a subline where hMLH1 expression is restored by chromosome 3 transfer (HCT-116+ch3). We have tested three alkylating minor groove binders (tallimustine, carzelesin and CC1065) and one non-covalent minor groove binder (PNU 151807). The HCT-116+ch3 subline was more sensitive than the parental line to the treatment with the three alkylating minor groove binders, while the non-alkylating compound had a similar activity in both cell lines. Further support for mismatch repair being involved in sensitivity of the minor groove alkylators is that two cisplatin-resistant sublines of the human ovarian adenocarcinoma cell line A2780 (A2780/CP70 and A2780/MCP-1) are defective in hMLH1 expression and are more resistant to these agents than the parental mismatch repair proficient cells. Furthermore, the restoration of hMLH1 activity in the A2780/CP70 cell line, by introduction of chromosome 3, was associated with an increased sensitivity to the three alkylating minor groove binders. Again, the non-covalent minor groove binder was equally effective in mismatch repair deficient and proficient clones. The data indicate that mismatch repair deficiency mediated by loss of hMLH1 expression is associated not only with drug-resistance to major groove binders, but also to minor groove binders. However, loss of mismatch repair does not mediate resistance to the non-covalent minor groove binder PNU 151807.     

DNA crosslinking, sister chromatid exchange and cytotoxicity of N-2-chloroethylnitrosoureas tethered to minor groove binding peptides

Chloroethylnitrosoureas (CENU) are clinically important chemotherapeuticagents whose mechanism of action involves the formation of interstrandDNA crosslinks via an ethane bridge between N1-G and N3-C. CENUgenerally alkylate G at the N7- and O⁶-positions, with the latterlesion being the precursor to the interstrand crosslink. Inprevious studies, we reported the synthesis of CENU appendedby a C₂H₄ linker to the N-terminus of DNA minor groove bindingdipeptides (lex, information reading peptides) based on N-methylpyrrole-carboxamidesubunits. Because of the dipeptide structure, these CENU-lex'sreact with DNA at adenines associated with lex equilibrium bindingsites. No other CENU has been reported to yield A adducts. Thebiological evaluation of these CENU-lex's show that they aresomewhat less cytotoxic than their simpler counterparts. Inaddition, in vitro studies show that the minor groove bindingCENU-lex's afford a lower level of sister chromatid exchange(SCE) in 9L cells that are sensitive to CENU. There is no differencebetween CENU-lex in SCE induction in 9L-2 cells that are resistantto CENU. Formation of DNA interstrand crosslinks from the CENU-lex'sis lower than for their non-affinity binding analogs in lowionic strength buffer, but similar in the same buffer containing200 mM NaCl. Salt inhibits crosslinking for all CENU, but distamycin,a competitive inhibitor of lex minor groove binding, uniquelyenhances crosslinks for the CENU-lex's. These results are consistentwith the novel minor groove adduction being a ‘detoxification’pathway for the CENU-lex's since this lesion is formed at theexpense of the cytotoxic major groove interstrand crosslink.     

Molecular mechanics and antibody binding in the structural analysis of polycyclic aromatic hydrocarbon-diol-epoxide--DNA adducts.

Analysis of polycyclic aromatic hydrocarbon (PAH)-DNA adducts using monoclonal antibodies raised against DNA that had been modified with (+-)-r-7-,t-8-dihydroxy-t-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene in an enzyme-linked immunosorbent assay, as well as analysis using human serum antibodies and antibodies raised in laboratory animals, have suggested the presence on these adducts of both common and unique immunological epitopes. The molecular mechanics studies reported here establish a model for the analysis of PAH-DNA adducts through the identification of energetically favored binding conformations and they further reveal structural alterations in DNA due to the presence of carcinogen adducts. The data explain the antibody reactivity patterns by defining different molecular presenting surfaces that are available for antibody binding. The preferred orientation of the aromatic portions of the adducts, which align either 3' or 5' in the minor groove, were found to be correlated with antibody reactivity patterns. Examination of the topographical characteristics of the adducts facilitated correlation of adduct-antibody recognition and adduct presenting surface. Significant differences were found between benzo[a]pyrene-diol-epoxide (BPDE)-DNA adducts, which align 5' in the minor groove, and benz[a]anthracene-diol-epoxide (BADE)-DNA and dibenz[a,c]anthracene-diol-epoxide-DNA adducts, which align 3' within the minor groove. Chrysene-diol-epoxide-DNA adducts were found to have only a weak preference for 5' alignment and therefore share topographical characteristics with both BPDE-DNA and BADE-DNA adducts.     

DNA minor groove binders: Back in the groove

With recent approval of the minor groove binding agent trabectidin in Europe for the treatment of patients with soft tissue sarcomas, there has been renewed interest in minor groove binders. Though previously considered to be without clinical value due to their initial significant toxicities, new minor groove binders are emerging which are challenging that perception. Toxicities in the most recently completed and ongoing trials have been easily manageable. These agents have demonstrable anti-tumor activity against a wide variety of tumor types including leukemias, sarcomas, melanomas, breast and ovarian cancers. Applying these agents according to a particular tumor’s context of vulnerability might reveal previously unconsidered applications for this diverse class of agents. This review provides a look at how minor groove binding agents have progressed from the lab through the clinic with particular emphasis on identifying the contexts of vulnerabilities of patient tumors which increase the effectiveness of these drugs.     

Effects of U-71,184 and several other CC-1065 analogues on cell survival and cell cycle of Chinese hamster ovary cells

CC-1065 is a very potent antitumor antibiotic which selectively binds in the minor groove of DNA with alkylation at N-3 of adenine. Since therapeutic doses of CC-1065 caused delayed deaths in mice, analogues were synthesized, some of which had significant antitumor activity. The effects of several of these analogues on inhibition of CHO cell survival, cell progression, and their phase-specific toxicity are reported. CC-1065, U-66,664, U-66,819, U-66,694, and U-71,184 all have a left hand segment with an intact cyclopropyl group but have different tail segments. Lethality of these compounds after 2 h drug exposure was in the following order (50% lethal dose in nM in parentheses): CC-1065 (0.06) greater than U-71,184 (1.3) greater than U-66,694 (3.2) greater than U-68,819 (171) greater than U-66,664 (greater than 1200). In general, these compounds did not inhibit progression from G1 to S but slowed progression through S and blocked cells in G2-M. The phase-specific toxicity of U-71,184 and U-66,694 was different from that of CC-1065. CC-1065 was most cytotoxic to cells in M and early G1 and toxicity decreased as cells entered late G1 and S. In contrast, U-66,694 and U-71,184 were most toxic to cells in late G1. The biochemical and cellular effects of U-71,184 were then studied in detail since it was the most active among these analogues. After a 2-h exposure to 3 ng/ml U-71,184, 90% cell kill or growth inhibition was observed whereas 100 ng/ml was needed for similar inhibition of DNA and RNA synthesis. This discrepancy between the doses suggested that inhibition of nucleic acid synthesis may not be causally related to lethality. Further studies showed that when drug was removed after 2 h exposure, DNA synthesis continued to be inhibited whereas RNA and protein synthesis reached levels higher than the control. Therefore, it is likely that at cytotoxic doses the low level of inhibition of DNA synthesis combined with the stimulation of RNA and protein synthesis leads to unbalanced growth and cell death.     

Receptor-mediated DNA-targeted photoimmunotherapy

We show the efficacy of a therapeutic strategy that combines the potency of a DNA-binding photosensitizer, UV(A)Sens, with the tumor-targeting potential of receptor-mediated endocytosis. The photosensitizer is an iodinated bibenzimidazole, which, when bound in the minor groove of DNA and excited by UV(A) irradiation, induces cytotoxic lesions attributed to a radical species resulting from photodehalogenation. Although reminiscent of photochemotherapy using psoralens and UV(A) irradiation, an established treatment modality in dermatology particularly for the treatment of psoriasis and cutaneous T-cell lymphoma, a critical difference is the extreme photopotency of the iodinated bibenzimidazole, approximately 1,000-fold that of psoralens. This feature prompted consideration of combination with the specificity of receptor-mediated targeting. Using two in vitro model systems, we show the UV(A) cytotoxicity of iodo ligand/protein conjugates, implying binding of the conjugate to cell receptors, internalization, and degradation of the conjugate-receptor complex, with release and translocation of the ligand to nuclear DNA. For ligand-transferrin conjugates, phototoxicity was inhibited by coincubation with excess native transferrin. Receptor-mediated UV(A)-induced cytotoxicity was also shown with the iodo ligand conjugate of an anti-human epidermal growth factor receptor monoclonal antibody, exemplifying the potential application of the strategy to other cancer-specific targets to thus improve the specificity of phototherapy of superficial lesions and for extracorporeal treatments.     

A DNA topoisomerase II-independent route for novobiocin-mediated resistance to DNA binding agents

Summary The coumermycin antibiotic novobiocin is currently under investigation as an agent that can modify the toxicity of various anti-cancer drugs, potentially via one of its many pharmacological effects: namely, the interference with type II DNA topoisomerase function. This paper investigates the ability of novobiocin to modify the cellular/nuclear accumulation and toxicity of two types of DNA binding agents (the minor groove ligand Hoechst 33342 and the intercalating anthracycline Adriamycin). We report that novobiocin reduces the cytotoxicity of both agents and that this can be attributed to a reduction in cellular and, consequently, nuclear accumulation of these agents rather than to any effect on cellular export. The antibiotic was also active (at non-toxic concentrations) in delaying the progression of cells into S phase and G₂ phase. This potential for novobiocin to effect rescue from toxicity by disturbance of the delivery of a drug to a potentially important intracellular target, together with the provision of an extended period of cellular recovery prior to the commitment of cells to G₂ + M phase, should be recognised in the design of combination chemotherapy.     

Ecteinascidin 743 induces protein-linked DNA breaks in human colon carcinoma HCT116 cells and is cytotoxic independently of topoisomerase I expression.

Ecteinascidin 743 (Et743; NSC 648766) is a potent antitumor agent presently in clinical trials. Et743 selectively alkylates guanine N2 from the minor groove of duplex DNA and bends the DNA toward the major groove. This differentiates Et743 from other DNA-alkylating agents presently in the clinic. To date, the cellular effects of Et743 have not been elucidated. Recently, Et743 DNA adducts have been found to suppress gene expression selectively and to induce topoisomerase I (top1) cleavage complexes in vitro and top1-DNA complexes in cell culture. In the present study, we characterized the DNA damage and the cell cycle response induced by Et743 in human colon carcinoma HCT116 cells. Alkaline elution experiments demonstrated that micromolar concentrations of Et743 produced comparable frequencies of DNA-protein cross-links and DNA single-strand breaks. The single-strand breaks were protein-cross-linked and were not associated with detectable DNA double-strand breaks. By contrast with camptothecin, these lesions persisted for several hours after drug removal and were not formed at 4 degrees C. Et743 treatment induced transient p53 elevation, dose-dependent cell cycle accumulation in G2-M and in G1- and S-phase, and inhibition of DNA synthesis. The sensitivity of camptothecin-resistant mouse leukemia P388/ CPT45 cells, which fail to express detectable top1, was similar to the sensitivity of wild-type P388 cells, suggesting that top1 is not a critical target for the antiproliferative activity of Et743.