具有抗肿瘤活性的DNA小沟区结合剂的研究新进展

随着近年来基因工程基础研究的迅速发展以及小分子配体与DNA相互作用的机制研究的逐步深入,DNA已成为抗肿瘤药物研究的一个重要的药理学作用靶点,DNA小沟区结合剂成为抗肿瘤药物研究的重要方向。目前已开发了大量结构新颖、靶点单一、高效低毒的有效药物,多种已处于临床研究阶段。该文对近5年来DNA小沟区结合剂的研究新进展进行简要综述。     

抗肿瘤药物DNA小沟区结合剂的研究进展

多靶点是抗肿瘤药物引起不良反应的主要原因之一,开发单一靶点药物是提高药物专属性的一种有效方法.以DNA小沟区为靶点的抗肿瘤药物是近年来研究的热点,它们主要是一些小分子化合物.目前已有多种小沟区结合剂处于基础和临床研究阶段,对近年来DNA小沟区结合剂的研究情况进行简要阐述.     

Effect of minor groove binding drugs on mammalian topoisomerase I activity

Three minor groove binding drugs, distamycin A, bisbenzimide (Hoechst 33258) and 4',6-diamidino-2-phenylindole (DAPI), were examined for their abilities to modulate the activity of topoisomerase I purified from L1210 cells. At 0.5 and 1.0 microM, distamycin stimulated topoisomerase I relaxation of supercoiled DNA by 38 and 13%, respectively, while increasing the drug concentration above 2.0 microM resulted in inhibition. Inhibition was reversible. Complete relaxation could be achieved even in the presence of inhibitory concentrations of distamycin if the incubation time with topoisomerase I was increased from 7.5 to 120 min. The velocity of topoisomerase I mediated relaxation was reduced by 2 microM distamycin at DNA levels ranging from 350 to 2000 ng/reaction. Hoechst 33258 and DAPI inhibited topoisomerase I relaxation in a concentration-dependent manner. Hoechst 33258 and distamycin were equivalent in their abilities to inhibit topoisomerase I, whereas DAPI had a lesser effect (e.g. relaxation was reduced by 50% with 2.7 microM distamycin and 2.8 microM Hoechst 33258 compared to 5 microM DAPI). This study suggests that ligand binding in the minor groove can be a factor in the regulation of topoisomerase I activity.     

Interaction of cis-diamminedichloroplatinum(II) with sensitive and resistant L1210 cell lines. Drug binding to nuclei and DNA

We used in parallel, to study the kinetics of cis-DDP cellular binding and distribution, a cL cell culture line established from L1210 murine leukemia ascites and its cLP derivative which acquired a 30-fold (ID50) resistance to cis-diamminedichloroplatinum(II). Cell cultures were incubated with 0.9 microgram/ml (3 microM) of the drug and after various incubation times up to 24 hr, the amount of platinum associated to whole cells, to isolated nuclei and to purified DNA was determined using atomic absorption spectrophotometry. For the first hours of incubation no significant difference in the rate of platinum association was observed between the two cell lines. After the first hours of incubation the amount of platinum associated to whole cells and to isolated nuclei was significantly higher in the drug sensitive cells. However, the rates of platinum association to the respective DNAs were quite similar in the two cell lines. Our study failed to demonstrate any significant quantitative modification of the overall drug-DNA association between the resistant and sensitive cell lines.     

Virtual screening of DNA minor groove binders

The utility of the two docking programs DOCK and AutoDock in studying the binding of small molecules to the minor groove of B-DNA is examined. The AutoDock program is found to be more effective in both pose prediction and ranking of known binders over random compounds, and this superior performance is shown to be because of the scoring functions rather than the sampling algorithms.     

Heterocyclic analogs of DNA minor groove alkylating agents

It is generally accepted that neoplastic transformation is related to genes alteration or oncogene activation. In particular, DNA minor groove binding drugs have been extensively studied through the years in order to influence the regulation of gene expression by means of specific interactions with DNA bases moieties. Pyrrolo[2,1-c],[1,4].benzodiazepines (PBDs), CC-1065 and distamycins are three classes of minor groove binders which showed interesting cytotoxicity profiles, refined through already reviewed processes of SAR studies. Among the modifications to the three families of antitumor compounds, heterocyclic substitutions have been extensively applied by many groups in order to either modify the reactivity profile or introduce extra interactions within the minor groove, thus changing the binding site or modulating the binding sequence. The updated material related to these modifications has been rationalised and ordered to offer an overview of the argument.     

血卟啉衍生物—光辐射对L1210白血病细胞核酸生物合成的影响

本文报道几种血卟啉衍生物—光辐射效应对L1210白血病细胞DNA及RNA生物合成有不同程度的抑制。对DNA生物合成抑制的IC₅₀,HA 308为2.1μg/ml,激光3号为2.5μg/ml,LF-019为3.6μg/ml,Photofrin Ⅱ>5μg/ml,对RNA生物合成抑制的IC₅₀ LF-019为3.7μg/ml,激光3号为5.7 μg/ml,HA 308为8.8 μg/ml,photofrin Ⅱ>10 μg/ml.其中激光3号对DNA聚合酶α的光动力学失活作用也较明显。     

Antitumor AHMA linked to DNA minor groove binding agents: synthesis and biological evaluation

DNA minor groove binder hybrid molecules, netropsin derivatives such as N-[2-(dimethylamino)ethyl]-1-methyl-4-aminopyrrolo-2-carboxamide (MePy) or its derivatives containing two units of N-methylpyrrolecarboxamide (diMePy) and bisbenzimidazole (Ho33258), were linked to the NH(2) function of AHMA or to the CH(2)OH group of AHMA-ethylcarbamate to form AHMA-N-netropsins (13-16) and AHMA-ethylcarbamate-O-netropsins (19-22), and AHMA-bisbenzimidazole (AHMA-Ho33258, 25), respectively. These conjugates' in vitro antitumor activity, inhibition of a variety of human tumor cell growth, revealed that AHMA-ethylcarbamate-O-netropsin derivatives were more cytotoxic than AHMA-N-netropsin compounds. In the same studies, all compounds bearing MePy were more potent than those compounds linked with diMePy. Moreover, AHMA-netropsin derivatives bearing a succinyl chain as the linking spacer were more potent than those compounds having a glutaryl bridge. Among these hybrid molecules, AHMA-ethylcarbamate-O-succinyl-MePy (19) was 2- to 6-fold more cytotoxic than the parent compound AHMA (5) in various cell lines, whereas compound 25 had very poor solubility and was inactive. Studies on the inhibitory effect against topoisomerase II (Topo II) and DNA interaction of these conjugates showed no correlation between the potency of DNA binding and inhibitory activity against Topo II.     

A prototype bioreductive DNA groove binding ligand.

Molecular mechanics calculations have been used to evaluate the potential bioreductive behaviour of several DNA minor groove binding ligands containing quinone/hydroquinone redox systems. The proposed structures are analogues of the Hoechst 33258 molecule with modifications of the benzimidazole rings. Binding energies of simple analogues indicate the reduced forms bind more strongly to the DNA minor groove. N-methylation of the imidazole ring(s) produces structures which can form extended quinone methides. These also show stronger binding in the reduced form and it is speculated that such structures might provide a basis for the design of groove binding ligands which will act as bioreductive alkylating agents.     

Ecteinascidin 743: a minor groove alkylator that bends DNA toward the major groove.

The ecteinascidins (Ets), which are natural products derived from marine tunicates, exhibit potent antitumor activity. Of the numerous Ets isolated, Et 743 is presently being evaluated in phase II clinical trials. Et 743 binds in the minor groove of DNA and alkylates N2 of guanine. Although structurally similar to saframycin, which exhibits poor activity in cellular assays, Et 743 has shown good efficacy as an antitumor agent. In this study, DNA structural distortions induced by Et 743 were examined to provide insight into the molecular basis for the antitumor activity of Et 743. Electrophoretic mobility shifts of ligated oligomers containing site-directed adducts were used to examine the extent and direction of the Et 743-induced bend. Surprisingly, we find that Et 743 bends DNA toward the major groove, which is a unique feature among DNA-interactive agents that occupy the minor groove.     

Recent developments in sequence selective minor groove DNA effectors

DNA is a well characterized intracellular target but its large size and sequential nature make it an elusive target for selective drug action. Binding of low molecular weight ligands to DNA causes a wide variety of potential biological responses. In this respect the main consideration is given to recent developments in DNA sequence selective binding agents bearing conjugated effectors because of their potential application in diagnosis and treatment of cancers as well as in molecular biology. Recent progress in the development of cross linked lexitropsin oligopeptides and hairpins, which bind selectively to the minor groove of duplex DNA, is discussed. Bis-distamycins and related lexitropsins show inhibitory activity against HIV-1 and HIV-2 integrases at low nanomolar concentrations. Benzoyl nitrogen mustard analogs of lexitropsins are active against a variety of tumor models. Certain of the bis-benzimidazoles show altered DNA sequence preference and bind to DNA at 5'CG and TG sequences rather than at the preferred AT sites of the parent drug. A comparison of bifunctional bizelesin with monoalkylating adozelesin shows that it appears to have an increased sequence selectivity such that monoalkylating compounds react at more than one site but bizelesin reacts only at sites where there are two suitably positioned alkylation sites. Adozelesin, bizelesin and carzelesin are far more potent as cytotoxic agents than cisplatin or doxorubicin. A new class of 1,2,9,9a-tetrahydrocyclo-propa[c]benz[e]indole-4-one (CBI) analogs i.e., CBI-lexitropsin conjugates arising from the latter leads are also discussed.A number of cyclopropylpyrroloindole (CPI) and CBI-lexitropsin conjugates related to CC-1065 alkylate at the N3 position of adenine in the minor groove of DNA in a sequence specific manner, and also show cytotoxicities in the femtomolar range. The cross linking efficiency of PBD dimers is much greater than that of other cross linkers including cisplatin, and melphalan. A new class of PBD-lexitropsin conjugates is also discussed. Certain functional models of the bleomycins (BLMs) show outstanding DNA cleavage activity comparable with that of and positionally distinct from natural BLM.     

Oligopyrrole carboxamides linked with a nucleobase as potential DNA minor groove binding ligands: synthesis, DNA binding and biological evaluation

The synthesis of a series of new oligopyrrole carboxamides closely related to netropsin and distamycin A, linked with a nucleobase is reported. The new compounds possess similar structure elements as the known peptide nucleic acids which are interesting sequence reading DNA ligands. Cytotoxicity in vitro, the DNA binding characteristics and the inhibition of topoisomerase I were studied. Four of the compounds, 27, 31, 33 and 37 bind to DNA probably at AT sequences like netropsin or distamycin A in the minor groove. Surprisingly, no cytotoxicity and no inhibition of topoisomerase I was found.     

Distamycin A as stem of DNA minor groove alkylating agents

Analogues of naturally occurring antitumor agents, such as distamycin A, which bind in the minor groove of DNA, represent a new class of anticancer compounds currently under investigation. Distamycin A has driven researcher's attention not only for their biological activity, but also for its non intercalative binding to the minor groove of double-stranded B-DNA, where it forms strong reversible complex preferentially at the nucleotide sequences consisting of 4-5 adjacent AT base pairs. The pyrrole-amide skeleton of distamycin A has been also used as DNA sequence selective vehicles for the delivery of alkylating functions to DNA targets, leading to a sharp increase of its cytotoxicity, in comparison to that, very weak, of distamycin itself. In the last few years, several hybrid compounds, in which known antitumor derivatives or simple active moieties of known antitumor agents have been tethered to distamycin frames, have been designed, synthesized and tested. Several efforts have been made to modify DNA sequence selectivity and stability of the distamycin and the structural modifications have been based on replacement of pyrrole by other heterocycles and/or benzoheterocycles obtaining a novel class of minor groove binding molecules called lexitropsins. The role of the amidino moiety, by means of the substitution with various groups, which includes ionizable, acid or basic, and non-ionizable groups, has been also studied. The synthesis of a hybrid deriving among the combination of the distamycin A and naturally occurring alkylating agent has been also reported. Several classes of distamycin derivatives that have been reported in the published literature have been described in this review article.     

Effects of Alkyl Amine Carboxyboranes on L1210 DNA Fragmentation and Nucleic Acid Metabolism

Amine-carboxyboranes with varying alkyi chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase α, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 μM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.     

Brostallicin: a new concept in minor groove DNA binder development

Brostallicin is a bromoacryloyl derivative of distamycin A, which has shown very promising preclinical activity against a variety of human tumors both in vitro and in vivo. The drug has a limited toxicity towards bone marrow precursor cells in vitro resulting in a therapeutic index much higher than those achieved with other distamycin A derivatives. It retains activity against cancer cells resistant to alkylating agents, topoisomerase I inhibitors and cells with mismatch repair deficiency. Brostallicin has a peculiar mechanism of action involving activation upon binding to glutathione (GSH) catalyzed by glutathione-S-transferase (GST). As a consequence, cells expressing relatively high GST/GSH levels are more susceptible to treatment with brostallicin. Considering that increased levels of GST/GSH are often found in human tumors, this could represent an advantage for the drug in the clinic. Initial clinical studies indicate the tolerability of the drug and allow the determination of the optimal dose for subsequent studies. Some partial response were obtained in these initial phase I studies. Altogether, the results suggest brostallicin to be a new promising anticancer agent with a new mechanism of action. It also raises the possibility to use it in combination with other anticancer drugs currently used.     

Tanshinone IIA triggers p53 responses and apoptosis by RNA polymerase II upon DNA minor groove binding

Our previous work has shown that tanshinone IIA (Tan IIA) is a DNA minor groove binder instead of an intercalator as previously thought. In this study, we have further demonstrated that the molecular antitumor pharmacology of Tan IIA is dependent on its groove-binding capability. First, we investigated the structure damage to duplex DNA upon Tan IIA binding using circular dichroism spectra. Subsequently, we performed western blot, flow cytometry analysis, chromatin immunoprecipitation, and quantitative real-time PCR to illustrate the RNAPII degradation, phosphorylation, and distribution along the transcribed gene in H22 cells exposed to Tan IIA. In addition, p53 activation and apoptosis induction in both cultured H22 cells and in mice bearing the ascitic-type H22 were measured following Tan IIA treatment. It was revealed that Tan IIA decreases the level of RNAPII by altering DNA structure. At the low dose range (0.2-4 μM) of Tan IIA exposure, the DNA structure damage results in the inhibition of RNAPII binding to DNA and the initiation of RNAPII phosphorylation, while higher concentrations of Tan IIA (4-20 μM) cause complete phosphorylation and degradation of RNAPII followed by p53 activation and apoptosis. A similar apoptosis induction by RNAPII was observed in animals. Apoptosis of tumor cells from ascitic fluid was not detected until RNAPII levels were downregulated by Tan IIA, which requires 40 mg/kg body weight of Tan IIA. It was concluded that DNA-conformational-damage-dependent RNAPII response upon groove binding is the molecular basis of the antitumor property of Tan IIA, in vivo and in vitro.     

Synthesis, DNA binding and cytotoxicity of isohelical DNA groove binding platinum complexes

The two platinum complexes [[Pt(dien)]2mu-dpzm]4+ and trans-[Pt(NH3)2(mu-dpzm)2]2+(dpzm = 4,4'-dipyrazolylmethane) have been synthesized and their DNA binding and cytotoxicity studied in order to evaluate the potential of square-planar platinum complexes as DNA groove binding anti-cancer agents. 1H-NMR spectroscopy was used to study the binding of both metal complexes to the dodecanucleotide d(CGCGAATTCGCG)2. For both platinum complexes, a considerable number of intermolecular NOE contacts were observed in NOESY spectra of the platinum complex bound dodecanucleotide. The NOE data demonstrated that each platinum complex bound in the minor groove at the central AATT region. Neither platinum complex appeared to induce any major DNA conformation change. In vitro cytotoxicity studies in the murine leukaemia cell lines L1210 and L1210/cisR showed that trans-[Pt(NH3)2(mu-dpzm)2]2+ had some activity (IC50: 64 and 32 microM respectively) while the [[Pt(dien)]2mu-dpzm]4+ complex showed no activity at all (>200 microM). The results indicate that it may be possible to synthesize platinum complexes that are useful as groove binding agents in the treatment of cancer.     

Design and development of sequence selective lexitropsin DNA minor groove binders

The concept of lexitropsins, or information-reading molecules, is introduced within the antigene strategy as an alternative to, and complementary with, the antisense approach for cellular intervention and gene control. Consideration is given to alternative approaches to the development of DNA sequence selective binding agents because of their potential applications in diagnosis and treatment of cancer as well as in molecular biology. The chemical, physical, and pharmacological factors involved in the design of effective lexitropsins are discussed and illustrated with experimental results. Among the factors contributing to the molecular recognition processes are: the presence and disposition of hydrogen bond accepting and donating groups, ligand shape, chirality, stereochemistry, flexibility, and charge. For longer ligands, such as are required to target unique sequences in biological systems (14–16 base pairs), the critical feature is the phasing or spatial correspondence between repeat units in the ligand and the receptor. The recently discovered 2:1 lexitropsin-DNA binding motif provides a further refinement in molecular recognition in permitting discrimination between GC and CG base pairs. The application of these factors in the design and synthesis of novel agents which exhibit anticancer, antiviral, and antiretroviral properties, and in the inhibition of critical cellular enzymes including topoisomerases, is discussed. The emerging evidence of a relationship between sequence selectivity of the new agents and the biological response they invoke is also described. © 1995 Wiley-Liss, Inc.