Anti-cancer drug design最新文献

Resistance to cis-diamminedichloroplatinum(II) (cisplatin), a DNA damaging agent, is a major obstacle for its clinical effectiveness. Multiple mechanisms may be involved in cisplatin resistance. Frequently cited mechanisms include reduced accumulation, elevated levels of glutathione (GSH) and metallothionein, and enhanced DNA repair. Alterations in oncogene expression and in signal transduction pathways involved in apoptosis have been associated with cisplatin resistance. Of these mechanisms, decreased accumulation of cisplatin is the most common finding. Efflux of cisplatin by an organic anion transporter has been proposed, and one of the organic anion transporters, canalicular multispecific organic anion transporter, is associated with cisplatin resistance. Sensitivity to cisplatin has been increased by inhibitors of DNA repair, agents that increase accumulation of cisplatin and depletion of GSH. None of the agents tested that modulate cisplatin sensitivity completely reverses cisplatin resistance. These observations indicate that multiple mechanisms of resistance arise in the same cell line when cells are selected in vitro.

Herein we review studies demonstrating resistance manifested at the multicellular level, a phenomenon referred to as intrinsic or acquired multicellular resistance (MR). In addition, due to the fact that such resistance can be recapitulated in vitro only when cells are adhered to one another in a three-dimensional culture context, we examine the roles of cell adhesion molecules and how they may contribute directly or indirectly to MR. Finally, we suggest an experimental approach to circumvent MR in the treatment of advanced, aggressive ascites tumors.

In this article we first very briefly review current approaches to the design of drugs that have specificity for the modulation of gene expression and selectivity for target cells at the transcription level by targeting DNA. We focus this review on our approaches to gaining selectivity by drug-induced architectural alteration in DNA structure, selectivity achieved by protein-induced changes in DNA structure or dynamics, and hijacking of nuclear receptors.

The role of base excision repair in the repair of alkylation damage produced by a series of sequence specific oligopyrrole-containing analogues of distamycin A that tether benzoic acid mustard (BAM) has been examined. Whereas BAM alkylates and cross-links in the major groove of DNA, attachment to pyrrole units produces monoalkylations in the minor groove of DNA at AT tracts. Both sequence specificity of alkylation and cytotoxicity increase from one to three attached pyrrole units (compounds 1-3), and with 3 alkylation is selective for purine-N3 in the sequence 5'-TTTTGPu (where Pu = guanine or adenine). In a model bacterial (Escherichia coli) system repair of the sequence specific minor groove alkylations produced by 2 and 3 does not appear to involve BER, since neither a formamidopyrimidine-DNA glycosylase repair deficient E. coli mutant (BH 20, fpg- mutant) nor a 3-methyladenine-DNA glycosylase repair deficient mutant (GC 4803, tag-alkA- mutant) showed increased cytotoxicity to 2 or 3 compared with the wild type, AB 1157. The monopyrrole compound 1 was, however, approximately 4-fold more cytotoxic to the GC 4803 mutant compared with wild type and BH 20, suggesting a role for the 3-methyladenine-DNA glycosylase in the recognition and excision of the adducts formed by 1. In contrast, increased sensitivity (> 10-fold) was observed for the conventional nitrogen mustard BAM in the BH 20 strain, suggesting a role for the formamidopyrimidine-DNA glycosylase in the repair of the lesions produced by the agent. In a cell-free system the E. coli 3-methyladenine-DNA glycosylase (AlkA) was shown to remove alkylations at 5'-TTTTGPu sequences. However, the efficiency in removing the adducts formed by the oligopyrrole compounds decreased dramatically from compound 1 to compound 3. Increasing the size of the DNA adduct formed in the minor groove therefore decreased the efficiency of recognition and removal of the adduct by the DNA glycosylase.

A series of cytotoxic polybenzamide mustards targeted to the minor groove of DNA were used to define structure-activity relationships for sequence-specific DNA alkylation. Compounds with an annular structure closely matched to the minor groove of DNA, and with concave-facing, potentially H-bonding NH groups, had a strong preference for alkylating adenines in sequences possessing four or more consecutive adenines. Two compounds whose annular structure matched that of the minor groove better when at least one carboxamide NH group faced outwards showed a high specificity for the consensus sequence (A/T)A(G/C) (A/T)N. Several compounds also alkylated specific guanines, presumably at the N3 position. Modelling studies suggest the most important contribution to sequence-specific alkylation is the H-bonds formed between these compounds and DNA, with factors such as the degree and positioning of cationic charge being less influential.

Four diphenylfuran derivatives possessing different dicationic terminal side chains were used to investigate sequence-specific binding to DNA and poisoning of human topoisomerase II. Footprinting experiments with a range of DNA substrates attest that all four drugs bind selectively to AT-rich sequences in DNA. However, the quantitative analysis of the footprinting profiles reveals significant differences in terms of AT-selectivity according to the nature of the basic side chains. Furimidazoline (DB60) shows a reduced capacity to interact selectively with A.T tetrads compared with furamidine (DB75) and the 3-pentyl-substituted diamidine analogue DB226. DB244, for which the two amidine ends are substituted with a cyclopentyl group, exhibits the most pronounced AT specificity. It binds tightly to sites composed of at least four adjacent AT base pairs, such as 5'-TAAT, AATT and TTTT. At low concentrations (< 2 microM) DB60 is also capable of forming stable complexes with AT sites but at higher concentrations the binding becomes totally non-specific due to additional intercalation of drug molecules into GC-rich sequences. Nevertheless, DB60 is the only drug is the series which stabilizes DNA-topoisomerase II covalent complexes. This compound effectively promotes DNA cleavage by topoisomerase II whereas DB75, DB226 and DB244 have practically no effect. The topoisomerase II poisoning activity of DB60 correlates with its ability to intercalate into GC sites in DNA whereas the three other diphenylfurans essentially behave as typical AT-selective minor groove binders. The study suggests that the antimicrobial activity of the diphenylfurans, which are active against the Pneumocystis carinii pathogen (PCP), depends essentially on their capacity to recognize AT-rich DNA sequences rather than their ability to interfere with topoisomerase II. In contrast, the cytotoxicity of drugs like DB60 would be connected with the formation of intercalation complexes and the stimulation of DNA cleavage by human topoisomerase II.

Both 5-hydroxy- and 5-amino-seco-CBI-TMI minor groove alkylators are very potent cytotoxins. The patterns of alkylation of the two enantiomers of both compounds were compared on a section of the gpt gene. All of the compounds alkylated only at adenines, with the amino compounds being slightly more selective. Consensus alkylation sequences for both S (natural) enantiomers were identical, but for the R (unnatural) enantiomers these varied slightly. The consensus sequences suggest that the S enantiomers bind lying in the 3'-->5' direction from the alkylated adenine, but there was no clear indication of which direction the R enantiomers lie on the DNA. Both S enantiomers were 10- to 100-fold more efficient alkylators than the R enantiomers, and the amino compounds were somewhat more efficient than the corresponding phenols. The S enantiomers were more cytotoxic then the R in both the phenol and amino series. The large amounts of end-labelled DNA required for this work was obtained by first end-labelling appropriate primer oligonucleotides, then amplifying by PCR. Compared with other methods in use, this is a simple and flexible one-step procedure for the preparation of labelled DNA of any sequence. An improvement in the synthesis of 5-hydroxy-seco-CBI-TMI is reported.

Certain DNA minor groove binding agents, distamycin, netropsin, and a series of anticancer bis-benzimidazoles can block DNA helicase activity by binding to duplex DNA at specific base sequences. DNA helicases are crucial to cell DNA replication, transcription and repair because these enzymes separate double-stranded DNA, thereby preparing the strands for enzymatic manipulation. From our studies we have developed a hypothesis that focuses on cellular DNA helicase action as a mechanistic site where these minor groove binders can act. A crucial aspect for modulation of DNA activity by drugs is for specificity and selectivity. A series of DNA-interactive bis-benzimidazole analogues of Hoechst 33258 was also prepared to explore the potential for anticancer activity mediated for certain of the drugs via bioreductive activation by endogenous NADH or NADPH. The biological endpoints examined included intracellular distribution in euoxic and hypoxic conditions observed by fluorescence microscopy; relative efficacy as antimetabolites determined by the MTT [tetrazolium salt, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay in euoxic and hypoxic conditions; and relative inhibitory activities on human DNA helicase, as determined by degree of dissociation of GC B6486 DNA. The intracellular distribution was unique to each of the test compounds. Compounds V-93 and V-153, the respective semiquinone and quinone derivatives, demonstrated the predicted enhanced cytotoxicity and anti-helicase activities, supporting the concept that preferential binding of DNA at 5'-CG and TG sequences provides a novel approach to anticancer drug development.

DACA is a DNA-intercalating agent and dual topoisomerase (topo) I/II inhibitor currently in clinical trial as an anticancer drug. Substitutions in the acridine ring of DACA have significant effects on biological activity, with 5-substituted analogues being more potent but relatively less active against cell lines that underexpress topo II, and the converse for 7-substituted analogues. A small series of 5,7-disubstituted analogues was therefore prepared and evaluated. The compounds were prepared by CDI-assisted coupling of the appropriate acridine acids. When these contained no or only one halogen atom, they could be prepared by Al/Hg amalgam reduction of the corresponding acridine acids. However, this method could not be used to prepare dihalogen-substituted acridine acids due to substantial dehalogenation, and these intermediates were synthesized via cyclization of the appropriate aldehydes to give the acridines directly. These compounds showed enhanced DNA binding compared with the parent DACA, indicating that the known favourable influence of 5-substituents on DNA binding is retained. Cell line studies showed that the 5,7-disubstituted compounds retained both the broad-spectrum effectiveness of the 7-monosubstituted analogues and the higher cytotoxic potency of the 5-monosubstituted analogues. The 7-chloro-5-methyl and 5-chloro-7-methyl analogues showed comparable in vivo antitumour activity to DACA in the subcutaneous colon 38 model, but were substantially more potent (optimal doses of 60 mg/kg compared with 200 mg/kg for DACA).

The design, synthesis, in vitro and in vivo activity against L1210 murine leukaemia of the dibromo nitrogen mustard derivative of 2, called PNU 157977, is described and the structure-activity relationship discussed. This dibromo derivative is almost two orders of magnitude more cytotoxic than the dichloro counterpart having the same oligopeptidic chain (IC50 2.7 ng/ml versus 225 ng/ml), and it showed in vivo an increased survival time which is 5- and 3-fold longer than that of tallimustine and 2 (and T/C 750 versus 133 and 213) respectively. Moreover PNU 157977 shows activity against the M5076 solid tumour markedly inferior to that of the closely analogous 2. Footprinting experiments conducted using the oestrogen receptor PCR probe as the footprinting target molecule show that PNU 157977 possesses a different sequence-specific alkylation and greater cleavage activity than either 2 or tallimustine.