Anti-cancer drug design最新文献

We have investigated the sequence selectivity, DNA binding site characteristics, interstrand cross-linking ability and cytotoxicity of four 4-anilinoquinoline aniline mustards related to the AT-selective minor groove-binding bisquaternary ammonium heterocycles. The compounds studied include two full mustards that differ in alkylating power, a half mustard and a quaternary anilinoquinolinium bismustard. We have also compared their cytotoxicity with their precursor diols and their toxicity and cross-linking ability with the classical alkylating agents melphalan and chlorambucil. We find that the anilinoquinoline aniline mustards weakly and non-specifically alkylate guanines in the major groove and that they bind strongly to AT-rich sequences in the minor groove, where they alkylate both adenines and guanines at the N3 position. The most preferred sites are classical minor groove binder AT-tracts to which all four ligands bind equally well. The remaining sites are AT-rich, but include GC base pairs, to which the ligands bind with preferences depending on their structure. The full mustards alkylate at the 3' ends of the binding site in an orientation that depends on the spatial disposition of the purines within the two strands. Generally speaking guanines are found to be much less reactive than adenines. The anilinoquinoline aniline mustards are interstrand cross-linking agents that are 60- to 100-fold more effective than melphalan, with the quaternary compound being the most efficacious. However, the type of binding site at which the cross-links occur is not clear, since distamycin challenge fails to antagonize them fully. The full mustards are 20- to 50-fold more cytotoxic than their diol precursors, are more cytotoxic than the half mustard and are 20- to 30-fold more active than melphalan and chlorambucil. The quaternary ligand is the most potent. Given the evidence to hand, it appears that antitumour activity correlates with capacity to cause interstrand cross-links at classical or near-classical AT-minor groove binder sites, rather than with ability to discriminate between the subsets of potential anilinoquinoline aniline mustard binding sites.

Ecteinascidin-743 (ET-743) is a tetrahydroisoquinoline alkaloid isolated from Ecteinascidia turbinata, a tunicate growing in mangrove roots in Caribbean. It has been shown to bind in the minor groove of DNA forming covalent adducts by reaction of the N2 of guanine with the carbinolamine moiety. We investigated ET-743 ability to inhibit the binding of different transcription factors to their consensus sequences by using gel shift assays. We have selected three types of factors: (i) oncogene products such as MYC, c-MYB and Maf; (ii) transcriptional activators regulated during the cell cycle as E2F and SRF; and (iii) general transcription factors such as TATA binding protein (TBP), Sp1 and NF-Y. We observed no inhibition of the binding of Sp1, Maf, MYB and MYC. Inhibition of DNA binding was observed for TBP, E2F, SRF at ET-743 concentrations ranging from 50 to 300 microM. The inhibition of binding of NF-Y occurs at even lower concentrations (i.e. 10-30 microM) when the recombinant subunits of NF-Y are preincubated with the drug, indicating that the inhibition of NF-Y binding does not require previous ET-743 DNA binding. Since NF-Y is a trimer containing two subunits with high resemblance to histones H2B and H2A, we have investigated the effect of ET-743 on nucleosome reconstitution. ET-743 caused a decrease of the nucleosomal band at 100 nM, with the complete disappearance of the band at 3-10 microM. These data suggest that the mode of action of this novel anticancer drug is related to its ability to modify the interaction between some DNA binding proteins and DNA.

We have used quantitative DNase I and hydroxyl radical footprinting with three DNA fragments to examine the sequence-specific recognition of DNA by five novel minor groove-binding ligands which contain a structural motif in which a para-disubstituted benzene ring is flanked by two meta-disubstituted benzene rings. The compounds are all AT-selective and bind better to (AT)6 than (AT)4 sites. The ligands bind more tightly to AATT and TAAT than TTAA, TATA and ATAT, and avoid sequences containing central TpA steps. Different side groups cause subtle changes to the sequence recognition properties of these ligands.

Multidrug resistance-associated protein gene MRP/MRP1, and its family genes, including MRP2/cMOAT, have been isolated and characterized. These ATP-binding cassette (ABC) superfamily transporter genes are differentially expressed in various normal tissues and multidrug-resistant cell lines. Transfection of MRP/MRP1 and MRP2/cMOAT cDNA confers drug resistance on different spectra of anticancer agents from that of MDR1 coding P-glycoprotein. Although it remains unclear how MRP/MRP1 and related family genes are specifically involved in drug resistance in clinical cancers, current knowledge of the MRP subfamily suggests the importance of this class of transporters as a molecular target for drug sensitivity to anticancer agents.

Resistance to antimitotic agents is caused by decreased accumulation, altered tubulin, altered microtubule-associated proteins and increased metabolism. Vinca alkaloids, paclitaxel and docetaxel are actively effluxed by P-glycoprotein and/or the MRP1. Decreased intracellular accumulation is one of the major determinants of resistance to antimitotic agents. Increased tubulin levels and a decreased polymerization ratio were observed in resistant cells. Increased acetylation of tubulin and altered intracellular distribution of tubulin were also observed in resistant cells; however, the relationship between the function of tubulin and resistance remains unclear. The expression of each beta-tubulin isotype (beta 1-beta 6) is altered in resistant cells, but the functional differences among the isotypes have not been clarified. Recent evidence has demonstrated the alteration of binding properties of antimitotic agents in resistant cells. Therefore, the altered expressions of tubulin isotypes and related molecules might influence the antimitotic action and adverse events by antimitotic agents. Taxanes are metabolized and inactivated by p450 isozymes, and this is related to drug-resistant to taxanes.

We have previously shown that the DNA topoisomerase II alpha (topo II alpha) gene is down-regulated in VP16/VM26-resistant cells at the transcriptional level. To determine the DNA elements responsible for down-regulation, the transcriptional activities of luciferase reporter constructs containing various lengths of the promoter sequences were investigated by transient transfection of two resistant cell lines, KB/VP2 and KB/VM4. The transcriptional activities of the full-length promoter (-295 to +85) and of three deletion constructs (-197, -154 and -74 to +85) were significantly down-regulated in resistant cells. In contrast, the transcriptional activity of the minimal promoter (-20 to +85) in resistant cells was similar to that in parental KB cells. Furthermore, introduction of a mutation in ICE1 abolished the down-regulation of the topo II alpha promoter activity in drug-resistant cells. In vivo footprinting analysis of topo II alpha gene promoter revealed several specific protein-binding sites, a GC box, ICE1, ICE2 and ICE3. In vivo footprinting analysis also identified a cluster of hypersensitive sites. However, there was no marked difference in protein-binding sites between parental and resistant cells. To confirm our previous results, we have established the VP16-resistant cell lines T12-VP1 and T12-VP2 from T12 cells derived from human bladder cancer T24 cells stably transfected with the chloramphenicol acetyltransferase reporter gene driven by the topo II alpha gene promoter. The expression to topo II alpha was down-regulated in both cell lines. We also found that CAT gene expression was significantly decreased to one-fifth of that in T12 parental cells. These results suggest that the expression of the topo II alpha gene requires the binding of multiple factors to the core promoter and is down-regulated at the transcriptional level, probably through binding of a negative factor to ICE1 in drug-resistant cells.

Most solid tumors show resistance to current chemotherapy. This drug resistance can be associated with the unique physiology of solid tumors. Solid tumors generally have regions of low oxygen (hypoxia), low pH and low levels of glucose, which are not observed in normal tissues. These tumor-specific conditions commonly cause the glucose-regulated stress response of cancer cells. Accumulating evidence shows that the stress response leads to induction of resistance to multiple drugs, such as etoposide, doxorubicin, camptothecin and vincristine. This type of drug resistance is reversible and decays rapidly when stress conditions are removed. The induction of drug resistance can be partly explained by cell cycle arrest at the G1 phase in stressed cells because most anticancer drugs are primarily effective against rapidly dividing cells. Specific mechanisms, such as the decreased expression of DNA topoisomerase (topo) II alpha for the resistance to topo II poisons, are also involved in the drug resistance. Stressed cells, however, become hypersensitive to cisplatin, one of the most effective drugs against solid tumors, suggesting that preferential cytotoxicity to stressed cells may be important for the clinical efficacy against solid tumors. Further characterization of stressed cells will provide a unique target to circumvent the drug resistance of solid tumors.

Mutations in the p53 gene were detected in 27 of the 107 (25%) cases of non-Hodgkin's lymphoma (NHL), examined by assaying the transcriptional activity of p53 in yeast. A relatively high mutation rate of p53 was observed in B-cell intermediate-grade NHL and in T-cell high-grade immunoblastic NHL, in contrast to the relatively low mutation rate observed in other pathological classifications. However, retrospective analyses of all 76 cases revealed that the survival profile and therapeutic responses were very similar in NHL patients bearing lymphomas with a mutant p53 or with the wild-type p53 even within the subclasses characterized by frequent p53 mutation. In patients with high-intermediate grade tumors, the median survival period was 24 months in mutated p53 cases and 14 months in wild-type cases. Complete remission (CR) was observed in 9 of the 17 patients (53%) with mutated forms of p53 and 18 of the 35 patients (51%) with wild-type p53 genes. Our analyses of NHL patients revealed that the presence of p53 mutations may influence pathological grades of NHL, but did not strongly correlate with poor prognosis or reduced chemo/radiosensitivity in NHL. Hence, mutations of p53 do not serve as a prognostic, or chemo/radiosensitivity marker in NHL.

New prodrugs consisting of a beta-D-glucuronic acid linked to a MDR reversal agent (verapamil, quinine or dipyridamole) through a self-immolative spacer were synthesized. Four of them were selected for their reduced cytoxicity and beta-glucuronidase enzymatic efficient hydrolysis. Combined use of these prodrugs with a beta-D-glucuronyl-spacer-doxorubicin (HMR1826) according to an ADEPT strategy restored in vitro the sensibility of a MDR resistant strain.

P-glycoprotein can extrude a variety of structurally diverse, toxic xenobiotic compounds from cells. It is believed to be one of key molecules which can cause multidrug resistance in cancer. This paper deals with recent progress in P-glycoprotein research, especially in its structure, mechanisms for substrate recognition and transport. The review also discusses specific modulators of multidrug resistance in cancer and gene therapy using the MDR1 gene.