Anti-cancer drug design最新文献

As is common with a newly discovered cancer-associated gene/protein, there is a lag between the elucidation of its cellular and molecular biology and appropriate therapeutic intervention. Telomerase represents an interesting and promising anticancer drug target but poses a particular drug discovery challenge. It is unclear at present what is the optimum means of targeting this complex ribonucleoprotein and associated telomeric DNA and binding proteins: various strategies are actively being explored. Some recent data (e.g. 2-5A antisense against telomeric RNA, targeting TRF2, introduction of dominant-negative hTERT into cells) has raised doubts over the previously presumption of a requirement for prolonged enzyme inhibition with gradual telomere erosion, especially in tumour cells with relatively short telomeres. Highly potent and selective in vivo inhibitors are required to validate the target and address these critical issues.

There is currently significant interest in the development of inhibitors of human telomerase for the treatment of cancer. We describe here the design and synthesis of a new class of mono-substituted small-molecule inhibitors of human telomerase based upon a tetracyclic structural motif. In contrast to the structurally related molecule 9-hydroxyellipticine, recently shown to inhibit telomerase activity in cell cultures but found to be inactive in a cell-free system, we demonstrate direct inhibition of the telomerase enzyme by the tetracyclic compounds in a modified cell-free TRAP assay. The most potent compounds exhibit activity in the low micromolar range and are thus comparable with some of the more active small-molecule telomerase inhibitors based on planar aromatic chromophores, previously described by ourselves and others. These compounds may represent useful leads for the development of more potent inhibitors of human telomerase.

N-[2-(Dimethylamino)ethyl]acridine-4-carboxamide (DACA), a DNA intercalating dual topoisomerase I/II poison, has high experimental antitumour activity, is able to overcome several forms of multidrug resistance, and is undergoing clinical trial. We prepared 3H-labelled DACA and investigated its uptake using cultured Lewis lung carcinoma cells (LLTC), P388 leukaemia cells and P/DACT cells that were multidrug resistant. The kinetics of uptake and efflux were very rapid and equilibrium was obtained within seconds of drug addition. Fluorescence microscopy of LLTC cells treated with DACA showed punctate fluorescence in the cytoplasm, consistent with uptake into vesicles. To investigate the role of lipophilicity in drug uptake, a fluorimetric assay was developed to measure uptake of a more hydrophilic derivative, 9-amino-5-sulphonylmethyl-DACA (as-DACA). The calculated n-octanol-water partition coefficient for as-DACA was 20-fold lower than that for DACA. On the other hand, as determined by ethidium displacement from DNA, as-DACA bound DNA 16-fold more strongly than did DACA. Uptake and efflux of DACA and as-DACA were very rapid and the uptake ratios in LLTC cells were 550 for DACA and 54 for as-DACA. At equitoxic concentrations (corresponding to the IC50 values), LLTC cell association was estimated to be approximately 1.6 x 10(8) molecules per cell for DACA and 3.0 x 10(6) molecules per cell for as-DACA. It is argued that DACA binds predominantly to lipophilic sites such as proteins and cellular membranes, while as-DACA associates predominantly with DNA. The high affinity of DACA for membranes may contribute to the rapidity of its uptake and efflux, as well as to its ability to overcome multidrug resistance.

The DNA-binding properties of a series of 2-aza-anthracenedione (benz[g]isoquinoline-5,10-dione) derivatives bearing two 3-dimethylaminopropylamino side chains at different (6,9, 7,9 and 8,9) positions of the planar ring system have been investigated. The affinity for the nucleic acid is dramatically affected by the substitution pattern, the 6,9-regioisomer being substantially more effective than the 7,9- or the 8,9-congeners. This cannot be ascribed to different binding mechanisms, as all compounds are shown to intercalate into the double helix. Instead, the geometry of intercalation into DNA and the site specificity are extensively affected by the substitution pattern. The site preference is CA (or AC) for the 6,9-regioisomer, whereas it is TA (or AT) for the 8,9-congener, the 7,9-analogue lying in between. Molecular modeling studies are in agreement with the experimental results. Although the 6,9-regioisomer was remarkably cytotoxic, it stimulated topoisomerase II-mediated cleavage of DNA very poorly. Hence, a different mechanism of DNA damage is probably operating in 2-aza-anthracenediones as the main cell-killing event. Changes in affinity for DNA, intercalation geometry and sequence specificity can explain the different cytotoxic responses exhibited by the test drugs.

A series of tricyclic aromatic carboxamides, and their corresponding dimeric analogues, were prepared and their growth-inhibitory properties were evaluated in a series of cell lines. The dimeric compounds were prepared by reaction of the appropriate acids with carbonyl-1,1'-diimidazole, isolating the resulting imidazolides, and reacting these with a stoichiometric amount of the diamine. The monomeric carboxamides containing a (CH2)2NMe2 side chain had widely differing inhibitory potencies, with the known nitronaphthalimide (mitonafide) and acridine-4-carboxamide (DACA) being the most potent. The corresponding bis analogues, linked by a (CH2)3NMe(CH2)3 chain, were generally more potent, with the largest increases (dimer/monomer ratio 20- to 30-fold) seen for the nitronaphthalimides and the phenazines. Based on the intrinsic cytotoxicity of the monomers and the highest degree of increase in cytotoxicity on dimerization, the most interesting chromophores appear to be the acridine-4-carboxamide and phenazine-1-carboxamide. Both of these compounds showed significant growth delays (approximately 6 days) in an in vivo colon 38 tumour model in mice.

An in vitro transcription assay was used to probe the sequence specificity of the binding of phenazine-tethered platinum complexes to DNA. It was found that when compared to cis-dichloro(ethylenediamine)platinum(II), the number of RNA polymerase blockage sites was increased by approximately 50% and the blockage sites were broadened by 1-3 nucleotides by the presence of the phenazine ligand. The rate of platination was also enhanced by the presence of the intercalator, and the increase in the kinetics of platination resulted in increased levels of adducts formed (i.e. high drug occupancy) as detected under conditions of active transcription. The level of platination by derivative 3 was 20-fold greater than that of the reference compound, which lacked a tethered intercalating phenazine group.

A series of cobalt (III) complexes, [Co(Racac)2(L)]+, have been prepared as potential hypoxia-selective prointercalator forms of the ligands L, where L is the cytotoxic DNA mono-intercalating ligands N-[2-[(aminoethyl)amino]ethyl]-phenazine-1-carboxamide and N-[5-[(aminoethyl)amino]pentyl]-phenazine-1-carboxamide or the potentially bis(intercalating) ligand bis[2-(phenazine-1-carboxamido)ethyl]-1,2-diaminoethane. The cobalt(III) complexes of the monointercalating ligands have significantly lower DNA binding affinity and cytotoxicity than the ligands themselves, indicating the potential utility of this prodrug approach for deactivation (and release under hypoxic conditions). However, the complexes showed only low hypoxic selectivity. The complex of the bis(intercalating) ligand also showed significantly lower DNA binding affinity than the free ligand, but in this case there was no attenuation of cytotoxicity.

Footprinting experiments with DNase I provide a starting-point for investigating the molecular basis of nucleotide sequence recognition by 2QN, a bis-quinoline derivative of the quinoxaline antibiotic echinomycin produced by directed biosynthesis in Streptomyces echinatus. Using tyrT DNA molecules variously substituted with inosine and/or 2,6-diaminopurine residues it is shown that the location of the 2-amino group of purine nucleotides in the minor groove of the double helix exerts a dominant influence in determining where the antibiotic will bind, as it does for echinomycin. However, newly created binding sites in DNA molecules substituted with diaminopurine (D), all located round TpD steps, bind 2QN with so much higher affinity than the canonical CpG steps that the latter fail completely to appear as footprints in D-substituted DNA; indeed CpG sequences appear in regions of enhanced susceptibility to nuclease cleavage as do CpI steps in doubly D + I-substituted DNA. Quantitative footprinting plots confirm that sequences surrounding TpD steps bind 2QN several hundred-fold more tightly than do CpG-containing sequences, with dissociation constants of the order of 25 nM. To test the hypothesis that differences in stacking interactions between the chromophores of the drug and the DNA base pairs could account for the differences in binding affinities, models of 2QN bound to two DNA hexamers containing either a central CpG or a central TpD step were built. Calculation of the molecular electrostatic potential (MEP) of 2QN in solution using a continuum method revealed a distinctive pattern that is considered relevant to DNA binding. When the MEPs calculated for the two DNA hexamers in the complexed state were compared, substantial differences were found in the major groove and in the space between the base pairs that is occupied by the chromophores of the drug upon binding. The modelling data support the notion that electrostatic stacking interactions underlie the considerably preferred binding of echinomycin and 2QN around TpD steps rather than CpG steps.

The platinum-iminoether complexes trans-[PtCl2[E - HN = C(OEt)Me]2] (1) and trans-[PtCl2[Z - HN = C(OEt)Me[2] (2), differing in the configuration of the iminoether ligands, were investigated for cytotoxicity towards human tumor cell lines, the involvement of DNA as a cytotoxic target, and their DNA binding mode. The cytotoxicity of isomer 1 was comparable to that of cisplatin, whereas isomer 2 was slightly less active. Excision-repair-deficient xeroderma pigmentosum group A cells were four times more sensitive to both isomers than normal cells, thus implicating cellular DNA as the cytotoxic target. Replication mapping experiments showed that both isomers interact preferentially with guanine residues at py-G-py sites. Oligodeoxyribonucleotides containing unique N7-guanine monofunctional adducts of the more cytotoxic isomer 1 were prepared and investigated for chemical reactivity, stability and DNA conformational alterations. The results showed that the ability of thiourea to labilize the monofunctional adducts depends upon the DNA secondary structure, but not upon the sequence context. Monofunctional adducts evolve to bidentate adducts in single-stranded oligonucleotides, but they are stable in double-stranded oligonucleotides and produce conformational distortions selectively located at the 5'-adjacent base pair. This study gives new insight into the mechanism of action of trans platinum-iminoether complexes, enabling for the first time comparison between different ligand isomers.

Methods have been developed to conjugate the antitumour imidazotetrazines mitozolomide and temozolomide to DNA minor and major groove-binding peptidic motifs by solid phase peptide synthesis. Side chain deprotection and resin cleavage steps were accomplished under acidic conditions to maintain the structural integrity of the imidazotetrazine nucleus. When mitozolomide was conjugated to the DNA minor groove-binding peptide (SPKK)2-NH2 (3) a strong preference for binding with [dA-dT]2 sequences was observed by circular dichroism studies, consistent with the construct making non-covalent interactions within the minor groove. This conjugate showed a > 100-fold DNA alkylating activity compared with the free imidazotetrazine as measured by a Taq polymerase assay. Unexpectedly, alkylation patterns of all conjugates were nearly identical to those elicited by the major groove interactive agents cisplatin and the unconjugated imidazotetrazines temozolomide and mitozolomide, indicating that covalent modification was restricted to guanine sites in the major groove of DNA irrespective of the targeting property of the peptidic ligand. The electrophilic reactive chloroethyldiazonium ion intermediate formed in the breakdown of the imidazotetrazine ring of mitozolomide (methyldiazonium ion from temozolomide) must be liberated from the DNA-bound conjugate prior to the alkylation event, and must diffuse to and react with more nucleophilic sites in the major groove.