Anti-cancer drug design最新文献

In a systematic effort to design inhibitors of the epidermal growth factor receptor (EGFR) family protein tyrosine kinases (PTK) as anti-cancer agents, we have constructed a three-dimensional homology model of the EGFR kinase domain and used molecular modeling methods for the structure-based design of analogs of the active metabolite of leflunomide (LFM) with potent and specific inhibitory activity against EGFR. These docking studies identified alpha-cyano-beta-hydroxy-beta-methyl-N-[4-(trifluoromethoxy)phenyl]-p ropenamide (LFM-A12) as our lead compound, which was predicted to bind to the EGFR catalytic site in a planar conformation. LFM-A12 inhibited the proliferation (IC50 = 26.3 microM) and in vitro invasiveness (IC50 = 28.4 microM) of EGFR positive human breast cancer cells in a concentration-dependent fashion. Similarly, the model of the EGFR binding pocket was used in combination with docking procedures to predict the favorable placement of chemical groups with defined sizes at multiple modification sites on another class of EGFR inhibitors, the 4-anilinoquinazoline. This approach has led to the successful design of a dibromo quinazoline derivative, WHI-P97, which had an estimated Ki value of 0.09 microM from modeling studies and a measured IC50 value of 2.5 microM in EGFR kinase inhibition assays. WHI-P97 effectively inhibited the in vitro invasiveness of EGFR-positive human cancer cells in a concentration-dependent manner. However, unlike LFM-A12, the quinazoline compounds are not specific for EGFR.

Thymidine phosphorylase (EC 2.4.2.4), identical to the angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF), is up-regulated in several tumour types. A similarity model of human thymidine phosphorylase was built, based on the crystal structure of the Escherichia coli enzyme. The high residue conservation between the two enzyme sources (39% sequence identity and 53% sequence similarity) aided model building. The human model was very similar to the E. coli enzyme's crystal structure, with the main tertiary structure difference being the destruction of helix 15 in E. coli by the presence of a loop in the human model. The model was used to rationalize the nature of the binding of the substrates thymine and thymidine, and of known inhibitors using a quantitative docking algorithm. Ab initio calculations on the nM inhibitor 5-chloro-6-(1-(2-iminopyrrolidinyl)methyl)uracil hydrochloride gave its conformation and distribution of charge. Subsequent quantitative docking studies have led to the suggestion, for the first time, that this inhibitor behaves as an oxycarbenium ion transition-state analogue, explaining its strong reported inhibition.

We have reported previously that unsymmetrical disulfide inhibitors of the human thioredoxin/thioredoxin reductase redox system (hTrx/TR) possess antitumor activity. We have broadened the search for more potent inhibitors and evaluated a large range of mono- and bis-disulfide compounds, prepared using parallel syntheses. Reaction of isothioisourea-HCI salts (R') or bis-salts (R) with aromatic or aryl thiols (R") in wells of 96-well plates produced >450 derivatives with the structures R"SSR' and R"SSRSSR". The excellent yield and purity of the disulfides provided sufficient material for evaluations of enzyme inhibition and cytotoxicity. Selection criteria based on the IC50 values for hTrx/TR inhibition and for cytotoxicities of the disulfides identified agents for subsequent scale-up syntheses and in vivo evaluations of antitumor activity. These scale-up studies confirmed the original activities of agents synthesized in the plates and validated the parallel synthetic approach. Structure-activity information derived from the hTrx/TR IC50 data allow for a number of generalizations. The most potent inhibitors of the Trx system contained two heteroatoms ortho to the disulfide moiety in an aromatic functionality. The thioalkylating moieties had greatest activity with one branch point alpha to the disulfide. In the absence of branching, more potent inhibition was observed with the electron withdrawing functionalities. Bis-disulfides showed patterns of activity which depended on chain length, with optimum activity observed when the disulfide units were separated by 3.9 A, a similar distance to that separating the thioredoxin active site cysteine residues. From the agents selected for scale-up syntheses, three disulfide compounds were studied for their antitumor activity in vivo against human tumor xenografts in scid mice. One of the analogues discovered through the combinatorial syntheses/screening for Trx inhibition, 1-phenylethyl 2-imidazolyl disulfide, N1 (ProlX agent PX-C5), has demonstrated excellent in vivo activity against the MCF-7 human breast cancer and the HL-60 human leukemia, thus validating this approach for novel drug discovery.

In a previous report we described the synthesis and biological properties of a group of pyrimido[4,5,6-kl]acridines 2, related to the pyrazolo[4,5,6-kl]acridines 1, promising antitumor agents possessing a broad spectrum of activity. Since the substitution of the pyrazole ring of the pyrazoloacridine chromophore with a pyrimidinone leads to derivatives that retain in vitro cytotoxic activity, we decided to further investigate the pyrimido[4,5 6-kl]acridines. Modifications at the ring system level, leading to chromophores with different characteristics, changes of substituent groups in position 6, simultaneous alteration of the chromophore and the introduction of a second cationic side chain in position 1 afforded 29 new pyrimido[4,5,6-kl]acridines, which were tested in vitro against the human colon adenocarcinoma HT29 cell line. Interesting structure-activity relationships could be drawn. Some selected derivatives were screened for their cytotoxic activity on the National Cancer Institute cell panel (60 human tumor lines).

The reverse transcriptase inhibitors (RTI) azidothymidine and carbovir can block telomerase function in various cells, whereas dideoxycytidine does not exhibit such activity. RTI induce senescence in 3T3 Swiss, NIH 3T3 cell cultures and in the clones of immortal spontaneously transformed mouse fibroblasts. The RTI-induced senescence of L6 rat myoblasts in culture resembles the senescence of fibroblasts, but the resulting cells acquire sharp morphological peculiarities. The artificial senescence of fibroblasts and myoblasts resulted in both the appearance of corresponding senescent cells and a small portion of cells with the signs of another type of differentiation. The blockade of telomerase function by RTI in the human tumour cell lines U-937 and MeWo leads to the shortening of telomeres, but does not result in senescence. These cells may undergo crisis and after a while the proliferation resumes and resistant cells appear. RTI inhibit spontaneous reactivation of telomerase in the process of spontaneous transformation of mouse embryonic fibroblasts, which leads to the formation of telomerase-free clones. A fraction of these clones may overcome the senescence via the acquisition of telomerase activity. Cells with a very high level of telomerase activity become resistant to RTI. Thus, the blockade of telomerase function in different cells can induce senescence, partial differentiation or crisis. In human tumour cells it induces mainly crisis.

Human telomeres, which consist of repeated TTAGGG sequences, have recently become the focus of intense and highly competitive biological research. This scientific interest lies in their unique biological functions: telomeres are essential for genome integrity and appear to play an important role in cellular aging and cancer. As telomerase appears to be selectively expressed in tumors versus normal cells, this enzyme represents a good target for inhibition. Different types of telomerase inhibitors have recently been described. We will present briefly the different strategies that have been proposed to achieve efficient telomerase inhibition, with a special emphasis on G-quartet ligands.

The binding of porphyrins to quadruplex DNAs provides a model system for the examination of drug binding to telomere, centromere, triplet repeat and other DNAs which may form quadruplex structures in vivo. Porphyrins, and certain other molecules that interact with quadruplex DNAs, have been shown to have significant biological activity. In this investigation the interactions of porphyrins with quadruplex DNAs have been examined by optical and NMR methods. The fluorescence of selected porphyrins can be used to discriminate between duplex and quadruplex DNAs. The fluorescence of the porphyrins can also be used to discriminate partially between the chair, basket and parallel stranded types of quadruplex DNA. At the relatively high DNA concentrations used in NMR, the porphyrins catalyze the conversion of both chair and basket type structures into parallel strand quadruplex DNAs. A DNA-porphyrin system has been found which appears to be a model for an intermediate of the catalytic pathway.

The discovery that the ribonucleoprotein telomerase is responsible for the immortality of human cancer cells represents a major advance in our quest to identify a distinguishing biochemical feature of the malignant phenotype that could be useful as a target for novel anti-cancer drug development. However, recent observations on telomere dynamics and cell lifespan using telomerase 'knockout' mouse models together with improved techniques to assay telomerase in normal human tissues have raised certain questions regarding potential side effects of anti-telomerase treatments. More importantly, such work has also demonstrated the propensity of mouse cell populations, in which telomerase has been experimentally inactivated, to generate immortal variants capable of maintaining their telomeres by alternative mechanisms. These recent findings and their implications for the potential success of anti-telomerase therapies are subjected to critical review. The wide differences between telomerase and telomere biology in mouse and human cells are highlighted, and the urgent need to obtain direct experimental evidence concerning the behaviour of a wide variety of human cancer cells under conditions of telomerase inhibition is stressed. It is concluded that, despite the caveats, the development of small molecule drugs that powerfully inhibit telomerase should remain a top priority area for those engaged in the rational design of novel cancer therapeutics.

Cationic porphyrins, which interact with guanine quadruplex (G4) telomeric folds, inhibit telomerase activity in human tumor cells. In this study, we have further examined effects of porphyrins and other telomere- and telomerase-interactive agents on proliferation rates and chromosome stability in a novel in vivo model, developing sea urchin embryos. We studied two porphyrins: (i) TMPyP4, a potent telomerase inhibitor; and (ii) TMPyP2, an isomer of TMPyP4 and an inefficient telomerase inhibitor, azidothymine (AZT), the reverse transcriptase inhibitor, antisense phosphorothioate oligonucleotide to telomerase RNA (TAG6) and a control scrambled sequence (ODN). TMPyP4, AZT and TAG6 (but not TMPyP2 or ODN) decreased the rates of cell proliferation and increased the percentage of cells trapped in mitosis. Nuclear localization of TAG6, but not of ODN, was demonstrated with 5'-fluoresceinated analogs of TAG6 and ODN. Formation of elongated chromosomes incapable of separating in anaphase, induced by TMPyP4, AZT and TAG6, closely resembled phenotypes resulting from telomerase template mutation or dominant negative TRF2 allele. Our data suggest that G4-interactive agents exert their antiproliferative effects via chromosomal destabilization and warrant their further development as valuable anticancer tools.