Cyclin-dependent kinase inhibitors for treating cancer.

Cyclin dependent kinases (Cdks) are essential enzymes for the control of cell cycle progression. Inhibitors of cyclin-dependent kinases are anticipated to possess therapeutic utility against a wide variety of proliferative diseases, especially cancer. The field of published small molecule Cdk inhibitors is briefly reviewed here as background to a summary of work on a class of pyrido[2,3-d]pyrimidine Cdk inhibitors. Compounds from this class are described that display potency against cyclin D/Cdk4 up to IC(50) = 0.004 microM. Good to moderate selectivity for cyclin D/Cdk4 is also reported for compounds in this structural class. Structure-activity relationship data are presented for substitution at the C2 and N8 positions and these data are interpreted in the context of a binding model that is based on the Cdk2 crystal structure. A representative cyclin D/Cdk4 inhibitor (compound 56) is demonstrated to selectively inhibit the proliferation of an Rb(+) cell line vs. a matched Rb(-) cell line and to produce a distinct G(1) block consistent with cyclin D/Cdk4 inhibition in cells.

Synthesis and biological evaluation of didemnin photoaffinity analogues.

The synthesis of four benzophenone-containing analogues of the antiproliferative natural product didemnin B is presented. In vitro protein biosynthesis inhibition potency and antitumor activity were evaluated. The results indicate that all four analogues are biologically active and could serve as photoaffinity reagents for the study of receptor-binding interactions of didemnins. These analogues could also be useful in studying antitumor effects of didemnins.

Inhibition of protein synthesis by didemnins: cell potency and SAR.

Synthetic and naturally occurring didemnins are potent and specific inhibitors of protein synthesis in vitro. Structure-activity analysis indicates a requirement for the intact macrocycle; however, the smaller ring size represented by the didemnin analogue, tamandarin A, is equipotent to didemnin B. Replacement of the N,O-dimethyltyrosine by a N-methylphenylalanine or N-methylleucine residue is also well-tolerated. The rank order for inhibition of protein synthesis in vitro appears to be retained in MCF-7 cells, albeit at much higher potency. This increase in potency is explained for the first time by data indicating that MCF-7 cells can accumulate didemnin B up to 2-3 orders of magnitude compared to the growth medium.

Inhibition of protein synthesis by didemnin B: how EF-1alpha mediates inhibition of translocation.

The antineoplastic cyclic depsipeptide didemnin B (DB) inhibits protein synthesis in cells and in vitro. The stage at which DB inhibits protein synthesis in cells is not known, although dehydrodidemnin B arrests translation at the stage of polypeptide elongation. Inhibition of protein synthesis by DB in vitro also occurs at the elongation stage, and it was shown previously that DB prevents EF-2-dependent translocation in partial reaction models of protein synthesis. This inhibition of translocation displays an absolute requirement for EF-1alpha; however, the dependence upon EF-1alpha was previously unexplained. It is shown here that DB binds only weakly to EF-1alpha/GTP in solution, but binds to ribosome. EF-1alpha complexes with a dissociation constant K(d) = 4 microM. Thus, the inhibition of protein synthesis by DB appears to involve an interaction with both EF-1alpha and ribosomes in which all three components are required. Using diphtheria toxin-mediated ADP-ribosylation to assay for EF-2, it is demonstrated that DB blocks EF-2 binding to pre-translocative ribosome.EF-1alpha complexes, thus preventing ribosomal translocation. Based on this model for protein synthesis inhibition by DB, and the proposed mechanism of action of fusidic acid, evidence is presented in support of the Grasmuk model for EF-1alpha function in which this elongation factor does not fully depart the ribosome during polypeptide elongation.

Inhibition of protein synthesis by didemnin B is not sufficient to induce apoptosis in human mammary carcinoma (MCF7) cells.

Didemnin B (DB) is one member of a class of natural cyclic depsipeptides that display potent cytotoxicity in vitro. The detailed mechanism of action of DB is unknown, although it appears to involve the inhibition of protein biosynthesis. Additional activities of DB have established DB as a rapid and potent inducer of apoptosis in HL-60 cells. Our aim was to determine if the induction of apoptosis by DB is mediated through inhibition of protein synthesis in MCF-7 human breast carcinoma cells. Apoptosis was observed only at > or = 100 nM DB, even though inhibition of protein synthesis occurred at much lower DB concentrations (IC50 = 12 nM). DB-induced apoptosis was mediated by caspase activation, since cleavage of the caspase substrate poly(ADPribose) polymerase was observed as early as 6 hr after DB exposure. Two additional protein synthesis inhibitors, cycloheximide (CHX) and emetine (ET), failed to induce apoptosis at concentrations that completely inhibited protein synthesis. Moreover, DB-induced apoptosis was enhanced only slightly by pre- and co-treatment with CHX and ET. Thus, inhibition of protein synthesis alone was not sufficient to induce apoptosis in these cells. As a measure of antiproliferative potential, DB (1-5 nM) inhibited the colony forming ability of MCF7 cells regardless of pretreatment with CHX. In conclusion, additional effects of DB, independent of protein synthesis inhibition, are proposed to account for its ability to induce apoptosis and prevent cell proliferation.

Mechanism of protein synthesis inhibition by didemnin B in vitro.

The cytotoxic and immunosuppressive marine depsipeptide didemnin B is a potent inhibitor of protein biosynthesis in intact cells. Here, didemnin B is shown to inhibit protein synthesis in vitro during the elongation cycle, by preventing eukaryotic elongation factor 2-(eEF-2-) dependent translocation. No inhibition of aminoacyl-tRNA delivery or of peptidyltransferase activity is observed. Didemnin B stimulates eEF-1 alpha-dependent aminoacyl-tRNA binding to rabbit reticulocyte ribosomes, and eEF-1 alpha is required for inhibition of the subsequent translocation of phenylalanyl-tRNA(Phe) from the A- to the P-site. These observations suggest that didemnin B prevents translocation by stabilizing aminoacyl-tRNA bound to the ribosomal A-site, similar to the antibiotic kirromycin, and consistent with the known affinity of didemnins for elongation factor eEF-1 alpha [Crews et al. (1994) J. Biol. Chem. 269, 15411]. Unlike kirromycin, didemnin B does not prevent peptide bond formation, so inhibition is observed only at the translocation step. Inhibition of translocation by didemnin B is attenuated by increasing concentrations of eEF-2.

Ligand recognition by influenza virus. The binding of bivalent sialosides.

Infection by influenza virus is initiated by a cellular adhesion event that is mediated by the viral protein, hemagglutinin, which is exposed on the surface of the virion. Hemagglutinin recognizes and binds to cell surface sialic acid residues. Although each individual ligand binding interaction is weak, the high affinity of influenza virus for cells that bear sialic acid residues is thought to result from a multivalent attachment process involving many similar recognition events. To evaluate such binding we have synthesized three series of compounds, each containing two sialic acid residues separated by spacers of different length, and have tested them as ligands for influenza hemagglutinin. No increased binding to the bromelain-released hemagglutinin ectodomain was seen for any of the bivalent compounds as determined by 1H NMR titration. In contrast, however, a spacer length between sialic acid residues of approximately 55 A sharply increases the binding of these bidentate species to whole virus as determined by hemagglutination inhibition assays. The most effective compound containing glycines in the linking chain displayed 100-fold increased affinity for whole virus over the paradigm monovalent ligand, Neu5Ac alpha 2Me.