The marine natural product didemnin B displays potent antiviral, antitumor and immunomodulatory effects in cultured cells and in vivo. It has entered phase-II clinical trails as an antitumor agent. The reasons why didemnin B displays such potent biological activity are not understood, however didemnin B has been shown to inhibit the biosynthesis of DNA, RNA and proteins in whole cells. We have demonstrated the inhibition of in vitro protein synthesis by didemnin B and didemnin A, using a Cell-free translation system derived from rabbit reticulocytes, and have shown that didemnin B is equally effective as an inhibitor of protein synthesis in wheat germ. Furthermore, this effect is attenuated by modification of didemnin B as its bis(O-acetyl)derivatives. In our preliminary results, we present data that support he hypothesis that didemnin B inhibits protein synthesis specifically at eh elongation stage. Further studies are proposed to elucidate the detailed mechanism of inhibition of protein synthesis by didemnin B in vitro. Specifically, experiments are described that will define the precise step of elongation that is affected (amino acid delivery, peptide bond formation, or translocation), based on the effect of didemnin B upon protein synthesis partial reactions in rabbit reticulocyte or yeast lysates. The cytosolic molecular target of didemnin B will be identified using radiolabeled didemnin derivatives, or by cross-linking with a didemnin ligand. The design and synthesis of a new cross-linking agent is described. The effect of didemnin B upon regulated via phosphorylation of several translation factors and ribosomal proteins. The phosphoproteins will be labeled with 32P and resolved by tow-dimensional gel electrophoresis. To identify which structural features of didemnin B are important for its inhibition of protein synthesis, didemnin analogs will be synthesized and tested as inhibitors of in vitro protein synthesis. The structure- function profile for the didemnins that is obtained from these studies will be used in the design of new potential inhibitors of translation in ukaryotes. It is hypothesized that other potent inhibitors of protein synthesis besides the didemnins will also be effective antitumor agents, and this hypothesis will be tested by the synthesis and evaluation of didemnin mimics and limited screening of related natural products.
| Beidler, D R; Ahuja, D; Wicha, M S et al. (1999) Inhibition of protein synthesis by didemnin B is not sufficient to induce apoptosis in human mammary carcinoma (MCF7) cells. Biochem Pharmacol 58:1067-74 |
