Small-molecule inhibition of protein and lipid biosynthesis.

摘要

A novel synthetic approach to natural product spirofungin A is described. The strategy is based on our new spiroketal assembly, which was successfully employed during the synthesis of bistramide A. Spirofungin A poseses a unique spiroketal moiety where one of the substituents is axially oriented, destabilizing molecule's structure and providing a synthetic challenge. A fully stereocontrolled assembly of a spiroketal subunit of spirofungin A, providing a unique and unprecedented solution to a highly challenging stereochemical problem was achieved. The total synthesis was completed in 20 chemical step in the longest linear sequence and 2% overall yield. Spirofungin A exhibits interesting biological properties. By testing the antiproliferative activity of this natural product in mammalian cells I established that spirofungin A inhibits the growth of several human cancer cell lines, including HL-60 (leukemia), HCT116 (colon), PC3 (prostate), and A549 (lung), with IC50 values of 1.0, 0.64, 1.9, and 6.4 &mgr;M, respectively. Based on the structural similarity of spirofungin A with reveromycin A, isoleucyl-tRNA synthetase was proposed as a target of spirofungin A. We showed that it inhibits activity of isoleucyl-tRNA in vitro by measuring tRNA aminoacylation using [3H]-labeled amino acids and HL-60 cell lysates. The results showed inhibition of isoleucyl-tRNA synthetase in a dose-dependent manner, while no effect on the activity of a homologous leucyl-tRNA synthetase. Furthermore, development of new yeast-based system where chemical libraries were screened to identify new drug candidates is developed. We have developed a practical strategy for rapid and efficient generation of new small-molecule libraries. The synthesis is based on miniaturization of the reaction scale, which enables rapid access to structural diversity in a combination of solution-phase high-throughput organic synthesis where each individual library member is purified in parallel using preparative thin layer chromatography. Libraries were screened using yeast strains in which the yeast gene encoding cytosolic ACC (ACC1) is replaced by one encoding an active form of human ACC1 or ACC2 in order to find compounds that prevent the growth of these strains, while permitting the growth of the wild-type yeast. Specificity of compounds identified using the yeast screening system as specific inhibitors for human ACC will be further confirmed using in vitro enzymatic assays. Several possible ACC2 inhibitors have been found so far. Two of these compounds have been confirmed as ACC2 inhibitors and will be studied further as lead compounds for the development of new drugs against obesity.