Yukimasa Nozaki

GS-164, a small synthetic compound, stimulates tubulin polymerization by a similar mechanism to that of Taxol

Purpose: During our search for new microtubule effectors as anticancer agents, we have found that a small synthetic molecule designated GS-164 interferes with the assembly of porcine microtubule proteins and has cytotoxic activity against a wide range of human tumor cell lines. In this study, we investigated mode of action of the compound in comparison with Taxol and colcemid. Methods: To gain an insight into the mode of action of GS-164, we used an in vitro microtubule polymerization assay and a flow-cytometric measurement technique. Microtubule organization and the level of tubulin polymerization in HeLa cells were also examined by immunofluorescence microscopy and cytoskeletal protein analyses, respectively. Results: GS-164 stimulated assembly of microtubule proteins in vitro in a concentration-dependent and a GTP-independent manner. Furthermore, as with Taxol, the microtubule polymerization induced by GS-164 was antagonized by podophyllotoxin, a tubulin polymerization inhibitor, and microtubules formed by GS-164 were resistant to disassembly by calcium or low temperatures. GS-164 in the micromolar range arrested the cell cycle of HeLa cells in the mitotic phase leading to cell death. GS-164 also increased the amounts of cellular microtubules in HeLa cells, resulting in the formation of microtubule bundles. Conclusion: These results indicate that GS-164 stimulates microtubule assembly by a similar mechanism to that of Taxol. A comparative conformational analysis of GS-164 and Taxol suggested that the structure of the former mimics the minimum essential sites of Taxol required to exert the Taxol-like activities described above. Although the cytotoxicity of GS-164 against human tumor cells was 1000-fold lower than that of Taxol and GS-164 was one-tenth as active as Taxol in vitro, these findings pave the way for synthesizing clinically useful anticancer agents using GS-164 as a lead compound.

SCE-963, a New Potent Cephalosporin with High Affinity for Penicillin-Binding Proteins 1 and 3 of Escherichia coli

A few biochemical activities of SCE-963, a new cephalosporin with potent antibacterial activities against gram-negative bacteria, were compared with those of several currently available cephalosporins against strains of Escherichia coli K-12. The minimum inhibitory concentrations of SCE-963, cefazolin, cephaloridine, cephalothin, and cephalexin were 0.2, 1.56, 3.13, 12.5, and 25 μg/ml, respectively. Affinities of these cephalosporins for the penicillin-binding protein (PBP) 1B of E. coli correlated well with their antibacterial activities; among tested cephalosporins, SCE-963 showed the highest affinity for PBP 1B. SCE-963 inhibited cross-linking of peptidoglycan in a cell-free system the most strongly suggesting that this inhibition results from its high affinity for PBP 1B. SCE-963 also showed the highest affinity for PBP 3; it caused filamentation of cells over a wide range of relatively lower concentrations. Thus its superior antibacterial activity is believed to be manifested through its high affinity for the PBPs. Images

Regulation of Glucosamine Utilization in Staphylococcus aureus and Escherichia coli

Glucosamine- or N-acetylglucosamine-requiring mutants of Staphylococcus aureus 209P and Escherichia coli K12, which lack glucosamine-6-phosphate synthetase [2-amino-2-deoxy-D-glucose-6-phosphate ketol-isomerase (amino-transferring); EC 5.3.1.19], were isolated. Growth of these mutants on glucosamine was inhibited by glucose, but growth on N-acetylglucosamine was not. Addition of glucose to mutant cultures growing exponentially on glucosamine inhibited growth and caused death of bacteria, though chloramphenicol prevented death. Uptake of glucosamine by S. aureus and E. coli mutants was severely inhibited by glucose whereas uptake of N-acetylglucosamine was only slightly inhibited. Uptake of glucose was not inhibited by either glucosamine or N-acetylglucosamine. In glucosamine auxotrophs, glucose causes glucosamine deficiency which interrupts cell wall synthesis and results in some loss of viability in the presence of continued protein synthesis.