Biomedical subjects
Publications and source records attributed to Richard H Himes.
An efficient synthesis of a C16 side chain benzophenone analogue of cryptophycin-24 using a crotylboration reaction and Heck coupling as key steps is described. In an in vitro tubulin assembly assay, the benzophenone analogue of the beta isomer (IC(50)=7.4 microM) is twice as active as cryptophycin-24 (IC(50)=15 microM).
The yeast Saccharomyces cerevisiae has two genes for alpha-tubulin, TUB1 and TUB3, and one beta-tubulin gene, TUB2. The gene product of TUB3, Tub3, represents approximately 10% of alpha-tubulin in the cell. We determined the effects of the two alpha-tubulin isotypes on microtubule dynamics in vitro. Tubulin was purified from wild-type and deletion strains lacking either Tub1 or Tub3, and parameters of microtubule dynamics were examined. Microtubules containing Tub3 as the only alpha-tubulin isotype were less dynamic than wild-type microtubules, as shown by a shrinkage rate and catastrophe frequency that were about one-third of that for wild-type microtubules. Conversely, microtubules containing Tub1 as the only alpha-tubulin isotype were more dynamic than wild-type microtubules, as shown by a shrinkage rate that was 50% higher and a catastrophe frequency that was 30% higher than those of wild-type microtubules. The results suggest that a role of Tub3 in budding yeast is to control microtubule dynamics.
The dynamic behavior of mammalian microtubules has been extensively studied, both in living cells and with microtubules assembled from purified brain tubulin. To understand the intrinsic dynamic behavior of mammalian nonneural microtubules, we purified tubulin from cultured HeLa cells. We find that HeLa cell microtubules exhibit remarkably slow dynamic instability, spending most of their time in an attenuated state. The tempered dynamics contrast sharply with the dynamics of microtubules prepared from purified bovine brain tubulin under similar conditions. In accord with their minimal dynamic instability, assembled HeLa cell microtubules displayed a slow treadmilling rate and a low guanosine-5'-triphosphate hydrolysis rate at steady state. We find that unlike brain tubulin, which consists of a heterogeneous mixture of beta-tubulin isotypes (beta(II), beta(III), and beta(IV) and a low level of beta(I)), HeLa cell tubulin consists of beta(I) tubulin ( approximately 80%) and a minor amount of beta(IV) tubulin ( approximately 20%). The slow dynamic behavior of HeLa cell microtubules in vitro differs strikingly from the dynamic behavior of microtubules in living cultured mammalian cells, supporting the idea that accessory factors create the robust dynamics that occur in cells.
Paclitaxel (Taxol) and the epothilones are antimitotic agents that promote the assembly of mammalian tubulin and stabilization of microtubules. The epothilones competitively inhibit the binding of paclitaxel to mammalian brain tubulin, suggesting that the two types of compounds share a common binding site in tubulin, despite the lack of structural similarities. It is known that paclitaxel does not stabilize microtubules formed in vitro from Saccharomyces cerevisiae tubulin; thus, it would be expected that the epothilones would not affect yeast microtubules. However, we found that epothilone A and B do stimulate the formation of microtubules from purified yeast tubulin. In addition, epothilone B severely dampens the dynamics of yeast microtubules in vitro in a manner similar to the effect of paclitaxel on mammalian microtubules. We used current models describing paclitaxel and epothilone binding to mammalian beta-tubulin to explain why paclitaxel apparently fails to bind to yeast tubulin. We propose that three amino acid substitutions in the N-terminal region and at position 227 in yeast beta-tubulin weaken the interaction of the 3'-benzamido group of paclitaxel with the protein. These results also indicate that mutagenesis of yeast tubulin could help define the sites of interaction with paclitaxel and the epothilones.
Microtubule dynamics are influenced by interactions of microtubules with cellular factors and by changes in the primary sequence of the tubulin molecule. Mutations of yeast beta-tubulin C354, which is located near the binding site of some antimitotic compounds, reduce microtubule dynamicity greater than 90% in vivo and in vitro. The resulting intrinsically stable microtubules allowed us to determine which, if any, cellular processes are dependent on dynamic microtubules. The average number of cytoplasmic microtubules decreased from 3 in wild-type to 1 in mutant cells. The single microtubule effectively located the bud site before bud emergence. Although spindles were positioned near the bud neck at the onset of anaphase, the mutant cells were deficient in preanaphase spindle alignment along the mother-bud axis. Spindle microtubule dynamics and spindle elongation rates were also severely depressed in the mutants. The pattern and extent of cytoplasmic microtubule dynamics modulation through the cell cycle may reveal the minimum dynamic properties required to support growth. The ability to alter intrinsic microtubule dynamics and determine the in vivo phenotype of cells expressing the mutant tubulin provides a critical advance in assessing the dynamic requirements of an essential gene function.
A library with 63 paclitaxel analogues modified at the C10 position of paclitaxel has been prepared using parallel solution phase synthesis. Most of the C10 analogues were slightly less active than paclitaxel in the tubulin assembly assay and had reduced potency in the B16 melanoma and MCF-7 cell line cytotoxicity assays. These modifications at C10, however, did not lead to the total loss of activity, indicating that the C10 moiety of paclitaxel may not be directly involved in the drug-microtubule interactions, but could influence its binding affinity to P-glycoprotein. Approximately 50% of the analogues demonstrated better activity against the drug resistant cell line MCF7-ADR. However, the increase in activity was 10-fold at most. This result demonstrates that the cytotoxicity against this drug resistant cancer cell line is sensitive to structural changes at the C10 position of paclitaxel. It was also found that the presence of a nitrogen atom in the C10 substituent might play a role in the interaction of analogues with microtubules.