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Richard H Himes

Publications and source records attributed to Richard H Himes.

18 recordsLinked to original sources
PubMed2006

Design, synthesis, and antiproliferative and CDK2-cyclin a inhibitory activity of novel flavopiridol analogues.

The design and synthesis of a small library of 8-amidoflavone, 8-sulfonamidoflavone, 8-amido-7-hydroxyflavone, and heterocyclic analogues of flavopiridol is reported. The potential activity of these compounds as kinase inhibitors was evaluated by cytotoxicity studies in MCF-7 and ID-8 cancer cell lines and inhibition of CDK2-Cyclin A enzyme activity in vitro. The antiproliferative and CDK2-Cyclin A inhibitory activity of these analogues was significantly lower than the activity of flavopiridol. Molecular docking simulations were carried out and these studies suggested a different binding orientation inside the CDK2 binding pocket for these analogues compared to flavopiridol.

Adenosine Triphosphate↗
PubMed2005

Single-site chemical modification at C10 of the baccatin III core of paclitaxel and Taxol C reduces P-glycoprotein interactions in bovine brain microvessel endothelial cells.

A single-site modification of paclitaxel analogs at the C10 position on the baccatin III core that reduces interaction with P-glycoprotein in bovine brain microvessel endothelial cells is described. Modification and derivatization of the C10 position were carried out using a substrate controlled hydride addition to a key C9 and C10 diketone intermediate. The analogs were tested for tubulin assembly and cytotoxicity, and were shown to retain potency similar to paclitaxel. P-glycoprotein interaction was examined using a rhodamine assay and it was found that simple hydrolysis or epimerization of the C10 acetate of paclitaxel and Taxol C can reduce interaction with the P-glycoprotein transporter that may allow for increased permeation of taxanes into the brain.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Synthesis and interactions of 7-deoxy-, 10-deacetoxy, and 10-deacetoxy-7-deoxypaclitaxel with NCI/ADR-RES cancer cells and bovine brain microvessel endothelial cells.

7-Deoxypaclitaxel, 10-deacetoxypaclitaxel and 10-deacetoxy-7-deoxypaclitaxel were prepared and evaluated for their ability to promote assembly of tubulin into microtubules, their cytotoxicity against NCI/ADR-RES cells and for their interactions with P-glycoprotein in bovine brain microvessel endothelial cells. The three compounds were essentially equivalent to paclitaxel in cytotoxicity against NCI/ADR-RES cells. They also appeared to interact with P-glycoprotein in the endothelial cells with the two 10-deacetoxy compounds having less interaction than paclitaxel and 7-deoxypaclitaxel.

Animals↗
PubMed2005

Chemical modification of paclitaxel (Taxol) reduces P-glycoprotein interactions and increases permeation across the blood-brain barrier in vitro and in situ.

The purpose of this work was to introduce a chemical modification into the paclitaxel (Taxol) structure to reduce interactions with the product of the multidrug resistant type 1 (MDR1) gene, P-glycoprotein (Pgp), resulting in improved blood-brain barrier (BBB) permeability. Specifically, a taxane analogue, Tx-67, with a succinate group added at the C10 position of Taxol, was synthesized and identified as such a candidate. In comparison studies, Tx-67 had no apparent interactions with Pgp, as demonstrated by the lack of enhanced uptake of rhodamine 123 by brain microvessel endothelial cells (BMECs) in the presence of the agent. By contrast, Taxol exposure substantially enhanced rhodamine 123 uptake by BMECs through inhibition of Pgp. The transport across BMEC monolayers was polarized for both Tx-67 and Taxol with permeation in the apical to basolateral direction greater for Tx-67 and substantially reduced for Taxol relative to basolateral to apical permeation. Taxol and cyclosporin A treatments also did not enhance Tx-67 permeation across BMEC monolayers. In an in situ rat brain perfusion study, Tx-67 was demonstrated to permeate across the BBB at a greater rate than Taxol. These results demonstrate that the Taxol analogue Tx-67 had a reduced interaction with Pgp and, as a consequence, enhanced permeation across the blood-brain barrier in vitro and in situ.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae.

Wild-type Saccharomyces cerevisiae tubulin does not bind the anti-mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for beta-tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule-stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated beta-tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel-sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel-treated cells showed evidence of a mitotic block, with an increase in large-budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three-fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic-like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae.

ATP-Binding Cassette Transporters↗
PubMed2004

Synthesis and anti-tubulin activity of a 3'-(4-azidophenyl)-3'-dephenylpaclitaxel photoaffinity probe.

The synthesis and biological evaluation of a novel paclitaxel photoaffinity probe is described. The synthesis involved the preparation of an azide-containing C13 side chain through a Staudinger cycloaddition followed by a lipase-mediated kinetic resolution to obtain the azetidinone in 99% ee. Coupling of the enantiopure side chain precursor to 7-TES-baccatin III and subsequent silyl ether deprotection afforded 3'-(4-azidophenyl)-3'-dephenylpaclitaxel, which was shown to be as active as paclitaxel in tubulin assembly and cytotoxicity assays.

Antineoplastic Agents↗
PubMed2004

Total synthesis and anti-tubulin activity of epi-c3 analogues of cryptophycin-24.

Epi-C3-cryptophycin-24, epi-C3-m-chlorobenzyl-cryptophycin-24, and the corresponding styrenes were synthesized and tested in vitro against the MCF-7 and multidrug-resistant MCF-7/ADR breast cancer cell lines and in an in vitro tubulin assembly assay. The results demonstrate that the S configuration at the C3 stereocenter is not required to induce potent cytotoxicity and the m-Cl substituent present on the C10 side chain did not induce any large change in activity.

Antineoplastic Agents↗
PubMed2004

Synthesis, modeling, and anti-tubulin activity of a D-seco paclitaxel analogue.

[reaction: see text] We have previously described a model of paclitaxel-microtubule binding that led to the prediction that analogues of paclitaxel lacking any D ring could stabilize microtubules as well as paclitaxel if the substituent present at C4 did not have unfavorable steric interactions with the binding pocket. We report the synthesis of a 4-methyl paclitaxel analogue, compound 1, which bears this prediction out. Compound 1 is as potent as paclitaxel at microtubule stabilization in vitro; however, it has only about one-four-hundredth the cytotoxicity of paclitaxel.

Antineoplastic Agents, Phytogenic↗
PubMed2004

Total synthesis and antitubulin activity of c10 analogues of cryptophycin-24.

The unsubstituted, 3'-Cl, 4'-C1, and 3',4'-diCl C10 analogues of cryptophycin-24 were prepared via total synthesis and tested in vitro for cytotoxicity against MCF-7 and multi-drug-resistant MCF-7/ADR breast cancer cell lines and in a tubulin assembly assay. The ED(50) values ranged from 7.2 to 15.8 microM in the tubulin assay and from 0.05 to 3.4 nM in the cell assays. The presence of a 3'-C1 and/or 4'-C1 substituent on the C10 phenyl ring increased cytotoxicity in the MCF-7 cell line compared to the unsubstituted phenyl ring. The most potent compound in this series possessed a 3'-C1 substituent on the C10 phenyl ring. The 3'-C1 analogue had ED(50) values of 50 and 580 pM in the MCF-7 and MCF-7/ADR cell lines, respectively. Its activity was very similar to the parent compound cryptophycin-24. Substitution of the 4'-MeO group in cryptophycin-24 with a 4'-C1 moiety did not significantly affect cytotoxicity against MCF-7 and MCF-7/ADR cells compared to the parent compound. These results demonstrated that the 4'-MeO group in cryptophycin-24 is not essential and can be replaced with 3'-C1 or 4'-C1 substituents.

Antineoplastic Agents↗
PubMed2003

Synthesis of cryptophycins via an N-acyl-beta-lactam macrolactonization.

An efficient and concise approach to the synthesis of the macrolide core of the cryptophycins has been developed. A novel macrolactonization utilizing a reactive acyl-beta-lactam intermediate incorporates the beta-amino acid moiety within the 16-membered macrolide core. This modular approach, involving a cyanide-initiated acyl-beta-lactam ring opening followed by cyclization, was successfully applied to the total synthesis of cryptophycin-24. The strategy was also used in an efficient synthesis of the 6,6-dimethyl-substituted dechlorocryptophycin-52. In this case, the cyanide-initiated ring opening of the bis-substituted 2-azetidinone followed by macrolactonization was achieved through a catalytic process.

Acylation↗
PubMed2003

Understanding tubulin-Taxol interactions: mutations that impart Taxol binding to yeast tubulin.

We have successfully used mutagenesis to engineer Taxol (paclitaxel) binding activity in Saccharomyces cerevisiae tubulin. Taxol, a successful antitumor agent, acts by promoting tubulin assembly and stabilizing microtubules. Several structurally diverse antimitotic compounds, including the epothilones, compete with Taxol for binding to mammalian microtubules, suggesting that Taxol and these compounds share an overlapping binding site. However, Taxol has no effect on tubulin or microtubules from S. cerevisiae, whereas epothilone does. After considering data on Taxol binding to mammalian tubulin and recent modeling studies, we have hypothesized that differences in five key amino acids are responsible for the lack of Taxol binding to yeast tubulin. After changing these amino acids to those found in mammalian brain tubulin, we observed Taxol-related activity in yeast tubulin comparable to that in mammalian tubulin. Importantly, this experimental system can be used to reveal tubulin interactions with Taxol, the epothilones, and other Taxol-like compounds.

Binding Sites↗
PubMed2003

Vaults bind directly to microtubules via their caps and not their barrels.

Vaults are large (13 Mda) ribonucleoprotein particles that are especially abundant in multidrug resistant cancer cells and have been implicated in nucleocytoplasmic drug transport. To understand how these large barrel-shaped complexes are transported through the cytosol, we examined the association of vaults with microtubules both in vitro and in vivo. Within cells, a subpopulation of vaults clearly associates with microtubules, and these vaults remain associated with tubulin dimers/oligomers when microtubules are disassembled by nocodazole treatment. In vitro, a microtubule-pull down assay using highly purified rat vaults and reassembled microtubules reveals that vaults exhibit concentration-dependent binding to microtubules that does not require the carboxyl terminal end of tubulin. Remarkably, negative staining for electron microscopy reveals that vault binding to microtubules is mediated by the vault caps; more than 82% of bound vaults attach to the microtubule lattice with their long axes perpendicular to the long axis of the microtubule. Five to six vault particles were bound per micron of microtubule, with no crosslinking of microtubules observed, suggesting that only one end of the vault can bind microtubules. Taken together, the data support the model of vaults as barrel-shaped containers that transiently interact with microtubules.

Animals↗
PubMed2003

The two alpha-tubulin isotypes in budding yeast have opposing effects on microtubule dynamics in vitro.

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.

Amino Acid Sequence↗
PubMed2002

Intrinsically slow dynamic instability of HeLa cell microtubules in vitro.

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.

Animals↗
PubMed2002

Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules.

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.

Animals↗
PubMed2002

beta-Tubulin C354 mutations that severely decrease microtubule dynamics do not prevent nuclear migration in yeast.

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.

Cell Cycle↗
PubMed2002

A systematic SAR study of C10 modified paclitaxel analogues using a combinatorial approach.

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.

Animals↗