Richard H. Himes

Interactions of the catharanthus (Vinca) alkaloids with tubulin and microtubules

The dimeric Vinca alkaloids represent a group of important anti-tumor compounds whose intracellular target is tubulin, the protein monomer of microtubules. In this review data on the binding of these drugs to tubulin and microtubules in vitro are examined. The binding to tubulin is linked to a protein self-association reaction described by Na and Timasheff (1986a) as a ligand-induced plus ligand-mediated isodesmic self-association reaction. The simplest model which fits the binding data is one in which there is one intrinsic site which is linked to the self-association process. Effects of solution variables on the binding and self-association explain the wide variation of reported apparent binding constants for Vinca alkaloids to tubulin. The Vinca drugs also bind to microtubules via a low number of sites at the ends of microtubules with apparent high affinity and which are involved in the inhibition of tubulin dimer addition to the microtubule ends, and to sites along the microtubule wall with apparent low affinity which are involved in the disruption of the microtubules into spiraled protofilaments. This review also compares available binding data for different natural and semi-synthetic Vinca alkaloids.

Rotenone inhibition of tubulin self-assembly

Rotenone effectively inhibits the in vitro formation of microtubules from tubulin containing or lacking microtubule-associated proteins. In both cases a concentration of rotenone equal to that of tubulin present completely blocks assembly. The inhibition can be reversed by the addition of dimethylsulfoxide or by removing rotenone with charcoal.

Kinetic studies of membrane (NA+-K+-Mg2+)-ATPase

Abstract Kinetic studies on a microsomal (Na+-K+-Mg2+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rat brain are reported. The results indicate that MgATP is the real substrate of the reaction and ATP is a weak competitive inhibitor. Kinetic studies in which MgATP is varied at several Na+ and K+ concentrations reveal that the order of addition of cations and substrate to the enzyme is random. Product inhibition experiments suggest that the release of products is ordered, with Pi being released before ADP. A double reciprocal plot of 1/v vs 1/s[S] at temperatures ranging from 7–37° produced a linear Arrhenius plot with an energy of activation of 23150 cal/mole. Studies of the effect of pH on the kinetic constants show that two ionizing groups with p K values of 7.1 and 7.6 control the catalysis by (Na+-K+-Mg2+)-ATPase.

Identification of a distinct class of vinblastine binding sites on microtubules

Vinblastine, at concentrations above approximately 1 to 2 microM, causes depolymerization of steady-state bovine brain microtubules in vitro by a fraying of microtubule ends into protofilament-like spirals. Microtubule depolymerization is associated with the binding of vinblastine in approximately molar stoichiometry to tubulin in microtubules with apparent low affinity, as determined by binding experiments with radiolabeled vinblastine and by the ability of vinblastine to inhibit DEAE-dextran decoration of microtubule surfaces. Our data suggest that depolymerization occurs by a propagated mechanism, initially involving binding of vinblastine to a limited number of available sites on microtubule surfaces. This appears to cause loosening of protofilament associations which results in the exposure of new vinblastine-binding sites. Additional vinblastine binding in turn results in further loosening of protofilament associations. Such loosening, when it occurs at microtubule ends, results in protofilament-like splaying and end-wise depolymerization. Microtubule depolymerization appears mechanistically distinct from inhibition of microtubule polymerization by the drug, which is associated with the binding of vinblastine to small numbers of high-affinity binding sites on tubulin at one or both microtubule ends.

β-Amyloid-Induced Neurodegeneration and Protection by Structurally Diverse Microtubule-Stabilizing Agents

Deposition of β-amyloid peptide (Aβ) and hyperphosphorylation of the τ protein are associated with neuronal dysfunction and cell death in Alzheimers disease. Although the relationship between these two processes is not yet understood, studies have shown that both in vitro and in vivo exposure of neurons to Aβ leads to τ hyperphosphorylation and neuronal dystrophy. We previously reported that the microtubule-stabilizing drug paclitaxel (Taxol) protects primary neurons against toxicity induced by the Aβ25-35 peptide. The studies in this report were undertaken to characterize the actions of paclitaxel more fully, to assess the effectiveness of structurally diverse microtubulestabilizing agents in protecting neurons, and to determine the time course of the protective effects of the drugs. Primary neurons were exposed to Aβ in the presence or absence of several agents shown to interact with microtubules, and neuronal survival was monitored. Paclitaxel protected neurons against Aβ1-42 toxicity, and paclitaxel-treated cultures exposed to Aβ showed enhanced survival over Aβ-only cultures for several days. Neuronal apoptosis induced by Aβ was blocked by paclitaxel. Other taxanes and three structurally diverse microtubule-stabilizing compounds also significantly increased survival of Aβ-treated cultures. At concentrations below 100 nM, the drugs that protected the neurons did not produce detectable toxicity when added to the cultures alone. Although multiple mechanisms are likely to contribute to the neuronal cell death induced by oligomeric or fibrillar forms of Aβ, low concentrations of drugs that preserve the integrity of the cytoskeletal network may help neurons survive the toxic cascades initiated by these peptides.

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.

Interaction of cryptophycin 1 with tubulin and microtubules

The cryptophycins are newly discovered antimitotic agents isolated from the cyanobacterium Nostoc. Previous studies using cultured cells demonstrated that microtubules are the target of these compounds. We have studied the interaction of cryptophycin 1 with tubulin and microtubules in vitro. Cryptophycin 1 is an effective inhibitor of tubulin polymerization, causes tubulin to aggregate, and depolymerizes microtubules to linear polymers somewhat similar to the spiral‐like structures produced by the Vinca alkaloids. Cryptophycin 1 also inhibits vinblastine binding to tubulin but not colchicine binding. Thus, it appears that the cryptophycins may bind to the Vinca site in tubulin or to a site that overlaps with the Vinca site.

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

The yeast Saccharomyces cerevisiae has two genes for α‐tubulin, TUB1 and TUB3, and one β‐tubulin gene, TUB2. The gene product of TUB3, Tub3, represents ∼10% of α‐tubulin in the cell. We determined the effects of the two α‐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 α‐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 α‐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.

Schotten-Baumann acylation of N-debenzoyltaxol; an efficient route to N-acyl taxol analogues and their biological evaluation

Abstract An efficient route to N-acyl taxol analogues is described utilizing N-debenzoyltaxol ( 11 ). Acylation of 11 with various acid chlorides under Schotten-Baumann conditions led to a facile one step synthesis of several N-acyl taxol analogues from a common intermediate.

Cellular uptake and tubulin binding properties of four vinca alkaloids

The in vitro effects of four Vinca alkaloids, vinblastine (VLB), vincristine (VCR), vindesine (VDS) and vinepidine (VPD), on B16 melanoma proliferation, binding to bovine brain tubulin and B16 melanoma cell extracts, and uptake by the B16 cells were compared. The relative binding affinities to bovine brain tubulin were VPD greater than VCR congruent to VDS greater than VLB with the Ka for VPD being about 4-fold higher than that for VLB. On the other hand, the relative effects on B16 cell proliferation were exactly the opposite. Differences were found in the degree of concentration of the four alkaloids by the cells: 100-fold for VLB, 50-fold for VCR and VDS, and 20-fold for VPD. At the extracellular concentrations of drugs which inhibit proliferation by 50%, the intracellular concentration would still be far less than the tubulin concentration. Thus, it is likely that all of the Vinca alkaloids would be bound to tubulin and difference in uptake rather than Ka values is the major factor in determining the relative effectiveness of the drugs. L cells showed 50% the sensitivity of B16 melanoma cells toward VLB and 30% the sensitivity toward VPD. The L cells also concentrated these drugs to a lesser extent than did the B16 cells.