dolastatin-10 and dolastatin-15

The dolastatins and some related compounds are antineoplastic pseudopeptides isolated from the sea hare Dolabella auricularia by the groups of G. R. Pettit and K. Yamada. Several groups including ours have contributed to the development of synthetic routes to most of these compounds. We recently described the synthesis of dolatrienoic acid, the lipidic component of dolastatin 14. Among all these metabolites, dolastatin 10 and dolastatin 15 exhibit the most promising antiproliferative properties and are currently under evaluation in clinical trials. These antimitotic agents seem to exert their activity by interacting with tubulin and inducing apoptosis. Research in this domain could greatly benefit from the recent elucidation of the atomic structure of tubulin. However, other targets cannot be excluded. Elucidation of the structure-activity relationships is an important step in the development of therapeutic agents. Parallel to the studies developed by other groups, our approach to exploring structural requirements for the antineoplastic activity of these compounds involved the determination of their preferred conformations in solution. Our study showed that dolastatin 10 exists in two different conformations corresponding to a cis-trans isomerization of a central amide bond. Such a situation was not demonstrated in the case of dolastatin 15. In view of elucidating the biological relevance of these findings, we elaborated hybrid molecules constituted of parts of both compounds. We also synthesized a cyclic analogue of dolastatin 10 which locked this compound in its cis conformation. Our results as well as those of others could be interpreted in terms of an existing structural model.

Topics: Antineoplastic Agents; Apoptosis; Depsipeptides; Oligopeptides; Proto-Oncogene Proteins c-bcl-2; Tubulin

Topics: Animals; Antineoplastic Agents; Depsipeptides; Oligopeptides; Snails

This paper summarizes published data on the interactions of tubulin with antimitotic compounds that inhibit the binding of vinca alkaloids to the protein. These are all relatively complex natural products isolated from higher plants, fungi and marine invertebrate animals. These agents are maytansine, rhizoxin, phomopsin A, dolastatins 10 and 15 and halichondrin B and their congeners. Effects on tubulin polymerization, ligand binding interactions and structure-activity relationships are emphasized.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Depsipeptides; Ethers, Cyclic; Lactones; Macrolides; Maytansine; Mycotoxins; Oligopeptides; Tubulin; Vinca Alkaloids

A highly sensitive and specific assay for the quantitation of the anticancer agent dolastatin-10 (DOL-10) in human plasma is described. The method was based on the use of electrospray ionization-high-performance liquid chromatography/mass spectrometry (ESP-LC/MS). The analytical procedure involved extraction of plasma samples containing DOL-10 and the internal standard (DOL-15) with n-butyl chloride, which was then evaporated under nitrogen. The residue was dissolved in 50 microl mobile phase and 10 microl was subjected to ESP-LC/MS analysis using a C18 microbore column. A linear gradient using water/acetonitrile was used to keep the retention times of the analytes of interest under 5 min. The method exhibited a linear range from 0.005 to 50 ng/ml with a lower limit of quantitation (LLQ) at 0.005 ng/ml. Absolute recoveries of extracted samples in the 85-90% range were obtained. The method's accuracy (< or =5% relative error) and precision (< or =10% CV) were well within industry standards. The analytical procedure was applied to extract DOL-10 metabolites from samples obtained following incubation of the drug with an activated S9 rat liver preparation. Two metabolic products were detected and were tentatively identified as a N-demethyl-DOL-10 and hydroxy-DOL-10. Structural assignments were made based on the fragmentation patterns obtained using the electrospray source to produce collision-induced dissociation (CID). The method was also applied to the measurement of DOL-10 in the plasma of patients treated with this drug. Preliminary investigation of the pharmacokinetics suggested that drug distribution and elimination may be best described by a three-compartment model with t1/2alpha = 0.087 h, t1/2beta = 0.69 h and t1/2gamma = 8.0 h. Plasma clearance was 3.7 l/h per m2.

Topics: Antineoplastic Agents; Atmospheric Pressure; Chromatography, High Pressure Liquid; Depsipeptides; Humans; Mass Spectrometry; Neoplasms; Oligopeptides; Reference Standards; Tissue Distribution

Antibody-drug conjugates (ADC) are emerging as powerful treatment strategies with outstanding target-specificity and high therapeutic activity in patients with cancer. Brentuximab vedotin represents a first-in-class ADC directed against CD30(+) malignancies. We hypothesized that its sustained clinical responses could be related to the stimulation of an anticancer immune response. In this study, we demonstrate that the dolastatin family of microtubule inhibitors, from which the cytotoxic component of brentuximab vedotin is derived, comprises potent inducers of phenotypic and functional dendritic cell (DC) maturation. In addition to the direct cytotoxic effect on tumor cells, dolastatins efficiently promoted antigen uptake and migration of tumor-resident DCs to the tumor-draining lymph nodes. Exposure of murine and human DCs to dolastatins significantly increased their capacity to prime T cells. Underlining the requirement of an intact host immune system for the full therapeutic benefit of dolastatins, the antitumor effect was far less pronounced in immunocompromised mice. We observed substantial therapeutic synergies when combining dolastatins with tumor antigen-specific vaccination or blockade of the PD-1-PD-L1 and CTLA-4 coinhibitory pathways. Ultimately, treatment with ADCs using dolastatins induces DC homing and activates cellular antitumor immune responses in patients. Our data reveal a novel mechanism of action for dolastatins and provide a strong rationale for clinical treatment regimens combining dolastatin-based therapies, such as brentuximab vedotin, with immune-based therapies.

Topics: Animals; Antibodies; Antigens; Brentuximab Vedotin; Cancer Vaccines; Cell Line; Cells, Cultured; CTLA-4 Antigen; Cytokines; Dendritic Cells; Depsipeptides; Humans; Immunoconjugates; Mice, Inbred C57BL; Mice, Transgenic; Neoplasms; Ovalbumin; Programmed Cell Death 1 Receptor; T-Lymphocytes; Tubulin Modulators; Tumor Burden

Combinatorial biosynthesis meets combinatorial pharmacology, cyanobacterial style: A new antimitotic natural product with features of both dolastatins 10 and 15 was isolated from the same Floridian Symploca sp. sample that produced the histone deacetylase inhibitor largazole. Both agents in combination are more effective in inhibiting cancer cell proliferation than either agent alone.

Topics: Antimicrobial Cationic Peptides; Antimitotic Agents; Antineoplastic Combined Chemotherapy Protocols; Cell Cycle; Cell Line, Tumor; Cyanobacteria; Depsipeptides; Histone Deacetylase Inhibitors; Histone Deacetylases; Humans; Peptides; Thiazoles

The marine ascidian Diazona angulata was the source organism for the complex cytotoxic peptide diazonamide A. The molecular structure of this peptide was recently revised after synthesis of a biologically active analog of diazonamide A in which a single nitrogen atom was replaced by an oxygen atom. Diazonamide A causes cells to arrest in mitosis, and, after exposure to the drug, treated cells lose both interphase and spindle microtubules. Both diazonamide A and the oxygen analog are potent inhibitors of microtubule assembly, equivalent in activity to dolastatin 10 and therefore far more potent than dolastatin 15. This inhibition of microtubule assembly is accompanied by potent inhibition of tubulin-dependent GTP hydrolysis, also comparable with the effects observed with dolastatin 10. However, the remaining biochemical properties of diazonamide A and its analog differ markedly from those of dolastatin 10 and closely resemble the properties of dolastatin 15. Neither diazonamide A nor the analog inhibited the binding of [3H]vinblastine, [3H]dolastatin 10, or [8-14C]GTP to tubulin. Nor were they able to stabilize the colchicine binding activity of tubulin. These observations indicate either that diazonamide A and the analog have a unique binding site on tubulin differing from the vinca alkaloid and dolastatin 10 binding sites, or that diazonamide A and the analog bind weakly to unpolymerized tubulin but strongly to microtubule ends. If the latter is correct, diazonamide A and its oxygen analog should have uniquely potent inhibitory effects on the dynamic properties of microtubules.

Topics: Animals; Antineoplastic Agents; Cell Division; Depsipeptides; Drug Screening Assays, Antitumor; Guanosine Triphosphate; Heterocyclic Compounds, 4 or More Rings; Humans; Hydrolysis; Microtubules; Mitosis; Oligopeptides; Oxazoles; Tubulin; Tumor Cells, Cultured

The antimitotic depsipeptide dolastatin 15 was radiolabeled with tritium in its amino-terminal dolavaline residue. Dolastatin 15, although potently cytotoxic, is a relatively weak inhibitor of tubulin assembly and does not inhibit the binding of any other ligand to tubulin. The only methodology found to demonstrate an interaction between the depsipeptide and tubulin was Hummel-Dreyer equilibrium chromatography on Sephadex G-50 superfine. The average apparent Kd value obtained in these studies was about 30 microM, with no difference observed when column size or tubulin concentration was varied. This relatively high dissociation constant is consistent with the apparent weak interaction of dolastatin 15 with tubulin demonstrated indirectly in the assembly assay. We attempted to gain insight into the binding site for dolastatin 15 on tubulin by studying inhibitory effects of other drugs when the gel filtration column was equilibrated with both [3H]dolastatin 15 and a second, nonradiolabeled drug. No inhibition was detected with either the colchicine site agent combretastatin A-4 or with an analog of the antimitotic marine peptide diazonamide A (both the analog and diazonamide A are potent inhibitors of tubulin assembly). Weak inhibition was observed with cemadotin, a structural analog of dolastatin 15, and with the depsipeptide cryptophycin 1. Moderate inhibition occurred with vinblastine and vincristine, and strong inhibition with maytansine, halichondrin B, and the peptides dolastatin 10 and phomopsin A. These observations suggest that the binding site(s) for peptide and depsipeptide antimitotic drugs may consist of a series of overlapping domains rather than a well-defined locus on the surface of beta-tubulin.

Topics: Animals; Binding Sites; Cattle; Chromatography, Gel; Colchicine; Depsipeptides; Heterocyclic Compounds, 4 or More Rings; Kinetics; Maytansine; Oligopeptides; Oxazoles; Protein Binding; Protein Structure, Tertiary; Tritium; Tubulin; Vinblastine; Vincristine

Topics: Antineoplastic Agents; Clinical Trials, Phase II as Topic; Depsipeptides; Heart; Humans; Hypertension; Neutropenia; Oligopeptides; Structure-Activity Relationship

The natural cytotoxic compounds dolastatins 10 and 15 exhibit great similarities in structure and in their biological activity profiles. Two compounds (1 and 2) formed by interchanging the dolaisoleuine residue of dolastatin 10 and the MeVal-Pro dipeptide of dolastatin 15 were synthesized in order to evaluate the possible equivalence of these units. These compounds can be considered as chimeras of dolastatins 10 and 15 formed by the N-terminal part of the former and the C-terminal part of the latter and vice versa. Both analogues exhibited a marked decrease in their cytotoxic activity but showed similar differential cytotoxicity with regard to the cell lines assayed compared with the parent compounds. HT-29 cell line was the least sensitive one. However, this activity was in the nanomolar level and close to that of vincristine. The differences in their effect on tubulin polymerization were less pronounced. We confirmed the already known crucial role of the Dil residue in this assay. The nonequivalence of the Dil unit and the MeVal-Pro dipeptide probably reflects modification in the relative positions of the N-dimethylamino and the phenyl moieties.

Topics: Animals; Antineoplastic Agents; Cattle; Cell Division; Depsipeptides; Drug Screening Assays, Antitumor; HT29 Cells; Humans; L Cells; Mice; Oligopeptides; Tubulin

Eight peptoids have been synthesized as peptidomimetics of the cytostatic Dolastatin 15, a depsipeptide isolated from the Indian sea hare Dolabella auricularia. The compounds have been tested against several human cancer cell lines and did not show any cytostatic properties.

Topics: Antineoplastic Agents; Depsipeptides; Drug Screening Assays, Antitumor; Humans; Molecular Mimicry; Oligopeptides; Peptides; Peptoids; Tumor Cells, Cultured

Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia that have been shown to interact with tubulin. Their growth-inhibitory properties were compared using panels of human ovarian and colon-carcinoma cell lines. Both agents were very potent inhibitors of cell growth, with dolastatin 10 being an average of 9.1-fold more potent than dolastatin 15 [mean 50% inhibitory concentrations (IC50 values) 2.3 x 10(-10) and 2.1 x 10(-9) M, respectively; P < 0.05] and more potent than paclitaxel or vinblastine. While neither dolastatin exhibited marked cross-resistance in cisplatin- or etoposide-resistant cell lines, contrasting effects were observed using an acquired doxorubicin-resistant (CH1doxR, 100-fold resistant, P-glycoprotein overexpressing) cell line. Resistance was significantly higher to dolastatin 15 (12.7-fold) than to dolastatin 10 (only 3.2-fold; P < 0.05) and was reversible in both cases by verapamil. In vivo, using a s.c. advanced-stage human ovarian carcinoma xenograft and equitoxic doses, greater activity was observed with dolastatin 10 (6.1-day growth delay) versus 0.4 days for dolastatin 15. A radioimmunoassay for dolastatin 10 (limit of detection in mouse plasma 5 ng/ml) was developed. The rabbit antiserum aslo cross-reacted by 65% with dolastatin 15. Comparative mouse pharmacokinetics following i.v. administration of 1 mg/kg showed that both compounds are rapidly eliminated, but with a shorter second-phase half-life (t1/2 beta) being observed for dolastatin 15 (being detectable for only up to 4 h post-administration), the t1/2 beta being 3 times longer for dolastatin 10. In addition, areas under the plasma concentration-time curve (AUC values) were 1.6-fold higher for dolastatin 10 (333 versus 208 ng ml-1 h). Plasma binding of dolastatin 10 exceeded 90%. The highly sensitive RIA will be useful for pharmacokinetic studies in conjunction with the planned phase I clinical trials of these novel, extremely potent, tubulin-binding agents, of which dolastatin 10 appears to possess the more promising preclinical features.

Topics: Animals; Antineoplastic Agents; Carcinoma; Cell Division; Colonic Neoplasms; Cross Reactions; Depsipeptides; Drug Resistance, Multiple; Female; Half-Life; Humans; Injections, Intravenous; Mice; Mice, Inbred BALB C; Neoplasm Transplantation; Oligopeptides; Ovarian Neoplasms; Rabbits; Radioimmunoassay; Random Allocation; Transplantation, Heterologous; Tumor Cells, Cultured; Verapamil

We have assessed the vascular effects of vinblastine and four other tubulin binding agents (dolastatin 10, dolastatin 15, combretastatin A1 and combretastatin A4), which are awaiting clinical evaluation. All five agents induce a reduction in tumour blood flow as measured by uptake of RbCI 24 h post drug administration. The degree of reduction ranged from 50% with combretastatin A1 to 90% with dolastatin 10. These reductions were similar to that seen with flavone acetic acid (FAA) and indicate that antivascular effects are a common feature of tubulin binding agents. We subsequently evaluated whether the blood flow reductions, induced by FAA and vinblastine, could be used to enhance the activity of the bioreductive drug tirapazamine. Since the kinetics and extent of blood flow reductions induced by the agents is comparable, similar therapeutic response was expected. Potentiation was only evident with FAA, indicating that this effect is not directly related to killing of hypoxic tumour cells induced as a consequence of blood flow reduction.

Topics: Animals; Antineoplastic Agents; Bibenzyls; Depsipeptides; Flavonoids; Mice; Mice, Inbred CBA; Neoplasms, Experimental; Oligopeptides; Regional Blood Flow; Stilbenes; Tirapazamine; Triazines; Tubulin; Tumor Necrosis Factor-alpha; Vinblastine

The effects of dolastatin 10 (Dol10) and dolastatin 15 (Dol15) alone, and after treatment with bryostatin 1 (Bryo1), on human diffuse large cell lymphoma cell line (WSU-DLCL2) were studied. At a concentration of 1.0 ng/ml Dol10 and Dol15 showed significant growth inhibition (p < 0.05). This inhibition was intensified when the cells were pretreated for 24 h with 200 nM Bryo1. Bryo1, Dol10 and Dol15 induced apoptosis which was seen on morphological examination, by flow cytometry and DNA fragmentation on agarose gel electrophoresis. Cells pretreated with Bryo1 and then exposed to Dol10 showed an increase in apoptosis compared with cells that were treated with the Dol10, Dol15 alone. Immunocytochemistry revealed that WSU-DLCL2 cells express the bcl-2 oncoprotein constitutively. bcl-2 expression was decreased when cells were treated with Bryo1, Dol10 or Dol15 and abolished with the Bryo1/Dol10 combination. WSU-DLCL2 cells were negative for p53 protein expression, upon treatment with Bryo1 or Dol10, the expression of p53 was weak and moderate with the Bryo1/Dol10 combination. The inverse correlation between bcl-2 and p53 oncoprotein expression seems to be related to induction of apoptosis in this lymphoma cell line.

Topics: Antineoplastic Agents; Apoptosis; Bryostatins; Cell Division; Depsipeptides; DNA, Neoplasm; Electrophoresis, Agar Gel; Flow Cytometry; Humans; Immunohistochemistry; Lactones; Lymphoma, Large B-Cell, Diffuse; Macrolides; Oligopeptides; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Tumor Suppressor Protein p53

Dolastatins 10 and 15 are small peptides isolated from the marine sea hare Dolabella auricularia. In vitro studies of these peptides have demonstrated antimitotic and antiproliferative activity and growth inhibition in hematopoietic progenitor cells.. The purpose of our in vitro study was to determine the biological effects of these marine peptides on growth of human lymphoma cell lines and to investigate mechanisms by which the dolastatins may act.. Cell lines DB, HT, RL, and SR were grown from the ascites or pleural effusion of four patients with lymphoma. The DB, HT, and RL cell lines are of B-cell origin, and the SR cell line appears to be a less differentiated lymphoid cell type. Cells from these lines were cultured in the presence of vincristine or dolastatin 10 or 15. [3H]Thymidine-uptake assays were used to measure effects on DNA synthesis. Cell cycle analysis using propidium iodide was performed to measure drug-induced cell-cycle arrest. DNA fragmentation was used as an assay for drug-induced apoptosis and was measured by agarose gel electrophoresis.. In the three B cell lines, dolastatin 10 was more effective than dolastatin 15. Values for concentrations required for inhibition of proliferation by 50% (IC50) were .00013-.0013 nM for dolastatin 10 in each cell line; values for dolastatin 15 were approximately .13 nM in DB and HT cells and .0013-.013 nM in RL cells. SR cells were more sensitive to dolastatin 15 than to dolastatin 10 (IC50 = .00013-.0013 nM versus .0013-.013 nM). Both dolastatins arrested more than 70% of cells in mitosis in all cell lines. This effect was reversed if the drug was removed by 4 hours, but by 8 hours of exposure, reversal was not possible. Both dolastatins 10 and 15 produced apoptosis in DB and HT cells but not in the other two cell lines.. We have demonstrated that dolastatins 10 and 15 have a profound antiproliferative effect on four different human lymphoma cell lines and that the dolastatins are approximately 3-4 logarithms more effective as antiproliferative compounds, on a molar basis, than vincristine--a clinically useful, antiproliferative agent. These data support the hypothesis that apoptosis, as measured by DNA fragmentation, appears to be a cell-specific response and may not be directly related to the antimitotic effect of the dolostatins.. Our results suggest that these compounds may be good candidates for development as antineoplastic agents.

Topics: Antineoplastic Agents; Apoptosis; Cell Division; Depsipeptides; Humans; Lymphoma; Mitosis; Oligopeptides; Tumor Cells, Cultured

Dolastatin 10 is a potent antimitotic peptide isolated from the marine mollusk Dolabella auricularia. Four of its five residues are modified amino acids (in sequence, dolavaline, valine, dolaisoleuine, dolaproine, dolaphenine). Besides inhibiting tubulin polymerization, dolastatin 10 non-competitively inhibits vinca alkaloid binding to tubulin, inhibits nucleotide exchange and formation of the beta s cross-link, and stabilizes the colchicine binding activity of tubulin. To examine the mechanism of action of dolastatin 10 we prepared six chiral isomers, one tri- and one tetrapeptide segment, and one pentapeptide analog of dolastatin 10, all of which differ little from dolastatin 10 as inhibitors of tubulin polymerization. However, only two of the chiral isomers were similar to dolastatin 10 in their cytotoxicity for L1210 murine leukemia cells and in their effects on vinblastine binding, nucleotide exchange, beta s cross-link formation, and colchicine binding. These were isomer 2, with reversal of configuration at position C(19a) in the dolaisoleuine moiety, and isomer 19, with reversal of configuration at position C(6) in the dolaphenine moiety. The pentapeptides with reduced cytotoxicity and reduced effects on tubulin interactions with other ligands were all modified in the dolaproine moiety at positions C(9) and/or C(10). The tripeptide and tetrapeptide segments which inhibited polymerization but not ligand interactions were the amino terminal tripeptide (lacking dolaproine and dolaphenine) and the carboxyl terminal tetrapeptide (lacking dolavaline). We speculate that strong inhibition of other ligand interactions with tubulin requires stable peptide binding to tubulin (i.e. slow dissociation), but that inhibition of polymerization requires only rapid binding to tubulin.

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Cell Division; Cell Survival; Colchicine; Depsipeptides; Isomerism; Mice; Molecular Sequence Data; Nucleotides; Oligopeptides; Stereoisomerism; Tubulin; Tumor Cells, Cultured; Vinblastine

Dolastatins are naturally occurring peptides isolated from marine animals and are known to be potent anti-neoplastic agents. Here, the three compounds dolastatin 10 (Dola 10), dolastatin 15 (Dola 15) and deo-dolastatin 10 (Deo-Dola 10) were added to cultures of two human chronic B-leukemia cell lines, JVM-2 and EHEB. The three dolastatins (Dola 10 > Dola 15 > Deo-Dola 10) inhibited cell proliferation and immunoglobulin production. Decreased cell growth and lack of accumulation of immunoglobulin was not caused by cytotoxicity as cell viability in the cultures remained high. The unchanged surface marker immunoprofiles during treatment and a predominance of treated cells in S and G2/M phase suggested cytostatic rather than directly cytotoxic effects. Exposure to these reagents increased quickly, albeit only short c-myc and bcl-2 mRNA expression. These results indicate that the three dolastatins are potent inhibitors of leukemic B-cell proliferation.

Topics: Antigens, Neoplasm; Antigens, Surface; Antineoplastic Agents; Cell Cycle; Cell Division; Cell Survival; Depsipeptides; Humans; Immunoglobulins; Leukemia, Lymphocytic, Chronic, B-Cell; Leukemia, Prolymphocytic; Oligopeptides; Proto-Oncogenes; Transcription, Genetic; Tumor Cells, Cultured

The effects of two natural peptides, dolastatin 10 and dolastatin 15, on growth and differentiation of hematopoietic cells were studied using freshly explanted leukemia cells and continuous leukemia cell lines. The proliferation of several myeloid cell lines and of growth-factor-stimulated peripheral blood cells from patients with acute myeloid leukemia (AML) was efficiently inhibited by the two agents at concentrations between 1 and 0.01 nM. Growth inhibition was dose-dependent and reversible. Neither of the dolastatins exhibited significant cytotoxicity on dividing cells, nor did they interfere with the viability of resting cells. The 12-O-tetradecanoylphorbol 13-acetate or bryostatin I induced differentiation of AML cells was not affected by the dolastatins. Short-term exposure to the phorbol ester conferred reduced sensitivity of the cell line HL-60 to the antiproliferative effect of the drugs. Our data suggest that the dolastatins alone or in combination with other drugs could exert a role in the treatment of human myeloid leukemia.

Topics: Antineoplastic Agents; Bryostatins; Cell Differentiation; Cell Division; Depsipeptides; Humans; In Vitro Techniques; Lactones; Leukemia; Macrolides; Oligopeptides; Tetradecanoylphorbol Acetate; Tumor Cells, Cultured

Dolastatin 15, a seven-subunit depsipeptide derived from Dolabella auricularia, is a potent antimitotic agent structurally related to the antitubulin agent dolastatin 10, a five-subunit peptide obtained from the same organism. We have compared dolastatin 15 with dolastatin 10 for its effects on cells grown in culture and on biochemical properties of tubulin. The IC50 values for cell growth were obtained for dolastatin 15 with L1210 murine leukemia cells, human Burkitt lymphoma cells, and Chinese hamster ovary (CHO) cells (3, 3, and 5 nM with the three cell lines, respectively). For dolastatin 10, IC50 values of 0.4 and 0.5 nM were obtained with the L1210 and CHO cells, respectively. At toxic concentrations dolastatin 15 caused the leukemia and lymphoma cells to arrest in mitosis. In the CHO cells both dolastatin 15 and dolastatin 10 caused moderate loss of microtubules at the IC50 values and complete disappearance of microtubules at concentrations 10-fold higher. Despite its potency and the loss of microtubules in treated cells, the interaction of dolastatin 15 with tubulin in vitro was weak. Its IC50 value for inhibition of glutamate-induced polymerization of tubulin was 23 microM, as compared to values of 1.2 microM for dolastatin 10 and 1.5 microM for vinblastine. Dolastatin 10 noncompetitively inhibits the binding of vincristine to tubulin, inhibits nucleotide exchange, stabilizes the colchicine binding activity of tubulin, and inhibits tubulin-dependent GTP hydrolysis (Bai et al., Biochem Pharmacol 39: 1941-1949, 1990; Bai et al. J Biol Chem 265: 17141-17149, 1990). Only the latter reaction was inhibited by dolastatin 15. Nevertheless, its structural similarity to dolastatin 10 indicates that dolastatin 15 may bind weakly in the "vinca domain" of tubulin (a region of the protein we postulate to be physically close to but not identical with the specific binding site of vinca alkaloids and maytansinoids), presumably in the same site as dolastatin 10 (the "peptide site").

Topics: Amino Acid Sequence; Animals; Antineoplastic Agents; Binding Sites; Cell Line; Depsipeptides; Glutamates; Guanosine Triphosphate; Microtubule-Associated Proteins; Microtubules; Mitotic Index; Molecular Sequence Data; Mollusca; Oligopeptides; Tubulin; Vinblastine