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. 2000 Jul;91(7):737–747. doi: 10.1111/j.1349-7006.2000.tb01007.x

Characterization of the Interaction of TZT‐1027, a Potent Antitumor Agent, with Tubulin

Tsugitaka Natsume 1, Jun‐ichi Watanabe 1, Satoru Tamaoki 1, Nami Fujio 1, Katsuhiko Miyasaka 1, Motohiro Kobayashi ✉
PMCID: PMC5926414  PMID: 10920282

Abstract

TZT‐1027, a derivative of dolastatin 10 isolated from the Indian Ocean sea hare Dolabella auricularia in 1987 by Pettit et al., is a potent antimicrotubule agent. We have compared the activity of TZT‐1027 with that of dolastatin 10 as well as the vinca alkaloids vinblastine (VLB), vincristine (VCR) and vindesine (VDS). TZT‐1027 and dolastatin 10 inhibited microtubule polymerization concentration‐dependently at 1–100 μM with IC50 values of 2.2±0.6 and 2.3±0.7 μM, respectively. VLB, VCR and VDS inhibited microtubule polymerization at 1–3 μM with IC50 values of 2.7±0.6, 1.6±0.4 and 1.6±0.2 μM, respectively, but showed a slight decrease in inhibitory effect at concentrations of 10 μM or more. TZT‐1027 also inhibited monosodium glutamate‐induced tubulin polymerization concentration‐dependently at 0.3–10 μM, with an IC50 of 1.2 μM, whereas VLB was only effective at 0.3–3 μM, with an IC50 of 0.6 μM, and caused so‐called “aggregation” of tubulin at 10 μM. Scatchard analysis of the binding data for [3H]VLB suggested one binding site (Kd 0.2±0.04 μM and Bmax 6.0±0.26 nM/mg protein), while that for [3H]TZT‐1027 suggested two binding sites, one of high affinity (Kd 0.2±0.01 μM and Bmax 1.7±0.012 nM/mg protein) and the other of low affinity (Kd 10.3±1.46 μM, and Bmax 11.6±0.83 nM/mg protein). [3H]TZT‐1027 was completely displaced by dolastatin 10 but only incompletely by VLB. [3H]VLB was completely displaced by dolastatin 10 and TZT‐1027. Furthermore, TZT‐1027 prevented [3H]VLB from binding to tubulin in a non‐competitive manner according to Lineweaver‐Burk analysis. TZT‐1027 concentrationdependently inhibited both [3H]guanosine 5′‐triphosphate (GTP) binding to and GTP hydrolysis on tubulin. VLB inhibited the hydrolysis of GTP on tubulin concentration‐dependently to a lesser extent than TZT‐1027, but no inhibitory effect of VLB on [3H]GTP binding to tubulin was evident even at 100 μM. Thus, TZT‐1027 affected the binding of VLB to tubulin, but its binding site was not completely identical to that of VLB. TZT‐1027 had a potent inhibitory effect on tubulin polymerization and differed from vinca alkaloids in its mode of action against tubulin polymerization.

Keywords: TZT‐1027‐Microtubule polymerization‐Tubulin binding‐Antimicrotubule agent‐ Dolastatin 10

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REFERENCES

  • 1. ) Pettit , G. R. , Kamano , Y. , Herald , C. L. , Tuinman , A. A. , Boettner , F. E. , Kizu , H. , Schmidt , J. M. , Baczynskyj , L. , Tomer , K. B. and Bontems , R. J.The isolation and structure of a remarkable marine animal antineoplastic constituent: dolastatin 10 . J. Am. Chem. Soc. , 109 , 6883 – 6885 ( 1987. ). [Google Scholar]
  • 2. ) Pettit , G. R. , Kamano , Y. , Herald , C. L. , Fujii , Y. , Kizu , H. , Boyd , M. R. , Boettner , F. E. , Doubek , D. L. , Schmidt , J. M. , Chapuis , J. C. and Michel , C.Isolation of dolastatin 10–15 from the marine mollusk Dolabella auricularia . Tetrahedron , 49 , 9151 – 9170 ( 1993. ). [Google Scholar]
  • 3. ) Pettit , G. R. , Singh , S. B. , Hogan , F. , Lloyd‐Williams , P. , Herald , D. L. , Burkett , D. D. and Clewlow , P. J.The absolute configuration and synthesis of natural (‐)‐dolastatin 10 . J. Am. Chem. Soc. , 111 , 5463 – 5465 ( 1987. ). [Google Scholar]
  • 4. ) Hamada , Y. , Hayashi , K. and Shioiri , T.Efficient stereoselective synthesis of dolastatin 10, an antineoplastic peptide from a sea hare . Tetrahedron Lett. , 32 , 931 – 934 ( 1991. ). [Google Scholar]
  • 5. ) Owellen , R. J. , Hartke , C. A. , Dickerson , R. M. and Hains , F. O.Inhibition of tubulin‐microtubule polymerization by drugs of the Vinca alkaloid class . Cancer Res. , 36 , 1499 – 1502 ( 1976. ). [PubMed] [Google Scholar]
  • 6. ) Himes , R. H. , Kersey , R. N. , Heller‐Bettinger , I. and Samson , F. E.Action of Vinca alkaloids vincristine, vinblastine and desacetyl vinblastine amide on microtubules in vitro . Cancer Res. , 36 , 3798 – 3802 ( 1976. ). [PubMed] [Google Scholar]
  • 7. ) Safa , A. R. , Hamel , E. and Felsted , R. L.Photoaffinity labeling of tubulin subunits with a photoactive analogue of vinblastine . Biochemistry , 26 , 97 – 102 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 8. ) Bai , R. , Pettit , G. R. and Hamel , E.Dolastatin 10, a powerful cytostatic peptide derived from a marine animal . Biochem. Pharmacol. , 39 , 1941 – 1949 ( 1990. ). [DOI] [PubMed] [Google Scholar]
  • 9. ) Bai , R. , Pettit , G. R. and Hamel , E.Binding of dolastatin 10 to tubulin at a distinct site for peptide antimitotic agents near the exchangeable nucleotide and Vinca alkaloid sites . J. Biol. Chem. , 265 , 17141 – 17149 ( 1990. ). [PubMed] [Google Scholar]
  • 10. ) Ludueña , R. F. , Roach , M. C. , Prasad , V. and Pettit , G. R.Interaction of dolastatin 10 with bovine brain tubulin . Biochem. Pharmacol. , 43 , 539 – 543 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 11. ) Li , Y. , Kobayashi , Y. , Hashimoto , Y. , Shirai , R. , Hirata , A. , Hayashi , K. , Hamada , Y. , Shioiri , T. and Iwasaki , S.Interaction of marine toxin dolastatin 10 with porcine brain tubulin: competitive inhibition of rhizoxin and phomopsin A binding . Chem. Biol. Interact. , 93 , 9175 – 9183 ( 1994. ). [DOI] [PubMed] [Google Scholar]
  • 12. ) Miyazaki , K. , Kobayashi , M. , Natsume , T. , Gondo , M. , Mikami , T. , Sakakibara , K. and Tsukagoshi , S.Synthesis and antitumor activity of novel dolastatin 10 analogs . Chem. Pharm. Bull. , 43 , 1706 – 1718 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 13. ) Kobayashi , M. , Natsume , T. , Tamaoki , S. , Watanabe , J. , Asano , H. , Mikami , T. , Miyasaka , K. , Miyazaki , K. , Gondo , M. , Sakakibara , K. and Tsukagoshi , S.Antitumor activity of TZT‐1027, a novel dolastatin 10 derivative . Jpn. J. Cancer Res. , 88 , 316 – 327 ( 1997. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14. ) Hyams , J. S. and Lloyd , C. W.“Microtubules ,” pp. 1 – 464 ( 1994. ). Wiley‐Liss; , New York . [Google Scholar]
  • 15. ) Hamm‐Alvarez , S. F.Microtubule‐based motor proteins: new targets for enhancing drug delivery ? Pharm. Res. , 13 , 489 – 496 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 16. ) Shelanski , M. L. , Gaskin , F. and Cantor , C. R.Microtubule assembly in the absence of added nucleotides . Proc. Natl. Acad. Sci. USA , 70 , 765 – 768 ( 1973. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17. ) Williams , R. C. , Jr. and Lee , J. C.Preparation of tubulin from brain . Methods Enzymol , 85 , 376 – 385 ( 1982. ). [DOI] [PubMed] [Google Scholar]
  • 18. ) Bradford , M. M.A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein‐dye binding . Anal. Biochem. , 72 , 248 – 254 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 19. ) Laemmli , U. K.Cleavage of structural proteins during the assembly of the head of bacteriophage T4 . Nature , 227 , 680 – 685 ( 1970. ). [DOI] [PubMed] [Google Scholar]
  • 20. ) Mandelbaum‐Shavit , F. , Wolpert‐DeFilippes , M. K. and Johns , D. G.Binding of maytansine to rat brain tubulin . Biochem. Biophys. Res. Commun. , 72 , 47 – 54 ( 1976. ). [DOI] [PubMed] [Google Scholar]
  • 21. ) Borisy , G. G.A rapid method for quantitative determination of microtubule protein using DEAE‐cellulose filters . Anal. Biochem. , 50 , 373 – 385 ( 1972. ). [DOI] [PubMed] [Google Scholar]
  • 22. ) Hamel , E. and Lin , C. M.Glutamate‐induced polymerization of tubulin: characteristics of the reaction and application to the large‐scale purification of tubulin . Arch. Biochem. Biophys. , 209 , 29 – 40 ( 1981. ). [DOI] [PubMed] [Google Scholar]
  • 23. ) Bai , R. , Friedman , S. J. , Pettit , G. R. and Hamel , E.Dolastatin 15, a potent antimitotic depsipeptide derived from Dolabella auricularia . Biochem. Pharmacol. , 43 , 2637 – 2645 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 24. ) Takahashi , M. , Iwasaki , S. , Kobayashi , H. , Okuda , S. , Murai , T. and Sato , Y.Rhizoxin binding to tubulin at the maytansine‐binding site . Biochim. Biophys. Acta , 926 , 215 – 223 ( 1987. ). [DOI] [PubMed] [Google Scholar]
  • 25. ) Li , Y. , Kobayashi , H. , Tokiwa , Y. , Hashimoto , Y. and Iwasaki , S.Interaction of phomopsin A with porcine brain tubulin. Inhibition of tubulin polymerization and binding at a rhizoxin binding site . Biochem. Pharmacol. , 43 , 219 – 224 ( 1992. ). [DOI] [PubMed] [Google Scholar]
  • 26. ) Bai , R. , Paull , K. D. , Herald , C. L. , Malspeis , L. , Pettit , G. R. and Hamel , E.Halichondrin B and homohalichondrin B, marine natural products binding in the vinca domain of tubulin. Discovery of tubulin‐based mechanism of action by analysis of differential cytotoxicity data . J. Biol. Chem. , 266 , 15882 – 15889 ( 1991. ). [PubMed] [Google Scholar]
  • 27. ) Bai , R. , Taylor , G. F. , Schimidt , J. M. , Williams , M. D. , Kepler , J. A. , Pettit , G. R. and Hamel , E.Interaction of dolastatin 10 with tubulin: induction of aggregation and binding and association reactions . Mol. Pharmacol. , 47 , 965 – 976 ( 1995. ). [PubMed] [Google Scholar]
  • 28. ) Huang , A. B. , Lin , C. M. and Hamel , E.Maytansine inhibits nucleotide binding at the exchangeable site of tubulin . Biochem. Biophys. Res. Commun. , 128 , 1239 – 1246 ( 1985. ). [DOI] [PubMed] [Google Scholar]
  • 29. ) Schiff , P. B. , Fant , J. and Horwitz , S. B.Promotion of microtubule assembly in vitro by taxol . Nature , 277 , 665 – 667 ( 1979. ). [DOI] [PubMed] [Google Scholar]
  • 30. ) Parness , J. and Horwitz , S. B.Taxol binds to polymerized tubulin in vitro . J. Cell Biol. , 91 , 479 – 487 ( 1981. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31. ) Manfredi , J. J. , Parness , J. and Horwitz , S. B.Taxol binds to cellular microtubules . J. Cell Biol. , 94 , 688 – 696 ( 1982. ). [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32. ) Bollag , D. M. , McQueney , P. A. , Zhu , J. , Hensens , O. , Koupal , L. , Liesch , J. , Goetz , M. , Lazarides , E. and Woods , C. M.Epothilones, a new class of microtubule‐stabilizing agents with a taxol‐like mechanism of action . Cancer Res. , 55 , 2325 – 2333 ( 1995. ). [PubMed] [Google Scholar]
  • 33. ) Mühlradt , P. F. and Sasse , F.Epothilone B stabilizes microtubuli of macrophages like taxol without showing taxol‐like endotoxin activity . Cancer Res. , 57 , 3344 – 3346 ( 1997. ). [PubMed] [Google Scholar]
  • 34. ) Kowalski , R. J. , Giannakakou , P. and Hamel , E.Activities of microtubule‐stabilizing agents epothilone A and B with purified tubulin and in cells resistant to paclitaxel (taxol) . J. Biol. Chem. , 272 , 2534 – 2541 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 35. ) Haar , E. , Kowalski , R. J. , Hamel , E. , Lin , C. M. , Longley , R. E. , Gunasekera , S. P. , Rosenkranz , H. S. and Day , B. W.Discodermolide, a cytotoxic marine agent that stabilizes microtubules more potently than taxol . Biochemistry , 35 , 243 – 250 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 36. ) Hung , D. T. , Chen , J. and Schreiber , S. L.(+)‐Discodermolide binds to microtubules in stoichiometric ratio to tubulin dimers, blocks taxol binding and results in mitotic arrest . Chem. Biol. , 3 , 287 – 293 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 37. ) Kowalski , R. J. , Giannakakou , P. , Gunasekera , S. P. , Longley , R. E. , Day , B. W. and Hamel , E.The microtubule‐stabilizing agent discodermolide competitively inhibits the binding of paclitaxel (taxol) to tubulin polymers, enhances tubulin nucleation reactions more potently than paclitaxel, and inhibits the growth of paclitaxel‐resistant cells . Mol. Pharmacol. , 52 , 613 – 622 ( 1997. ). [PubMed] [Google Scholar]
  • 38. ) Knick , V. C. , Eberwein , D. L. and Miller , C. G.Vinorelbine tartrate and paclitaxel combinations: enhanced activity against in vivo P388 murine leukemia cells . J. Natl. Cancer Inst. , 87 , 1072 – 1077 ( 1995. ). [DOI] [PubMed] [Google Scholar]
  • 39. ) Photiou , A. , Shah , P. , Leong , L. K. , Moss , J. and Retsas , S.In vitro synergy of paclitaxel (taxol) and vinorelbine (navelbine) against human melanoma cell lines . Eur. J. Cancer , 33 , 463 – 470 ( 1997. ). [DOI] [PubMed] [Google Scholar]
  • 40. ) Chang , A. Y. and Garrow , G. C.Pilot study of vinorelbine (navelbine) and paclitaxel (taxol) in patients with refractory breast cancer and lung cancer . Semin. Oncol. , 22 , 66 – 71 ( 1995. ). [PubMed] [Google Scholar]
  • 41. ) Retsas , S. , Mohith , A. and Mackenzie , H.Taxol and vinorelbine: a new active combination for disseminated malignant melanoma . Anticancer Drugs , 7 , 161 – 165 ( 1996. ). [DOI] [PubMed] [Google Scholar]
  • 42. ) Speicher , L. A. , Barone , L. and Tew , K. D.Combined antimicrotubule activity of estramustine and taxol in human prostatic carcinoma cell lines . Cancer Res. , 52 , 4433 – 4440 ( 1992. ). [PubMed] [Google Scholar]
  • 43. ) Nishida , E. and Sakai , H.Calcium‐sensitivity of the microtubule reassembly system. Difference between crude brain extract and purified microtubular proteins . J. Biochem. , 82 , 303 – 306 ( 1977. ). [DOI] [PubMed] [Google Scholar]
  • 44. ) de Arruda , M. , Cocchiaro , C. A. , Nelson , C. M. , Grinnell , C. M. , Janssen , B. , Haupt , A. and Barlozzari , T.LU103793 (NSC D‐669356): a synthetic peptide that interacts with microtubules and inhibits mitosis . Cancer Res. , 55 , 3085 – 3092 ( 1995. ). [PubMed] [Google Scholar]

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