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Intracellular Activation and Deactivation of Tasidotin, an Analog of Dolastatin 15: Correlation with Cytotoxicity

Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland (R.B., M.C.E., E.H.); Genzyme Corporation, San Antonio, Texas (P.L.B.); Laboratory of Protein Dynamics and Signalling, Center for Cancer Research, National Cancer Institute at Frederick, National Institutes of Health, Frederick, Maryland (T.D.C.); Department of Chemistry and Biochemistry, Arizona State University, Tempe, Arizona (G.R.P.); and Department of Biochemistry, University of Texas Health Sciences Center, San Antonio, Texas (R.F.L.)

Tasidotin, an oncolytic drug in phase II clinical trials, is a peptide analog of the antimitotic depsipeptide dolastatin 15. In tasidotin, the carboxyl-terminal ester group of dolastatin 15 has been replaced by a carboxy-terminal tert-butyl amide. As expected from studies with cemadotin, [³H]tasidotin, with the radiolabel in the second proline residue, was hydrolyzed intracellularly, with formation of N,N-dimethylvalyl-valyl-N-methylvalyl-prolyl-proline (P5), a pentapeptide also present in dolastatin 15 and cemadotin. P5 was more active as an inhibitor of tubulin polymerization and less active as a cytotoxic agent than tasidotin, cemadotin, and dolastatin 15. [³H]P5 was not the end product of tasidotin metabolism. Large amounts of [³H]proline were formed in every cell line studied, with proline ultimately becoming the major radiolabeled product. The putative second product of the hydrolysis of P5, N,N-dimethylvalyl-valyl-N-methylvalyl-proline (P4), had little activity as either an antitubulin or cytotoxic agent. In seven suspension cell lines, the cytotoxicity of tasidotin correlated with total cell uptake of the compound and was probably affected negatively by the extent of degradation of P5 to proline and, presumably, P4. The intracellular enzyme prolyl oligopeptidase probably degrades tasidotin to P5. When CCRF-CEM human leukemia cells were treated with N-benzyloxycarbonylprolylprolinal (BCPP), an inhibitor of prolyl oligopeptidase, there was a 30-fold increase in the IC₅₀ of tasidotin and a marked increase in intracellular [³H]tasidotin. BCPP also caused a 4-fold increase in the IC₅₀ of P5, so the enzyme probably does not convert P5 to P4. Inhibiting degradation of P5 should have led to a decrease in the IC₅₀ obtained for P5 in the presence of BCPP.

Received for publication August 6, 2008.

Accepted for publication October 16, 2008.

Address correspondence to: Ernest Hamel, Building 469, Room 104, National Cancer Institute at Frederick, Frederick MD 21702. E-mail: hamele{at}mail.nih.gov

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