Tubulin Photoaffinity Labeling with Biotin‐Tagged Derivatives of Potent Diketopiperazine Antimicrotubule Agents |
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| Authors: | Yuri Yamazaki Kyoko Kohno Dr Hiroyuki Yasui Prof Yoshiaki Kiso Prof Miki Akamatsu Prof Benjamin Nicholson Dr Gordafaried Deyanat‐Yazdi Dr Saskia Neuteboom Dr Barbara Potts Dr G Kenneth Lloyd Dr Yoshio Hayashi Prof |
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| Affiliation: | 1. Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192‐0392 (Japan), Fax: (+81)?42‐676‐3275;2. Department of Medicinal Chemistry, Center for Frontier Research in Medicinal Science and 21st Century COE Program, Kyoto Pharmaceutical University, Kyoto 607‐8412 (Japan), Fax: (+81)?75‐595‐4787;3. Department of Molecular Genetics, Kyoto Pharmaceutical University, Kyoto 607‐8412 (Japan);4. Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Kyoto 607‐8414 (Japan);5. Graduate School of Agriculture, Kyoto University, Kyoto 606‐8502 (Japan);6. Nereus Pharmaceuticals, Inc. 10480 Wateridge Circle, San Diego, CA 92121 (USA) |
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| Abstract: | NPI‐2358 ( 1 ) is a potent antimicrotubule agent that was developed from a natural diketopiperazine, phenylahistin, which is currently in Phase I clinical trials as an anticancer drug. To understand the precise recognition mechanism of tubulin by this agent, we focused on its potent derivative, KPU‐244 ( 2 ), which has been modified with a photoreactive benzophenone structure, and biotin‐tagged KPU‐244 derivatives ( 3 and 4 ), which were designed and synthesized for tubulin photoaffinity labeling. Introduction of the biotin structure at the p′‐position of the benzophenone ring in 2 exhibited reduced, but significant biological activities with tubulin binding, tubulin depolymerization and cytotoxicity in comparison to the parent KPU‐244. Therefore, tubulin photoaffinity labeling studies of biotin‐derivatives 3 and 4 were performed by using Western blotting analysis after photoirradiation with 365 nm UV light. The results indicated that tubulin was covalently labeled by these biotin‐tagged photoprobes. The labeling of compound 4 was competitively inhibited by the addition of diketopiperazine 1 or colchicine, and weakly inhibited by the addition of vinblastine. The results suggest that photoaffinity probe 4 specifically recognizes tubulin at the same binding site as anticancer drug candidate 1 , and this leads to the disruption of microtubules. Probe 4 serves well as a useful chemical probe for potent antimicrotubule diketopiperazines, much like phenylahistin, and it also competes for the colchicine‐binding site. |
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| Keywords: | alkaloids anticancer agents photoaffinity labeling tubulin vascular‐disrupting agent |
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