Tubulin photoaffinity labeling with biotin-tagged derivatives of potent diketopiperazine antimicrotubule agents.
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.
Yamazaki
Yuri
Y
Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo, Japan.
Kohno
Kyoko
K
Yasui
Hiroyuki
H
Kiso
Yoshiaki
Y
Akamatsu
Miki
M
Nicholson
Benjamin
B
Deyanat-Yazdi
Gordafaried
G
Neuteboom
Saskia
S
Potts
Barbara
B
Lloyd
G Kenneth
GK
Hayashi
Yoshio
Y
D000970:Antineoplastic Agents / Q000737:chemistry* / Q000494:pharmacology; D001710:Biotin / Q000737:chemistry* / Q000378:metabolism; D045744:Cell Line, Tumor; D003078:Colchicine / Q000737:chemistry / Q000494:pharmacology; D054659:Diketopiperazines / Q000737:chemistry* / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D019920:Photoaffinity Labels / Q000138:chemical synthesis / Q000737:chemistry; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism / Q000528:radiation effects; D050257:Tubulin Modulators / Q000737:chemistry* / Q000494:pharmacology
D000970:Antineoplastic Agents; D054659:Diketopiperazines; D019920:Photoaffinity Labels; D014404:Tubulin; D050257:Tubulin Modulators; D001710:Biotin; D003078:Colchicine