Interaction of the tumor inhibitor IKP-104, a 4(1H)-pyridinone derivative, with microtubule proteins.

The effects of a mitotic arrestant, IKP-104, which has an antitumor activity, on the in vitro polymerization and depolymerization of rat brain microtubules were investigated. IKP-104 inhibited microtubule polymerization at concentrations greater than 0.71 x 10(-6) M, and its IC50 value was determined to be 1.31 x 10(-6) M by probit analysis. Fifty-two percent of pre-polymerized microtubules depolymerized at 1.31 x 10(-6) M IKP-104. Electron micrographs of microtubules taken immediately after treatment with 1 x 10(-3) M IKP-104 revealed a fraying of microtubule ends into elongated coil-like filaments, which were composed of 2 or 3 protofilaments. When microtubule protein treated with 1 x 10(-3) M IKP-104 was cleaved by trypsin, fragments of 41, 36, 34, 23, 21, 19 and 16 kilodaltons (kDa) derived from alpha-tubulin were produced. In particular, the 19, 23, and 34 kDa fragments were characteristically observed in the trypsin cleavage of microtubules tested with IKP-104, and these fragments were not observed with untreated microtubules. The effects of IKP-104 on microtubule protein mentioned above were mostly similar to those of vinblastine (VLB) and we suggest that IKP-104 bound to the site or sites near "VLB-binding site or sites" of alpha-tubulin subunit, resulting in induction of conformational changes.

colchicine

vinblastine sulfate

‐(N‐morpholino)ethanesulfonic acid

ethyleneglycol‐O, O′‐bis(β‐aminoethyl ether)‐N, N, N′,N′‐tetraacetic acid

guanosine‐5′‐triphosphate

phenylmethylsulfonyl fluoride

2‐nitro‐4‐car‐boxyphenyl‐N, N‐diphenyl carbamate

sodium dodecyl sulfate

bromophenol blue

trichloroacetic acid

polyvinylidene difluoride

3,3′‐diaminobenzidine

50% inhibitory concentration

guanyl‐5′‐methylene diphosphonate