guanosine-triphosphate and rhazinilam

The most potent microtubule assembly inhibitor of newer diphenylpyridazinone derivatives examined was NSC 613241. Because NSC 613241 and (-)-rhazinilam also induce the formation of similar 2-filament spirals, these aberrant reactions were compared. Spiral formation with both compounds was enhanced by GTP and inhibited by GDP and by 15 other inhibitors of microtubule assembly. Similarly, microtubule assembly induced by paclitaxel or laulimalide is enhanced by GTP and inhibited by GDP and assembly inhibitors, but neither [(3)H]NSC 613241 nor [(3)H](-)-rhazinilam bound to microtubules or inhibited the binding of [(3)H]paclitaxel or [(3)H]peloruside A to microtubules. Differences in the pitch of aberrant polymers were found: NSC 613241-induced and (-)-rhazinilam-induced spirals had average repeats of 85 and 79-80 nm, respectively. We found no binding of [(3)H]NSC 613241 or [(3)H](-)-rhazinilam to αβ-tubulin dimer, but both compounds were incorporated into the polymers they induced in substoichiometric reactions, with as little as 0.1-0.2 mol compound/mol of tubulin, and no cross-inhibition by NSC 613241 or (-)-rhazinilam into spirals occurred. Under reaction conditions where neither compound induced spiral formation, both compounds together synergistically induced substantial spiral formation. We conclude that (-)-rhazinilam and NSC 613241 bind to different sites on tubulin that differ from binding sites for other antitubulin agents.

Topics: Alkaloids; Binding Sites; Dose-Response Relationship, Drug; Glutamic Acid; Guanosine Diphosphate; Guanosine Triphosphate; Humans; Indolizines; Lactams; Ligands; Microscopy, Electron; Protein Binding; Pyridazines; Pyridines; Stereoisomerism; Temperature; Tubulin

(-)-Rhazinilam was spontaneously generated from a natural product during isolation. In cultured cells, it causes microtubule bundle formation, like those caused by paclitaxel. With tubulin, (-)-rhazinilam causes formation of an aberrant spiral polymer. Using glutamate and GTP, we developed an assay for spiral formation and applied it to 17 new (+/-)-rhazinilam analogs with either a modified side chain or a different size D ring. There was reasonable correlation between spiral formation and inhibition of human MCF-7 breast carcinoma cell growth. Only one side chain analog was as active as (+/-)-rhazinilam. During these studies, we observed that omitting GTP from the reaction mixture caused a major change in the morphology of the (-)-rhazinilam-induced polymer, with half the observed polymer being microtubule-like and half being spirals. This mixed polymer slowly disassembled at 0 degrees C, but there was no apparent difference in the lability of the microtubules versus the spirals.

Topics: Alkaloids; Animals; Antimitotic Agents; Cell Line, Tumor; Cell Proliferation; Centrifugation; Glutamates; Guanosine Triphosphate; Humans; Indolizines; Lactams; Microtubules; Protein Binding; Tubulin

We have investigated the effects of the microtubule poison rhazinilam on microtubule assembly in vivo and in vitro. In mammalian cells, rhazinilam mimics the effects of taxol and leads to microtubule bundles, multiple asters, and microtubule cold stability. In vitro, rhazinilam protected preassembled microtubules from cold-induced disassembly, but not from calcium ion-induced disassembly. Moreover, both at 0 degrees C and at 37 degrees C, rhazinilam induced the formation of anomalous tubulin assemblies (spirals). This process was prevented by maytansine and vinblastine, but not by colchicine. Preferential saturable and stoichiometric binding of radioactive rhazinilam to tubulin in spirals was observed with a dissociation constant of 5 microM. This binding was abolished in the presence of vinblastine and maytansine. In contrast, specific binding of radioactive rhazinilam to tubulin assembled in microtubules was undetectable. These results demonstrate that rhazinilam alters microtubule stability differently than taxol, and that the overall similar effects of rhazinilam and taxol on the cellular cytoskeleton are the consequence of two distinct mechanisms of action at the molecular level.

Topics: Alkaloids; Animals; Calcium; Cell Line; Guanosine Triphosphate; Indolizines; Lactams; Macropodidae; Maytansine; Mice; Microtubule-Associated Proteins; Microtubules; Mitosis; Paclitaxel; Temperature; Tubulin; Tumor Cells, Cultured; Vinblastine