Demonstration of microtubule-like structures formed with (-)-rhazinilam from purified tubulin outside of cells and a simple tubulin-based assay for evaluation of analog activity.
(-)-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.
Edler
Michael C
MC
Toxicology and Pharmacology Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer Institute at Frederick, National Institutes of Health, Frederick, MD 21702, USA.
Yang
Guangli
G
Katherine Jung
M
M
Bai
Ruoli
R
Bornmann
William G
WG
Hamel
Ernest
E
D000470:Alkaloids / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D000818:Animals; D050256:Antimitotic Agents / Q000138:chemical synthesis / Q000737:chemistry* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D002498:Centrifugation; D005971:Glutamates / Q000494:pharmacology; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D007212:Indolizines / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D007769:Lactams / Q000138:chemical synthesis / Q000737:chemistry / Q000494:pharmacology; D008870:Microtubules / Q000737:chemistry*; D011485:Protein Binding / Q000187:drug effects; D014404:Tubulin / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*
D000470:Alkaloids; D050256:Antimitotic Agents; D005971:Glutamates; D007212:Indolizines; D007769:Lactams; D014404:Tubulin; C009170:rhazinilam; D006160:Guanosine Triphosphate