R-253 disrupts microtubule networks in multiple tumor cell lines.
OBJECTIVE
The design and development of synthetic small molecules to disrupt microtubule dynamics is an attractive therapeutic strategy for anticancer drug discovery research. Loss of clinical efficacy of many useful drugs due to drug resistance in tumor cells seems to be a major hurdle in this endeavor. Thus, a search for new chemical entities that bind tubulin, but neither are a substrate of efflux pump, P-glycoprotein 170/MDR1, nor cause undesired side effects, would potentially increase the therapeutic index in certain cancer treatments.


METHODS
A high-content cell-based screen of a compound library led to the identification of a new class of compounds belonging to a thienopyrimidine series, which exhibited significant antitumor activities. On structure-activity relationship analysis, R-253 [N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno[3,2-d]pyrimidin-4-yl)hydrazine carbothioamide] emerged as a potent antiproliferative agent (average EC(50), 20 nmol/L) when examined in a spectrum of tumor cell lines.


RESULTS
R-253 is structurally unique and destabilizes microtubules both in vivo and in vitro. Standard fluorescence-activated cell sorting and Western analyses revealed that the effect of R-253 on cell growth was associated with cell cycle arrest in mitosis, increased select G(2)-M checkpoint proteins, and apoptosis. On-target activity of R-253 on microtubules was further substantiated by immunofluorescence studies and selected counter assays. R-253 competed with fluorescent-labeled colchicine for binding to tubulin, indicating that its binding site on tubulin could be similar to that of colchicine. R-253 neither is a substrate of P-glycoprotein 170/MDR1 nor is cytotoxic to nondividing human hepatocytes.


CONCLUSIONS
Both biochemical and cellular mechanistic studies indicate that R-253 could become a promising new tubulin-binding drug candidate for treating various malignancies.
Gururaja
Tarikere L
TL
Rigel Pharmaceuticals, Inc., South San Francisco, California 94080, USA. tgururaja@rigel.com
Goff
Dane
D
Kinoshita
Taisei
T
Goldstein
Eileen
E
Yung
Stephanie
S
McLaughlin
John
J
Pali
Erlina
E
Huang
Jianing
J
Singh
Rajinder
R
Daniel-Issakani
Sarkiz
S
Hitoshi
Yasumichi
Y
Cooper
R D G
RD
Payan
Donald G
DG
D000230:Adenocarcinoma; D000970:Antineoplastic Agents / Q000494:pharmacology; D017209:Apoptosis / Q000528:radiation effects; D001859:Bone Neoplasms; D002453:Cell Cycle / Q000528:radiation effects; D045744:Cell Line, Tumor / Q000187:drug effects*; D002470:Cell Survival / Q000187:drug effects*; D003110:Colonic Neoplasms; D005434:Flow Cytometry; D006367:HeLa Cells; D006801:Humans; D008175:Lung Neoplasms; D008870:Microtubules / Q000528:radiation effects*; D008970:Molecular Weight; D012516:Osteosarcoma; D011743:Pyrimidines / Q000737:chemistry; D013876:Thiophenes / Q000494:pharmacology*; D013890:Thiourea / Q000031:analogs & derivatives* / Q000494:pharmacology
D000970:Antineoplastic Agents; C513198:N-cyclopropyl-2-(6-(3,5-dimethylphenyl)thieno(3,2-d)pyrimidin-4-yl)hydrazine carbothioamide; D011743:Pyrimidines; D013876:Thiophenes; D013890:Thiourea