nsc-83265 and dimethylenastron

Determining the mechanism of action of drugs and their target specificity in cells remains a major challenge. Here we describe the use of cell lines expressing two point mutations in the allosteric inhibitor binding pocket of the mitotic kinesin Eg5 (D130A, in the loop L5 region and L214A in helix α3), which following transfection, were selected for their ability to proliferate normally in the presence of STLC, a well known Eg5 inhibitor. The cell lines were used to discriminate the mechanism of action of other chemically distinct small molecule inhibitors of Eg5 that differ in their mode of action. The STLC resistant cells were capable of continuous proliferation in the presence of ATP uncompetitive inhibitors, such as K858 and dimethylenastron, but were still sensitive to ATP competitive inhibitors that are thought to bind to a distinct site on Eg5 than the allosteric binding pocket. The STLC resistant cell lines can therefore be used as a filter to distinguish Eg5 loop L5 binding drugs from drugs binding to other pockets without prior structural information. Additionally, the cells can be used to analyze whether inhibitors of Eg5 are specific to this potential drug target or whether they have additional targets in dividing cells.

Topics: Adenosine Triphosphatases; Adenosine Triphosphate; Allosteric Site; Antineoplastic Agents; Binding Sites; Cell Line, Tumor; Cell Proliferation; Cysteine; Drug Resistance, Neoplasm; Humans; Kinesins; M Phase Cell Cycle Checkpoints; Point Mutation; Quinazolines; Thiadiazoles; Thiones

The inhibition of kinesin Eg5 by small molecules such as monastrol is currently evaluated as an approach to develop a novel class of antiproliferative drugs for the treatment of malignant tumours. Therefore, we studied the effects of the new monastrol analogues enastron, dimethylenastron and vasastrol VS-83 on the proliferation of human glioblastoma cells in the kinetic crystal violet assay. Compared to monastrol, the new cell cycle specific compounds showed an at least one order of magnitude higher anti proliferative activity against U-87 MG, U-118 MG, and U-373 MG glioblastoma cells. The compounds were neither inactivated by hydrolysis nor by binding to serum proteins. Moreover, we demonstrated the characteristic monoaster formation after incubation of cells with the new compounds by confocal laser scanning microscopy. We also showed that the arrangement of beta-actin and tubulin, vital components of the cyto-skeleton of mitotic and quiescent cells, were not affected by the new compounds. Due to the necessity of overcoming the blood-brain barrier in the treatment of brain tumours, we investigated if the new monastrol analogues are modulators or substrates of the p-glycoprotein (p-gp) 170 by a flow cytometric calcein-AM efflux assay. The tested compounds showed no modulating effects on the p-gp function. With respect to the treatment of primary and secondary CNS tumours, the results of our experiments suggest that the new monastrol analogues represent an interesting class of potential anticancer drugs, predicted to be less neurotoxic in comparison to classical tubulin inhibitors.

Topics: Acridines; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cell Proliferation; Cell Survival; Cysteine; Dose-Response Relationship, Drug; Flow Cytometry; Fluoresceins; Glioblastoma; Humans; Insecticides; Kinesins; Molecular Structure; Paclitaxel; Pyrimidines; Quinazolines; Rotenone; Spindle Apparatus; Tetrahydroisoquinolines; Thiones; Time Factors; Tubulin; Tubulin Modulators; Vinblastine