Synthesis and biological evaluation of l-cysteine derivatives as mitotic kinesin Eg5 inhibitors

Bioorganic and Medicinal Chemistry Letters

Volumn 17, Issue 14, 2007, Pages 3921-3924

  Ogo, Naohisa ^a   Oishi, Shinya ^b   Matsuno, Kenji ^a   Sawada, Jun ichi ^a   Fujii, Nobutaka ^b   Asai, Akira ^a  

^a UNIVERSITY OF SHIZUOKA   (Japan)

^b KYOTO UNIVERSITY   (Japan)

Author keywords

Anticancer; Eg5; Inhibitor; Mitotic kinesin; S Trityl l cysteine

Indexed keywords

ADENOSINE TRIPHOSPHATASE; CYSTEINE; KINESIN; MONASTROL; PROTEIN EG5; PROTEIN EG5 INHIBITOR; PROTEIN INHIBITOR; S TRITYLCYSTEINE; UNCLASSIFIED DRUG;

ANTINEOPLASTIC ACTIVITY; ARTICLE; CELL CYCLE M PHASE; CONTROLLED STUDY; CYTOTOXICITY; DRUG SYNTHESIS; ENZYME INHIBITION; HELA CELL; HUMAN; HUMAN CELL; INHIBITION KINETICS; MEASUREMENT; MITOSIS INHIBITION; STRUCTURE ACTIVITY RELATION;

CYSTEINE; HELA CELLS; HUMANS; KINESIN;

EID: 34250328223     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2007.04.101     Document Type: Article

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9. Assays for Eg5 ATPase activity were carried out in 96-well half-area plates. Our typical reaction contained 38 nM of recombinant Eg5, 30 μM ATP, and 350 nM taxol-stabilized microtubules in 15 μl of the reaction buffer with 20 μM of small molecules. The microtubule-activated Eg5 ATPase activity was evaluated using Kinase-Glo luminescent kinase assay (Promega).
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15. Other kinesins' inhibitory activity was assessed by measuring the amount of ATP resulting from in vitro reaction using Kinase-Glo Plus luminescent kinase assay (Promega). The in vitro ATPase assay was performed as described as Eg5.
16. Mitotic arrest was measured by assessing the mitosis-specific phosphorylation of Histone H3 using anti-phospho-Histone H3 (Ser10) antibody (Upstate). The positive cells to the antibody were counted under the microscope while total cell number was measured by assessing the DAPI-stained cells.
17. HeLa cells treated with STLC derivatives were fixed by the conventional paraformaldehyde/methanol method, and stained immunologically. Mitotic chromatin was stained by anti-phospho-Histon H3 (Ser10) antibody (Upstate). Microtubules and DNA/chromatins were stained by monoclonal anti-a-tubulin (DM1A) antibody (Sigma) and DAPI, respectively. Fluorescent images were observed under Olympus IX71 microscope with DP30BW CCD camera.