Synthesis and evaluation of potential inhibitors of eIF4E cap binding to 7-methyl GTP

Bioorganic and Medicinal Chemistry Letters

Volumn 15, Issue 8, 2005, Pages 2177-2180

  Ghosh, Phalguni ^a   Park, Chunkyung ^a   Peterson, Mark S ^b   Bitterman, Peter B ^b   Polunovsky, Vitaly A ^b   Wagner, Carston R ^a  

^a UNIVERSITY OF MINNESOTA   (United States)

^b UNIVERSITY OF MINNESOTA MEDICAL SCHOOL   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

7 METHYLGUANOSINE TRIPHOSPHATE; CAPPED RNA; GUANOSINE TRIPHOSPHATE; HYDROXYL GROUP; INITIATION FACTOR 4E; PHOSPHONIC ACID DERIVATIVE; UNCLASSIFIED DRUG;

ANIMAL CELL; ARTICLE; BINDING AFFINITY; BINDING ASSAY; CONTROLLED STUDY; DRUG SCREENING; DRUG SYNTHESIS; IMMUNOASSAY; INHIBITION KINETICS; MOUSE; NONHUMAN; RNA TRANSLATION;

EID: 15944412913     PISSN: 0960894X     EISSN: None     Source Type: Journal    
DOI: 10.1016/j.bmcl.2005.01.080     Document Type: Article

References (13)

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10. Experimental detail of the cap binding assay: Up to 6 compounds were tested in triplicate in each run and compared to the reference compound, 7 methyl GTP, to assess their relative activity. Cell lysate generated from 3T3/4E cells using a freeze-thaw lysis method was prepared in advance and aliquots stored at -70°C. Each sample required 20 μL of packed 7-methyl GTP agarose beads (Amersham Biosciences) pre-equilibrated with freeze-thaw lysis buffer. Beads were placed into microcentrifuge tubes and 7-methyl GTP was added to form a set of standards. Test compounds were added in parallel to other sets of tubes. Freeze-thaw lysis buffer was added to the tubes to bring the volume in each tube to 150 μL. At the start of the binding step 50 μL of 3T3/4E cell lysate at a concentration of 3 mg/mL was added to each tube, which was placed in a horizontal rotating platform to mix for 2 h at 4°C. Following the binding step, tubes were centrifuged to form a pellet, which was washed with 0.5 mL of freeze-thaw lysis buffer and centrifuged. The wash step was repeated once. Bound eIF4E was eluted from the beads by the addition of 250 μL of elution buffer (150 mM KCl, 25 mM Tris pH 7.5, and 100 μM 7-methyl GTP). The beads were centrifuged to form a pellet and 200 μL of each eluate was removed and spotted onto a nitrocellulose membrane using a vacuum dot blotting manifold. The membrane was blocked for 1 h with 5% NFD milk in TTBS buffer and probed with mouse anti-eIF4E (1:500, B D Biosciences) for 1 h. The blot was washed three times for 5 min each and then incubated with HRP conjugated Goat anti mouse IgG (1:2000, Sigma Chemical) for 1 h, rewashed and signal was visualized on film using ECL reagents (Amersham Biosciences). Densitometric analysis was performed by scanning and quantitating spot densities with Molecular Analyst software (Bio-Rad). Values were expressed in arbitrary optical density (OD) units
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