| Assay Method Information | |
| | Biochemical Assay for Inhibitors of FLT3 Kinase Activity |
| Description: | i. Enzyme The assay was performed using FLT3 cytoplasmic domain product and purified in house as GST fused protein. The FLT3 protein (1 microM) was pre activated with 800 microM ATP for 1 hour at 28° C. in order to obtain a linear kinetic.ii. FLT3 Kinase Buffer (KB) Kinase buffer was composed of 50 mM HEPES pH 7.9 containing 4 mM MgCl2, 1 mM DTT, 10 microM Na3VO4, and 0.2 mg/mL BSAiii. Assay conditions The FLT3 kinase assay was run with a final pre activated enzyme concentration of 2 nM, in the presence of 254 microM ATP (residual ATP from KIT pre activation step is negligible), 8 nM 33P-γ-ATP and 55 microM of substrate BioDB n*24 (Aminoacidic sequence: GGKKKVSRSGLYRSPSMPENLNRPR SEQ ID NO: 1). The peptide was purchased from American Peptide Company (Sunnyvale, Calif.).Compound Testingi. Compound Dilution For IC50 determination, test compounds were received as a 1 mM solution in 100% DMSO, distributed into 96 well plates: compounds were then plated into the first column of a microtiter plate (A1 to G1), 100 microL/well. An automated station for serial dilutions (Biomek FX, Beckman) was used for producing 1:3 dilutions in 100% DMSO, from line A1 to A10, and for all the compounds in the column. Moreover, 4-5 copies of daughter plates were prepared by reformatting 5 microL of this first set of 100% DMSO dilution plates into 384 deep well-plates: one of these plates with the serial dilutions of test compounds was thawed the day of the experiments, reconstituted at a 3× concentration with water and used in the IC50 determination assays. In a standard experiment, the highest concentration (3×) of all compounds was 30 microM, while the lowest one was 1.5 nM.Each 384 well-plate contained at least one curve of the standard inhibitor staurosporine and reference wells (total enzyme activity vs. no enzymatic activity) for the Z′ and signal to background evaluation,ii. Assay Scheme 384-well plates, V bottom (test plates) were prepared with 5 microL of the compound dilution (3×) and then placed onto a PlateTrak 12 robotized station (Perkin Elmer; the robot had one 384-tip pipetting head for starting the assay plus one 96-tip head for dispensing the resin) together with one reservoir for the Enzyme mix (3×) and one for the ATP mix (3×). At the start of the run, the robot aspirated 5 μl of ATP mix, made an air gap inside the tips (3 microL) and aspirated 5 microL of Enzyme mix. The following dispensation into the plates plus 3 cycles of mixing, done by the robot itself, started the kinase reaction. At this point, the correct concentrations were restored for all the reagents. The robot incubated the plates for 60 minutes at r.t., and then stopped the reaction by pipetting 60 microL of dowex resin suspension into the reaction mix. In order to avoid tip clogging, wide bore tips were used to dispense the resin suspension. Three cycles of mixing were done immediately after the addition of the resin. Another mixing cycle was performed after all the plates were stopped, this time using normal tips: the plates were then allowed to rest for about one hour in order to allow resin sedimentation. At this point, 27 microL of the supernatant were transferred into 384-Optiplates (Perkin-Elmer), with 50 microL of Microscipt 40 (Perkin-Elmer); after 5 min of orbital shaking the plates were read on a Perkin-Elmer Top Count radioactivity counter.iii. Data Fitting Data were analyzed by an internally customized version of the SW package Assay Explorer that provided sigmoidal fitting of the ten-dilutions curves for IC50 determination in the secondary assays/hit confirmation routines. |
| Affinity data for this assay | |
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