2.3. Biological evaluation of captured hits

The bioassay was conducted using LanthaScreen Eu kinase binding assay (Invitrogen-Life Technologies, USA).⁴⁵ This assay is based on the ability of the potential kinase inhibitor under evaluation to bind and displace a proprietary “tracer” molecule (known as Alexa Fluor 674 conjugate) from the catalytic site of the targeted kinase. In case the tested molecule is of low affinity to the targeted kinase, and thus fails to displace the “tracer” molecule, then the tracer remains within the kinase binding site and maintains effective florescent interaction with certain Eu-labeled anti-tag antibody attached at the kinase surface, thus emitting significant fluorescence (time-resolved fluorescence energy transfer, TR-FRET). On the other hand, if the potential inhibitor binds tightly to the kinase, then it will displace the “tracer” molecule from the binding site causing loss of the TR-FRET interaction, i.e., resulting from tracer/Eu interaction, with concomitant loss of fluorescence. Stock solutions of hit molecules were prepared in DMSO, and then serially diluted in assay buffer 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl₂, 1 mM EGTA to yield final hit concentrations of 10 μM. The reaction of the test compound solution, kinase antibody mixture and tracer were mixed and incubated over 1 hour at room temperature, and then the fluorescence was read at λs 665 and 615 nm.⁴⁶ DMSO did not exceed 1% in the final kinase reaction. Hits that inhibited CLK4 more than 75% at 10 μM were further tested at 1.0, 0.10 and 0.01 μM to determine their IC₅₀ values. Staurosporine was used as standard (positive control) with IC₅₀ value = 7.45 nM. IC₅₀ values were calculated using nonlinear regression of the log(concentration) vs. inhibition percentage values using GraphPad Prism 5.0.