Quantification of Binding of Small Molecules to Native Kinases by Flow Cytometry Reveals Divergence from Biochemical...

Precise measurements of small molecule-protein interactions are critical for drug discovery. However, most biochemical profiling platforms measure binding using recombinant kinase domains or proteins in cell lysates, which can miss conformational regulation present in intact living cells. Here, we used flow cytometry-based fluorescent probe cellular binding assays (FPCBA) to demonstrate that the anticancer drug dasatinib binds native, untagged ABL1 kinase with 3-6-fold higher affinity than NanoLuc- or mVenus-tagged constructs in living cells. We further used this method for in-cell profiling of 25 native kinases, revealing conformational regulatory mechanisms, including SRC autoinhibition and membrane-dependent conformational states of DDR1, DDR2, and EPHA4 that are absent or attenuated in biochemical assays. For these studies, coumarin-dasatinib probes spanning a range of fluorophore acidity (pK_a 4.1-7.3) were optimized for intracellular target engagement. To enhance sensitivity of detection, we found that uptake of acidic probes can be promoted by expression of the organic anion transporter OATP1B3. Quantitative flow cytometry with NIST-standardized beads established that intracellular concentrations of an intermediate-acidity 6FC-dasatinib probe approximated extracellular concentrations in HEK293T cells at equilibrium. Cellular K_i values of dasatinib and imatinib for 25 kinases by FPCBA were broadly concordant with kinobead LC/MS measurements in cancer cell lysates but diverged substantially from recombinant KINOMEscan values, with divergences attributable to competition with ATP, autoinhibition, and membrane-dependent conformational states in living cells. FPCBA enables profiling of native protein-small molecule interactions in a physiologically relevant cellular context.