Concentration dependency of the unbound partition coefficient Kp uu and its application to correct for exposure related discrepancies between biochemical and cellular potency of KAT6A inhibitors

The unbound partition coefficient Kp allows the estimation of intracellular target exposure from free extracellular drug concentrations. Although the active mechanisms controlling Kpuu are saturable, Kp is commonly determined at a single concentration which may not be appropriate in cases where drug concentrations can largely vary, e.g. in plasma in vivo or in vitro IC50 assays. We examined the concentration dependency of Kp in vitro using KAT6A inhibitors with varying potency drop-off in ZR75-1 breast cancer cells to account for exposure related discrepancies between cellular and biochemical IC50. Considering saturability resulted in a better quantitative bridge between both IC50 values and gave way to a simplified method to determine Kp which is suitable for the prediction of unbound cytosolic drug concentrations without the need to generate f estimates from binding studies in cell homogenates. As opposed to the binding method which destroys cellular integrity, this approach provides an alternative f estimate and directly reflects the fraction unbound in the cytosol (f ) of intact cells. In contrast to the binding method, prediction of intracellular KAT6A exposure with this more physiologic approach was able to bridge the average exposure gap between biochemical and cellular IC50's from 73 down to only 5.4-fold. The concept of concentration dependent Kp provides a solid rationale for early drug discovery to discriminate between pharmacology and target exposure related IC50 discrepancies. The attractiveness of the approach also lies in the use of the same assay format for cellular IC50, f and Kp determination. SIGNIFICANCE STATEMENT: Examination of the yet unexplored concentration dependency of the unbound partition coefficient Kp led to a new experimental approach that resulted in more reliable predictions of intracellular target exposure and is well suitable for routine drug discovery projects.