The assessment of a ligand's activity is typically established by measuring its binding affinity in a biochemical assay, often expressed as Ka or Kd values. Further validation of its biological activity is achieved through cellular assays. There is frequently an inconsistency between the activity values obtained from those assays, which could delay research progress as well as drug development. Factors such as the permeability, solubility, specificity, and stability of active compounds are usually held responsible for this discrepancy. Even when these values are known, inconsistencies in activity measurements remain challenging to explain. This is not surprising since intracellular physicochemical conditions are undoubtedly different from the simplified conditions used in most in vitro biochemical assays. It is therefore reasonable to assume that these differences would be minimized if biochemical measurements were performed under conditions that more accurately mimic the intracellular environment. These physicochemical conditions can alter the Kd values. While the cellular environment has been extensively studied for decades, more recent efforts have focused on obtaining equilibrium and kinetic data directly from in-cell environments. Clarifying molecular crowding, salt composition, and lipophilic parameters inside the cell and thus their effect on molecular equilibrium is a crucial step toward replicating the intracellular environment.
Keywords: PBS; assay conditions; buffer; crowding; cytoplasmic viscosity; in vitro; ionic strength; pH; phosphate-buffered saline; physicochemical microenvironment.