Reviewing the experimental and mathematical factors involved in tight binding inhibitors Ki values determination: The bi-functional protease inhibitor SmCI as a test model

Abstract

Different methodologies for determining the dissociation equilibrium constant (K_i) of protein tight binding inhibitors are frequently found in the scientific literature. Taking into account that the K_i value is the main parameter characterizing the inhibition strength, its determination often represents the first step during the characterization of a potential drug. The purpose of this review is to summarize the current information related to tight binding inhibitors K_i values determination and discuss about the importance of different factors as the enzyme concentration, the inhibitor concentration dilution series, the enzyme-inhibitor incubation time and the dose-response data mathematical fitting. For this aim, the bi-functional SmCI protease inhibitor is used as a tool for exemplifying the experimental and mathematical steps performed during tight binding inhibitors K_i values determination. In addition, the natural and the different recombinant forms of SmCI were used to go deeply into the comparison of some mathematic approaches that are frequently used in the literature. Finally, other biochemical techniques that could be potentially used for tight binding inhibitors K_i values determination are also commented.

Keywords: K(i) value determination; Kinetic characterization; Morrison's equation; Tight binding inhibitors.