Comparative Analysis of the Techniques for the Determination of Binding Affinity between a Small Molecule Inhibitor and a Protein Target

Abstract

The binding affinity constant (KD) between a small molecule inhibitor and a protein target is pivotal parameter for target identification and early drug discovery. Despite the extensive applications of three major techniques, namely SPR, ITC and MST, the KD values for a specifically defined binding measured by these techniques are drastically different, which poses a remarkable difficulty to deal with these ambiguous data. Here, we report the evaluation of the accuracy of KD values from SPR, ITC and MST compared with enzyme kinetics. To enable an objective comparison, we theoretically proved that enzyme competitive inhibition constant (Ki) could directly reflect the binding affinity. Using purine nucleoside phosphorylase, its substrate inosine and competitive inhibitor immucillin-H, we determined respective KD and Ki values to make a direct comparison. Moreover, we found that the KD value measured by SPR is more relevant to its Ki value. This study highlights the urgent need on the development of new technologies for the determination of binding affinity between small molecule inhibitors and protein targets.