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

Abstract

AbstractThe 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, theKDvalues 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 ofKDvalues 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 respectiveKDandKivalues to make a direct comparison. Moreover, we found that theKDvalue measured by SPR is more relevant to itsKivalue. 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.