Domain-specific interactions between MLN8237 and human serum albumin estimated by STD and WaterLOGSY NMR, ITC, spectroscopic, and docking techniques

Hongqin Yang,Jiuyang Liu,Bin Tang,Hui Li,Shanshan Li,Rui Gao,Jiawei He,Yanmei Huang

doi:10.1038/srep45514

Abstract

Alisertib (MLN8237) is an orally administered inhibitor of Aurora A kinase. This small-molecule inhibitor is under clinical or pre-clinical phase for the treatment of advanced malignancies. The present study provides a detailed characterization of the interaction of MLN8237 with a drug transport protein called human serum albumin (HSA). STD and WaterLOGSY nuclear magnetic resonance (NMR)-binding studies were conducted first to confirm the binding of MLN8237 to HSA. In the ligand orientation assay, the binding sites of MLN8237 were validated through two site-specific spy molecules (warfarin sodium and ibuprofen, which are two known site-selective probes) by using STD and WaterLOGSY NMR competition techniques. These competition experiments demonstrate that both spy molecules do not compete with MLN8237 for the specific binding site. The AutoDock-based blind docking study recognizes the hydrophobic subdomain IB of the protein as the probable binding site for MLN8237. Thermodynamic investigations by isothermal titration calorimetry (ITC) reveal that the non-covalent interaction between MLN8237 and HSA (binding constant was approximately 105 M−1) is driven mainly by favorable entropy and unfavorable enthalpy. In addition, synchronous fluorescence, circular dichroism (CD), and 3D fluorescence spectroscopy suggest that MLN8237 may induce conformational changes in HSA.

Highlights

Aurora A kinase (AAK) belongs to a family of oncogenic serine/threonine protein kinases that are associated with centrosome maturation and separation, thereby regulating spindle assembly and stability[1]
Saturation transfer difference (STD) and water–ligand observed through gradient spectroscopy (WaterLOGSY) nuclear magnetic resonance (NMR) experiments were first conducted to validate the fine details of the MLN8237-Human serum albumin (HSA) interaction
Direct NMR spectroscopy methods, such as STD and WaterLOGSY assay, molecular docking, isothermal titration calorimetry (ITC), and conventional spectroscopy methods have been applied to investigate the interaction between MLN8237 and HSA

Summary

Introduction

Aurora A kinase (AAK) belongs to a family of oncogenic serine/threonine protein kinases that are associated with centrosome maturation and separation, thereby regulating spindle assembly and stability[1]. Human serum albumin (HSA) is the most widely distributed protein in human blood plasma. It binds to a variety of endogenous and exogenous compounds with moderate to high association constant (104–106 M−1)[11] and acts as a transporter for drugs and other organic molecules to their targets[12]. The investigation of the binding nature of MLN8237 with HSA is of interest, is helpful in understanding the mechanism of antitumor and anticancer activities of the drug, and can provide support for the continued clinical investigation of the drug

Methods

Results

Conclusion