Interaction of new kinase inhibitors cabozantinib and tofacitinib with human serum alpha-1 acid glycoprotein. A comprehensive spectroscopic and molecular Docking approach

Highlights

• Cabozantinib (CBZ) and Tofacitinib (TFB) are kinase inhibitors and anticancer drugs, binding process to human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions were studied.

• Alpha-1 acid glycoprotein (AAG) is a major acute phase reactant, a major glycoprotein of the human plasma.

• Binding mode is static. Binding constants for the two drugs with AAG lie in the order of 10⁴, K_sv and K_b values decreased with increasing temperature suggesting static mode of fluorescence quenching is operative in the two drugs.

• Protein binding of the two drugs caused the tertiary structure alterations in the protein. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein.

• Overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

Abstract

In the current study we have investigated the interaction of newly approved kinase inhibitors namely Cabozantinib (CBZ) and Tofacitinib (TFB) with human Alpha-1 acid glycoprotein (AAG) under simulated physiological conditions using fluorescence quenching measurements, circular dichroism, dynamic light scattering and molecular docking methods. CBZ and TFB binds to AAG with significant affinity and the calculated binding constant for the drugs lie in the order of 10⁴. With the increase in temperature the binding constant values decreased for both CBZ and TFB. The fluorescence resonance energy transfer (FRET) from AAG to CBZ and TFB suggested the fluorescence intensity of AAG was quenched by the two studied drugs via the formation of a non-fluorescent complex in the static manner. The molecular distance r value calculated from FRET is around 2 nm for both drugs, fluorescence spectroscopy data was employed for the study of thermodynamic parameters, standard Gibbs free energy change at 300K was calculated as − 5.234 kcal mol^− 1 for CBZ-AAG interaction and − 6.237 kcal mol^− 1 for TFB-AAG interaction, standard enthalpy change and standard entropy change for CBZ-AAG interaction are − 9.553 kcal mol^− 1 and − 14.618 cal mol^− 1K^− 1 respectively while for AAG-TFB interaction, standard enthalpy and standard entropy change was calculated as 4.019 kcal mol^− 1 and 7.206 cal mol^− 1K^− 1 respectively. Protein binding of the two drugs caused the tertiary structure alterations. Dynamic light scattering measurements demonstrated the reduction in the hydrodynamic radii of the protein. Furthermore molecular docking results suggested the Hydrophobic interaction and hydrogen bonding were the interactive forces in the binding process of CBZ to AAG while in case of TFB only hydrophobic interactions were found to be involved, overlap of the binding site for two studied drugs on the AAG molecule was revealed by docking results.

Introduction

Alpha-1 acid glycoprotein (AAG) is a major acute phase reactant [1] and a major glycoprotein of the human plasma [2]. AAG is a single polypeptide chain of 41kD molecular weight constructed from 183 amino acids forming the backbone [3]. AAG molecule contains five complex N linked oligosaccharide chains and two disulphide bonds, carbohydrate content accounts for about 45% of total molecular weight [4], [5]. AAG is normally synthesized in the liver but can also be generated from leukocytes, monocytes and granulocytes at the site of infection [6], AAG has also been found in the synovial fluids. It has a normal range of 0.6–1.2 mg/mL (1–3% of plasma proteins) [7]. In both chronic and acute inflammation the plasma concentration increases from 2 to 5 folds [8]. The precise function of AAG is still unknown but binding of basic and neutral drugs and immunomodulation are two important attributes of this protein [1], [9]. Functional diversity arises from the variability in attachment site of the oligosaccharide moiety. Moreover, the linkage pattern of different monosaccharide subunits, degree of branching, extent of sialylation and glycosylation may result in different glycoforms of protein and the rise in particular isoform concentration which can be an indication of particular pathophysiological state like cancers and immune disorders [10].

CBZ (CBZ) is a new multi targeted tyrosine kinase inhibitor (TKI) agent against mesenchymal–epithelial transition factor (MET) and vascular endothelial growth factor receptor types 1 (VEGFR-1), 2 (VEGFR-2) and 3 (VEGFR-3) [11], [12]. In both preclinical and clinical studies CBZ has been shown to inhibit the tumor angiogenesis, invasiveness and metastases [13]. CBZ was approved in 2011 by United States of America, federal drug administration department (U.S. FDA) for the treatment of progressive, metastatic medullary thyroid cancer [14].

Recently in November 2012, TFB (TFB). A Janus kinase inhibitor approved by the FDA for the treatment of rheumatoid arthritis [15], [16].TFB inhibits JAK- STAT pathway which ultimately inhibits DNA transcription in the cells [17]. TFB also showed promising activity in patients with active ulcerative colitis [18]. TFB is being investigated for approval in the treatment of adult patients with moderate to severe chronic plaque psoriasis who are candidates for systemic therapy or phototherapy [19].

Various methods such as UV–visible [20], [21], fluorescence [22], [23], [24] and circular dichroism (CD) [25], [26] spectroscopy, equilibrium dialysis [27] and potentiometry [28], [29] have been used to investigate the binding of ligands (drugs, dyes, electrolytes, etc.) to proteins. Among these methods, equilibrium dialysis is used widely; but it requires the analysis of free and total drug concentration and takes a long time [30]. In potentiometric method, ion selective electrodes are used. These electrodes have the lack of selectivity for many ligands such as drug molecules [28], [29]. Fluorescence techniques are very helpful in protein-drug interactions due to their high sensitivity, rapidity and ease of implementation. The fluorescence measurements can provide some useful information about the binding of small molecules to protein such as binding mechanism, binding mode, binding constants and binding numbers [30].

The rapid pace of development of such small kinase inhibitor molecules as therapeutic candidates for several life threatening diseases urges research into their binding process to important human proteins such as alpha 1- acid glycoprotein. Therefore this study is sought to investigate the interaction of CBZ and TFB with AAG using spectroscopic techniques. The changes in topology of AAG in the absence and presence of CBZ and TFB were studied by using dynamic light scattering measurements. Furthermore molecular docking was performed to get insight into the binding process and binding site location.