Spectroscopic studies and molecular docking on the interaction of organotin antitumor compound bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) with human cytochrome P450 3A4 protease

Highlights

• We investigated the relationship between the DFDPT and human CYP3A4 proteaset.

• Fluorescence spectroscopy, circular dichroism and molecular docking were used.

• The binding mechanisms and the conformation changes were identified.

• We found the predicted binding mode of DFDPT into CYP3A4.

Abstract

A novel organotin DFDPT was synthesized and characterized by elemental analysis, IR, ¹H, ¹³C, ¹¹⁹Sn, NMR techniques,etc. In order to investigate profoundly the relationship between DFDPT with human CYP3A4 proteaset and anticancer molecular mechanism of DFDPT, the intercalative mode of binding of DFDPT with CYP3A4 under physiological conditions were comprehensively evaluated using steady state, synchronous, three-dimensional fluorescence spectroscopy,circular dichroism and molecular docking. Fluorescence emission data showed that CYP3A4 fluorescence affected by DFDPT was a static quenching procedure, which implied that DFDPT-CYP3A4 complex had been formed. Apparent binding constants K_b of CYP3A4 with compound at 298 and 310 K were 2.51 × 10⁷ and 3.09 × 10⁵, respectively. The binding sites number n was 1.64 and 1.22, respectively. The thermodynamic parameters ΔH and ΔS of the DFDPT-CYP3A4 complex were negative, which indicated that their interaction was driven mainly by hydrogen bonding and van der Waals force. The binding of DFDPT-CYP3A4 was spontaneous process in which ΔG was negative. The synchronous results showed DFDPT induced conformational changes of CYP3A4 protein. Three-dimensional fluorescence and circular dichroism spectra results also revealed conformation of CYP3A4 protein had been possible changed in the presence of DFDPT. Molecular docking was used to study the interaction orientation between DFDPT and CYP3A4 protease. The results indicated that DFDPT interacted with a panel of amino acids in the active sites of CYP3A4 protein mainly through formation of hydrogen bond. Furthermore, the predicted binding mode of DFDPT into CYP3A4 appeared to adopt an orientation with interactions among Arg105, Ser119 and Thr309.

Introduction

In the past several decades, organotin (IV) compounds were studied as the probable antitumor drug [1]. Our research group synthesized and structurally analyzed a potential organotin candidate for the clinical application, bis[2,4-difluoro-N-(hydroxy-⟨κ⟩O)benzamidato-⟨κ⟩O]diphenyltin(IV) (DFDPT) which exhibited the strong antitumor activity against seven human cancer cell lines including HepG-2, SHSY5Y, HEC-1-B, EC, T24, HeLa and A549 along with human liver HL-7702, a human normal hepatocytes cell [2]. The mechanism of great anti-cancer activity of DFDPT may be relevant to the inhibition effect on CYP3A. The studies of our previous reports suggested both mRNA and protein expression of CYP450 were inhibited by organotin compounds. The inhibition of the key isoenzyme CYP3A could cause the change of metabolism. Cytochrome P450 (CYP) proteins play significant roles in metabolism, both the endogenous molecules and exogenous substances are detoxified by these enzymes. A crucial fraction of the CYP family is CYP3A4, which composes up to 30% of the total liver CYP enzyme pool in humans [3]. The interaction between proteins and organotin compound was imperative for investigating the pharmacodynamics, pharmacokinetics and activity of organotin compound. Moreover, we can further clarify the relationship between the effective antitumor activity and the interaction of DFDPT with CYP3A4. The mechanism of interactions of organotin compounds and CYP3A4 was studied in present work. The structure of DFDPT and is shown in Fig. 1 A, and the structure of CYP3A4 protease is shown in Fig.1 B, which is divided into two regions, namely α helix and β fold region. Heme is the binding site of oxygen and substrate in the oxidation reaction. Central iron atom of heme is the non-covalent binding form in the CYP3A4 molecule.

Various techniques had been employed to investigate the complex-protein interaction, including nuclear magnetic resonance (NMR), UV–Vis spectrophotometry, fourier infrared spectrophotometry (FT-IR), fluorescence [4], [5], [6], [7], [8], circular dichroism (CD) and molecular docking and so on. Among these methods, fluorescence spectroscopy has a variety of superior advantages over other techniques, including its potency for sensing any minimal changes in the local environment of a fluorophore. Fluorescence spectroscopy is an effective method to reveal the interaction between small molecules and proteins [9]. In present work, we use fluorescence spectroscopy to investigate the quenched mechanism, the binding constant, the binding site and the thermodynamic parameters [10], [11], [12], [13]. The synchronous fluorescence and three-dimensional fluorescence were performed to confirm the changes of conformation [14], [15]. The CD spectrum can effectively reflect the secondary structure changes of protein. The present paper would deal with the binding mechanism of organotin compound DFDPT with CYP3A4 protein by fluorescence, CD and the molecular docking measurement which was reported for the first time. Molecular docking can be used to study the metabolic behavior of the compounds through docking the compounds into the activity sites of drug-metabolizing enzymes. In addition, understanding the interaction of DFDPT with CYP3A4 could contribute to the clinical employment of the organotin compounds and the relief of the organotin pollutant. Therefore, the aim of the present study is to determine the binding of DFDPT towards the activity cavity of CYP3A4 protein. Through these data, the metabolic behavior of DFDPT by CYP3A4 protease could be deeply understood.