X-Ray Crystal structure of Aminothymoquinone and its Interaction with Human Serum Albumin at physiological Conditions

that NF- κ B was inactivated in animal tumors pretreated with TQ followed by gemcitabine and/ or oxaliplatin [12]. The growth inhibitory properties of TQ was shown to be mediated by down-regulation of NF- κ B 73, while other mechanisms like decrease in phosphorylation of Akt, reduction of p73-dependant cell cycle checkpoint signaling and increase in cellular concentration of p53 and p21 proteins have also found to be involved in TQ-induced apoptosis in cancer cells [13]. TQ was Abstract Aminothymoquinone (ATQ) which exhibits five times more potent anticancer activity against pancreatic cancer has been crystallized and its single crystal structure has been reported in the present work. The crystal structure analysis of ATQ reveals that it crystallises in a monoclinic P21/c group. Additionally its interaction with the major transport protein of human blood circulation human serum albumin (HSA) has been examined using a multitude of spectroscopic techniques and molecular docking studies. Analysis of the fluorescence quenching data showed a moderate binding affinity between ATQ and HSA with a 1:1 stoichiometry. Fluorescence analysis of the binding data and molecular simulation results suggested involvement of hydrophobic and van der Waals forces, as well as hydrogen bonding in the complex formation. The number of binding sites and binding constant estimated from the fluorescence studies yielded values of 0.716 and 3.0334 x 105 mol L-1 respectively. Competitive drug displacement and molecular docking results suggested the binding site of ATQ on HSA as Sudlow’s site I, located at subdomain IIA which was supported by the molecular modelling data.

In order to enhance the significance of TQ several analogs are synthesized which include disubstituted benzoquinones10 and terpene conjugates [17]. Recently, we have reported the synthesis and characterization of novel TQ analogs amongst which aminothymoquinone exhibited promising activity against pancreatic cancer cells.
Since the efficiency of the drug is highly influenced by its interaction with human serum albumin (HSA) which is present in ample amount in the circulatory system. HSA performs a number of functions in colloid osmotic pressure of plasma [18] and also acts as a reservoir for signaling nitric oxide molecules [19]. The chief function of HSA has been to transport a number of endogenous compounds like fatty acids, hormones like thyroxine and number of renal toxins. Numbers of metabolites compete in binding the drug to the protein [20]. Ligand binding to HSA is a spontaneous exchange between solution and the protein pocket. HSA has the noteworthy ability to bind to a range of compounds including metabolites and drugs under similar physiological conditions and hence is of great pharmaceutical interest in the drug discovery process. The binding properties of 66kDa monomer HSA have been studied with great interest on a significant number of drugs but have been hampered by the flexible nature of the protein and its multiple binding sites [18,19]. In the current paper we explain the crystal structure of ATQ along with its interaction with the most abundant protein human
All chemicals were used without further purification. The solution of HSA (molecular weight = 66,500 Dalton) was prepared in 50mM tris-HCl buffer. All other reagents used were analytical grade and the aqueous solutions were made in double distilled water for all the experimental measurements. ATQ was synthesized according to procedure described by us earlier [18]. By refluxing the mixture of TQ (1mmole, 0.164g), sodium azide (1.3mmole, 0.084g) and 3ml glacial acetic acid in ethanol for three hours [18]. The stock solution of ATQ was prepared in dimethyl sulphoxide.

Absorption Spectroscopy
The absorption spectra were recorded using a JASCO V-630 Spectrophotometer. Aqueous solution of human serum albumin (5mM) was prepared in 50mM Tris -HCl buffer (pH 7.4). The ATQ (10mM) solution was prepared in dimethyl sulphoxide solvent followed by successive dilutions in distilled water. The absorption spectrum of ATQ was recorded followed by successive spectra with increased concentration of HSA (0.2mM to 2.8mM). Absorbance spectra were also recorded for the aqueous solution of ATQ and varied concentrations of ATQ-HSA complex [21].

Fluorescence Spectroscopy
The fluorescence experiments were carried out on FP-8200 spectrofluorimeter (JASCO) using a quartz cuvette of 1cm path length. The excitation and emission bandwidth were 5nm each and the excitation wavelength was set at 285nm. The fluorescence spectra are measured within the range of 200-750nm. The fluorescence measurements were carried out by titrating 100µM HSA in 3ml Tris-HCL buffer with 100µL of ATQ (50mM) solutions.
The titrations were done using micropipettes of varied volume [22]. To elucidate the binding site of ATQ in HSA competitive site binding experiments were carried out using bilirubin (specific site marker for site I) [23] and ibuprofen (specific site marker for site I) [24]. The concentration of HSA, bilirubin and ibuprofen were kept 100µM. Equimolar solution of HSA and bilirubin were prepared and titrated with ATQ solution to record the spectra. The excitation wavelength for HSA was adjusted at 285 nm and the spectra were recorded within the range 200 to 500nm. The ibuprofen and HSA DOI: 10.26717/BJSTR.2021. 38.006144 complex is prepared by mixing equimolar concentration of both the constituents and then titrating the complex with ATQ solution to observe the binding site [24].

Molecular Docking Studies
Molecular docking, visualization and drawing simulation were performed using AutoDock 4.2 [25] and AutoDockTools 1.5.4(ADT) [26] and PYmol. The structure of ATQ was drawn using ChemDraw and was copied as smilies while its PDB structure was obtained from

Results and Discussion
Crystal Structure of ATQ ATQ crystallises in a monoclinic P21/c group and the corresponding ORTEP plot is shown in the ( Figure 2). The relevant crystallographic parameters are provided (Table 1) and selected bond lengths, bond angles and torsional angles are listed in Table 2.

UV-Visible Spectra
The absorption spectrum of drugs mostly varies on addition of proteins due to the binding of the drug to the protein [29][30]. The absorption spectrum of ATQ shows absorption maxima at 325nm where ∆A is change in the absorbance with and without the protein, Dε the differential extinction coefficient, K is the association in subdomain IIIA. In addition a secondary binding cleft has been found for ibuprofen located at the interface between subdomains IIA and IIB [32][33][34].
To identify the binding site of ATQ on HSA, the site marker competitive experiments were carried out using drugs (bilirubin and ibuprofen) that specifically bind to known sites or region on HSA. The information about the ATQ binding site can be achieved by monitoring the changes in the fluorescence of ATQ bound HSA that are brought about by site I (bilirubin) and site II (ibuprofen) markers ( Figure 5). In the site marker competitive experiment, ATQ was gradually added to the solution of HSA and site markers mixed in equimolar concentrations (1.0×10 -5 mol·L -1 ). As shown in Figure   5A with the addition of bilirubin into HSA, the maximum emission wavelength of HSA undergoes an obvious red shift. Consequently, with the addition of ATQ, the fluorescence intensity of the HSA decreased gradually accompanied by an increase of wavelength emission maximum in the albumin spectrum. This suggests an increased polarity of the region surrounding the tryptophan site (Trp-214) [35] and indicating that the binging of ATQ to HSA was affected by addition of bilirubin. Figure 5B shows the comparison of the fluorescence spectra of the ATQ-HSA system in the absence and presence of ibuprofen. By contrast, with the addition of ibuprofen, the fluorescence intensity of the ATQ-HSA system was almost the same as in the absence of ibuprofen, which indicated that ibuprofen does not prevent the binding of ATQ in its usual binding location.
To support the competitive site binding experiments, molecular docking study was done and is discussed in the further section (Table 3).   -ATQ was analysed due to the fact that 1BM0 has the highest resolution [38]. The binding energy of ATQ with 1BM0 and 2BXD is -6.4Kcal/mole. The binding energy of 2BXF is higher than 1BM0 and 2BXD.This can be very well seen in Table 4. ATQ forms a variable number of hydrogen bonds in the HSA cavity. There are number of hydrophobic interactions between hydrophobic residues of the protein and the benzene rings of the ligand which are believed to add towards the stability of the docking conformation of ATQ inside this binding pocket. However, the interaction between ATQ and HSA cannot be presumed to be exclusively hydrophobic in nature, since there were several polar residues in the vicinity of the bound ligand that may participate in the polar interactions with the hydrophilic groups of ATQ. Hence, it can be concluded that ATQ binds to a hydrophobic pocket located in the subdomain IIA.

Conclusion
In present study we report the monoclinic P2 1/c group crystallization of ATQ and also provide a quantitative analysis of ATQ-HSA interaction at physiological conditions. The fluorescence and molecular modelling data suggests involvement of van der Waals forces as well as hydrophobic and hydrogen bonding interactions in the complexation between ATQ and HSA. Alterations in the protein conformation upon ATQ binding were evident from the multiple spectroscopic results. The binding site of ATQ on HSA was confirmed as site I based on competitive ligand displacement results as well as docking analysis. The biological implication of this work lies in understanding the interaction of ATQ with HSA, which will be vital for the future designing of ATQ-derived drugs.