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. 2020 Jan 27;5(5):2267–2279. doi: 10.1021/acsomega.9b03479

Privileged Scaffold Chalcone: Synthesis, Characterization and Its Mechanistic Interaction Studies with BSA Employing Spectroscopic and Chemoinformatics Approaches

Nidhi Singh †, Neeraj Kumar †, Garima Rathee †, Damini Sood †, Aarushi Singh †, Vartika Tomar †, Sujata K Dass ‡, Ramesh Chandra †,§,*
PMCID: PMC7016911  PMID: 32064388

Abstract

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Chalcone, a privileged structure, is considered as an effective template in the field of medicinal chemistry for potent drug discovery. In the present study, a privileged template chalcone was designed, synthesized, and characterized by various spectroscopic techniques (NMR, high-resolution mass spectrometry, Fourier transform infrared (FT-IR) spectroscopy, UV spectroscopy, and single-crystal X-ray diffraction). The mechanism of binding of chalcone with bovine serum albumin (BSA) was determined by multispectroscopic techniques and computational methods. Steady-state fluorescence spectroscopy suggests that the intrinsic fluorescence of BSA was quenched upon the addition of chalcone by the combined dynamic and static quenching mechanism. Time-resolved spectroscopy confirms complex formation. FT-IR and circular dichroism spectroscopy suggested the presence of chalcone in the BSA molecule microenvironment and also the possibility of rearrangement of the native structure of BSA. Moreover, molecular docking studies confirm the moderate binding of chalcone with BSA and the molecular dynamics simulation analysis shows the stability of the BSA–drug complex system with minimal deformability fluctuations and potential interaction by the covariance matrix. Moreover, pharmacodynamics and pharmacological analysis show good results through Lipinski rules, with no toxicity profile and high gastrointestinal absorptions by boiled egg permeation assays. This study elucidates the mechanistic profile of the privileged chalcone scaffold to be used in therapeutic applications.

Introduction

Serum albumins are one of the major soluble protein components present in the circulatory system that perform numerous physiological functions, including regulation of osmotic pressure, maintenance of blood pH, and distribution and transportation of various endogenous and exogenous molecules such as drugs, food additives, etc.1−7 The approximate concentration of serum albumin in human blood is 3.6–5.2 g/dL, which can be increased up to double.8 The drug or ligand molecule binds with albumin either weakly or strongly. A stronger binding of a drug molecule with albumin leads to a decrease of concentration of the drug in plasma, while the weakly bounded drug has a shorter lifetime and poor distribution in plasma.9,10 The degree of interaction between drug and serum albumin is an important factor for any molecule being a commercial drug as the binding interaction study decides the drug lifetime, its solubility, and distribution in plasma.11

Bovine serum albumin (BSA) is about 76% sequential analogs to human serum albumin (HSA).12 BSA is considered as a model protein for deciphering the interaction between different small ligand molecules and albumins due to its low cost, easy availability, and structure homology with HSA.13−15 BSA consists of three structurally homolog domains (I–III), and each domain is further split into two subdomains, named A and B.9 The drug-binding sites of serum albumin are commonly located in the hydrophobic cavity of subdomains IIA and IIIA, which are known as Sudlow’s sites I and II, respectively.16,17 X-ray crystallographic data reveal that the major difference between BSA and HSA is that HSA contained only one tryptophan-Trp-214, while BSA consists of two tryptophans (Trp-134 and Trp-213). Trp-134 is positioned on the surface of the protein and is present in subdomain IB, while Trp-213 is trapped within the hydrophobic pocket of the protein and is present in subdomain IIA.1

Chalcone is a simple and common chemical scaffold of many biologically active compounds isolated from natural sources. This privileged structure has attracted research attention for a century.18 The common scaffold present in chalcones is 1,3-diaryl-2-propen-1-one, commonly called chalconoid, which exists in two isomeric forms (cis and trans), with the trans form found to be more thermodynamically stable.19,20 There are two phenyl rings in chalcone derivatives. In this research article, the phenyl ring which is attached to the carbonyl group is named ring A, while the other benzene ring is defined as ring B (Figure 1).

Figure 1.

Figure 1

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The structure of chalcone.

Chalcones belong to a class of potential lead compounds that act as an effective template in novel drug discovery in medicinal chemistry. The synthetic protocol for developing a new chalcone is very easy and environmentally feasible. Synthetic and natural chalcones show various therapeutic applications such as antidiabetic, anti-inflammatory, anticancer, antioxidant, anti-infective, or antiproliferative activities.21−27 However, the exact mechanism of action for various pharmacological effects of chalcones is not discovered yet.

In 2006, Kuo-Hsiung Lee and his group reported that the 2-hydroxy-3-methoxy chalcone showed significant activity against the human tumor cancer cell line (lung carcinoma A549).28 A simple chalcone molecule possessing only hydroxyl and methoxy substituents exhibits a good NF-kB inhibitory activity, and thus acts as a potential anticancer agent.29 The replacement of hydrogen by halogen can effectively change the biological property of the drug. The substitution of hydrogen by chlorine in trimethoxy chalcone significantly improves the anticancer activity, which is due to the electromeric effect provided by the chlorine group being located at the 4′ position (para position to the carbonyl group) of the phenyl ring.30 Various quinolinyl chalcone derivatives were tested for biological activity against the Plasmodium falciparum strain. The hypoxanthine uptake by strain of P. falciparum (chloroquine-resistant strain) was mostly inhibited by quinolinyl chalcone bearing chloro-substituted benzoyl ring.31 More research is needed to develop potent therapeutic agents, which can prove effective against multidrug-resistant strains.

By considering the structure–activity relationship study of chalcones reported in the literature, we have designed to synthesize the chalcone “(E)-1-(2,4-dichlorophenyl)-3-(2-hydroxy-3-methoxyphenyl)prop-2-en-1-one”. To the best of my knowledge, only single-crystal X-ray diffraction (XRD) study of this compound has been reported so far.32 It is essential to study the biodistribution of the drug in blood plasma, which directs the development of effective strategies for their safe medical use.33 The binding mechanism between drug and albumin is an effective tool to understand the pharmacokinetic property of the drug properly. Some previous studies have reported the mode of binding interaction of chalcone with BSA.34−38 In the present study, a new chalcone derivative incorporating a chlorine substituent in ring A and hydroxyl and methoxy substituents in ring B was designed, synthesized, and well characterized by various spectroscopic techniques (NMR, high-resolution mass spectrometry (HRMS), FT-IR spectroscopy, UV spectroscopy, and single-crystal XRD). The interaction study of chalcone with BSA has been investigated by employing various multispectroscopic techniques such as UV–vis, steady-state fluorescence, time-resolved fluorescence, FT-IR, and circular dichroism (CD) spectroscopy. Molecular modeling studies, including molecular docking and molecular dynamics simulation studies, are also employed to assess the potency and binding mechanism of the drug with target proteins.39−41 Theoretical studies are well employed to assess the potency and absorption studies of the drug.42,43 Also, pharmacokinetic and pharmacodynamic analyses have been carried out for the complete assessment of drugs for high absorption, excretion, and no toxicity profiles or side effects. Computational values are reported in many studies to follow the Lipinski rule of five and drug likeliness. The pharmacological and pharmacodynamic profiles of compounds are well converted to develop effective therapeutic agents.44−46

Results and Discussion

Single-Crystal XRD Analysis

The chalcone C16H12Cl2O3 belongs to the monoclinic system with space group P21/n. The observed cell parameters are a = 11.5245(10) Å, b = 3.9894(3) Å, c = 31.742(2) Å, α = 90°, β = 96.235(7)°, γ = 90°, and V = 1450.7(2) Å3. The data which relate the structural refinement to information about the collection of data are presented in Table 1. The ORTEP diagram obtained from the XRD data is presented in Figure 2, which closely resembles the diagram reported in the literature.32 The obtained bond length and bond angle are listed in Table S1.

Table 1. Crystal Data and Structural Refinement Parameter of C16H12Cl2O3.

empirical formula C16H12Cl2O3
crystal shape/color needle/yellow
formula weight 323.16
temperature (K) 293(2)
crystal system monoclinic
space group P21/n
cell parameters a = 11.5245(10) Å
b = 3.9894(3) Å
c = 31.742(2) Å
α = 90°
β = 96.235(7)°
γ = 90°
volume 1450.7(2) Å3
Z 4
density (g/cm3) 1.480
μ (mm–1) 0.454
F(000) 664
temperature (K) 293
crystal size (mm3) 0.07 × 0.06 × 0.01
radiation Mo Kα (λ = 0.71073)
absorption correction multiscan
index ranges –14 ≤ h ≤ 14, −4 ≤ k ≤ 4, −39 ≤ l ≤ 39
reflections collected 17 651
independent reflections 2940 (Rint = 0.0466, Rsigma = 0.0345)
data/restraints/parameters 2940/0/238
goodness of fit on F2 1.091
final R indexes (I ≥ 2σ (I)) R1 = 0.0469, wR2 = 0.1000
final R indexes (all data) R1 = 0.0662, wR2 = 0.1114
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Figure 2.

Figure 2

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Molecular structure of C01 is presented in the ellipsoid style at the 50% probability level. The hydrogen atom is shown as a fixed sphere of radius 0.17 Å, and the bond style is of stick type with radius 0.1 Å.

UV–Visible Absorption Spectroscopy Analysis

The UV–visible absorption measurement is a very simple and valuable technique to analyze the formation of a complex between drug and protein. The absorption spectra of pure BSA have an only characteristic peak around 280 nm, which represents the π–π* transition due to the presence of aromatic amino acids (tyrosine, tryptophan, and phenylalanine).47 The peak intensity of BSA increased with the addition of chalcone (Figure 3), which inferred the interaction between chalcone and BSA.

Figure 3.

Figure 3

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UV–visible absorption spectra of pure BSA and BSA in the presence of different concentrations of C01. [BSA] = 15 μM, [C01] = (a) 0 μM, (b) 1.5 μM, (c) 3.0 μM, (d) 4.5 μM, (e) 6.0 μM, (f) 7.5 μM, (g) 9.0 μM.

Fluorescence Spectroscopy Analysis

Steady-state fluorescence spectroscopy is the most effective and valuable technique to decipher the mechanism of interaction between protein (HSA/BSA) and drug molecule.48−50 The fluorescent nature of the BSA molecule, which is mainly due to the presence of the tryptophan, phenylalanine, and tyrosine residues, is often used as a fluorescent probe to study the alteration in the conformation of BSA on the addition of the drug.51,52 Out of the three residues, Trp has the highest fluorescence intensity, which contributes more to fluorescence quenching of the protein. The two Trp residues, Trp-134 and Trp-213, are present in the BSA molecule. The former one is present on the surface, while the latter is located in the hydrophobic pocket of the BSA molecule.53

The fluorescence spectra of the BSA solution in the absence and presence of chalcone are shown in Figure 4. The BSA solution showed the emission maximum at 340 nm, when the excitation wavelength was kept at 280 nm. The fluorescence emission intensity of the BSA solution decreased regularly on the successive addition of chalcone without any significant change in the emission maximum wavelength. This observation suggests the changes in the BSA molecule microenvironment due to interaction with chalcone, and the chalcone binds into the binding cavity of the BSA molecule.

Figure 4.

Figure 4

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Steady-state fluorescence emission spectra of BSA and its fluorescence quenching spectra in the presence of varying concentration of C01 at 298 K. [BSA] = 15 μM, [C01] = 0, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 μM.

The fluorescence quenching of the protein is classified into three quenching mechanisms, namely, static quenching mechanism, dynamic quenching mechanism, and the combination of the static and dynamic quenching mechanisms. The static quenching mechanism refers to the ground-state complex between the drug and protein, the dynamic quenching mechanism resulted from the collision of protein with the drug molecule, and the combined quenching refers to both collision encounter of the protein with the drug and the complex formation between the protein and drug.53−56

The Stern–Volmer plot and the double-logarithmic plot of the BSA–C01 complex system are shown in Figure 5A,B, respectively. The Stern–Volmer constant (KSV) was determined by using eq 4. The quenching rate constant (kq) of the BSA–C01 complex system is simply calculated by considering the τo value equal to 10–8 s.57 The calculated value of kq ≫ 2.0 × 1010 M–1 s–1 indicates the presence of the static quenching mechanism (the maximum scattering collision quenching rate constant value is 2.0 × 1010 M–1 s–1).58

Figure 5.

Figure 5

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(A) Stern–Volmer plot of F0/F versus [C01] for fluorescence quenching spectra of BSA upon addition of C01 at 298 K. (B) Plot of log[(F0 – F)/F] versus log[C01] for the BSA–C01 complex system at 298 K.

The value of binding constant (Kb) and the binding stoichiometry (n) of chalcone in the protein molecule are calculated using eq 5 and are represented in Table 2. The binding constant value (Kb) is found to be 1.60 × 105 M–1, and the binding stoichiometry (n) is nearly equal to 1.

Table 2. Quenching Constant (kq), Binding Constant (Kb), and the Binding Stoichiometry (n) of Chalcone in the BSA Molecule Microenvironment at 298 K.

code T (K) KSV (M–1) kq (M–1 s–1) r2 Kb (M–1) n r2
C01 298 7.96 × 104 7.96 × 1012 0.9934 1.60 × 105 1.06 0.9899
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Time-Resolved Fluorescence Spectroscopy Analysis

The lifetime decay measurement is considered as an ideal nanoscale detection method. The emission measurement of the BSA molecule is highly influenced by the presence of other interacting molecules in the lifetime measurement.59 Time-resolved measurement is used to distinguish between different modes of quenching: static, dynamic, and mixed quenching modes. The fluorescence decay profile was recorded for the BSA solution in the absence and presence of chalcone (C01) (Figure 6). The lifetime value of the fluorescence decay profile of BSA in the absence and presence of C01 is presented in Table 3. The dynamic quenching constant (KD) is obtained from lifetime measurement by using eq 1

graphic file with name ao9b03479_m001.jpg 1

where τo is the lifetime of the BSA solution in the absence of C01, τ is the lifetime of the BSA solution in the presence of C01, and the concentration of C01 is represented by [Q].

Figure 6.

Figure 6

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Lifetime decay profile of pure BSA and BSA in the presence of C01 in phosphate-buffered saline (PBS) (pH = 7.4). [BSA] = 15 μM and [C01] = 8 μM.

Table 3. Lifetime Obtained from the Fluorescence Decay Profile of BSA and the BSA–C01 System.

concentration τ1 (ns) τ2 (ns) a1 a2 τav (ns)
BSA 2.57 6.39 25.21 74.79 5.43
BSA + 8 μM C01 2.12 5.95 35.69 64.31 4.58
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The value of dynamic quenching constant obtained from eq 1 is 2.3 × 104 M–1. Equation 2 is used to calculate the value of static quenching constant.60

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The plot of the graph [(F0 – F)/F]/[Q] versus [Q] gives the value of static quenching constant. The value of KS (3.7 × 104 M–1) is greater than that of KD. The difference between KS and KD is not too large. Hence, this result indicates that C01 did not induce the quenching of protein (BSA) by a single quenching mechanism. The combined static and dynamic quenching exists in the C01–BSA system. A change in the absorption spectra of BSA was observed upon addition of C01, and the value of the quenching rate constant (7.96 × 1012 M–1 s–1) is greater than the maximum collision quenching rate constant (2.0 × 1010 M–1 s–1). All such observations indicate that the predominately static quenching exists in the BSA–C01 system.

FT-IR Spectra Analysis

To probe a deep insight into the binding interaction of chalcone with BSA, FT-IR spectra were analyzed. Proteins exhibit two bands in the infrared region. The amide I band in the region 1600–1700 cm–1 is mainly due to C=O stretching vibration of the amide moiety, whereas the amide II band, which lies in the region 1500–1600 cm–1, is due to the C–N stretching vibration in combination with the N–H bending mode. The amide I band is normally more susceptible to change in the secondary structure of protein (BSA) than the amide II band.61 While adding C01 into the BSA solution, the transmittance intensity of the IR spectra of BSA decreased slightly and the peak positions of the amide I and II bands showed minor shifts from 1640 to 1638 and from 1541 to 1539, respectively (Figure 7). This result indicated the presence of chalcone in the BSA microenvironment.

Figure 7.

Figure 7

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FT-IR spectra of (A) pure BSA solution and (B) BSA in the presence of C01. [BSA] = 1 × 10–4 M, [C01] = 1 × 10–4 M.

CD Analysis

Circular dichroism analysis has been performed to observe the secondary structure conformational changes in BSA upon addition of chalcone (C01). Figure 8 shows the CD spectra of pure BSA and the BSA:C01 complex. The circular dichroism spectra of pure BSA are obtained in phosphate buffer (pH = 7.4) at 298 K. The spectra exhibited two negative minima located at 208 and 222 nm due to the characteristic α helical structural unit present in the protein.54 The bands at 208 and 222 nm correspond to the π → π* and n → π* transitions of the α helix.57,62 Upon the addition of chalcone to the BSA solution, the intensity of the minima at 208 and 222 nm in the CD spectrum of pure BSA decreased slightly. The α-helix content residing in the secondary structure of BSA shows a very less decrease upon addition of chalcone (Table 4), which indicates the little change in the secondary structure of BSA.63 In other words, the protein BSA has retained its native secondary α- helical structure even after binding the drug.53

Figure 8.

Figure 8

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Circular dichroism spectra of BSA in the absence and presence of different concentrations of C01 in phosphate-buffered saline (PBS) (pH = 7.4). [BSA] = 5 μM and [C01] = 5 μM.

Table 4. Value of α-Helix % Obtained from the Interaction between BSA and Chalcone (C01).

concentration α-helix (%)
pure BSA 32.7
BSA–C01 (ratio 1:1) 31.5
BSA–C01 (ratio 1:5) 32.2
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Three-Dimensional (3D) Crystal Structure Retrieval of Bovine Serum Albumin and Assessment

Three-dimensional (3D) crystal structure of BSA was retrieved from the Protein Data Bank with PDB ID 4OR0 and 2.58 Å resolution. The BSA structure consists of two chains of A and B of size 583 amino acid residues and in conjugation with drug naproxen. Structural parameters and stereochemical properties of the BSA structure were evaluated by the Ramachandran plot, which provides information about the dihedral angles and the amino acid residues region, whether in favorable/unfavorable regions. The result showed that 92.8% of amino acid residues lie in the allowed region and 7.2% lie in the additionally allowed region with no amino acids in the outlier region (Figure 9). Local quality estimation showed that the BSA structure is stable with many fluctuations (0.38–0.98 Å deviations) with native structures. Also, verified 3D server elucidated the high quality of the BSA crystal structure by depicting that the BSA structure has more than 80% amino acid residues with optimal 3D/1D profiles (Figure S1). Furthermore, Errat server also confirmed the high quality of BSA with major residues underlying the warning region and showed the high resolution of the structure. Assessment results of the BSA structure confirmed its high quality to be used for further studies.

Figure 9.

Figure 9

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(A) Three-dimensional crystal structure of bovine serum albumin, retrieved from the Protein Data Bank, (B) Ramachandran plot of BSA protein, which showed that 92.7% amino acid residues lie in the favorable region and 7.2% lie in the additionally allowed region. (C) Local quality estimation plot of the BSA protein, showing minimal fluctuations of residues (<1 Å).

Molecular Docking Studies of Chalcone with BSA

Molecular docking was performed to evaluate the binding potential of C01 with the BSA protein. Autodock was set on the interaction of 3D conformations of the target BSA protein, and the grid dimensions were made to 0.6 Å with a receptor and ligand range of 180°, which was followed by a BSA protein flip and twist range of 360°. The molecular docking process followed the translational steps to determine the high energy scoring functions for various ligand-binding conformations to the target protein BSA. Among the resulting 1000 complex systems, the top five clusters with high binding energy scoring functions were assessed. Chalcone interacts with the binding groove of the BSA protein at high-energy confinements, and the highest docking score was found to be −5.79 kJ/mol by Autodock (Figure 10). Moreover, to validate our results, we have redocked the C01 with BSA using the Hex 8.0 software. It was set to shape and steric conformations interaction study using the fast Fourier mode. BSA grid dimensions were set at 0.6 Å, as provided by the docking manual and followed by the BSA protein flip and a twist range of 360°. From the output 25 interacting complexes, one with the highest docking score (−192.99 kJ/mol) was found, which was further analyzed to determine the involved molecular interactions using the Ligplot (Figure 11). Ligplot showed the strong hydrophobic interaction of the C01 with BSA with the involvement of amino acids: Leu 24, Lys 132, His 145, Leu 189, Ser 128, Ile 455. These results suggested the moderate binding of C01 under study to the BSA protein at two regions: one at 24–189 and the second at the 428–455 amino acid region of BSA.

Figure 10.

Figure 10

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Binding energy conformations of drug to the BSA protein with scoring functions. (A) The closest binding with the highest docking score of −5.79 kJ/mol, (B) binding of drug with the second most high energy score of −5.73 kJ/mol, (C) binding energy conformations of drug to BSA with −5.50 kJ/mol, (D) binding conformations of drug to BSA with a score of −5.47 kJ/mol, (E) binding conformations with a score of −5.39 kJ/mol, and (F) binding conformations with minimal interaction of drug to the BSA with a score of −5.37 kJ/mol. The drug is shown in gray, and BSA is shown in orange.

Figure 11.

Figure 11

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(Left) Depiction of molecular interactions of C01 to the binding groove of the BSA protein and (right) depiction of molecular interactions involved in the C01–BSA complex.

Molecular Dynamics Simulation Study of the C01–BSA Complex

The C01–BSA complex was assessed for large-scale flexibility and its stability using the normal mode analysis (NMA). I-Mod simulation server was employed to study the internal coordinating molecules of the complex. The resulted complex trajectory was analyzed for the deformation to define its stability. Trajectory showed that the C01–BSA complex is stable with minimal deformability fluctuations (0.1–1 Å) by protein hinge distortion analysis. Trajectory files are also found to be similar to the normal mode analysis reference protein with minimal atomic fluctuations (Figure 12). The Eigen score is obtained to be 1.354970 × 10–5, and the variance of normal mode is the inversion of this score (Figure S2), which signified the rigidity of the C01–BSA complex. After that, an elastic network and covariance matrix analysis was performed using the complex trajectory. The covariance matrix stated the coupling of C01-protein atoms; correlated, noncorrelated, and uncorrelated atomic fluctuations are shown by red, blue, and white colors in the figure, respectively. The elastic model shows the atomic pairs through the strings of complex, individual dots show the one sting by degree of stiffness with the corresponding atomic pairs, and dark gray stings show the rigidity and stability of the C01–BSA complex.

Figure 12.

Figure 12

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Simulation trajectory analysis of C01–BSA complex. (A) Plot depicting the atomic fluctuations and showing the minimal deformability complex. (B) Comparison of a complex system with normal mode analysis complex as a reference, indicating fewer atomic fluctuations. (C) Covariance matrix and (D) elastic network analysis; the results signify the higher rigidity of the complex system and higher stability of the complex with a minimal deformation index.

Bioavailability Absorption Analysis of Drug by Boiled Egg Permeation

Boiled egg permeation assay was performed to assess the drug efficacy for high bioavailability and high gastrointestinal absorption. It is an intuitive graphical analysis for the passive absorption of the compound through the intestine and blood–brain barrier. The result showed that chalcone is falling inside the yellow ellipse (yolk), which indicated high values for permeation through the blood–brain barrier. Moreover, chalcone was found to pass through the white ellipse, which suggested the high intestinal absorption of the chalcone, according to the defined algorithm of permeation assay (Figure 13).

Figure 13.

Figure 13

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(A) Depiction of parameters of the Lipinski rule of five for chalcone. (B) Graphical representation of chalcone absorption through the boiled egg permeation assay.

Pharmacological and Toxicity Profiling of the Chalcone

Pharmacological analysis of chalcone was performed to assess the physicochemical potency and toxicity profile. The result showed that chalcone followed the Lipinski rule of five (requisite parameters for druggability). Chalcone has a molecular weight of 323.17 g/mol, partition coefficient value (log P) of 4.603, three hydrogen-bond acceptors, one hydrogen-bond donor group, and a surface area of 46.53 A2. Importantly, the compound was obtained to be highly absorbed in intestine with 91.91% oral absorption values with a good water solubility; it is a primary site of absorption of the drugs, and absorption <30% is considered to be poorly absorbed.64 The skin permeability value was obtained to be −2.80 log Kp. The volume of distribution parameter was also studied to determine the drug concentration in plasma, and it showed that chalcone is less absorbed with a score of −0.017 and an available unbound fraction of 0.055 fraction unit.65 Moreover, the maximum recommended daily dose of chalcone was assessed through the local weighed approach, which showed that chalcone has a high tolerated daily dose with a score of 0.489 log mg/kg/day. Also, toxicity profiling and side-effects analyses were performed, as these are important aspects to consider during the design and development of the drug. Toxicity profile analysis showed that chalcone is noncarcinogenic, as confirmed by the negative output of the Ames test. Moreover, the relative toxicity of chalcone was analyzed by assessing the acute toxicity lethal dosage value (LD50). The LD50 value demonstrates the concentration of drug dose that may cause the death of 50% of animals under investigation. The results showed an LD50 value of 2.346 log mg/kg and the oral rat chronic toxicity score of 1.403 log mg/kg per day with no skin sensations. These results illustrated the no toxicity and side effects associated with administration of chalcone.

Conclusions

This study summarizes the synthesis, characterization, and mechanistic interaction of chalcone with serum protein (BSA) using spectroscopic and chemoinformatics approaches. Chalcone effectively quenched the intrinsic fluorescence of BSA by a combined static and dynamic quenching mechanism. The quenching of fluorescence intensity of BSA was mainly caused by the complex formation of chalcone with BSA. FT-IR and CD spectroscopy experiments revealed the potential interaction of chalcone and BSA with no disturbance to the native structure of BSA and the physiological function of the serum protein. Moreover, in silico studies validated the moderate binding of chalcone with BSA, in correlation with spectroscopic data. Molecular docking and molecular dynamic simulation studies suggested the stabile binding of chalcone with the BSA microenvironment. The pharmacological and pharmacodynamics results add on druggability of the chalcone to be used in therapeutic applications.

Materials and Methods

Chemicals and Reagents

All of the chemicals are used as obtained commercially without any purification. 1H and 13C NMR spectra were recorded on a Jeol JNM ECX-400P spectrometer at 400 and 100 MHz, respectively. NMR spectra of the compound were obtained in CDCl3, considering tetramethylsilane as an internal standard. High-resolution mass spectra (HRMS) were recorded on an Agilent 6520 Q-TOF mass spectrometer. Single-crystal XRD was obtained using an X-Calibur instrument. The UV–vis absorption measurement was carried out using a CARY 300 Conc UV–visible spectrophotometer. Fluorescence spectra were recorded on a Horiba PTI QM-8450-11-C. The lifetime decay spectra were recorded using Horiba PTI QM-8450-11-C. The FT-IR spectral analysis was carried out using an IR Affinity-1S, Shimadzu Fourier transform infrared spectrophotometer. A JASCO J-185 CD spectrometer was used to record the CD spectra.

Synthesis of Chalcone (C01)

The synthetic route of the target chalcone is presented in Scheme S1 using ref (66). To a solution of 2,4-dichloroacetophenone (0.01 mol) and ortho-vanillin (0.01 mol) in ethanol (13 ml) was added an aqueous solution of 40% NaOH (3.0 ml) in a dropwise manner. Then, the reaction mixture was refluxed at 60 °C. After 12 h, the solution was poured into ice-cold water, then the suspension was quenched with 2 M HCl to make the mixture acidic. The organic layer was extracted with ethyl acetate, then washed with water and brine. The organic layer was dried over anhydrous sodium sulfate, concentrated in vacuo, and then purified by silica gel column chromatography to give the target compound C01 (yellow needle-shaped crystal). 1H NMR (400 MHz, CDCl3): δ 7.72 (1H, d, J = 16.2 Hz), 7.46 (1H, d, J = 1.9 Hz), 7.42 (1H, d, J = 8.2 Hz), 7.33 (1H, dd, J = 8.2, 2.0 Hz), 7.25 (1H, d, J = 16.2 Hz), 7.12 (1H, dd, J = 7.4, 2.1 Hz), 6.83–6.90 (2H, m), 6.19 (1H, s), 3.90 (3H, s). 13C NMR (101 MHz, CDCl3): δ 193.35, 146.93, 145.94, 141.89, 137.74, 136.72, 132.49, 130.51, 130.24, 127.25, 126.98, 121.21, 120.72, 119.94, 112.52, 56.32. HRMS (EI) m/z Calculated for C16H12Cl2O3 322.0163; Observed (M + H)+ 323.0228. UV-309 nm. The 1H NMR, 13C NMR, HRMS, UV, and FT-IR spectra are provided in the supporting information (Figures S3–S7). The scanning electron microscopy (SEM) images of chalcone (C01) and normal chalcone without modification are also provided in the Supporting Information (Figure S8).

X-ray Crystallography

Single-crystal data of chalcone (C01) were collected by using the X-Calibur instrument of Oxford Diffraction Ltd. (λ = 0.71073 Å, Mo Kα radiation). OLEX 2 software was used to determine all of the geometric parameters, and mercury software packages were used to obtain the image of the compound.67,68

Binding of Chalcone with BSA

Preparation of Solutions

The purity of the BSA was verified by determining its absorbance at 280 nm. The stock solution of 15 μM BSA was prepared in 10 mM phosphate-buffered saline (PBS, pH = 7.4). The stock solution of 2.5 mM chalcone was prepared in acetonitrile. The volume/volume ratio of acetonitrile/PBS was less than 1%, and at this volume percentage, the acetonitrile does not affect the structure of BSA.69 All of the spectroscopic experiments were carried out at 298 K.

Absorbance Measurements

The UV–vis absorption measurement for both pure BSA solution and the BSA–C01 complex were recorded in the range of 200–400 nm at 298 K. The reference solution used in the UV measurement was phosphate-buffered saline (PBS, 10 mM, pH = 7.4). The concentration of BSA was made constant at 15 μM, whereas the concentration of C01 was varied from 0 to 9 μM with an interval of 1.5 μM.

Steady-State Fluorescence Measurement

The fluorescence emission spectra of both BSA and the BSA–C01 complex were obtained in the wavelength range of 290–500 nm at 298 K. The excitation–emission slit width was kept at 3 nm, and the excitation wavelength was fixed at 280 nm for all of the emission measurements. For the titration, the concentration of BSA was made constant at 15 μM, and the C01 varied from 0 to 9 μM with an interval of 1 μM for the BSA–C01 complex. While the C01 solution was being added into the BSA solution, the fluorescence quenching of BSA was observed.

The fluorescence quenching of serum protein (BSA) was also attributed to the absorbance of ultraviolet radiation by the drug at the excitation wavelength (280 nm) and emission wavelength (340 nm). This inner filter effect from the drug was compensated by correcting the steady-state fluorescence spectra using eq 3(70,71)

graphic file with name ao9b03479_m003.jpg 3

where Fcor and Fobs are the corrected and observed fluorescence intensity values, respectively, while Aex and Aem are the absorbance values of the drug at excitation and emission wavelengths, respectively.

The binding mechanism of the BSA–C01 complex was determined by the Stern–Volmer equation (eq 4)

graphic file with name ao9b03479_m004.jpg 4

where F0 is the fluorescence intensity of the pure BSA solution, F is the fluorescence intensity of the BSA solution after the addition of chalcone, [Q] denotes the concentration of chalcone, KSV is the Stern–Volmer association constant, kq is the quenching rate constant of the biomolecular reaction, and τo is the average lifetime of the BSA in the absence of the chalcone.

The fluorescence quenching data of the BSA were evaluated to determine the binding parameters like binding constant (Kb) and the number of binding sites (n) for chalcone in the BSA environment by using eq 5

graphic file with name ao9b03479_m005.jpg 5

Time-Resolved Fluorescence Measurement

The time-correlated single photon counting technique is utilized to perform the time-resolved fluorescence measurement to record the fluorescence decay profile with a high resolution.72 The BSA molecule was excited using a nanosecond pulsed light-emitting diode source (pulse width, 1.2 nm; pulse repetition rate, 1 MHz).43 Fluorescence lifetime is derived from the fluorescence decay profile.73 The fluorescence decay profile was recorded for pure BSA solution (15 μM) and BSA solution in the presence of chalcone. The excitation and emission wavelengths were fixed at 280 and 340 nm, respectively. The scattering of Ludox solution was measured regularly to determine the instrument response function.74

FT-IR Measurement

The IR spectra of pure BSA and BSA–C01 system (1:1 ratio) were acquired in the wavelength range of 1000–1900 cm–1, while the FT-IR spectra of pure chalcone were obtained in the wavelength range of 480–4000 cm–1 at 298 K. The IR spectra of the free BSA solution were obtained by subtracting the spectra of the buffer solution from the spectra of the protein solution. The difference absorbance spectra of BSA were obtained after subtracting the spectra of the pure chalcone solution from the spectra of the chalcone–BSA solution.

Circular Dichroism Measurement

The CD spectra of the BSA solution in the absence and presence of different concentrations of chalcone were obtained in the wavelength range of 200–260 nm at 298 K. The concentration of BSA was kept constant at 5 μM.

Molecular Docking Studies

The lead drug C01 was assessed for its binding with the serum protein BSA to analyze its physiological profile. The three-dimensional (3D) BSA crystal structure was retrieved from the Protein Data Bank with PDB ID 4OR0. The crystal structure was analyzed by using the multistep program of the preparation wizard of the maestro server. The 3D structure was processed, energy-minimized, and optimized. Also, it was checked if any ligand or any other unwanted molecules are bound to the structure. Also, the structure was evaluated for stereochemical parameters through the Ramachandran plot and other computational avenues, including the Verify 3D, Swiss model structure assessment suite, and saves server.75−77 Before molecular docking, the BSA 3D structure was optimized and prepared. The BSA structure was prepared to employ the protein preparation wizard of WhatIf server (https://swift.cmbi.umcn.nl/servers). Water molecules were also discarded from the 3D structure to analyze the dry trajectories. Chalcone was drawn using the Chemdraw software and saved in the desired format (.sdf and.mol) for molecular docking.

Molecular docking is an interactive assessment approach for drugs with target receptors. We have executed the molecular docking of C01 with the BSA protein using the Autodock tool. Open-screen endeavor MTiAutodock server was employed to perform molecular docking studies. The prepared receptor BSA protein and ligand files of C01 were provided. Autodock performs the docking of ligand file to set off grids with the target BSA protein, and the scoring calculation on GPU was done for these grid binding. Autodock outputs 1000 energy conformation of interaction, out of which the top complex system was considered and further evaluated.

Moreover, to strengthen our results, we have redocked the C01 with BSA using the molecular docking software HEX 8.0. Hex 8.0 works by the fast Fourier transformations algorithm to give the interacting energy conformations through the electrostatic potentials and steric shapes. The resulting docked complex was analyzed to identify the molecular interaction using the Ligplot, and binding conformations were analyzed using the chimera molecular modeling suite.78

Molecular Dynamic Simulations Studies

The docked complex system of BSA–C01 was assessed through the molecular dynamics simulation. The stability of the complex was analyzed by the complex dynamics of normal modes of protein using the iMod server.79,80 This server works by determining the direction of motions and the range of motion of the docked complex system in terms of B-factors, deformability, and covariance scores. A minimal deformability plot will show the stability of the complex with very little fluctuation by analyzing the drug’s ability to stabilize or deform the complex rigidity. Besides, Eigen scoring function was calculated, which signifies the atomic motion composed of rigidity and complexity of the docked system. It has a direct relation with the energy level for complex stability and deformation (a low Eigen score leads to deformation), and the Eigen score of BSA residues is shown in the covariance matrix unit by independent component analysis approach.

Bioavailability Absorption Analysis of Drug by Boiled Egg Permeation

For designing and development of potential drugs, it is needed to assess the bioavailability for pharmacokinetics and gastrointestinal absorption at different levels. We have assessed the gastrointestinal absorption and brain penetration capacity through the estimated permeation method (boiled egg) using the accurate predictive model algorithm. It works by analyzing the lipophilicity (Log P) and the polar nature of the drug (TPSC) from a large drug dataset with 93% accuracy.81,82

Pharmacological Analysis of the Chalcone

The lead compound C01 was studied for its pharmacokinetic and pharmacodynamic studies through many computational servers: ACD/I-Lab, pkCSM, SwissADME, and Molinspiration. Pharmacological parameters were assessed based on the Lipinski rule of five and drug-likeness.

Acknowledgments

The authors are grateful to DST-SERB (EEQ/2016/000489) for providing financial assistance to Prof. Ramesh Chandra. They are also grateful to Council of Scientific and Industrial Research (CSIR) for necessary funds. They would like to acknowledge University of Delhi for providing support and necessary facilities to carry out the research work. Nidhi Singh is grateful to CSIR-SRF for providing the fellowship.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b03479.

  • Claisen–Schmidt condensation reaction for chalcone synthesis (Scheme S1); cell parameters: bond lengths and bond angles of C16H12Cl2O3 (Table S1); structural estimation of bovine serum albumin (Figure S1); local stability estimation of the drug–BSA complex and eigenvalue calculation (Figure S2); 1H NMR spectra of C01 (Figure S3); 13C NMR spectra of C01 (Figure S4); HRMS of C01 (Figure S5); FT-IR spectra of C01 (Figure S6); UV–vis absorption spectra of C01 (Figure S7); and SEM images of chalcone with and without modification (Figure S8) (PDF)

Author Contributions

N.S., N.K., D.S., and R.C. designed and performed the experimental studies. N.S. and N.K. carried out the in silico experiments. The manuscript was written by N.S., G.R., A.S., V.T., and S.K.D.

The authors declare no competing financial interest.

Supplementary Material

ao9b03479_si_001.pdf (879.2KB, pdf)

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