In this study, the interaction between the coumarin derivative:
Coumarin and its derivatives have been known for a long time, yet their vital role in plant and animal systems has not been thoroughly analyzed. Many coumarins and their derivatives exert antitumor, antiviral, anticoagulant, anti-inflammatory, antioxidant, vasorelaxant, antimicrobial, and enzyme inhibition properties. These compounds are precisely known to exert an antitumor effect and can cause significant changes in the regulation of the immune response, cell growth, and differentiation. Coumarins have recently attracted much attention due to their broad pharmacological activities as precursor molecules for the synthesis of some synthetic anticoagulant drugs [
(a) Crystal structure of human serum albumin (PDB ID: 4L8U). (b) Chemical structure of the coumarin derivative. (c) Pymol view of the energy-minimized structure of the coumarin derivative.
Serum albumins (Figure
In the present study, synthesized
The emission spectroscopy study was performed using a spectrofluorometer (ISA FluoroMax-2; Jobin Yvon-Spex, Edison, NJ); the spectral bandpass was fixed at 5 nm for monochromators of both wavelengths of excitation and emission, and the recorded wavelength region is 300–500 nm at 280 nm excitation. The lifetime fluorescence measurements of the coumarin derivative and human serum albumin complex were detected using Fluorolog (Horiba Jobin Yvon IBH, UK) with a fast response sensitive PMT detector (Hamamatsu Photonics, Japan) for an excitation wavelength of 280 nm to 450 nm. The decays were sliced and normalized each wavelength for HSA and HSA-coumarin complex. The excitation-emission matrix (EEM) was mapped with the following two conditions: the emission wavelength was recorded between 200 and 700 nm and the initial excitation wavelength was set to 200 nm with an increment of 10 nm. In EEM measurements, the spectral bandpasses were kept at 5 nm for both excitation and emission. In addition to that, the Forster resonance energy transfer analysis between the donor and acceptor and further single-point polarization studies between human serum albumin and the coumarin derivative complex were also carried out.
Fourier-transform infrared spectra (FTIR) of the human serum albumin solutions were administered at temperature by using an FTIR spectrometer (Interspec 2020; Estonia) in the attenuated total reflection (ATR) mode with a resolution of 4 cm−1 and 32 scans. The secondary structural analysis was carried out using circular dichroism (CD) for free human albumin and complex using a spectropolarimeter (Jasco J715; MD, USA) employing a quartz cell of 0.1 cm beam bath.
The human serum albumin structure was taken from the Protein Data Bank (PDB ID: 4L8U), and the coumarin derivative is drawn using the ChemDraw software. Proteins and small molecules are energetically minimized using the OPLS 2005 force field (Schrödinger LLC) used for molecular docking [
Energy minimization has been performed using the density functional theory (DFT) methodology with B3LYP functional and 6-311G∗∗ basis sets for both coumarin molecule and cofactor with active site amino acids of HSA using the Gaussian 03 software. The electrostatic potential countermaps were pictured using the GView software [
The cytotoxic assay for the drug was obtained using the Cell Counting Kit-8 (CCK-8) (Dojindo Laboratories, Osaka, Japan). The cell density in the 96-well plate was 5000 cells/well, 24 hours later; the drug was added to the culture plate and incubated for another 24 h. After drug exposure, the cells were incubated with 10
The steady-state fluorescence spectroscopy is the most effective technique to understand the further binding mechanism of the HSA-coumarin derivative complex. In general, the human serum albumin emission will be at 350 nm due to the presence of tryptophan residue and other phenylalanines; tyrosine residues emission is considered to be less compared to tryptophan residues emission. The emission spectrum of HSA (1
(a) Steady-state emission fluorescence spectroscopy studies of human serum albumin (1
To know more about the type of quenching, we performed Stern–Volmer (Equation (
Fluorescence binding parameters for HSA-coumarin derivatives (pH 7.4).
Compound |
|
|
|
|
Δ |
Δ |
Δ |
---|---|---|---|---|---|---|---|
Coumarin derivatives | 293 | 7.41 | 6.04 | 1.15 | −25.34 | ||
298 | 5.82 | 5.67 | 1.11 | −10.35 | −25.76 | 86.51 | |
303 | 4.38 | 5.48 | 1.10 | −26.19 |
The thermodynamic study is important for the drug-binding mechanism to calculate the binding stability of the drug with the macromolecule complex. Thermodynamics parameters Δ
According to Ross and Subramanian [
Time-resolved emission spectroscopy is one of the highly noninvasive techniques to understand the real-time dynamics of biomolecules. Figure
(a) Time-resolved emission spectroscopy (TRES) studies of HSA (1
Fluorescence lifetime profiles of HSA and increasing concentration of coumarin derivatives at different excitation wavelengths.
Wavelength (nm) |
|
|
|
|
|
|
|
χ2 |
---|---|---|---|---|---|---|---|---|
|
||||||||
280 | 0.00098 ± 0.012 | 0.74 ± 5.13 | 0.00015 ± 0.02 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 1.13 | 0.5 |
290 | 0.017 ± 0.024 | 0.61 ± 5.60 | 0.00106 ± 0.05 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 2.55 | 1.03 |
300 | 0.037 ± 0.033 | 0.29 ± 5.34 | 0.0024 ± 0.07 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 2.84 | 1.42 |
310 | 0.037 ± 0.036 | 0.21 ± 5.13 | 0.0047 ± 0.086 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 3.40 | 1.19 |
320 | 0.037 ± 0.042 | 0.15 ± 5.31 | 0.010 ± 0.013 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 4.24 | 1.12 |
330 | 0.035 ± 0.046 | 0.093 ± 5.34 | 0.016 ± 0.013 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 4.89 | 1.09 |
340 | 0.032 ± 0.048 | 0.042 ± 5.24 | 0.020 ± 0.014 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.23 | 1.00 |
350 | 0.027 ± 0.05 | 0.027 ± 5.23 | 0.024 ± 0.015 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.56 | 1.00 |
360 | 0.026 ± 0.051 | 0.00054 ± 5.0 | 0.026 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.69 | 1.05 |
370 | 0.024 ± 0.050 | 0.0018 ± 5.0 | 0.027 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.79 | 0.96 |
380 | 0.023 ± 0.052 | 0.0061 ± 5.1 | 0.029 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.87 | 1.00 |
390 | 0.022 ± 0.051 | 0.0031 ± 5.1 | 0.028 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.91 | 0.91 |
400 | 0.022 ± 0.051 | 0.016 ± 5.0 | 0.029 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.90 | 1.00 |
410 | 0.024 ± 0.051 | 0.007 ± 5.0 | 0.027 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.81 | 1.00 |
420 | 0.028 ± 0.051 | 0.035 ± 5.0 | 0.026 ± 0.016 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.66 | 1.00 |
430 | 0.029 ± 0.053 | 0.029 ± 4.9 | 0.024 ± 0.015 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.53 | 1.00 |
440 | 0.030 ± 0.050 | 0.030 ± 4.9 | 0.023 ± 0.015 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.50 | 1.00 |
450 | 0.031 ± 0.050 | 0.038 ± 4.9 | 0.023 ± 0.015 | 2.12 ± 0.008 | 0.0097 ± 0.002 | 6.82 ± 0.006 | 5.47 | 0.99 |
|
||||||||
|
||||||||
280 | 0.0015 ± 0.011 | 0.00004 ± 0.0013 | 0.815 ± 5.5 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 0.65 | 0.34 |
290 | 0.0051 ± 0.016 | 0.00021 ± 0.0021 | 0.806 ± 5.9 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 1.55 | 0.73 |
300 | 0.029 ± 0.030 | 0.0015 ± 0.0054 | 0.499 ± 6.0 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 2.51 | 1.07 |
310 | 0.037 ± 0.037 | 0.0004 ± 0.0073 | 0.374 ± 6.1 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 3.17 | 1.16 |
320 | 0.038 ± 0.042 | 0.0008 ± 0.0096 | 0.20 ± 5.9 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 4.03 | 1.11 |
330 | 0.041 ± 0.049 | 0.015 ± 0.012 | 0.095 ± 5.9 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 4.69 | 1.07 |
340 | 0.037 ± 0.050 | 0.019 ± 0.013 | 0.025 ± 5.6 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.11 | 0.98 |
350 | 0.035 ± 0.053 | 0.024 ± 0.014 | 0.012 ± 5.6 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.41 | 1.01 |
360 | 0.030 ± 0.053 | 0.025 ± 0.014 | 0.020 ± 5.4 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.59 | 0.97 |
370 | 0.029 ± 0.055 | 0.029 ± 0.015 | 0.035 ± 5.5 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.73 | 1.09 |
380 | 0.026 ± 0.054 | 0.029 ± 0.015 | 0.049 ± 5.3 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.80 | 1.03 |
390 | 0.026 ± 0.055 | 0.030 ± 0.016 | 0.072 ± 5.2 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.83 | 0.98 |
400 | 0.024 ± 0.054 | 0.029 ± 0.016 | 0.075 ± 5.0 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.89 | 1.13 |
410 | 0.027 ± 0.055 | 0.031 ± 0.016 | 0.11 ± 5.0 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.85 | 1.06 |
420 | 0.028 ± 0.054 | 0.028 ± 0.016 | 0.15 ± 4.7 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.76 | 1.26 |
430 | 0.030 ± 0.054 | 0.027 ± 0.016 | 0.17 ± 4.5 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.67 | 1.15 |
440 | 0.033 ± 0.054 | 0.026 ± 0.016 | 0.19 ± 4.4 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.50 | 1.17 |
450 | 0.032 ± 0.053 | 0.025 ± 0.015 | 0.21 ± 4.2 | 1.9 ± 0.0082 | 6.8 ± 0.0065 | 0.009 ± 0.002 | 5.53 | 1.04 |
The study is used to further understand the conformational change of HSA more carefully during the interaction with the coumarin derivative. Figure
Excitation-emission matrix spectra of (a) free HSA (1
In general, the FTIR spectral technique is performed to understand the biochemical changes; here, the FTIR spectra of HSA were recorded to investigate the conformational changes to understand the binding mechanism of the coumarin derivative. Figure
(a) FTIR spectra analysis of HSA (1
To understand the conformational changes of the protein secondary structure, CD spectral analysis was carried out. From Figure
Secondary structure calculations were carried out using the DichroWeb (
The molecular docking method is useful to understand the binding insight mechanism between the macromolecule and the small molecule at the atomic level [
Binding energy and hydrogen bonding interaction of the coumarin derivative with HSA active site residues.
Compound name | Hydrogen bond (D-H...A) | Distance (Å) | Binding energy (kJ/mol) |
---|---|---|---|
Coumarin derivative | N–H⋯O (LEU 185) | 3.0 | −28.32 |
N–H⋯O (ARG 117) | 2.9 |
(a) Pymol view of HSA-coumarin derivative complex hydrogen bonding interaction with active site residues. (b) Charge distribution (DFT) analysis plot for the HSA-coumarin derivative complex of active site residues.
The Mulliken atomic charges were analyzed for both coumarin derivative and cofactor with interaction amino acids of HSA. The MPA charges are presented in Table
The calculated Mulliken population analysis (MPA) of the coumarin molecule with active site amino acids of HSA.
MPA | MPA cofactor | ||||
---|---|---|---|---|---|
1N −0.38 | 51C −0.06 | 101C −0.06 | 1N −0.43 | 51O −0.28 | 101C 0.06 |
2C −0.05 | 52C −0.05 | 102C 0.32 | 2C −0.11 | 52C −0.18 | 102C −0.16 |
3C 0.16 | 53C −0.13 | 103C 0.08 | 3C 0.15 | 53C −0.09 | 103C 0.14 |
4O −0.24 | 54C −0.09 | 104C −0.10 | 4O −0.30 | 54C −0.05 | 104C 0.29 |
5C −0.20 | 55C 0.12 | 105C −0.14 | 5C −0.16 | 55C −0.05 | 105N −0.38 |
6C −0.27 | 56O −0.38 | 106C −0.03 | 6C −0.26 | 56C −0.09 | 106C 0.09 |
7C −0.09 | 57H 0.18 | 107C −0.09 | 7C −0.09 | 57C −0.06 | 107C −0.09 |
8N −0.44 | 58H 0.20 | 108C −0.09 | 8N −0.46 | 58C 0.12 | 108C 0.02 |
9C 0.68 | 59H 0.12 | 109C −0.09 | 9C 0.65 | 59O −0.41 | 109C −0.21 |
10N −0.45 | 60H 0.12 | 110C −0.06 | 10N −0.47 | 60H 0.25 | 110C 0.44 |
11N −0.46 | 61H 0.09 | 111C −0.07 | 11N −0.49 | 61H 0.19 | 111O −0.41 |
12H 0.15 | 62H 0.08 | 112C −0.09 | 12H 0.23 | 62H 0.12 | 112C 0.01 |
13H 0.10 | 63H 0.15 | 113C −0.09 | 13H 0.19 | 63H 0.08 | 113C 0.04 |
14H 0.09 | 64H 0.09 | 114C −0.10 | 14H 0.13 | 64H 0.12 | 114C 0.39 |
15H 0.11 | 65H 0.26 | 115C 0.00 | 15H 0.09 | 65H 0.08 | 115O −0.49 |
16H 0.14 | 66N −0.38 | 116H 0.11 | 16H 0.10 | 66H 0.08 | 116O −0.49 |
17H 0.12 | 67C −0.08 | 117H 0.12 | 17H 0.13 | 67H 0.10 | 117O −0.37 |
18H 0.14 | 68C 0.23 | 118H 0.17 | 18H 0.12 | 68H 0.08 | 118C −0.21 |
19H 0.14 | 69O −0.16 | 119H 0.11 | 19H 0.32 | 69H 0.10 | 119C −0.26 |
20H 0.27 | 70C −0.22 | 120H 0.13 | 20H 0.28 | 70H 0.19 | 120O −0.37 |
21H 0.25 | 71C −0.12 | 121H 0.13 | 21H 0.25 | 71N −0.45 | 121N −0.52 |
22H 0.26 | 72C −0.03 | 122H 0.15 | 22H 0.20 | 72C −0.07 | 122H 0.21 |
23H 0.24 | 73C −0.04 | 123H 0.26 | 23H 0.15 | 73C 0.19 | 123H 0.08 |
24H 0.28 | 74C −0.13 | 124H 0.15 | 24H 0.11 | 74O −0.28 | 124H 0.09 |
25N −0.49 | 75C −0.12 | 125H 0.07 | 25H 0.11 | 75C −0.21 | 125H 0.08 |
26C −0.05 | 76C 0.16 | 126H 0.09 | 26H 0.21 | 76C −0.06 | 126H 0.07 |
27C 0.03 | 77O −0.37 | 127H 0.09 | 27N −0.43 | 77C −0.04 | 127H 0.12 |
28O −0.30 | 78H 0.18 | 128H 0.09 | 28C −0.11 | 78C −0.07 | 128H 0.10 |
29C −0.24 | 79H 0.19 | 129H 0.10 | 29C 0.22 | 79C −0.12 | 129H 0.15 |
30C −0.20 | 80H 0.15 | 130H 0.08 | 30O −0.29 | 80C −0.08 | 130H 0.23 |
31C −0.27 | 81H 0.12 | 131H 0.09 | 31C −0.22 | 81C 0.13 | 131H 0.11 |
32C −0.26 | 82H 0.08 | 132H 0.09 | 32C −0.20 | 82O −0.37 | 132H 0.10 |
33H 0.16 | 83H 0.08 | 133H 0.09 | 33C −0.25 | 83H 0.24 | 133H 0.22 |
34H 0.14 | 84H 0.09 | 134H 0.11 | 34C −0.26 | 84H 0.19 | 134H 0.22 |
35H 0.10 | 85H 0.12 | 35H 0.10 | 85H 0.12 | 135H 0.08 | |
36H 0.09 | 86H 0.25 | 36H 0.20 | 86H 0.07 | 136H 0.11 | |
37H 0.11 | 87C −0.09 | 37H 0.13 | 87H 0.11 | 137H 0.09 | |
38H 0.10 | 88N −0.42 | 38H 0.08 | 88H 0.07 | 138H 0.10 | |
39H 0.09 | 89O −0.31 | 39H 0.12 | 89H 0.08 | ||
40H 0.10 | 90C −0.08 | 40H 0.09 | 90H 0.09 | ||
41H 0.10 | 91O −0.39 | 41H 0.11 | 91H 0.12 | ||
42H 0.10 | 92C −0.10 | 42H 0.10 | 92H 0.14 | ||
43H 0.10 | 93O −0.35 | 43H 0.18 | 93H 0.19 | ||
45N −0.39 | 94C 0.18 | 44H 0.11 | 94C −0.07 | ||
46C −0.08 | 95O −0.37 | 45H 0.10 | 95C −0.08 | ||
47C 0.24 | 96C −0.19 | 46H 0.10 | 96C −0.06 | ||
48O −0.16 | 97C 0.00 | 47H 0.10 | 97C 0.10 | ||
49C −0.20 | 98C 0.39 | 48N −0.44 | 98C −0.15 | ||
50C −0.15 | 99C −0.25 | 49C −0.06 | 99C 0.20 | ||
100C 0.36 | 50C 0.19 | 100N −0.39 |
The global reactivity descriptors [
The calculated global reactivity properties of the coumarin derivative molecule.
Global reactivity descriptors | DFT (SP) of the coumarin derivative | DFT (SP) of the cocrystal drug |
---|---|---|
Active site | Active site | |
Energy (eV) | Energy (eV) | |
Band gap of LUMO-HOMO | 4.857 | 4.825 |
HOMO energy | −6.501 | −7.245 |
LUMO energy | −1.644 | −2.422 |
Ionization potential |
6.501 | 7.245 |
Electron affinity |
1.644 | 2.422 |
Global hardness |
4.857 | 4.823 |
Electrophilicity |
6.829 | 9.688 |
Electronegativity |
8.145 | 9.667 |
The electrostatic potential countermap of the (a) coumarin derivative and (b) cofactor molecules with active site amino acids of HSA.
Fluorescence anisotropy is widely used to measure the binding constants and kinetics of reactions that cause a change in the rotational time of the fluorescent molecules [
Anisotropy studies of the coumarin derivative with the HSA complex.
The Forster energy transfer method is used to understand the energy distance between the donor and acceptor of biomolecules. The overlap absorbance spectrum of the drug and emission spectrum of human serum albumin are shown in Figure
The distance between HSA and coumarin derivative.
The energy transfer efficiency (
The cytotoxicity of a coumarin derivative, which was used to evaluate its biocompatibility, was studied using the HeLa cell as a model cell by the CCK-8. Figure
Cytotoxicity studies of the coumarin derivative.
In this context, the synthesized coumarin derivative molecules have been subjected to various spectroscopy and computational techniques for understanding the interaction mechanism in the HSA molecule. From the UV-Vis absorbance result, it can be seen that the HSA-coumarin derivative complex may be due to the ground state complex; further fluorescence technique reveals that the coumarin derivative in the HSA complex is in the static quenching mode, and also, the thermal parameters calculated show good binding results. Time-resolved emission spectroscopy results show that the HSA molecule could be able to carry the coumarin derivative, and the calculated average lifetime value found for free HSA is 4.83 ns and for the HSA-coumarin derivative complex is 4.67 ns. Energy transfer analysis also calculated between donor and acceptor and
The data used to support the findings of this study are available from the corresponding author upon request.
The authors declare that they have no conflicts of interest.