Determination of modes of interactions of lanosterol with bovine serum albumin (BSA) using different spectroscopic techniques and molecular docking

doi:10.21203/rs.3.rs-3890410/v1

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Determination of modes of interactions of lanosterol with bovine serum albumin (BSA) using different spectroscopic techniques and molecular docking

https://doi.org/10.21203/rs.3.rs-3890410/v1

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Graphical abstract Abstract Lanosterol is a natural steroidal molecule which is used as an anti-cataract agent. In the present work, binding interactions of lanosterol with bovine serum albumin (BSA) were determined with different spectroscopic techniques, including UV-spectrophotometry, fluorimetry, circular dichroism spectroscopy, nanoDSF, and molecular docking. UV absorption spectroscopy showed the formation of a ground-state complex between lanosterol and bovine serum albumin (BSA). Fluorimetric analysis showed that lanosterol quenched the intrinsic fluorescence of BSA through a static quenching mechanism with a binding constant of 6.19×107 M-1 at 297 K. Thermodynamic parameters showed that the reaction was spontaneous, and the main interacting forces of this complex were found to be hydrophobic. Circular dichroism showed stable changes in the secondary structural contents of BSA while binding with lanosterol. This indicated the conformational changes in the structure of the protein during the binding of this compound. NanoDSF studies showed an increase in protein stability in the presence of 9.75 µM to 78 µM concentration of lanosterol. Docking studies showed the binding of lanosterol in site I through hydrophobic interactions. Since no information is available so far regarding binding studies between lanosterol and BSA, this study may provide initial insights about lanosterol-BSA interactions that can be used for further to investigate pharmacological properties.

Lanosterol

Anti-cataract agent

Bovine serum albumin

Quenching

Circular dichroism

Over 50 percent of the blindness cases worldwide are caused by cataracts, and the only proven cure is the surgical removal of the clouded lens. Furthermore, cataracts have a significant morbidity in developing countries where the availability of surgical care is poor [1]. The most significant cause of cataract development is the aggregation of protein. The α-, β- and γ-crystallin make up the crystallin superfamily of proteins [2], whose high concentration in lens fibers contributes to the transparency and refractive qualities [3]. Any mutation in these crystallin proteins results in protein aggregation which may be responsible for congenital cataracts and age-related cataracts [4].

Lanosterol is an organic steroid that helps to clear up cataracts in the eyes. The first report of this discovery was published in July 2015. The intermediary in the manufacture of cholesterol, lanosterol is found in non-saponified lanolin and is produced by lanosterol synthase (LSS) [5]. In vitro and intracellular investigations on human genetics reveal its ability to solubilize the crystallin clumps. It has also been shown to restore lens clarity in dogs and rabbits [6]. Lanosterol prevents the aggregation of human crystallin by attaching to the C-terminus region of D-crystallin proteins at the interface of hydrophobic dimerization. Lanosterol is also suggested to increase proteasome activity and aid in the removal of aggregated or misfolded proteins [5, 7]. Additionally, oxidative damage-induced UV-B exposure can denature crystallin and cause apoptosis of lens epithelium, and lanosterol can act as a protective agent during these early stages [8].

Any drug which is transported through blood regardless of the method of administration, can bind to several blood components. The albumins being the most significant due to their small size and high concentrations (35–50 mg/mL) [9]. The two serum albumins that are most crucial for binding various active compounds are human serum albumin (HSA) and bovine serum albumin (BSA). HSA and BSA are highly similar to one another, sharing 76% of the tertiary structure and 80% of the sequence. They each weigh about 66 kDa. Because it is readily available and inexpensive, BSA is typically utilized for binding investigations [10]. The two primary binding sites for both HSA and BSA are found in subdomains IIA (site I) and IIIA (site II). Binding at site I is primarily mediated by hydrophobic interactions, while at site II is mediated by a mix of hydrophobic, hydrogen-binding, and electrostatic interactions [11].

Serum albumin is the major plasma protein found in humans as well as in other mammals [12]. It is an α-helical non-glycosylated protein responsible for maintaining the colloid osmotic pressure of the plasma and transporting and storing many endogenous as well as exogenous molecules [13]. Drug pharmacokinetics, as well as pharmacodynamics, can be affected by binding to a plasma protein because when a drug is absorbed into the blood, it binds to varying degrees to the plasma proteins through noncovalent interactions between them, and these interactions are the results of their physicochemical and structural properties. The equilibrium is established between the concentration of free and bound fraction of a drug [14] which affects its distribution to the site of action and thus can alter the duration as well as intensity of its response within the body [15]. Therefore, it is crucial to investigate how the drug binds to serum albumin before being administered as a medication.

In the present work, binding interactions between lanosterol (an anti-cataract agent) and bovine serum albumin (BSA) were determined utilizing various spectroscopic techniques, including UV-spectrophotometry, fluorimetry, circular dichroism spectroscopy, and nanoDSF. At low concentrations, UV-Vis and fluorescence spectroscopy can offer both quantitative and qualitative data about a potential mechanism of interaction between the drug and protein [16]. Serum albumins have maximum absorption peak wavelengths around 280 nm. When excited at 280 nm, the intrinsic fluorescence of serum albumins is visible at 340 nm which is caused by three aromatic residues, tryptophan (Trp), tyrosine (Tyr), and phenylalanine (Phe). It is a very effective method for analyzing protein-drug interactions because the intrinsic fluorescence properties are highly sensitive to changes in the local environment of the serum albumins or the microenvironment of fluorescent residues, such as biomolecular binding, denaturation, and conformational transition [17]. Circular dichroism (CD) is the most useful absorption spectroscopic tool to examine the conformational changes of biomolecules, particularly proteins and polypeptides. Protein secondary structure, such as α-helixes, β-sheets, random coils, and unordered structure, is often quantified by CD spectroscopy by using far-UV range (i.e., 190–250 nm) [18]. Furthermore, for the investigating the thermal stability of BSA-lanosterol complex, nano differential scanning fluorimetry (nanoDSF), has been used [19]. It is possible to detect changes in dual-UV intrinsic fluorescence caused by the thermal unfolding of proteins in limited volume with great precision using nanoDSF. Molecular docking makes use of the atomic-level binding mechanisms of small or large molecules that encounter protein [20] and used to further validate spectroscopic results [21]. Based on UV spectra, fluorescence spectra, and CD spectra, the conformational changes of proteins were monitored. This study will give an insight of molecular interactions and binding properties of lanosterol with BSA. Furthermore, the findings of these studies may offer a new knowledge regarding the structural properties of pharmaceuticals that use BSA as a drug carrier which affect their dosage forms and therapeutic efficacy.

2.1. Materials

Bovine serum albumin (BSA, Cat. No. P6154), was purchased as a lyophilized powder, from Biowest. Lanosterol (Cat. No. 102083) was purchased from MP Biomedical, Inc. France. All other chemicals were obtained from Sigma Aldrich. Stock solution of BSA (30 µM) was freshly prepared in 20 mM Tris-HCl buffer of pH 7.4 containing 100 mM NaCl. Stock solutions (10 mM) of lanosterol were prepared in 20 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl and stored at 4°C. The stock solutions were further diluted with buffer to make working solutions of the required concentration. For CD spectroscopic analysis, all solutions were prepared in deionized water.

2.2. Methods

2.2.1. UV-vis spectroscopy

Thermo Scientific Multiskan-GO spectrophotometer was used to measure UV-vis absorption spectra of BSA (15 µM) in the presence of lanosterol (0 µM, 7.5 µM, 15 µM, and 50 µM) over the wavelength range of 200–400 nm at 297 K. SkanIt software was used to analyze the spectra, using 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl buffer as blank.

2.2.2. Fluorescence quenching study

Varioskan Lux Microplate Reader (VLMR) with a slit width of 5 nm was used to measure the intrinsic fluorescent spectra of BSA (15 µM) in the absence and presence of lanosterol (0 µM to 50 µM) at different temperatures (297 K, 304 K, and 311 K) by using 96 well standard fluorescent plates, to investigate the binding of lanosterol with BSA through fluorescence quenching. The protein was excited at 280 nm and each emission spectrum was recorded at a selected wavelength range of 300–450 nm. SkanIt software was used to analyze the spectra, using the 50 mM Tris-HCl buffer (pH 7.4) containing 100 mM NaCl buffer as a blank solution. BSA would not experience any structural degradation due to the narrow temperature range of fluorescence experiments, hence the enthalpy change of the lanosterol-BSA interaction can be considered as constant [22]. The data were further analyzed using Stern-Volmer equation to explain the quenching mechanism [23].

$$\frac{{\text{F}}_{0}}{\text{F}}+{K}_{SV }\left[\text{Q}\right]=1+Kq{\tau }_{0} \left[\text{Q}\right]$$

where F and F₀ represent the fluorescence intensities of BSA with and without quencher i.e., lanosterol, K_SV represents Stern-Volmer constant, τ₀ represents average lifetime of the fluorophore without quencher (10^− 8 s for BSA) [24], K_q is the biomolecular quenching rate constant, and [Q] is the quencher concentration. The values of F₀ / F against [Q] were plotted, and K_SV was calculated from slope of the equation by applying linear regression fit using GraphPad Prism. K_q was calculated using following equation.

$${K}_{q}= \frac{{K}_{SV}}{{\tau }_{0}}$$

The binding parameters such as binding constant (K_b) and number of binding sites (n) for the lanosterol-BSA complex were determined using the double logarithm equation assuming that independent binding sites are present in BSA [25].

$$\frac{{log} ({F}_{0} -F)}{F} =log{K}_{b}+n{log} \left[Q\right]$$

where logK_b represents the binding constant and n represents the number of binding sites per BSA molecule. The values of log (F₀-F) / F against log [Q] were plotted, and K_b and n were obtained from the intercept and slope of the equation by applying linear regression fit by using GraphPad Prism.

Thermodynamic parameters (enthalpy change (ΔH^ο), entropy change (ΔS^ο), and Gibbs free energy change (ΔG) were calculated using Van't Hoff equation based on binding constant K_b at different temperatures.

$$ln{K}_{b}= -\frac{\varDelta \text{H}}{\text{R}\text{T}} + \frac{\varDelta \text{S}}{\text{T}}$$

$$\varDelta \text{G}={\text{R}\text{T}lnK}_{b}$$

where K_b represents binding constant at three different temperatures (T) of 297K, 304K, and 311K and R represents the universal gas constant (R = 8.314 J/mol). The values of lnK_b against 1/T were plotted to calculate enthalpy change (ΔH^ο) and entropy change (ΔS^ο) and determined from slope and intercept of the equation through applying linear regression fit by using GraphPad Prism. ΔG was calculated using (equation no.5).

2.2.3. Circular dichroism

CD spectra of BSA (1 µM) in the absence and presence of lanosterol (10 µM and 20 µM) were recorded using the Jasco Spectropolarimeter (J810) in a quartz cell of 1 mm path length. The spectra were recorded in the far-UV (190–250 nm) region at room temperature under constant nitrogen flush with scan rate of 50 nm/min and step resolution of 0.5 nm. Jasco's Spectra Manager™ software was used to control the instrument using deionized water as blank solution. Online Bestsel software was used to estimate the percentage of secondary structural elements.

2.2.4. Nano differential scanning fluorimetry (NanoDSF)

Thermal shift assay was done on the Promenthus NT.48 nano DSF instrument (Nano Temper Technologies GmbH, Munchen, Germany) in triplicates. Sample solutions of BSA (5 µM) in a buffer (50 mM Tris-HCl pH 7.5) and in the presence of lanosterol (0, 4.8, 9.7, 19.5, 78, and 156 µM) were allowed to heat from 20°C to 90°C in the capillaries at heating rate of 1.5 ◦C/min. Protein was excited at 280 nm and the fluorescence emission intensity was monitored at 330 nm and 350 nm. The measured fluorescence ratio 350 nm / 330 nm and its first derivative was plotted as a function of temperature. The melting temperatures were obtained through instrument manufacturer provided software. All spectra and graphs were plotted using GraphPad Prism software.

2.2.5. Molecular docking

Docking study was carried out to investigate the interaction between lanosterol and BSA using AutoDock software 4.2 to predict the binding modes of lanosterol to the main drug binding sites (site I and site II). From the Protein Data Bank (PDB), the crystal structure of BSA (6QS9) was obtained. Three-dimensional structure of lanosterol was obtained as PDB file format from UCSF Chimera software using smiles string. Protein and ligand files were then optimized using MGL tools. For ligand and protein preparation, loading of ligand file and protein file in PDB format was done individually using MGL tools, and docking parameters were then optimized. Individual output files of ligand and of protein were saved in PDBQT file format and run the docking. Analysis of results were done using MGL tools. LigPlot + and UCSF Chimera were used to study interactions of BSA-lanosterol complex.

3.1. Effect of lanosterol on the UV absorption spectra of BSA

UV absorption spectroscopy was used to investigate ground state complex formation of protein with small molecules. UV absorption spectra of BSA in the presence and absence of lanosterol is shown in Fig. 1. UV-vis spectra showed that absorption maxima of BSA was found at 280nm. The aromatic amino acids Trp, Tyr, and Phe are responsible for the peak that BSA exhibits near 280 nm due to π–π*transition [16, 24]. This figure showed that absorbance intensity of the BSA at 280 nm started to increase with the successive addition of lanosterol. This suggested the ground state complex formation of BSA-lanosterol which causes a change in BSA structure around aromatic residues when interacted with lanosterol.

3.2. Fluorescence spectroscopic studies

3.2.1. Fluorescence quenching mechanism of BSA

Fluorescence spectroscopy was used to study the binding interactions of BSA with lanosterol through intrinsic fluorescence quenching mechanism. Aromatic amino acids (phenylalanine, tyrosine, and tryptophan) contribute to the intrinsic fluorescence of proteins. The majority of these residues that contribute to intrinsic fluorescence are the Trp and Tyr residues [26]. Interactions between small molecules and protein results in reduction in the intensity of protein fluorescence. This phenomenon is known as fluorescence quenching [27]. Fluorescence spectra of BSA with increasing concentration of lanosterol is shown in Fig. 2. Fluorescence spectra showed that maximum fluorescence emission of BSA was appeared at 340 nm, while lanosterol did not show any fluorescence. This figure showed that intrinsic fluorescence of BSA was quenched with successive addition of lanosterol. The isobestic point was found to be at 450 nm demonstrating the stable complex formation between BSA and lanosterol. These findings suggested that lanosterol and BSA had a significant interaction.

Fluorescence quenching can be either dynamic, static or a hybrid of the two [28] and can result via the creation of ground state complexes, collision, or energy transfer [29]. Dynamic quenching utilizes diffusion-controlled collisions between an excited fluorophore and a quencher that do not result in photon emission, while a non-fluorescent ground-state complex is formed between the fluorophore and the quencher in static quenching [30]. Molecular contact is required for both mechanisms. The quenching constants are supposed to increase in dynamic quenching and decrease in static quenching with increasing temperature [23].

The estimated Stern-Volmer constant K_SV and biomolecular quenching rate constant K_q at three different temperatures using Stern-Volmer plots are shown in Table 1. The estimated value of K_SV decreased with increasing temperature and K_q value was greater than the maximum scattering collision quenching constant (2 × 10¹⁰ M^− 1 s^− 1) showing that quenching was mediated through static process.

Table 1

Stern-Volmer quenching constants and biomolecular quenching rate constants of BSA-lanosterol interaction at three different temperatures.
Temperature (K)	K_SV ± SD×10⁴ (L mol^− 1)	K_q×10¹² (L mol^− 1s^− 1)	R²
297	1.2917 ± 0.01	1.2917	0.9950
304	0.9337 ± 0.01	0.9337	0.9919
311	0.6976 ± 0.01	0.6976	0.9865

3.2.2. Binding parameters of lanosterol-BSA complexes

The values of estimated binding constant (K_b) and number of binding sites n are presented in Table 2, which were obtained using the double logarithm plot shown in Fig. 3. Table 2 showed that values of n were very close to 1 at all three temperatures which indicated the presence of single binding site of lanosterol on BSA. Estimated values of K_b were in the order of 10⁷ M^− 1 indicating the significant binding between lanosterol and BSA. Values of K_b were shown to increase might be due to the enhanced stability of complex with rise in temperature [31].

Table 2

Binding constants and the relative number of binding sites for the BSA-lanosterol interaction at three different temperatures.
Temperature (K)	K_b ± SD×10⁷(L mol^-1)	n	R²
297	6.19 ± 0.07	0.83	0.9894
304	15.41 ± 0.13	0.91	0.9711
311	13.89 ± 0.08	1.03	0.9914

3.2.3. Binding model and thermodynamic parameters

Hydrogen bonding, electrostatic, hydrophobic, and van der Waals interactions are the main interacting forces involved in the protein-drug binding process. These interactions can be confirmed significantly based on magnitudes and signs of enthalpy change (ΔH₀), entropy change (ΔS₀), and Gibbs free energy change (ΔG₀). These can be interpreted as: if ΔH and ΔS are both greater than zero (+), hydrophobic force is the main force of interaction. If ΔH and ΔS are both less than zero (-), hydrogen bond and the van der Waals force are the main force of interaction. If ΔS is greater than zero (+) and ΔH is almost zero, electrostatic interaction is the main force of interaction [25]. Binding parameters for the binding interaction and energetics of the reaction are presented in Table 3, which were obtained using the Van't Hoff's plot (Fig. 4). Table 3 showed that ΔH and ΔS were both greater than zero (+). According to the Ross theory [32], the positive ΔH and ΔS values of the interaction of lanosterol and BSA implied that the binding was mostly entropy-driven, and that the enthalpy was unfavorable for it [33, 34], and that the hydrophobic forces played an important part in the interaction indicating that the hydrophobic force was the main interacting force in lanosterol-BSA binding process. Furthermore, the negative value of ΔG demonstrated that the binding process was spontaneous.

Temperature (K)	ΔG⁰ (kJ mol^− 1)	ΔS⁰ (kJ mol^− 1)	ΔH⁰ (kJ mol^− 1)
297	-19.3052	333.05	80.13
304	-20.6948
311	-23.6406

3.3. Effect of lanosterol binding with BSA on CD spectra

Secondary structural elements of any globular proteins are α-helix, β-sheet, unordered fraction and turns. According to the literature, the secondary structure of BSA is dominated by -helix motifs [16, 35]. Circular dichroism spectroscopy was used to measure the change in secondary structural elements of BSA interacting with varying concentration of lanosterol. CD spectra in the absence and presence of different concentration of lanosterol are presented in Fig. 5, and percentage of their secondary structural elements are presented in Table 4. Figure 5 showed that there were two negative bands in far UV region at 210 and 223 nm for BSA in the absence of lanosterol confirming an alpha-helical nature of the protein and is explained by the n → π* transition in the peptide bond along the α-helixes [36]. Decreased intensities of both negative bands with successive addition of lanosterol indicating a loss in α -helical content, while the other structure contents of the protein were increased. It is possible that this is related to the complex formation between BSA and lanosterol. Table 4 showed reduction in α-helical structural content and increased percent of other structural parameters of BSA in the presence of increased concentration of lanosterol. As the pattern of the spectra remained constant confirming the stability of protein. These findings offered preliminary evidence for a small change in BSA structure, which was compatible with fluorescence and UV-vis absorbance data.

Table 4

Secondary structural elements of BSA in the absence and presence of varied molar ratio of BSA:lanosterol (1:10 and 1:20).
Protein + drug	Alpha-Helix (% )	Antiparallel ( % )	Parallel ( % )	Turn ( % )	Others ( % )
BSA	35.8	3.9	24.3	1.1	34.9
BSA + Lanosterol (1:10)	30.2	8.6	24.9	0.3	37.7
BSA + Lanosterol (1:20)	27.2	10.4	26.6	0.8	35

3.3. Effect of lanosterol on thermal stability of BSA

Tryptophan (Trp) fluorescence is the primary source of intrinsic protein fluorescence. Fluorescence of tryptophan is highly dependent on the polarity of its microenvironment. When Trp is exposed to a non-polar environment, it fluoresces at 330 nm, and when it is exposed to a polar environment, it fluoresces at 350 nm upon excitation at 280 nm. When a protein unfolds, Trp residues that are typically buried in the hydrophobic core of the protein are exposed to water, which causes their fluorescence to decrease and their emission maximum to move to longer wavelengths. Protein melting temperature (Tm) can thus be determined in a dye-free manner by monitoring variations in Trp fluorescence intensity. NanoDSF employs this strategy [37]. Melting temperatures (Tm) and temperature at which unfolding begins (T_onset) of BSA in the absence and presence of different lanosterol concentration was used to assess the effect of lanosterol on thermal stability of the protein (Table 5). Tm, which is determined from the thermal denaturation profiles, is the temperature at which the protein coexists in both its folded and unfolded states at equilibrium. Changes in Tm can reveal information about the interactions between proteins and drug and signify modification in protein stability [38]. For nanoDSF, the intensity ratio of the fluorescence at 350 nm/330 nm is measured. The thermal transition signals can be either positive or negative. When buried tryptophan residues are unfold and exposed to a buffer, they cause a red shift. This means that when tryptophan (Trp) residues are exposed to more polar settings, their maximal emission intensity moves to longer wavelengths, producing positive thermal transition signals in thermograms. Conversely, tryptophans exposed to the surface exhibit either a blue shift or no shift upon unfolding, meaning that when residues are less exposed to polar environments or there is little change in the polar environments, maximum emission intensity shifts to shorter wavelengths or does not shift, leading to negative thermal transition signals and no signals in the nanoDSF thermograms [19]. Fluorescence signal ratio of 350 nm/330 nm and its first derivative in the presence and absence of lanosterol is shown in Fig. 6. It showed two transition temperature of unfolding (Tm₁) and (Tm₂). The first is negative transition thermal signal and the second is of positive transition thermal signal. These two transition signals may be due to the presence of two tryptophan residues in two different domains and hence exposed to different microenvironment.

Table 5 showed Tm₁ decreased initially at low concentration of lanosterol (4.875 µM and 9.75 µM) showing destabilizing effect but increased at moderate concentration of lanosterol (19.5 µM and 78 µM) showing stabilizing effect. Further increment in lanosterol concentration to 156 µM caused Tm₁ to decrease. This shows that protein was thermally unstable at very low and at very high concentration of lanosterol but stable at moderate concentration of the drug. While Tm₂ decreased only at low concentration 4.875 µM but increased at all moderate and higher concentration from 9.75 µM to 156 µM showing stabilizing effect of the drug. T_onset also showed increased thermal stability of BSA in the presence of lanosterol concentration from 9.75 µM to 156 µM.

Table 5

Melting temperatures (Tm₁ and Tm₂) and T_onset of BSA in the presence and absence of lanosterol
Lanosterol conc. (µM)	Tm₁	Tm₂	T_onset
0	56.17	78.84	22.18
4.875	40.43	53.36	20.0
9.75	51.66	89.59	39.53
19.5	65.15	86.28	39.54
78	71.85	82.16	74.12
156	49.65	85.55	27.64

3.4. Molecular docking

Hydrophobic forces were the primary forces stabilizing the BSA-lanosterol complex with 1:1 binding stoichiometry showed by the thermodynamic study. Molecular docking was used to further validate the theoretical computations in this investigation. Docking was done to predict the binding modes of lanosterol to the primary ligand binding sites of BSA. Docking study of the BSA-lanosterol complex at the binding site I identified eight distinct conformational clusters. As cluster 1 had the lowest binding energy of ‒10 kcal·mol − 1 (Table 6), and hence this cluster was selected as the docking result (Fig. 7). Figure 8 shows the presence of lanosterol in the binding site I. It showed the hydrophobic interactions between lanosterol and hydrophobic amino acid residues including PHE 205, ARG 208, ALA 209, ALA 212, ASP-323, LEU 326, GLY-327, LEU 330, LEU 346, ALA 349, LYS 350, LEU 480 and SER-479.Figure 8. Hydrophobic interactions between lanosterol and amino acid residues of binding site I. (A) showing 3-D view, and (B) showing 2-D view

Table 6 Molecular docking results of BSA-lanosterol complex

Cluster Rank	Binding Energy	Cluster size
1	‒ 10.00	1
2	‒ 9.99	3
3	‒ 9.09	2
4	‒ 8.55	1
5	‒ 8.28	1
6	‒ 8.19	1
7	‒ 6.09	1

As binding affinity of a drug to serum albumin determine overall drug pharmacokinetics. This study was conducted to reveal important information regarding magnitude and types of binding interactions involved in the formation of lanosterol-BSA complex as well as change in secondary structural components in vitro, by using combination of different spectroscopic methods. UV absorption spectroscopy showed the formation of a ground-state complex. Fluorimetric analysis showed significant binding strength between lanosterol and BSA as shown by 10⁷ orders of binding constant (K_b) with binding stoichiometry of 1:1, and the mechanism of fluorescence quenching was found to be static process. Main binding force of interaction was found to be hydrophobic interactions. Docking studies further confirmed the binding of lanosterol in drug binding site I through hydrophobic interactions. CD analysis showed slightly unfolded but stable BSA structure upon binding with lanosterol and loss in alpha-helicity was observed while binding with the drug. NanoDSF studies showed that thermal stability of BSA was increased in the presence of 9.75 µM to 78 µM concentration of lanosterol. The biological importance of this finding is evident because albumin functions as a drug carrier molecule and the binding interactions of lanosterol with albumin have not been characterized so far. As a result, this report is significant in drug design methodology and drug pharmacology.

Author Contribution

Dr. Malik Shoaib Ahmad gave the idea and supervised the research. Sahar Khursheed has performed an experimental work of the research. Rameen Raza and Zeeshan Akbar helps in preparing the manuscript of the research.

Zhao L, Chen XJ, Zhu J, Xi YB, Yang X, Hu LD, Ouyang H, Patel SH, Jin X, Lin D, Wu F, Flagg K, Cai H, Li G, Cao G, Lin Y, Chen D, Wen C, Chung C, Wang Y, Qiu A, Yeh E, Wang W, Hu X, Grob S, Abagyan R, Su Z, Tjondro HC, Zhao XJ, Luo H, Hou R, Jefferson J, Perry P, Gao W, Kozak I, Granet D, Li Y, Sun X, Wang J, Zhang L, Liu Y (2015) Yan, and K. Zhang. Lanosterol reverses protein aggregation in cataracts. Nature 523(7562):607–611
Moreau KL, King JA (2012) Protein misfolding and aggregation in cataract disease and prospects for prevention. Trends Mol Med 18(5):273–282
Bloemendal H, de Jong W, Jaenicke R, Lubsen NH, Slingsby C, Tardieu A (2004) Ageing and vision: structure, stability and function of lens crystallins. Prog Biophys Mol Biol 86(3):407–485
Jing X, Zhu M, Lu X, Wei P, Shi L, Zhang BY, Xu Y, Tang YP, Xiang DM (2023) Gong. Cataract-causing Y204X mutation of crystallin protein CRYβB1 promotes its C-terminal degradation and higher-order oligomerization. J Biol Chem 299(8):104953
Kang H, Yang Z (2018) Lanosterol Disrupts Aggregation of Human γD-Crystallin by Binding to the Hydrophobic Dimerization Interface. 140(27): p. 8479–8486
Nagai N, Fukuoka Y, Sato K (2020) The Intravitreal Injection of Lanosterol Nanoparticles Rescues Lens Structure Collapse at an Early Stage in Shumiya Cataract Rats. 21(3)
Hu L-D, Wang J, Chen X-J (2020) Yan. Lanosterol modulates proteostasis via dissolving cytosolic sequestosomes/aggresome-like induced structures. Biochim et Biophys acta Mol cell Res 1867(2):118617
Hua H, Yang T, Huang L, Chen R, Li M, Zou Z, Wang N, Yang D, Liu Y (2019) Protective Effects of Lanosterol Synthase Up-Regulation in UV-B-Induced Oxidative Stress. Front Pharmacol 10:947
Kratz F, Elsadek B (2012) Clinical impact of serum proteins on drug delivery. J Control Release 161(2):429–445
Carter DC, He XM (1990) Structure of human serum albumin. Science 249(4966):302–303
Dufour C, Dangles O (2005) Flavonoid–serum albumin complexation: determination of binding constants and binding sites by fluorescence spectroscopy. Biochimica et Biophysica Acta (BBA) - General Subjects, 1721(1): p. 164–173
Neumann E, Frei E, Funk D, Becker MD, Schrenk HH, Müller-Ladner U (2010) Fiehn. Native albumin for targeted drug delivery. Expert Opin Drug Deliv 7(8):915–925
Barbero N, Barni E, Barolo C, Quagliotto P, Viscardi G, Napione L, Pavan S, Bussolino F (2009) A study of the interaction between fluorescein sodium salt and bovine serum albumin by steady-state fluorescence. Dyes Pigm 80(3):307–313
Seyfinejad B, Ozkan SA, Jouyban A (2021) Recent advances in the determination of unbound concentration and plasma protein binding of drugs. Anal methods Talanta 225:122052
Kratz F (2008) Albumin as a drug carrier: design of prodrugs, drug conjugates and nanoparticles. J Control Release 132(3):171–183
Negrea E, Oancea P, Leonties A, Ana Maria U, Avram S, Raducan A (2023) Spectroscopic studies on binding of ibuprofen and drotaverine with bovine serum albumin. J Photochem Photobiol A 438:114512
Naveenraj S, Anandan S (2013) Binding of serum albumins with bioactive substances – Nanoparticles to drugs. J Photochem Photobiol C 14:53–71
Chaturvedi SK, Siddiqi MK, Alam P, Khan RH (2016) Protein misfolding and aggregation: Mechanism, factors and detection. Process Biochem 51(9):1183–1192
Wen J, Lord H, Knutson N, Wikström M (2020) Nano differential scanning fluorimetry for comparability studies of therapeutic proteins. Anal Biochem 593:113581
Singh S, Bani Baker Q, Singh DB (2022) Chapter 18 - Molecular docking and molecular dynamics simulation, in Bioinformatics, D.B. Singh and R.K. Pathak, Editors. Academic Press. p. 291–304
Gu J, Liu H, Huang X, Ma Y, Zhang L (2023) Investigation of the separate and simultaneous bindings of warfarin and fenofibrate to bovine serum albumin. Int J Biol Macromol 236:123978
Ying M, Huang F, Ye H, Xu H, Shen L, Huan T, Huang S, Xie J, Tian S, Hu Z, He Z, Lu J (2015) Zhou. Study on interaction between curcumin and pepsin by spectroscopic and docking methods. Int J Biol Macromol 79:201–208
Wu J, Bi S-Y, Sun X-Y, Zhao R, Wang J-H (2019) Zhou. Study on the interaction of fisetholz with BSA/HSA by multi-spectroscopic, cyclic voltammetric, and molecular docking technique. J Biomol Struct Dynamics 37(13):3496–3505
Wang P-Y, Yang C-T, Chu L-K (2021) Differentiating the protein dynamics using fluorescence evolution of tryptophan residue(s): A comparative study of bovine and human serum albumins upon temperature jump. Chem Phys Lett 781:138998
Shi J-H, Wang Q, Pan D-Q, Liu T-T, Jiang M (2017) Characterization of interactions of simvastatin, pravastatin, fluvastatin, and pitavastatin with bovine serum albumin: multiple spectroscopic and molecular docking. J Biomol Struct Dynamics 35(7):1529–1546
Zhang R-J, Kou S-B, Hu L, Li L, Shi J-H, Jiang S-L (2022) Exploring binding interaction of baricitinib with bovine serum albumin (BSA): multi-spectroscopic approaches combined with theoretical calculation. J Mol Liq 354:118831
M M. and R. H.D. Interpretation of the binding interaction between bupropion hydrochloride with human serum albumin: A collective spectroscopic and computational approach. Spectrochim Acta Part A Mol Biomol Spectrosc, (2019) 209: p. 264–273
Kou S-B, Lin Z-Y, Wang B-L, Shi J-H, Liu Y-X (2021) Evaluation of the binding behavior of olmutinib (HM61713) with model transport protein: Insights from spectroscopic and molecular docking studies. J Mol Struct 1224:129024
Jameson DM (2014) Introduction to fluorescence. Taylor & Francis
Hudson EA, de Paula HMC, Ferreira GMD, Ferreira GMD, Hespanhol MdC, da Silva LHM (2018) d.S. Pires. Thermodynamic and kinetic analyses of curcumin and bovine serum albumin binding. Food Chem 242:505–512
Huang F, Chen C (2023) Insights into the interaction between the kusaginin and bovine serum albumin: Multi-spectroscopic techniques and computational approaches. J Mol Recognit 36(3):e3003
Ross PD, Subramanian S (1981) Thermodynamics of protein association reactions: forces contributing to stability. Biochemistry 20(11):3096–3102
Hu Y-J, Liu Y, Shen X-S, Fang X-Y (2005) Qu. Studies on the interaction between 1-hexylcarbamoyl-5-fluorouracil and bovine serum albumin. J Mol Struct 738(1):143–147
Zhang W, Han B, Zhao S, Ge F, Xiong X, Chen D, Liu D, Chen C (2010) Study on the Interaction between Theasinesin and Bovine Serum Albumin by Fluorescence Method. Anal Lett 43(2):289–299
Iovescu A, Băran A, Stîngă G, Cantemir-Leontieş AR, Maxim ME, Anghel DF (2015) A combined binding mechanism of nonionic ethoxylated surfactants to bovine serum albumin revealed by fluorescence and circular dichroism. J Photochem Photobiol B 153:198–205
Saranya V, Mary PV, Vijayakumar S, Shankar R (2020) The hazardous effects of the environmental toxic gases on amyloid beta-peptide aggregation: A theoretical perspective. Biophys Chem 263:106394
NT-PR A.N. F, Bleffert V, Misetic M, Jerabek K-E, Jaeger, Kovacic F Thermal stability of membrane-bound proteins
Alves MMS, Araújo JMM, Martins IC, Pereiro AB (2021) Archer. Insights into the interaction of Bovine Serum Albumin with Surface-Active Ionic Liquids in aqueous solution. J Mol Liq 322:114537

No competing interests reported.

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Determination of modes of interactions of lanosterol with bovine serum albumin (BSA) using different spectroscopic techniques and molecular docking

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1. Materials

2.2. Methods

2.2.1. UV-vis spectroscopy

2.2.2. Fluorescence quenching study

2.2.3. Circular dichroism

2.2.4. Nano differential scanning fluorimetry (NanoDSF)

2.2.5. Molecular docking

3. Results and Discussion

3.1. Effect of lanosterol on the UV absorption spectra of BSA

3.2. Fluorescence spectroscopic studies

3.2.1. Fluorescence quenching mechanism of BSA

3.2.2. Binding parameters of lanosterol-BSA complexes

3.2.3. Binding model and thermodynamic parameters

3.3. Effect of lanosterol binding with BSA on CD spectra

3.3. Effect of lanosterol on thermal stability of BSA

3.4. Molecular docking

4. Conclusion

Declarations

Author Contribution

References

Additional Declarations

Supplementary Files

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