Tenofovir antiviral drug solubility enhancement with β-cyclodextrin inclusion complex and in silico study of potential inhibitor against SARS-CoV-2 main protease (M^pro)

Graphical abstract

Abstract

Tenofovir (TFR) is an antiviral drug commonly used to fight against viral diseases infection due to its good potency and high genetic barrier to drug resistance. In physiological conditions, TFR is less water soluble, more unstable, and less permeable, limiting its effective therapeutic applications. In addition to their use in treating the Coronavirus disease 2019 (COVID-19), cyclodextrins (CDs) are also being used as a molecule to develop therapies for other diseases due to its enhance solubility and stability. This study is designed to synthesize and characterization of β-CD:TFR inclusion complex and its interaction against SARS-CoV-2 (M^Pro) protein (PDB ID;7cam). Several techniques were used to characterize the prepared β-CD:TFR inclusion complex, including UV–Visible, FT-IR, XRD, SEM, TGA, and DSC, which provided appropriate evidence to confirm the formation. A 1:1 stoichiometry was determined for β-CD:TFR inclusion complex in aqueous medium from UV–Visible absorption spectra by using the Benesi–Hildebrand method. Phase solubility studies proposed that β-CD enhanced the excellent solubility of TFR and the stability constant was obtained at 863 ± 32 M⁻¹. Moreover, the molecular docking confirmed the experimental results demonstrated the most desirable mode of TFR encapsulated into the β-CD nanocavity via hydrophobic interactions and possible hydrogen bonds. Moreover, TFR was validated in the β-CD:TFR inclusion complex as potential inhibitors against SARS-CoV-2 main protease (M^pro) receptors by using in silico methods. The enhanced solubility, stability, and antiviral activity against SARS-CoV-2 (M^Pro) suggest that β-CD:TFR inclusion complexes can be further used as feasible water-insoluble antiviral drug carriers in viral disease infection.

1. Introduction

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus responsible for the new coronavirus disease (COVID-19) across the globe and effective treatments are in urgent need [1]. Globally, researchers are concentrating their efforts on hastening the clinical development and introduction of COVID-19 pharmacological therapies. One option for lowering COVID-19 patients is to continue using existing drugs until a vaccination has been found and is ready to help [2]. Antiviral combination treatments have been successfully used to treat COVID-19 [3]. However, formulation issues, most notably the active compound poor solubility in water, can hinder the development of antiviral drugs [4]. To ensure bioavailability and subsequently the effectiveness of oral antiviral treatments, the solubility of a drug must be adequate. Formulation issues, especially the low solubility and more instability of active chemicals in physiological conditions, might impede the development of novel drugs [5].

Tenofovir (TFV) is an antiretroviral drug, specifically a nucleotide analogue reverse transcriptase inhibitor, approved by the Food and Drug Administration and recommended by the World Health Organization for its use as a first-line drug for the treatment of HIV [6], [7], [8], [9], [10]. A [(2R)-1-(6-amino-9H-purin-9-yl)propan-2-yl]oxy group is used to substitute one of the methyl hydrogens in TFR, a methyl phosphonic acid that belongs to the same class as other phosphonic acids [6], [7], [8], [9], [10]. Additionally, it functions as an antiviral medication, a pharmacological metabolite, and an inhibitor of HIV-1 reverse transcription [7], [9]. There has not been any research on the parent drugs rather low bioavailability (25–30%) reported to date. 300 mg of TFR is given orally once each day [11]. Therefore, cyclodextrins (CDs) are an appropriate drug delivery strategy for directing antiviral drugs into infected cells because the virus localizes in these cells, improving oral bioavailability, virus therapy effectiveness, and patient compliance [12]. The virus COVID-19 belongs to have a lipid envelope that connects with the host cell by endocytosis and internalizes its parts in the cell [13]. In the fight against COVID-19, CDs, and their complexes with antiviral drug molecules are crucial [14]. CDs are cyclic oligosaccharides that are connected by seven glucose units in alpha-1,4 glycosidic linkages [15]. CDs are produced by enzymes during the starch-to-enzyme conversion process. After structural modification, CDs may be utilized as virucidal agents or for the containment of infections [16]. Complexes containing macromolecules and CDs are frequently employed for various purposes due to their versatility in shaping inclusion [17]. It can successfully function as a safe, enabling excipient for improving the solubility of antiviral drugs, stabilizing therapeutic monoclonal antibodies, and acting as an adjuvant in vaccines [12]. Natural or modified CDs have been demonstrated by Loftsson to be able to shield unstable molecules and boost their bioavailability by making them more soluble in physiological conditions [18]. During the last decade, our rsearch group have focusd mainly on nanomaterials, drug complexation properties and other applications [19], [20], [21], [22], [23], [24], [25], [26], [27], [28]. In vitro cell cultures have demonstrated that cholesterol depletion by CDs disturbs the lipid content of the host cell membrane, limiting the virus's ability to bind to protein receptors [29], [30], [31]. It has been proven that SARS-CoV-2 infections that contain the surface glycoprotein S of SARS-CoV-2 are inhibited by cholesterol depletion utilizing CDs.

Grancher et al. were successful in improving ribavirin antiviral effectiveness in recent antiviral drug tests after complexing it with CDs [32]. Finance et al. were successful in synthesizing the inclusion complex of ganciclovir with CDs, which increased the antiviral activity of ganciclovir [33]. In a recently published report, Carrouel et al. coupled mouthwash with CDs and Citrox to reduce SARS-CoV-2 salivary transmission [34]. The SARS-CoV-2 spike (S) receptor and major protease (M^pro) are attractive therapeutic targets, according to the most current studies [35], [36]. The primary protease then cut these proteins into 16 nonstructural proteins (M^pro). This scenario suggests that M^pro is crucial for viral transcription and replication. Therefore, it would be beneficial to cure COVID-19 by preventing M^pro and (S) protein from doing their functions. To date, no research has been observed on the potential of CDs as a delivery system for COVID-19 antiviral medications, particularly Tenofovir (TFR). This study will use molecular docking to evaluate the relationship between β-cyclodextrin (β-CD) and TFR and antiviral activity against SARS-CoV-2 (M^Pro).

This work purposes to the attention of researchers the essential to explore the influence of established β-CD:TFR inclusion complex against SARS-CoV-2 (M^Pro). Structural confirmation of the β-CD:TFR inclusion complexes was done by different spectroscopic and microscopic techniques such as a UV–Visible, FT-IR, XRD, SEM, TGA, and DSC and phase solubility diagram. It is well known that changes in physicochemical properties can be observed when a TFR antiviral drug molecule is incorporated inside the β-CD nanocavity. Finally, the resulting β-CD:TFR inclusion complexes were potential inhibition antiviral activities against SARS-CoV-2 (M^Pro) main protease as confirmed by in silico studies. The findings showed that β-CD complexation considerably enhanced TFR water solubility, and β-CD:TFR inclusion complex had excellent protein interaction against the SARS-CoV-2 (M^Pro) main protease.

2. Experimental section

2.1. Materials

β-cyclodextrin (purity ≥ 98%) were purchased from Merck, South Korea. Tenofovir (purity ≥ 98%) was purchased from TCI chemical company. Other reagents used were of analytical reagent grade without further purification. Deionized water was used for aqueous solutions the throughout manuscript.

2.2. Preparation of β-CD:TFR inclusion complex

Inclusion complex of β-CD:TFR was prepared by co-precipitation method comprising TFR and β-CD (1:1 M ratio) by following as previously reported [37], [38], [39], [40]. Briefly, 1.0 g of β-CD and 0.2528 g of TFR were diluted in deionized water (30 mL) and DMSO (20 mL), followed by 24 h stirring in the magnetic stirrer at room temperature. After cold precipitation, β-CD:TFR inclusion complex was obtained via vacuum-filtration. Then the mixture was refrigerated at −20 °C and freeze-dried for 24 h to obtain β-CD:TFR inclusion complex. The resulting β-CD:TFR inclusion complex powder was stored at 4 °C for further analysis.

2.3. Phase solubility studies

The phase solubility studies of β-CD:TFR inclusion complex were performed by Higuchi and Connors method. Briefly, 1.0 g of TFR was mixed with 25 mL β-CD aqueous solution containing various concentrations (0.002, 0.004, 0.006, 0.008, 0.010, and 0.012 M) in a flask. Then the conical flask containing the suspension was magnetically stirred at 100 rpm at 37 °C for 72 h to balance the dissolution, and filtered with a 0.22 μm membrane filter. The amount of TFR dissolved in each filtrate was measured by spectrophotometry with a 3220UV spectrometer (Optizen). All the experiments were carried out in triplicate. The apparent stability constant (Ks) for the β-CD:TFR inclusion complex were evaluated from the phase solubility analysis along with the following equation (1). Complexation efficiency (CE) was evaluated to determine the optimum conditions for β-CD:TFR inclusion complex, 0.9904 as it reflects the ability of β-CD to TFR and is independent of intrinsic TFR drug solubility in the following equation (2).

$${\mathrm{S}\mathrm{t}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}\mathrm{c}\mathrm{o}\mathrm{n}\mathrm{s}\mathrm{t}\mathrm{a}\mathrm{n}\mathrm{t}(\mathrm{K}}_{\mathrm{s}})=\frac{\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}}{{\mathrm{S}}_0\left(1-\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}\right)}$$ (1)

where S₀ is the solubility of the TFR drug in absence of β-CD.

$$\mathrm{C}\mathrm{o}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\mathrm{x}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{e}\mathrm{f}\mathrm{f}\mathrm{i}\mathrm{c}\mathrm{a}\mathrm{c}\mathrm{y}\left(\mathrm{C}\mathrm{E}\right)=\frac{\left[\frac{\mathrm{\beta }-\mathrm{C}\mathrm{D}}{\mathrm{T}\mathrm{F}\mathrm{R}}\right]}{\left[\mathrm{\beta }-\mathrm{C}\mathrm{D}\right]}=\frac{\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}}{1-\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{p}\mathrm{e}}$$ (2)

2.4. Statistical analysis

In this study, the experimental results were expressed as mean ± standard deviation. The mean values and standard deviation were calculated using Excel 2016 (Microsoft, USA). Significant difference p values at 0.05 were obtained using one-way analysis of variance (ANOVA).

3. Results and discussions

3.1. Optical properties of β-CD:TFR inclusion complex

The stability of TFR was affected by its poor solubility in water (2 mg/mL). To overcome this problem, an inclusion complex between TFR and β-CD was formed with 1:1 M ratio. Fig. 1 a depicts the TFR absorption spectra as a function of β-CD concentration at pH 7.4 aqueous solutions. Increasing the concentration of β-CD from 0.002 M to 0.012 M increased the absorbance of TFR (1 mM; DMSO) [41]. The absorption maximum of TFR shows a little blue shift from 268 nm to 265 nm with the increase in β-CD concentration [42]. The β-CD used improved the TFR low water solubility, demonstrating to some extent the complexation of its β-CD:TFR inclusion in aqueous solutions. From the results of UV–Visible absorption studies (Fig. 1a and Table 1 ), it was evident that β-CD helped in improving the aqueous solubility of TFR. The formation of β-CD:TFR inclusion complex system showed a 9.31 times enhancement in the solubility compared to the pure TFR; this can be attributed to the enhanced solubility of TFR by β-CD. The formation of the inclusion complex is demonstrated by these changes in maximum absorption intensity, which may also be utilized to calculate the binding constant and stoichiometry of the complex. The formation of 1:1 inclusion complex between TFR and β-CD, at equilibrium can be written as the following equation (3);

$$\mathrm{\beta }-\mathrm{C}\mathrm{D}+\mathrm{T}\mathrm{F}\mathrm{R}\rightleftharpoons \mathrm{\beta }-\mathrm{C}\mathrm{D}:\mathrm{T}\mathrm{F}\mathrm{R}$$ (3)

Fig. 1.

(a) UV–Visible absorption spectra of TFR (DMSO; 1 mM) at pH 7.4 in different concentrations of β-CD: (1–7) 0 to 0.012 M and (b) Benesi–Hildebrand plots of 1/[A-A₀] vs. 1/[β-CD].

Table 1.

Absorption spectral maxima of TFR with various concentrations of β-CD at pH 7.4.

β-CD concentration	Absorbance	[1/β-CD]	[1/A-A0]
0	0.118	0	0
0.002	0.283	500	6.06
0.004	0.416	250	3.35
0.006	0.570	166.66	2.21
0.008	0.733	125	1.62
0.010	0.870	100	1.32
0.012	1.099	83.33	1.01
Binding constant (K)			84.94 M−1
Gibbs free energy (ΔG)			−11.18 kJ/mol

The Benesi-Hildebrand method linear fit is shown in Fig. 1b along with the reciprocal of the absorbance change as a function of the inverse of the β-CD:TFR complex concentration. From the Benesi-Hildebrand equation, the apparent binding constant (K) value for the inclusion complex with a ratio of 1:1 stoichiometry can be calculated (Eq. (4)) [43].

$$\frac{1}{[\mathrm{A}-{\mathrm{A}}_0]}=\frac{1}{{\mathrm{\Delta }}_{\mathrm{\epsilon }}}+\frac{1}{{\mathrm{K}[\mathrm{T}\mathrm{F}\mathrm{R}]}_0{\mathrm{\Delta }}_{\mathrm{\epsilon }}{[\mathrm{\beta }-\mathrm{C}\mathrm{D}]}_0}$$ (4)

where A and A₀ denote the absorbance of TFR in the presence and absence of β-CD, Δε is the difference between the molar absorption coefficient of TFR and β-CD, while [TFR]₀ and [β-CD]₀ are the initial concentrations of TFR and β-CD, respectively. An excellent linear curve was produced from plotting 1/[A-A₀] as a function of β-CD concentration, as shown in Fig. 1b. The slope and intercept of the linear curve were used to calculate the K (84.94 M⁻¹) and Gibbs free energy (ΔG) (-11.18 kJ/mol) of the β-CD:TFR complex and the corresponding values are shown in Table 1.

3.2. FTIR spectra of β-CD:TFR inclusion complex

FT-IR spectroscopy makes it simple to determine whether inclusion complexes between TFR and β-CD. This technique helps in determining the change in the bands of vibrations of the individual moiety in the process of complex formation. Peak shifting, intensity changes, peak disappearance, and peak intensity changes are typical indicators of inclusion complex formation. Fig. 2 displays the FTIR spectra for the β-CD, TFR, and β-CD:TFR inclusion complex in the range of 4000 to 400 cm⁻¹. According to the FTIR spectrum for β-CD shown in Fig. 2, there are notable absorption bands at 3385 cm⁻¹ (O—H stretching vibrations), 1418 cm⁻¹ (O—H bending vibrations), and 2932 cm⁻¹ (the stretching and bending vibrations band for C—H in the CH₂ group). Additionally, ether linkage at 1031 cm⁻¹ (C—O—C bending vibration) and a significant absorption band at 1161 cm⁻¹ (for C—O stretching vibration) were both noted [44]. The band at roughly 1698 cm⁻¹, which corresponds to C O stretching, is prominent in the FTIR spectrum of TFR. Additionally, it has bands at 3194 and 3298 cm⁻¹, which stand for the C—H and N—H bonds, respectively. The P O stretching vibration, C N stretching vibration, C-N deformation, and NH₂ stretching vibration, respectively, have large maxima at 1678, 1448, 1249, and 1566 cm⁻¹ [7]. The FTIR spectra of the β-CD:TFR inclusion complex revealed an approximated superimposition of the two patterns. The inclusion complexes FTIR spectra revealed that extremely strong and wide β-CD bands completely blocked the TFR bands. That β-CD covers the bulk of the TFR significant absorption bands. Some of the distinctive peaks of β-CD, such as the peak of the C—H stretching vibration at 2969 cm1 and the peak of the solitary —OH bending vibration at 1211 cm⁻¹, are still present in the β-CD:TFR inclusion complex. The TFR absorption bands at 1627, 1360, 1250, and 811 cm⁻¹ were displaced and lost some of their intensity. A new phase is generated between TFR and β-CD as a result of the benzene ring of the TRF molecule entering the cavity of β-CD, as evidenced by the mild redshift of the absorption of C N at 1451 cm⁻¹ of TFR.

Fig. 2.

FTIR spectra of β-CD, TFR, and β-CD:TFR inclusion complex.

3.3. PXRD pattern of β-CD:TFR inclusion complex

A study was carried out to assess the crystallinity of the β-CD, TFR, and β-CD:TFR inclusion complex, and an efficient method for the Powder XRD. As illustrated in Fig. 3 , the typical diffraction peaks of β-CD are seen at 2θ = 10.9°, 13.1°, 15.5°, 18.7°, 21.2°, 23.1°, 26.9°, and 34.9° [44]. The PXRD spectrum of TFR revealed several distinct, strong, and sharp peaks, which support the crystalline nature of TFR [8]. The PXRD spectra of the β-CD:TFR inclusion complex showed that there are no prominent distinctive peaks corresponding to TFR 2θ values at 14.8°, 15.6°, 18.0°, 22.4°, 23.3°, and 24.6° indicating that TFR may be incorporated in the cavity of β-CD during inclusion complex formation. Additionally, the PXRD pattern of the β-CD:TFR inclusion complex was entirely different from that of the β-CD and TFR alone, providing clear evidence for the β-CD:TFR inclusion complex formation.

Fig. 3.

XRD pattern of β-CD, TFR, and β-CD:TFR inclusion complex.

3.4. SEM analysis of β-CD:TFR inclusion complex

SEM analysis could provide detailed surface images to investigate the structures and the morphology of intact TFR before and after β-CD:TFR inclusion complex formation. While TFR was seen as an irregular fiber-like crystal [45], β-CD was seen as irregular crystalline particles of various sizes and shapes in the SEM image [44]. The original surface morphology of TFR and β-CD was lost, according to SEM analysis, in the β-CD:TFR inclusion complex, which appeared as particles with a smooth surface (Fig. 4 ). It appears that the TFR molecules were incorporated into the cavities contained in β-CD to create the β-CD:TFR inclusion complex.

Fig. 4.

SEM images of (a) β-CD, (b) TFR, and (c) β-CD:TFR inclusion complex.

3.5. Thermal analysis of β-CD:TFR inclusion complex

Thermogravimetric analysis (TGA) and Differential scanning calorimetry (DSC) analyses were used to further study the thermal stability of β-CD, TFR, and the β-CD:TFR inclusion complex. The TGA was carried out to identify the changes in weight percent concerning temperature change. The TGA evaluation of β-CD exhibited three stages of weight loss ranges as shown in Fig. 5 a. A small initial weight loss of 6.5 %, up to 100 °C, related to the loss of water. The effective degradation occurs in a fast process at approximately 326 °C, whereas ∼62% weight loss occurs above 350 °C in the later stage, which is associated with the decomposition of the macrocycles [46]. The TGA curve of the thermal decomposition of TFR exhibited two-mass loss steps starting from 85 °C, the maximum mass loss rate was at 288 °C [10]. The β-CD:TFR inclusion complex clearly shows three steps of degradation at 79 °C, 255 °C, and 294 °C. The first stage results from the dehydration of water molecules from the β-CD. The observed second ∼43% of weight loss occurred in the range from 243 °C to 291 °C owing to the decompositions of both the backbone chain of TFR and β-CD ring in β-CD:TFR inclusion complex. Finally, a third stage from 294 °C to 380 °C around 53% mass loss can be related to the degradation of the β-CD and TFR in the β-CD:TFR inclusion complex. This phenomenon suggests that the formation of β-CD:TFR inclusion complex decrease the thermal stability of β-CD.

Fig. 5.

(a) TGA and (b) DSC analysis of β-CD, TFR, and β-CD:TFR inclusion complex.

To investigate any potential thermal transition brought on by TFR inclusion in the β-CD nanocavity, DSC tests were carried out. Melting, boiling, and sublimation points are moved to different temperatures or vanish as a guest molecule is inserted into β-CD nanocavity. The DSC profiles of β-CD, TFR, and β-CD:TFR inclusion complex is shown in Fig. 5b. The DSC curve of β-CD exhibited a wider endothermic peak water loss at 83℃ followed by a sharp peak at 326℃, which could be due to the dehydration of water molecules from the β-CD cavity [44]. The three distinctive TFR peaks may be seen in Fig. 5b [10]. A prominent endotherm was visible on TFR thermograms at 365 °C, 268 °C, and 75 °C, which corresponded to their melting and decomposition temperatures, respectively. The thermograms of the β-CD:TFR inclusion complex demonstrate that the two TFR peaks have completely disappeared, which would suggest that the active ingredient has been molecularly encapsulated inside the cavity of the β-CD. Additionally, the TFR drug exhibits a pronounced endothermal peak at 275 °C, while the β-CD exhibits no peak at all. These findings showed that the β-CD nanocavity was protecting the active TFR drug.

3.6. Phase solubility of β-CD:TFR inclusion complex

The phase solubility of TFR in the β-CD suspension was investigated in triplicate at 25 °C to calculate the stability constant (Ks) and complexation efficiency (CE) of the β-CD:TFR inclusion complex and assess its molecular stoichiometry. An aqueous solution containing β-CD at various concentrations ranging from 0 to 0.012 M was mixed with equal volumes of TFR (pH 7.4). A calibration curve was used to spectrophotometrically assess the concentration of the dissolved TFR (Fig. 6 ). Equation (1) was used to calculate the Ks of the β-CD:TFR inclusion complex and the linear portion of the plot is represented by the values y = 120.34 + 0.1437x and R² = 0.9904.

Fig. 6.

Phase solubility of β-CD:TFR inclusion complex.

The development of the β-CD:TFR inclusion complex (1:1) suggests an increase in the ligand solubility, and the Ks were calculated as 863.32 M⁻¹. Calculating their CE, or the concentration ratio between β-CD in a β-CD:TFR inclusion complex and free β-CD, is a more accurate way of determining the solubilizing efficiency of β-CD [47]. The phase-solubility diagrams slope is used to determine CE, which is separate from S₀ (Eq. (2)). The solubilizing capability of β-CD toward the TFR medication was found to be 0.038 according to the CE values. As can be seen, the concentration of β-CD rose along with the solubility of TFR. This demonstrates that the β-CD:TFR inclusion complex was formed, increasing the solubility of TFR in the aqueous solution and resulting at the same time reducing the usage of organic solvents.

3.7. Molecular docking study of β-CD:TFR inclusion complex against SARS-CoV-2 M^pro

To investigate the potential configurations of the inclusion complexation between β-CD and TFR molecules of molecular dynamics simulation (See supplementary information) and molecular docking was accomplished. Three-dimensional stick structures of the β-CD, TFR, and docked poses of the best-ranked docking score of TFR with β-CD stick and sphere structures of the β-CD:TFR inclusion complex as shown in Fig. 7 . Table 2 lists the molecular docking and data-derived lowest energy model and greatest complementary docking score [48], [49], [50]. The molecular docking studies showed that TFR was immersed in the cavity of the β-CD, indicating that TFR potentially may form inclusion complexes with β-CD. The –NH₂ moiety on the benzene ring of TFR initially entered the active site of β-CD in the model with the highest docking score (3274), and the relative position of the amide group also favors the interaction with the main hydroxyls of β-CD [51].

Fig. 7.

Three-dimensional stick structures of the β-CD, TFR and docked poses of the best-ranked docking score of TFR with β-CD stick and sphere structures of the β-CD:TFR inclusion complex. TRF; Tenofovir, β-CD; β-Cyclodextrin, and β-CD:TFR; inclusion complex of β-Cyclodextrin:Tenofovir. (Atom color; C-sandal, H-white, O-red, N-blue, and P-orange). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Table 2.

Molecular modeling of the top 5 docked models of β-CD:TFR inclusion complex (A) and β-CD:TFR inclusion complex interaction against SARS-CoV-2 (M^Pro) protease inhibitors (B).

(A)	β-CD:TFR
	Patchdock server			FireDock server
S. No.	Scorea	Area (Å2)b	ACE kcal/molc	Global Energy kcal/mold	Attractive VdW kcal/mole	Repulsive VdW kcal/mole	ACE kcal/molf
1	3274	386.10	−234.04	–33.73	−12.76	2.35	−10.44
2	3260	379.30	−242.05	–33.69	−12.98	1.31	−10.00
3	3200	373.50	−239.37	–33.46	−12.31	1.38	−10.27
4	3194	377.10	−221.22	–33.41	−12.21	1.60	−10.25
5	3156	331.60	−206.78	–33.21	−12.33	2.15	−10.29
(B)	β-CD:TFR/SARS-CoV-2 (MPro) protease inhibitors
1	5586	657.29	−198.97	−6.22	−10.27	3.54	−9.84
2	5237	708.63	−132.87	−11.37	−11.29	4.39	−3.86
3	5318	718.24	−149.27	−9.86	−9.89	4.12	−3.17
4	5407	696.87	−186.03	−8.43	−6.38	3.97	−4.19
5	5163	712.27	−190.12	−16.87	−14.37	6.83	−6.57

^aGeometric shape complementarity score.

^bApproximate interface area size of the complex.

^cAtomic contact energy.

^dIndicating binding energy of the solution.

^eRepresenting contribution of the van der Waals energy to the global binding energy.

^fACE shows the contribution of the atomic contact energy (ACE) to the global binding energy.

Additionally, the PatchDock and FireDock servers were used to research the protein and small molecule interactions of the β-CD:TFR inclusion complex with SARS-CoV-2 (M^Pro) protease inhibitors [52], [53], [54], [55], [56]. The results are shown in Fig. 8 and Table 2. Interesting protein-protein interactions with SARS-CoV-2 (M^Pro) protease inhibitors were seen in Fig. 8 against the β-CD:TFR inclusion complex. The β-CD:TFR/SARS-CoV-2 (M^Pro) protease inhibitors showed the highest docking score (5563) and atomic contact energy (−198 kcal/mol) in the PatchDock calculations. Additionally, the FireDock calculations were used to determine the lowest global energy (-6.22 kcal/mol), attractive van der Waals energy (-10.27 kcal/mol), repulsive van der Waals energy (3.54 kcal/mol) to the global binding energy, and atomic contact energy (-9.84 kcal/mol) [46]. Binding contributions of important residues (PHE8, ALA7, PHE246, ASP295, MET6) from the active site or near the active site regions with ≥1.0 kcal/mol suggest a potent binding of the inhibitors [57], [58]. Based on the results from the current study, it is expected that several valuable insights will be gained regarding the design of significantly efficient anti-SARS-CoV-2 M^pro drugs [59], [60]. According to Table 2  findings, the atomic contact energy of the β-CD:TFR inclusion complex was lower than that of the latter, suggesting that the β-CD:TFR inclusion complex preferentially binds to SARS-CoV-2 (M^Pro) protease inhibitors [60]. This β-CD:TFR inclusion complex was created based on a previously identified drug that is effective against SARS-CoV-2 (M^Pro) [57], [58], [59], [60]. The SARS-CoV-2 (M^Pro) protease receptor may therefore be inhibited by the β-CD:TFR inclusion complex.

Fig. 8.

Schematic representation of the energetically most favorable complexes. Docking studies of β-CD:TFR complex interaction with SARS-CoV-2 (M^Pro) protease inhibitors. Protein is shown in cartoon and hydrophobic rendering representation. Molecular surface of β-CD:TFR/SARS-CoV-2 (M^Pro) with a neutral surface area is highlighted by white and β-CD:TFR complex is round by red mark, maximum hydrophobic regions colored red; maximum hydrophilic indicated in blue. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

4. Conclusions

In summary, we report a new β-CD:TFR inclusion of complex molecules acting as inhibitors of the main protease (M^pro) of the SARS‐CoV‐2 virus. In this study, UV–Visible analyses showed an enhanced solubility of TFR in the presence of β-CD and the results suggested the formation of β-CD:TFR inclusion complex at 1:1 stoichiometric ratio and apparent binding constant were calculated based on the Benesi-Hildebrand plot. The spectral results obtained via FTIR, PXRD, SEM, TGA, and DSC analysis proved the formation of an inclusion complex in which TFR entered the cavity of β-CD. Both DSC and TGA of the inclusion complexes showed the presence of endothermic peaks between 250 °C and 270 °C, attributed to a β-CD:TFR inclusion complexation phenomenon. Phase solubility studies revealed that β-CD significantly improved the solubility of TFR and the stability constant was obtained at 863 ± 32 M⁻¹ through A_L-type diagrams. The experimental and computational modeling results showed that the phenyl ring with amide group of TFR was deeply inserted into hydrophobic β-CD nanocavity forming β-CD:TFR inclusion complex. The β-CD:TFR inclusion complex is bound strongly to the active sites of the protein target of SARS-CoV-2 (M^Pro) protease inhibitors, with predicted the highest docking score at 5586 and atomic contact energy of −198 kcal/mol. Therefore, the new inclusion complex enhanced solubility, stability, and antiviral activity against SARS-CoV-2 (M^Pro) suggesting that β-CD:TFR inclusion complexes can be further used as feasible water-insoluble antiviral drug carriers in viral diseases infection. Further investigations should focus on validating and finalizing effective antiviral drugs for COVID-19 beyond preliminary in silico and in vivo screening.

CRediT authorship contribution statement

Sonaimuthu Mohandoss: Conceptualization, Methodology, Writing – original draft. Kuppu Sakthi Velu: Methodology, Investigation. Thambusamy Stalin: Methodology, Investigation. Naushad Ahmad: Methodology, Investigation. Suliman Yousef Alomar: Methodology, Investigation. Yong Rok Lee: Supervision, Writing – review & editing.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following are the Supplementary data to this article:

Data availability

Data will be made available on request.