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. 2020 Mar 18;5(12):6472–6480. doi: 10.1021/acsomega.9b04095

Interaction between the Antimalarial Drug Dispiro-Tetraoxanes and Human Serum Albumin: A Combined Study with Spectroscopic Methods and Computational Studies

Priyanka Yadav 1, Bhawana Sharma 1, Chiranjeev Sharma 1, Preeti Singh 1, Satish K Awasthi 1,*
PMCID: PMC7114135  PMID: 32258882

Abstract

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Dispiro-tetraoxanes, a class of fully synthetic peroxides which can be used as an antiplasmodial remedy for multiple drug-resistant strains of Plasmodium falciparum, were selected for the interaction study with human serum albumin (HSA). The insight into the interaction of the two chemically synthesized, most potent antimalarial tetraoxane analogues (TO1 and TO2) and HSA has been scrutinized using distinct spectroscopic techniques such as. UV–visible absorption, fluorescence, time-resolved fluorescence, and circular dichroism (CD). Fluorescence quenching experiments divulged the static mode of quenching and binding constants obtained (∼104) indicated the moderate affinity of the analogues to HSA. CD confirmed the conformational changes in the serum albumin upon interaction with these analogues. Molecular docking validated the empirical results as these two analogues bind through hydrophobic interactions and hydrogen bonding with HSA. Present work first defined the binding mechanism of dispiro-tetraoxanes with HSA and thus provides a fresh insight into the drug transportation and metabolism. The present study could direct toward designing more potent tetraoxane analogues for their use in the biomedical field.

1. Introduction

Artemisinin and its semisynthetic analogues remain at the vanguard of antimalarial chemotherapy because of their safety, high potency, and their capability of rapid devaluation of the parasite biomass than any other clinically used class.1 However, recent reports witnessed the emergence and development of resistance to the artemisinin drug which has been first documented in Western Cambodia.2 Resistance to first-generation analogues foster risk to decimate recent achievements attained in reducing malaria burden worldwide and threatens the progress made on the elimination and future control of malaria on a global level.3 Another relevant issue related to the widespread application of aforementioned drugs is their limited availability, high cost, and short plasma half-life that result in recrudescence of the malarial parasite.1 These circumstances pushed efforts toward the advancement of completely synthetic endoperoxide alternatives with improved potency and pharmacokinetic aspects. Consequently, researchers have sought to replace the semisynthetic artemisinin components of ACTs with totally synthetic derivatives in order to lessen the ambiguity regarding cost and supply and to discover structurally simplified molecules with excellent bioavailability. This has led to the congregation of some fully synthetic pharmacophores which carry the critical endoperoxide bond that imparts activity to artemisinin such as 1,2,4-trioxolanes and 1,2,4,5-tetraoxanes.4

Recently, based on the ozonide structure, a series of synthetic molecules were developed among which OZ277 displayed very intriguing antimalarial activity profiles in vitro and in vivo.5 However, this molecule was found to be unstable during a phase II dose-ranging study in the plasma of malaria patients.6 For this, knowledge of the mode of action of the currently used drugs, understanding of regional drug resistance patterns, and gratitude of cross-resistance between drugs are vital to wisely design an individualized persuasive drug policy in all malaria-affected countries.

Human serum albumin (HSA) is the most copious and versatile carrier protein in the human body. It is a nonglycosylated polypeptide chain comprising 585 amino acids, with a molecular weight of 65 kDa and is responsible for the probable solubility of hydrophobic drugs in plasma.7 Serum albumin incorporates three homologous α-helical domains (I–III) which is further divided into two subdomains A and B. According to the crystallographic data, the hydrophobic cavities located in subdomain IIA (site I) and subdomain IIIA (site II) are two primary ligand binding sites in the albumin.8 These diversified binding sites emphasize the exceptional ability of serum albumin to act as a major depot and transport protein, which is adept of binding, transporting various endogenous and exogenous substances in the circulatory system to their target organs, and also contributing to the plasma colloid osmotic pressure.9 The binding interaction between drugs and plasma HSA influences its allocation and efficacy through clasping the drug in the plasma and confining its clearance, thus enhancing the pharmacokinetic half-life; however, this complexation decreases the drug distribution within the tissues and can restrict the contact of the drug with the biological target.10 Therefore, it is necessary to study the mode of interaction of the drugs with HSA in order to comprehend the transportation and distribution of drugs in the body on a molecular level. Further, it will also provide theoretical direction and valuable knowledge for designing more effective drugs.

Our research group has synthesized a series of dispiro-tetraoxane analogues and screened for their in vitro and in vivo antimalarial activities.11 Based on the outstanding antimalarial activity and pharmacokinetic profile, TO1 and TO2 from a pool of 37 compounds were selected as the lead candidates (Figure 1). These molecules exhibited an IC50 value of 8.43 ± 0.71 and 10.54 ± 0.81 nM against the chloroquine-sensitive 3D7 strain of Plasmodium falciparumin vitro (Table 1). At a dose level of 60 mg/kg, TO1 showed a complete curative behavior in Plasmodium berghei within 72 h in the in vivo study, whereas, TO2 displayed complete suppression of parasites after a 5-day treatment regimen.

Figure 1.

Figure 1

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Chemical structures of synthesized tetraoxanes.

Table 1. In Vitro Anti-Plasmodial Activity of Synthesized Tetraoxanes (TO1 and TO2).

  TO1 TO2
mean (IC50 ± SE) nM 8.43 ± 0.71 10.54 ± 0.81
CC50 value >100 μg/mL >100 μg/mL
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To extend our research, the optimal binding models of the tetraoxane–HSA complex were simulated through molecular docking, UV–vis absorption, fluorescence spectroscopy, and circular dichroism (CD) spectroscopy. This study supports in-depth insights into the mechanism of tetraoxane–serum albumin binding interaction, which may be invaluable for improving applications of tetraoxane in clinical research, and standardized screening in pharmaceutical firms and clinical research. The result of the present work has great significance in the study of the process of storage and transportation of dispiro-1,2,4,5-tetraoxane in the body and its mechanism of action and pharmacokinetics.

2. Results and Discussion

2.1. UV–Vis Absorbance

UV–vis absorption spectroscopy is a widely employed technique for the analysis of drug–protein interactions. The absorption spectra of HSA exhibits a strong peak at 280 nm because of the π–π* transition of Trp, Tyr, and Phe amino acids.12 Here, the UV spectra of free TOs as well as protein–TO complexes were recorded. It was observed that TOs do not absorb in the range of protein absorption (Figure 2b inset). The spectra of free protein and in the presence of various concentrations of TO1 and TO2 was taken to understand the interaction mechanism. It was noticed that absorption of HSA increased with successive addition of TO1–2 without any shift in the absorption peak (Figure 2). The observed hyperchromicity in both the cases implied toward the stable ground-state complex formation between HSA and TO1–2.13 Further, the absence of blue or red shift in the absorption spectra implied toward the binding of TO1–2 to HSA via the π–π stacking type of noncovalent interactions.14 This change in absorption spectra also confirmed the static nature of quenching as absorption spectra are unaffected by dynamic quenching.15

Figure 2.

Figure 2

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UV–visible absorbance spectra of HSA (10 μM) in the absence and presence of different concentrations of (a) TO1 and (b) TO2 (pH = 7.4, T = 298 K). The upward arrow shows an increasing intensity upon the addition of TO1 and TO2. Absorbance spectra of TO1 and TO2 (90 μM) are presented in the inset of Figure 2b.

2.2. Fluorescence Quenching

Fluorescence spectroscopy being a sensitive technique is used to analyze the structural and conformational changes during the complex formation between the protein and the ligand. HSA has a unique property of intrinsic fluorescence because of the three aromatic amino acid residues tryptophan, tyrosine, and phenylalanine.16 HSA showed an emission peak at 340 nm with excitation at 280 nm.17 The fluorescence of the free tetraoxanes TO1 and TO2 (150 μM) was also recorded with an excitation wavelength at 280 nm to check whether these analogues produce peak in the region of protein emission. It was observed that these analogues do not show any fluorescence. Further, to elucidate the interaction mechanism, emission spectra of HSA (10 μM) were recorded by varying the concentration of TO1–2 to it (Figure 3). It was observed that fluorescence was quenched by the successive addition of TO1–2 to HSA. Thus, it can be deduced that the Trp residue of HSA is situated at or nearby to the ligand binding site.18 The decrease in fluorescence intensity indicated the binding of TO1–2 to the protein and also it can be deduced that binding is concentration dependent.19

Figure 3.

Figure 3

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Fluorescence quenching of HSA (10 μM) in the presence of different concentrations of (a) TO1 and (b) TO2 excited at 280 nm at 298 K. Inset: fluorescence spectra of (a) TO1 and (b) TO2 (150 μM) excited at 280 nm.

The blue shift observed in the fluorescence emission maxima reflected toward the change in polarity around the chromophore unit of the protein. This shift indicated that the amino acid residues are located in a more hydrophobic environment.20 The fluorescence quenching can be of two types: static quenching and dynamic quenching. So, the mechanism of quenching was assessed by evaluating the fluorescence spectra by means of Stern–Volmer equation

2.2. 1

Here, the symbol F0 represents the fluorescence intensity of pure HSA, F represents the fluorescence intensity of HSA in the presence of the quencher, [Q] denotes the quencher concentration, and kq is the quenching constant. The constant KSV or kqτ0 is the Stern–Volmer constant.21 Here, τ0 was taken 10–8 s for calculating kq. In our case, we found that the quenching constant (kq) is larger than the maximum diffusion rate constant of the biomolecule (2 × 1010 L mol–1 s–1) (Table 2). So, it can be concluded that the mechanism of fluorescence quenching was due to complex formation between HSA and TO1–2.22 The Stern–Volmer plots of titration of HSA with TO1–2 are shown in Figure 4.

Table 2. Stern–Volmer Quenching Constants (KSV) and the Binding Parameters for TO–HSA Complexes at 298 K.

  HSA
T = 298 K KSV (×103 L mol–1) kq (×1012 L mol–1 s–1) Kb (L mol–1) n
TO1 4.21 ± 0.0032 4.21 ± 0.0032 6.233 × 104 1.274
TO2 2.56 ± 0.0035 2.56 ± 0.0035 1.0727 × 104 1.104
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Figure 4.

Figure 4

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Stern–Volmer plots of HSA with (a) TO1 and (b) TO2 at 298 K (λex = 280 nm, pH = 7.4). Data presented are mean values ± standard deviations of three independent experiments; some error bars are within symbol.

Fluorescence results were further analyzed to determine the number of binding sites and binding constants by the following modified Stern–Volmer equation23

2.2. 2

The values of the binding constant Kb (intercept) and n (slope) were calculated by plotting log[(F0 – F)/F] against log[Q] as shown in Figure 5. In this case, the value of the slope (n) came out was close to 1 for both the cases (for TO1 and TO2), indicating 1:1 binding stoichiometry.24 The calculated values of the binding constant (Kb) as shown in Table 2 are of the order 104 (L mol–1) for HSA–TOs showing a moderate interaction in high affinity.24b Hence, by the fluorescence quenching experiment and the calculated values of binding parameters, it can be deduced that TO1–2 forms a static complex with HSA in 1:1 stoichiometry.

Figure 5.

Figure 5

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Modified Stern–Volmer plots of HSA with (a) TO1 and (b) TO2 at 298 K (λex = 280 nm, pH = 7.4). Data presented are mean values ± standard deviations of three independent experiments; some error bars are within symbol.

2.3. Time-Resolved Fluorescence

The steady-state fluorescence quenching concluded the static nature of quenching of HSA by TO1–2. Further to differentiate between static and dynamic quenching and to affirm the mechanism of quenching precisely, time-resolved fluorescence measurements were performed (Figure 6). As fluorescence lifetime does not depend on the quencher concentration in case of static quenching, it decreases with the increasing quencher concentration for dynamic quenching.10 Thus, the lifetime values of HSA in the absence and presence of varying concentrations of TO1–2 were investigated. As can be seen from Figure 6, the fluorescence decay profiles of HSA remained almost unaffected even after the addition of a varying concentration of TO1–2.

Figure 6.

Figure 6

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Fluorescence decay profile of HSA in the absence and presence of varying concentration of (a) TO1 and (b) TO2 at 298 K.

The average fluorescence lifetime (τ) of HSA was determined on the basis of the decay time and relative amplitude (α), using the below equation

2.3. 3

The attained values for the relative fluorescence life time of HSA in the presence and absence of TOs are shown in Table 3. The average fluorescence life time of HSA reduced from 4.25 to 4.21 ns in the presence of TO1. This negligible decrease in the fluorescence life time implied toward the static nature of the quenching mechanism.25 Thus, the steady state fluorescence experiment as well as time resolved measurements proved the static mode of quenching.

Table 3. Fluorescence Decay Profile of TO–HSA Systems with Different Concentrations of TO1–2.

sample τ1 (ns) τ2 (ns) α1 α2 τav (ns)
free HSA 1.85 6.62 49.55 50.44 4.25
[TO1]/[HSA] = 1:1 1.84 6.70 49.09 49.90 4.24
[TO1]/[HSA] = 2:1 1.97 6.63 52.01 47.98 4.20
[TO1]/[HSA] = 3:1 1.87 6.6 50.43 49.56 4.21
free HSA 2.47 6.95 43.13 56.86 5.01
[TO2]/[HSA] = 1:1 2.36 6.88 42.31 57.68 4.96
[TO2]/[HSA] = 2:1 2.36 6.9 43.12 56.87 4.94
[TO2]/[HSA] = 3:1 2.34 6.83 43.05 56.94 4.89
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2.4. CD Spectroscopy

CD being a sensitive and informative tool, which is frequently used to investigate the conformational changes in protein upon drug–protein interaction. CD spectroscopy of HSA in phosphate-buffered saline (pH 7.4) was carried out in the far-UV region (190–240 nm), which reflected two significant negative minima at 208 and 222 nm. These two negative minima at 208 and 222 nm belongs to the π–π* and n−π* transition of the α helix and random coil of the protein.20a

Figure 7 shows that on subsequent addition of TOs to HSA, by regular increment of 1 μM each time from a molar concentration ratio (HSA/TOs) 1:0 to 1:1, the decrease in intensity and gradual shift of the 208 peak toward 222 nm was observed in all the cases. This decrease in intensity reflected toward the disruption in the secondary-structure conformation of the protein.26 Further, it was also observed that on successive addition of TO1–2 to HSA, the content of the α-helix decreases abruptly from 56.31 to 22.13 and 24.58% in case of TO1 and TO2, respectively (calculated from K2D3 software). Thus, unfolding of the α-helices is predominant with increasing concentration of TO1–2, which is in good agreement with UV-absorption and fluorescence studies.

Figure 7.

Figure 7

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CD spectra of HSA (10 μM) in the absence and presence of different concentrations of (a) TO1 and (b) TO2 (HSA/TOs ratio = 1:0 → 1:1).

2.5. Computational Studies

Theoretical calculations for both TO1–2 were done using density functional theory (DFT) studies. the hybrid functional DFT/B3LYP/6-31+G(d) method was employed using Gaussian software for excellent precision and speed of calculations.27 The TO1–2 geometries were optimized in the ground electronic state, and the energies for these were minimized. These optimized TO1–2 geometries were further used for calculating the energies of frontier molecular orbitals, that is, the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO). Owing to the importance of these frontier molecular orbitals and their associated properties, to conclude the reactivity and charge delocalization within the molecule, GaussView 5.0 was used for visualizing and constructing the shapes of frontier molecular orbitals of TO1–2.

It is reported that the molecules having large and small HOMO–LUMO gaps correlates to the less and more reactive species, respectively.20a Here, HOMO–LUMO gaps of TO1–2 were found of approximately similar values. Nevertheless, the lower HOMO–LUMO gap obtained for TO1 supports the more bioactive molecule in comparison to TO2, which is in good agreement with the experimental in vitro antiplasmodial activity.11

The HOMO and LUMO plots of TO1–2 (Figure 8) show the charge delocalization within these molecules upon excitation. In case of TO1 (Figure 8a), it can be seen that the HOMO is completely delocalized over the sulfonyl and attached side chain, while the LUMO is completely delocalized over the tetraoxane ring in the molecule. Hence, the HOMO–LUMO plots display the charge transfer toward the cyclic ring within the molecule.

Figure 8.

Figure 8

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Frontier molecular orbital diagram of (a) TO1 and (b) TO2 calculated at the B3LYP/6-31+G(d) level.

2.6. Molecular Docking Studies

Before the molecular docking assay, the HSA 3D crystal structure (PDB ID: 1E78) was downloaded from the Protein Data Bank.28 Molecular docking between tetraoxanes (TO1–2) and HSA was achieved using AutoDock to find out the interacting residues of the protein. AutoDockTools version 1.5.6 which is the graphical user interface of the AutoDock equipped with MGLTools was employed to find the interaction modes between TOs and HSA. The three-dimensional structure of ligands (TO1–2) was constructed using ChemBio3D Ultra12.0. The structures of tetraoxanes (TO1–2) were optimized using Gaussian 09W software. All bound water molecules and heteroatoms were removed from the HSA crystal structure, and essential hydrogen atoms and Gasteiger charges were added to the protein using the AutoDockTools. The implemented Lamarckian genetic algorithm (LGA) in AutoDock was adopted to check out the feasible conformation of the tetraoxanes upon the binding with HSA. Docking of both TO1–2 with whole of the protein was achieved in order to ascertain the comparative affinity of binding sites of proteins for these ligands, adopting the identical grid size of 256 Å along both three (X, Y, and Z) axes, covering all the protein and grid spacing of 0.375 Å.29 By docking results, 50 conformers were assessed and the conformer having lowest binding energy was chosen for further examination (Tables S1 and S2, Supporting Information). The results obtained after visualizing using PyMol are shown in Figure 9. Docking results showed that TO1 bind within the binding pocket of the subdomain IIA of HSA through H-bonding (with Asp324 and Lys212) and hydrophobic interactions with various amino acid residues. Similarly, TO2 forms H-bonding with Lys212 and surrounded by various hydrophobic amino acid residues of HSA (Table 4). Further, from Figure 9, it can be seen that the Trp-214 residue is in close vicinity in both the cases. These docking results further support the adequate fluorescence quenching of serum albumin in the presence of these analogues, that is, TO1 and TO2. From the cluster analysis, using a root-mean-square (rms) deviation tolerance of 2.0 Å, different conformational clusters were found for both TO1 and TO2 as shown in Figure 10.

Figure 9.

Figure 9

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Depiction of structural details obtained by the molecular modeling method of (a) TO1 and (b) TO2 with HSA. Ligands are represented as cylindrical models. Amino acid residues present in the binding pocket are expressed as wireframe models.

Table 4. Docking Calculations Depicting Interacting Residues and Residues Involved in H-Bonding along with the Binding Energy Obtained.

ligand receptor interacted residues residues involved in H-bonding H-bond distance (Å) binding energy (kcal/mol)
TO1 1E78 GLU208, ARG209, ALA210, PHE211, ALA213, ALA215, VAL216, ALA217, SER232, VAL235, THR236, ALA322, VAL325, PHE326, LEU327, MET329, PHE330, LEU331, TYR332 ASP324 3.50 –6.83
        3.54  
      LYS212 2.94  
        3.33  
TO2 1E78 GLU208, ARG209, ALA210, PHE211, ALA213, TRP214, ALA215, VAL216, THR236, SER232, VAL325, GLY328, PHE330, LEU331, TYR332 LYS212 2.77 –6.23
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Figure 10.

Figure 10

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Cluster analysis of the AutoDock docking runs of TO1 and TO2 in the binding site of HSA.

3. Conclusions

The present study includes a sequential analysis of interaction mechanism of two potent antimalarial tetraoxane analogues to HSA, using different spectroscopic and computational techniques. The hyperchromicity obtained in UV–vis spectra displayed the interaction of tetraoxanes with HSA. Fluorescence quenching results confirmed the static nature of the quenching mechanism as the values obtained for the quenching constant (∼1012) were greater than scatter collision (∼1010). The values of the binding constant (∼104) demonstrated a moderate and reasonable binding of TO1–2 to HSA, divulging that their clearance from kidneys and diffusion into the tissues decreases. Also, the tetraoxane (TO1), having a longer alkyl side chain bearing a Cl atom exhibited a greater value of the binding constant (Kb) as compared to the other one (TO2). The results obtained from CD confirmed that these tetraoxanes induced conformational changes in the secondary structure of HSA upon interaction. Further, molecular docking studies concluded that these analogues bind with strong affinity to HSA at the Sudlow site IIA in close proximity to Trp-214 through hydrophobic interaction and H-bonding.

4. Materials and Methods

4.1. Materials

All the chemicals and materials used in the experiments were of analytical grade. Fatty-acid-free HSA (A3782-1G) was procured from Sigma-Aldrich, India. The stock solution of HSA (10 μM) was prepared using phosphate-buffered saline (pH 7.4, 10 mM) and stored in a refrigerator to a maintain temperature of 2–6 °C. The stock solutions of TO1 and TO2 (10 mM) were prepared in 1% dimethyl sulfoxide. Further, working solutions of TO1 and TO2 were prepared by dilution of the stock solution with buffer. Millipore water was used to prepare the buffer.

4.2. UV–Visible Absorption Spectrum Experiments

The UV–visible absorption spectra at room temperature was recorded by a double-slit UV–visible spectrophotometer (JASCO V-670) using a quartz cuvette of dimension 1.0 × 1.0 cm. The HSA concentration was kept constant (10 μM), while the concentrations of the ligands were varied from 0 to 90 μM in intervals of 10 μM.

4.3. Fluorescence Spectroscopy Experiments

The fluorescence spectra of free HSA in the presence of TOs were recorded using a Varian Cary Eclipse spectrophotometer arrayed with a 1.0 cm quartz cell, in the range 290–500 nm. An excitation wavelength of 280 nm was set, and excitation and emission bandwidths were kept at 5 nm. A scanning speed of 200 nm/min was set. HSA concentration was kept constant (10 μM), and the molar concentration ratios of TO1–2 to HSA were varied.

4.4. Time-Resolved Fluorescence Spectroscopy

The HORIBA Yvon lifetime spectrophotometer was used to carry out the life time decay experiment. The time-correlated single-photon counting technique was used to evaluate the lifetime decay owing to its high optical efficiency and temporal resolution. An excitation pulse of 1.2 nm at a repetition rate of 1 MHz was sourced from NanoLED. Time-resolved fluorescence spectra were recorded for native HSA and in the presence of different concentrations of TO1–2.

4.5. CD Spectroscopy

A CD spectrometer (JASCO-810, Japan) furnished with a thermo-stated cell holder at a room temperature was operated using a quartz cuvette of 10 mm path length and 190–250 nm spectral range was selected for CD spectroscopy. Nitrogen was regularly purged into the spectropolarimeter in order to absorb the condensed water throughout the experiment. Each scan was obtained after subtracting scans of the buffer for baseline correction. The scan speed was fixed at 100 nm/min with 1 s response time and 1 nm bandwidth. The final spectra correspond to the average of three accumulations. Each spectra was analyzed by the web-based software package, K2D3, to evaluate the percentage of the α-helix.30

4.6. Computational Analysis

Gaussian 09W software was used to optimize the geometries of tetraoxanes, and these optimized geometries were used for DFT calculations. The AutoDock 1.5.6 program was employed to achieve molecular docking of tetraoxanes (TOs) and HSA. The HSA crystalline structure with the entry code 1E78 was obtained from the Protein Data Bank online database for molecular docking. All the water molecules and heteroatoms were deleted. Thus, polar hydrogens and Kollman charges were added, and Gasteiger charges were calculated. Ligand (TO1 and TO2) PDB files were introduced into AutoDockTools (ADT), and the rigid roots and rotatable bonds were defined. The grid size was set to 256 × 256 × 256 along three X, Y, and Z axes with 0.375 Å spacing. LGA was used as the search engine with population size: 150, maximum number of energy evolutions: 2,500,000, maximum number of generations: 27,000, an elitism of 1, mutation rate: 0.02, and crossover rate: 0.8. The resulting 50 conformations were clustered by setting rms to 2.0 Å. All the docked simulations are extracted and analyzed and aimed to determine least energy conformation. The best scored rank results are given in Tables S1 and S2 (Supporting Information). The PyMol molecular graphics system (DeLano Scientific, San Carlos, USA, version 0.99rc6) was used to analyze the docked conformations.

Acknowledgments

P.Y. and P.S. are thankful to UGC for financial support. B.S. and S.K.A. are thankful to the SERB, New Delhi (SERB/F/9974T20-17) and the University of Delhi, Delhi, India, for financial assistance. Authors are also thankful to the University Science and Instrumentation Centre (USIC), University of Delhi, India, for providing the instrumental facilities. Authors are also thankful to CIF, ACBR, University of Delhi, Delhi, for the CD instrument facility.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.9b04095.

  • Docking summary of TO1 with HSA using AutoDock program generating different ligand conformers and docking summary of TO2 with HSA using AutoDock program generating different ligand conformers (PDF)

The authors declare no competing financial interest.

Supplementary Material

ao9b04095_si_001.pdf (70KB, pdf)

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