Molecular dynamics simulations and experimental studies reveal differential permeability of withaferin-A and withanone across the model cell membrane

Poor bioavailability due to the inability to cross the cell membrane is one of the major reasons for the failure of a drug in clinical trials. We have used molecular dynamics simulations to predict the membrane permeability of natural drugs—withanolides (withaferin-A and withanone) that have similar structures but remarkably differ in their cytotoxicity. We found that whereas withaferin-A, could proficiently transverse through the model membrane, withanone showed weak permeability. The free energy profiles for the interaction of withanolides with the model bilayer membrane revealed that whereas the polar head group of the membrane caused high resistance for the passage of withanone, the interior of the membrane behaves similarly for both withanolides. The solvation analysis further revealed that the high solvation of terminal O5 oxygen of withaferin-A was the major driving force for its high permeability; it interacted with the phosphate group of the membrane that led to its smooth passage across the bilayer. The computational predictions were tested by raising and recruiting unique antibodies that react to withaferin-A and withanone. The time-lapsed analyses of control and treated cells demonstrated higher permeation of withaferin-A as compared to withanone. The concurrence between the computation and experimental results thus re-emphasised the use of computational methods for predicting permeability and hence bioavailability of natural drug compounds in the drug development process.

www.nature.com/scientificreports/ transport across artificial membranes 28 and transport across cultured epithelial cell monolayer 29,30 have been the valuable tools. However, these models do not take into account the molecular properties of membrane that play an important role in governing the permeability of a solute. Computational methods, on the other hand, hold remarkable potential to investigate the structure-permeability relationships. Nonetheless, full atom computational models of membranes such as POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine), POPS (1-palmitoyl, 2-oleoyl-sn-glycero-3-phosphoserine) etc. have been used to study the interactions of small molecules with the membrane and their permeability characteristics. These membrane models exhibit the structural and dynamic features of membrane bilayer, which have been investigated and verified by MDS 31,32 .
Cholesterol is one of the major components of eukaryotic cell membrane and also reported to have a large effect on the membrane permeability 33 . Generally solute permeability across the high cholesterol content membranes is low [34][35][36] . By measuring the potential of mean force (PMF) barriers of dynamic drugs, hypericin and its derivatives, it has been reported that cholesterol significantly influences the permeation of these molecules 37 . It was shown that the calculated rate of permeation is lower at high cholesterol content due to increase in PMF barrier. Therefore, adding cholesterol molecules to POPC lipid membrane helps in bringing the simulation environment closer to the actual cell membrane system. Such mixed membrane models have been used successfully to predict the permeability of drugs such as ibuprofen, cimitedine, six-β-blocker, aliphatic amine, and carboxylic acid drugs using the MDS 38,39 .
The partition of a drug could be either predicted by long nano-second classical MDS 40,41 , or by the Potential of Mean Force (PMF) methods 42,43 . Using MDS on various fluorescent azaaromatic probes on water-membrane, it has been earlier shown that the probe localization is determined by the electrostatic dipole-dipole and van der Waals interactions 44 . Further, it was also shown that 2,6 Bis (1H-Benzimidazole-2-yl) pyridine (BBP) preferred to locate around 15-16 Å from the DPPC (1,2dipalmityol-sn-glycero-3-phosphatidycholine) membrane center 42 . A MDS study of 17 amino acids in lipid bilayer showed that the partitioning of charged and polar side chain amino acids were accompanied by water defects 45 . Furthermore, the solute size, polarizability and free energy of transfer from water to membrane also affected the transfer of solute from water into membrane.
In the present study, we have elucidated the mechanism of permeation of two natural drug molecules (Wi-A and Wi-N) through POPC bilayer using computer simulations, which were further validated by recruiting unique withanolide-recognizing antibodies in cell-based assays. In-spite of having very little difference in their structures, Wi-A molecules successfully crossed the bilayer with low free energy associated with it, while the passage of Wi-N was associated with high free energy barrier. We present here a computational assay which would be useful for predicting permeation and bioavailability of drug compounds in drug development process.

Methods
Parameterization and placement of withanolides. The geometry optimization and the charge of Wi-A and Wi-N molecules were calculated at 6-31G* level by using GAMESS-US 40 and further refinement was carried out by using Restrained Electrostatic Potential Fit (RESP) using the RED-Tool package 41 . In each system, one drug molecule was placed in the aqueous phase by aligning the the plane of the molecule with the plane of the membrane (Fig. 1).

Molecular dynamics (MD) simulations.
Three MD simulations-(1) POPC membrane, (2) POPC membrane having Wi-A, and (3) POPC membrane with Wi-N, were carried out. The model membrane was composed of POPC and cholesterol molecules, where, the starting structures were taken from a 2 μs long equilibration simulation data 46 . The bilayer membrane model consisted of 70 POPC and 35 randomly placed cholesterol molecules per layer or leaflet. All simulations were run using TIP3P water models 47 and amber Lipid14 force field parameter 48 . It has been earlier shown that Lipid14 is a good choice for molecule permeability calculations 49 , all the structures were converted to the Lipid14 naming convention by using charmmlipid2amber.py script, available with Amber Tools v18. The prepared models were then completely solvated using 7499 TIP3P water molecules. To attain the charge neutrality, adequate numbers of Na + and Clions were added in each simulation box.
Equilibration and production procedure. The solvated membrane in the simulation box was then equilibrated using a slightly modified version of the multi-step protocol 50 . Briefly the procedure consisted of altering cycles of steepest descent (1000 steps) and conjugate gradient energy minimization (1000 steps) followed by position restrained MD with strong harmonic restraints on all non-hydrogen atoms of the bilayer membrane and solvent. In successive steps, the positional restraints were gradually relaxed. The detailed description of the minimization and equilibration protocol can be found in the supplementary material S1. All the energy minimization and MD simulations were run using the SANDER modules of the AMBER18 program suite 51 . All the three systems (Control, POPC-Wi-A, and, POPC-Wi-N) were equilibrated using a slightly modified version of the multi-step protocol The production simulations for all the systems were run for 600 ns in NPT ensemble using GPU accelerated version of the PMEMD program of AMBER simulation suite 52,53 . The temperature and pressure of the systems were kept restrained to 310 K above the crystalline fluid/liquid phase transition temperature and 1 atm respectively using the Langevin thermostate, while the aniosotrophic Berendsen barostat 50 was used for pressure control. All bonds involving hydrogen atoms were constrained using the SHAKE algorithm. Short range electrostatics were calculated up to 10 Å and the Particle Mesh Ewald 51 algorithm was used to calculate the long-range electrostatics. An integration time step of 2 fs was used, and structures were saved every picosecond resulting in around 6,00,000 structures. Since the transition of withanolides from bulk phase to lipid starts at ~ 33 ns, the analysis was performed on the last 570 ns of the datausing the CPPTRAJ module of Amber suite. www.nature.com/scientificreports/ Potential of mean force (PMF) simulations. The potential of mean force (PMF) free energy profiles for the partitioning of drug compounds were calculated using umbrella-sampling simulations. The withanolides were placed on the water phase on top of the membrane. Using a pulling rate of 1 Å/ns, the drugs were then pulled out from the aqueous phase towards the center of bilayer membrane system for a total of 38 Å (force constant of 1.5 kcal/mol/Å 2 ) in the NPT ensemble with semi-isotropic pressure coupling scheme. During the simulations, snapshots were saved every 1 Å from the top (z = 38) of membrane to center (z = 0) generating 38 windows. The results were calculated for one bilayer, and due the symmetry of membrane it was assumed that the other half behaves similarly. This was achieved by reflecting the data along z axis and adding 37 or 38 windows depending on whether or not the window at z = 0 Å was reflected as well. Each umbrella sampling window was run for 30 ns of production run with the force constant of 3.0 kcal/mol/Å 2 , totaling ~ 1 μs of sampling per permeant. Configurations were saved every 1 ps. Further, to calculate the PMF, the biased distributions were reweighted using Weighted Histogram Model (WHAM) 54,55 . Diffusion and resistance calculations. Theoretically, the permeability coefficient of a molecule can be computed from an atomistic simulation-based PMF approach via the inhomogeneous solubility diffusion permeability model 56 . Here, the permeation is divided into a three-step process: (a) involving the partitioning of a permeant from the aqueous phase on the side of leaflet, (b) diffusion across the bilayer and (c) partitioning from the other side of bilayer into aqueous phase.
where, R(z) is the resistivity of every "slice" of the membrane at position "z". w(z) is the PMF, D(z) is the local position specific diffusion coefficient, which can be calculated by the Hummer's positional autocorrelation extension of Wolf-Roux estimator 57 . A detailed discussion on the computation of local diffusion coefficient D(z) has been reviewed elsewhere [58][59][60] .
Area per lipid (APL). Molecular packing of a lipid bilayer is described by APL. In molecular dynamics simulation, a pure lipid bilayer has the normal along z direction. The APL can be calculated by following equation: where,L x , L y is the box length in x, y direction. N lipid indicates the total number of lipid molecules in one leaflet.
Order parameter. The ordering of lipid acyl chains was determined by the calculation of order parameter S CD . This quantity can be directly compared to experimental S CD values obtained by 2 H NMR or 1 H-13 C NMR. Since S CD is also a measure of relative orientation of the C-D bonds with respect to the bilayer normal, it can be calculated according to the following equation: where θ is the angle between the bilayer normal and vector joining C-D (actually C-H in the current simulation), and < > represents an ensemble average.

Statistical geometry.
To investigate the effect of drug permeation on the structure of membrane, the statistical geometry approach was used. The analysis was initiated by partitioning the space contained in the simulation box by means of Delaunay Tessellation 61 . The detailed description of the method can be found elsewhere 50,60,61 .
In this study, the Delaunay tetrahedron was formed between the heavy atoms of POPC (Phosphate), cholesterol (Oxygen) and water (Oxygen). The local structure of a system can be quantified by the distortion of Delaunay tetrahedra, which has been defined in terms of a parameter called tetrahedrality (T) defined as follows 62 : where l i , l j, l 2 are the edge length and the mean length of a tetrahedra, respectively. For a perfectly regular tetrahedron, the tetrahedrality is 0; an increasing deviation from regularity causes a corresponding increase in the parameter. The free energy, diffusion and resistivity were calculated using umbrella sampling, while the other properties were derived by classical all-atom MDS data.

Generation of anti-Withaferin-A and anti-Withanone antibodies.
Wi-A and Wi-N were extracted from dried Ashwagandha leaves as described in a previous study 15 . Mixture of Wi-A/Wi-N (1:1) along with Freund's adjuvant was used as antigen to immunize the mice for monoclonal antibody generation. The resulting clones (~ 30) were screened with affinity ELISA and one clone L7C3-6 (WiNA Ab) was isolated that reacted to Wi-A and Wi-N in fixed cells. The clone was established as hybridoma. Affinity purified antibody was used for this study.

Results and discussion
Membrane architecture. In order to validate our simulation protocol, few parameters like Area Per Lipid (APL), electron density, Lipid order and Tetrahedrality were calculated in control as well as in presence of Wi-A and Wi-N molecules, which were compared to the experimentally observed values. Electron density profile. Generally, thickness of the bilayer is determined by the electron density profile measured by the X-ray scattering of liquid-crystalline membranes 64,66,67 . The electron density profiles (EDP) were calculated by assuming an electron charge equal to the atomic number minus the atomic partial charge located at the center of each atom. The electron density profile decomposed into various groups as water, drug molecules, choline (CHOL), phosphate (PO 4 ), glycerol, carbonyl (COO), methylene (CH 2 ), unsaturated CH=CH and terminal methyl's (CH 3 ) is plotted in Fig. 2. The density profile for POPC bilayer in control is shown in Fig. 2A.

Area per lipid (APL
Almost similar trends were obtained in case of Wi-A and Wi-N ( Supplementary Fig. 1). From Fig. 2A, it is clear that in case of all atom density, three distinct domains are visible. While the flat region >|25| Å represents an aqueous phase, the unimodal distribution peak at ~ 15 Å corresponds to the interface region, containing lipid head-groups and water. Similar peaks were also observed for the phosphate atom, choline and carbonyl groups. The drop in the density represents the inner part of the bilayer, where the alkyl chains of the phospholipid reside. The decrease in water density, from bulk to middle of bilayer, indicates that no water molecule has traversed the lipid tail. In each case ( Fig. 2A, Supplementary Fig. 1), the electron density profiles were symmetrical, almost overlapping, with water penetrating up to the carbonyl groups, leaving the terminal methyl groups dehydrated in agreement to the experimental results. The simulated electron density profile indicated that the parameters chosen for the MD simulations reproduces the membrane geometry at a good level. On the contrary, the electron Voronoi/delaunay tessellation and interspatial arrangement. Traversing the membrane by a drug molecule is likely to result in the distortion of the spatial arrangement of the bilayer. Statistical geometry analysis provides an elegant approach to investigate the local geometrical arrangement of the molecules. Delaunay tessellation portions the 3D space into series of non-overlapping tetrahedra. In this study, the tetrahedrality distribution for control, Wi-N and Wi-A was uni-modal that peaked at ~ 0.5 depicting a regular arrangement of atoms, which was not perturbed by the traversing of bilayer by the drug molecules (Fig. 4). However, the long tail of tetrahedrality distribution, associated with low probability, indicated the formation of few irregular tetrahedral arrangements. Taken together, the variation of APL, electron density, Lipid order and Tetrahedrality variation suggested that the traversing of drug molecules had no effect on the over all architecture of the bilayer. Fraction of Native Contacts (Q) between drug and POPC lipid ( Supplementary Fig. 4) showed that both the drug molecules had interaction preferences for the lipid tail region as compared to the polar head region. This behavior could be attributed to the hydrophobic nature of the drugs as well as lipid molecules. Further, Wi-A was observed to interact more with lipid tail region in comparison to Wi-N.
Orientation and insertion depth of withanolides in the membrane. Withanolides are overall hydrophobic molecules (Fig. 1, Table 1). However, they carry different functional groups at various positions that influence their orientation and position of steroids in the membrane. The probability distribution for angle of Wi-A was calculated. It is defined by the atoms C18 "head" and C25 "tail" respectively that connected the two opposite positions with respect to bilayer normal. Similarly, for Wi-N, the vector was drawn between the C19 and C25 atoms. The Fig. 5 shows the tilt angle between membrane normal and steroidal axis, where both the drug molecules adapt different orientations. The multimodality of Wi-A tilt angle distribution suggested that it does not assume a single well-defined orientation, but instead has a wide distribution of orientation. The tilt    www.nature.com/scientificreports/ angle distribution of Wi-N was unit-modal (that peaked at ~ 100°) that indicated a well-defined orientation. For instance, the conformation of Wi-A is imposed by the polar hydroxyl groups at the C13, C24, C27 atoms and methyl groups at C14, C16, C21, C27 position (Fig. 1). This particular configuration imposed a specific vertical orientation (i.e. parallel to bilayer normal) position, such that the hydrophobic groups were solvated by the lipid tails and the polar groups (O5 and O4) can form hydrogen bonds with other polar parts of the membrane 72 . In addition to orientation, another major degree of freedom was given by the positions of the withanolides relative to the membrane center of mass, which is the insertion depth in the membrane ( Table 2). As expected, because of their overall lipophilic nature, these molecules tend to localize below or near the ester groups of POPC at the interface between polar or apolar regions of the membrane. Similar locations for other steroids are reported in literature 72 . The different orientations acquired by Wi-A and Wi-N while passing through the lipid membrane is shown in Supplemetary Figs. 2 and 3, respectively.

Potential of mean force (PMF).
In the solubility-diffusion model, the calculation of PMF is a critical component for the estimation of membrane permeability. It corresponds to the relative solubility of a permeant in solution versus the membrane interior. The respective permeability of Wi-A and Wi-N from water phase to the interior of membrane is shown in Fig. 6. Considering the first part of the curves, from bulk water region to the polar head group, the high free energy values for Wi-N as compared to Wi-A is clearly evident. This indicated that the bilayer is a more preferable location for Wi-N than Wi-A. However, the portioning in bilayer was critical. While proceeding deeper in the bilayer that corresponds to the glycerol region, both the drug molecules displayed a free energy decrease, with a deep free energy minima at ~ 20 Å (Wi-A) and ~ 8 Å (Wi-N). Therefore, this region could be considered as the preferably partitioned region for both the withanolides. The rightmost part of Fig. 6 shows the change in free energy characterizing the permeants in hydrocarbon tail region (bilayer core). Here, both drugs showed an increase in free energy, highlighting the barrier effect inserted by the lipid hydrocarbon tail. However, the differences in free energy values were small for both the drug molecules. From the PMF, it is clear that the bulk region is unfavorable for Wi-N. The ΔG value between the bilayer midplane and for free energy minimum for Wi-N is also smaller than that for Wi-A. This implied a smaller barrier for passing from one leaflet to the other for Wi-N when compared to Wi-A. The variation in free energy value for both permeants moving from bulk water phase into the membrane could be attributed to the increase in density (Fig. 2B). Density profile clearly showed that the preferable locations for withanolides were symmetric and close to lipid head groups (the densest region of bilayer). This location, as well as the tilt angle distribution (Fig. 5, Table 2) was in good agreement with the minima of the PMF implying that they were energetically favorable. For hydrophilic molecules, the lipid tail represented the main barrier to permeation, while for hydrophobic molecules, partition is more favored in the middle of a bilayer than at lipid/ water interface. The difference in the PMF values can be attributed to the hydrophobicity of the drug molecules. logP is the measure of lipophilicity of a molecule in un-ionized water, while logD is the measure of the same in Table 2. Distance of the drug molecules from the phosphate atom of membrane (mean and standard deviation in 500 ns).

Membrane location
Wi-A 29.60 ± 8.12 Wi-N 31.16 ± 8.01  (Table 1). As majority of known drugs are likely to be charged at physiological pH, using logP to describe their lipophilicity could be misleading. logP alone describes the partition coefficient of a neutral (uncharged) molecule, hence logP offers an advantage in such (neutral stage) cases. The difference in the value of logP and logD can also be used as a measurement for understanding the effect of charge distribution on the surface of molecule. Because POPC membrane is highly chemically diverse, its hydrophilic layers provided charged environment to the incoming molecules. The logP value of Wi-A (logP = 3.8) was slightly higher than that of Wi-N (logP = 3.1), but logD value of Wi-A (logD = 3.58) was much higher than that of Wi-N (logD = 2. Diffusion and resistivity. The second key component of the solubility-diffusion model is the diffusivity/ Resistivity. The above results indicated that transfer of Wi-A across the bilayer tail region is less energy-driven and hence favorable as compared to Wi-N. Further, to check the resistivity exhibited by the bilayer on the drug molecules, the resistance exerted by the bilayer on both the natural drug molecules were calculated and shown in Fig. 7. The variation in resistivity showed a high resistance for Wi-N molecule in the lipid head region, while the resistance experienced in the bilayer tail region is almost similar for both Wi-A and Wi-N drug molecules. From the electron density (Fig. 2B), PMF (Fig. 6) and resistivity variation (Fig. 7), it is evident that the head part of the membrane was primarily resisting the passage of natural drug molecule across the bilayer, while the hydrophilic region was offering almost similar barrier/resistance for both the withanolides. Between Wi-A and Wi-N, the lipid head group region seemed to play a key role in determining the easier passage of Wi-A drug molecule as compared to Wi-N drug molecule.
Solvation and hydrogen bond. Though both the withanolides possess similar structure, the results indicated a favorable passage for Wi-A across the bilayer polar region. To understand the differences associated with this phenomenon, the average numbers of water molecules in the first hydration shell (number of water molecules in the 3.5 Å radius of oxygen atom) were calculated for the drug molecules. The boxplot representation showed a higher solvation of Wi-A as compared to Wi-N, where the average solvation of epoxy oxygen is similar for both the permeants (Fig. 8). Though subtle differences could be observed for other oxygen atoms, in Wi-N, the average water solvation was ~ 6 water molecules for rest of five oxygen atoms. However, for Wi-A, all the oxygen atoms were more solvated with ~ 7 water molecules. Further to get a clear picture of water dynamics around the drug molecules, the hydrogen bond lifetime between water molecule and drug oxygen atoms was investigated. Oxygen of water has a natural tendency to form hydrogen bond with other electronegative atom and hydrogen atom of water has the similar tendency to form hydrogen bond. Both the drug molecules have several -OH group that can form hydrogen bonds with water molecules; both water and drug molecules could act as hydrogen bond donor and acceptor in the formation of hydrogen bond. Depending upon the accessibility as well as the ambient environment, the interaction between the water and drug molecules may differ. Therefore, the calculation of hydrogen bond lifetime was expected to provide a measure for the elucidation of water dynamics around drug molecules leading to the favorable passage of Wi-A when compared to Wi-N. The maximum lifetime (in picoseconds) of hydrogen bond formed between water and the terminal O4 (~ 4.98) and O5 (~ 5.10)  Table 3. While, in case of Wi-N the average maximum lifetime was found to be ~ 3.27 ps and ~ 3.69 ps respectively for O4 and O5 atoms.

Radial distribution function (RDF).
This analysis was carried out to track the passage of Wi-A molecule, which forms hydrogen bond with water, phosphate and oxygen atoms of the POPC atoms. The RDF between the oxygen atoms of Wi-A and lipid phosphate (Fig. 9A) and lipid nitrogen (Fig. 9B) was plotted. The nearest neighbor peak was clearly visible at ~ 3.5 Å and 4 Å for the phosphorus and nitrogen groups respectively. In case of phosphorus, the second nearest-neighbor peak was also pronounced at ~ 5 Å. Among all the oxygen atoms of Wi-A, a strong interaction with lipid phosphate was observed with O5 followed by O1 and O4 atom. Similarly, the O5 and O4 atoms again showed a high interaction with the lipid nitrogen atom. In case of Wi-N, the epoxy oxygen seemed to interaction with the lipid phosphorus and nitrogen atoms; however, the strength of this interaction was small as compared to Wi-A (Fig. 9C,D). Though both the molecules possess similar structure, Wi-A could still cross the bilayer proficiently but not Wi-N. The difference between two molecules actually lies in the distribution of single hydroxyl group. In Wi-A, the hydroxyl group is attached to the terminal lactone ring, whereas in Wi-N, hydroxyl group is attached to the middle of steroidal ring. Though the number of total polar atoms in Wi-A and Wi-N remains the same, the position of polar atoms makes a big difference in the charge distribution over their surface (Fig. 1, log D values). The terminal arrangement of polar atoms in Wi-A provided it an advantage over the distributed polar atoms in Wi-N. Grouping of the polar atoms at just the terminals made the middle surface of Wi-A continuously non-polar favoring interactions with both hydrophilic and hydrophobic environment. Grouped polar atoms are more capable to face the hydrophobic environment as their own interactions can reduce the total exposed surface area. From the high solvation (Fig. 8) of terminal polar groups (O4 and O5) in Wi-A as well as the strong hydrogen bond lifetime (Table 3) of same atoms along with the RDF analysis (Fig. 9), it can be interpreted that the interaction of terminal O5 and O4 atoms of Wi-A with polar atoms (water, lipid nitrogen, and lipid phosphate) of system enables the smooth transverse of Wi-A. While for Wi-N, no such favorable interactions were found; hence the passage of Wi-N across the normal membrane was associated with high-energy cost.

Experimental evidence to the differential absorption of Wi-A and Wi-N by normal cells.
In order to provide the experimental evidence to the above membrane permeability analysis, we generated, for the first time, antibodies to Wi-A and Wi-N. As shown in Supplementary Fig. 5, 3/96 clones that initially showed reactivity to Wi-A and Wi-N were developed into hybridoma and were examined for their reactivity to Wi-A and/or Wi-N. We found that the one clone (L7C3-6) was able to detect Wi-A and Wi-N in cells treated with either purified compounds or i-Extract containing Wi-A and Wi-N as major components. The clone was designated as WiNA antibody. We next used anti-WiNA antibody to detect Wi-A/Wi-N in human cancer cells treated with purified Wi-A or Wi-N; isotype matched secondary antibodies were used as negative control. As shown in Fig. 10A, higher level of both Wi-A and Wi-N were detected in cells treated for 48 h as compared to 24 h. We extended the analysis to human normal cells. Wi-A, but not Wi-N, was detected in the normal cells incubated with the purified compounds for 48 h (Fig. 10B). Furthermore, we found that Wi-A and Wi-N staining in the nucleus, which was confirmed by confocal microscopy (Fig. 10C). Wi-A was detected clearly in the nucleus of cancer as well as in normal cells. In contrast, Wi-N was detected in the nucleus of cancer cells only; normal cells showed weak pancytoplasmic staining (Fig. 10C). In order to further support these findings, we examined the expression of several proteins that have been shown to be involved in anticancer activity of Wi-A and Wi-N. As expected, Wi-A caused stronger up-regulation of γH2AX and p53, markers of DNA damage response in cancer cells (shown in Fig. 10D,E). Furthermore, only the cells treated with Wi-A showed decrease in CARF protein signifying apoptosis (Fig. 10E). The same was not observed in Wi-N treated cells. In light of these findings that endorsed reliable detection of Wi-A/Wi-N in cells with our antibodies and in order to further validate the computational predictions on their differential permeability, we expanded the analysis in human normal cells. We found that Wi-A showed strong nuclear staining both in TIG-1 and MRC5 cells and associated with activated DNA damage response as determined by increase in γH2AX foci, nuclear pATR and pCHK1 (Fig. 10F,G). In contrast, Wi-N showed faint pancytoplasmic staining. Of note, increase in the DNA-damage marker proteins were not detected in normal cells. These data confirmed the MD predictions for the differential permeability of Wi-A and Wi-N through cell membrane and suggested a mechanism for their differential activity, at least, in part.
In this study, a detailed computational analysis characterizing the permeation of Wi-A and Wi-N, two natural drug molecules across the lipid bilayer was conducted. We report that the permeation of Wi-A and Wi-N does not affect the membrane integrity. The values obtained by area per lipid, electron density and order parameter, and tetrahedrailty were almost similar for bilayer in control and as well as in presence of drug molecules, suggesting no change in the overall architecture of the bilayer. Next, the PMF profile showed that the permeation of Wi-A across the bilayer was associated with low energy barrier and therefore, could be considered as a favorable transverse for Wi-A as compared to Wi-N. While the lipid head group region appeared as a determining region www.nature.com/scientificreports/ for the movement of drugs across the bilayer, the variation of resistivity profile confirmed the same. The polar head group region appeared to exert a high resistance on the passage of Wi-N as compared to Wi-A. Further, to elucidate the mechanics for this behavior, the solvation, RDF and hydrogen bond dynamics were calculated, which clearly indicated that the high solvation of terminal hydroxyl group (O5) along with tight interaction with polar head group atoms (phosphate/nitrogen) of the membrane, facilitate a smooth passage for Wi-A. The logP and logD values associated with the Wi-A and Wi-N supported these results. In spite of the fact that Wi-N also has similar structure like Wi-A, the lipophilicity of the molecule was less as compared to Wi-A that may account for its weak permeation through the membrane. This study revealed that the subtle difference associated with the positioning of functional groups can play a major role in transportation of drug molecules across the bilayer. The experimental data using unique Wi-A and Wi-N antibodies demonstrated that Wi-A indeed proficiently entered the cells and was detected in the nucleus by immunostaining. All together the calculated free energy profile for the permeation of Wi-A and Wi-N were qualitatively matching with the experimental observation. Further studies enrolling varying drug concentrations and more membrane models including (varying composition and density, representing different normal and cancer cell types and intracellular structures) are warranted. The study is an important step towards understanding the molecular basis of permeability of natural drug molecules.