Assessing gastric toxicity of xanthone derivatives of

Biophysical Chemistry 220 (2017) 20–33

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Assessing gastric toxicity of xanthone derivatives of anti-inflammatory activity using simulation and experimental approaches Michal Markiewicz a, Tadeusz Librowski b, Anna Lipkowska b, Pawel Serda c, Krzysztof Baczynski a, Marta Pasenkiewicz-Gierula a,⁎ a b c

Department of Computational Biophysics and Bioinformatics, Jagiellonian University, Krakow, Poland Department of Radioligands, Jagiellonian University, Krakow, Poland Department of Crystal Chemistry and Crystal Physics, Jagiellonian University, Krakow, Poland

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• MD simulations of bilayers containing xanthones were carried out for 500 ns. • The xanthones have properties typical for nonsteroidal anti-inflammatory drugs. • Such drugs often affect hydrophobic barrier properties of the gastric mucosa. • The model bilayer contained lipids typical for the gastric mucosa. • The studied xanthones had little effect on the hydrophobicity of the model bilayer.

a r t i c l e

i n f o

Article history: Received 17 July 2016 Received in revised form 12 October 2016 Accepted 26 October 2016 Available online 05 November 2016 Keywords: Lipid bilayer Gastric mucosa Hydrophobic barrier Hydrogen bonds MD simulation

a b s t r a c t Xanthones are tricyclic compounds of natural or synthetic origin exhibiting a broad spectrum of therapeutic activities. Three synthetic xanthone derivatives (KS1, KS2, and KS3) with properties typical for nonsteroidal antiinflammatory drugs (NSAID) were objects of the presented model study. NSAIDs are in common use however; several of them exhibit gastric toxicity predominantly resulting from their direct interactions with the outermost lipid layer of the gastric mucosa that impair its hydrophobic barrier property. Among the studied xanthones, gastric toxicity of only KS2 has been determined in previous pharmacological studies, and it is low. In this study, carried out using X-ray diffraction and computer simulation, a palmitoyloleoylphosphatidylcholine-cholesterol bilayer (POPC-Chol) was used as a model of a hydrophobic layer of lipids protecting gastric mucosa as POPC and Chol are the main lipids in human mucus. X-ray diffraction data were used to validate the computer model. The aim of the study was to assess potential gastric toxicity of the xanthones by analysing their atomic level interactions with lipids, ions, and water in the lipid bilayer and their effect on the bilayer physicochemical properties. The results show that xanthones have small effect on the bilayer properties except for its rigidity whereas their interactions with water, ions, and lipids depend on their protonation state and for a given state,

Abbreviations: NSAID, nonsteroidal anti-inflammatory drug; POPC, palmitoyl-oleoyl-phosphatidylcholine; Chol, cholesterol; MD, molecular dynamics; H-bond, hydrogen bond; RDF, radial distribution functions. ⁎ Corresponding author at: Department of Computational Biophysics and Bioinformatics, Faculty of Biochemistry, Biophysics, and Biotechnology, Jagiellonian University, ul. Gronostajowa 7, 30-387 Krakow, Poland. E-mail address: [email protected] (M. Pasenkiewicz-Gierula).

http://dx.doi.org/10.1016/j.bpc.2016.10.007 0301-4622/© 2016 Elsevier B.V. All rights reserved.

M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

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are similar for all the xanthones. As gastric toxicity of KS2 is low, based on MD simulations one can predict that toxicity of KS1 and KS3 is also low. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The basic chemical structure of a xanthone molecule consists of the pyrone system condensed with two benzene rings [1]. The molecule has eight places to accept substituents. Unsubstituted xanthone does not manifest strong physiological action, but plays an important role as a pharmacophore. Substituted xanthones or xanthen-9H-one derivatives, both of natural and synthetic origin, constitute a class of compounds with high therapeutic potential. Natural xanthone derivatives extracted from herbs (e.g. Gentiana lutea, Polygala nyikensis, Garcinia livingstonei, Garcinia mangostana, Mesua ferrea) are known to possess anti-bacterial [2], mycotoxic [3] and cytotoxic [4] properties. In addition, their anti-inflammatory [5–7], antimalarial [8], antiarrhythmic [9], analgesic [10], hypotensive [11], antiplatelet [5], antiasthmatic [12], hepatoprotective [13], antidiabetic [14], antiallergic, HIV-inhibition [15] and other [16,17] activities have been reported. Also, they are potent agents against multi-drug resistant cancer cells [18]. Synthetic xanthone derivatives, similarly to the natural ones, display a broad spectrum of therapeutic activities, in particular anti-inflammatory [19], cytostatic [20], antimycotic [21], cardiovascular [22,23], antiepileptic [24] and anticancer [25]. As in the case of most drugs (for comprehensive overview, see Ref. [26]), biological activity of a xanthone derivative depends on the chemical character of its substituents and on their positions in the molecule, thus the xanthone platform offers a rich variety of isomeric compounds to investigate [27]. Xanthone derivatives examined in this study show anti-inflammatory and analgesic properties that are typical for nonsteroidal anti-inflammatory drugs (NSAIDs). Drugs belonging to this group act through enzyme cyclooxygenase, inhibiting prostaglandin synthesis [28]. Unfortunately, a long-term uptake of some NSAIDs is connected with adverse side effects, mainly from the gastrointestinal track. The aetiology of these effects has previously been linked to the lack of prostaglandins protective effects on the gastric mucosa [29]. Recently, however, the side effects of these NSAIDs have been associated with their direct interactions with gastric mucosa lipids [30,31]. Several experimental results suggest that they disturb the hydrophobicity of the outermost lipid layer of gastric mucosa, which results in its increased permeability, making it more vulnerable to the

gastric acid [32–34]. On the microscopic scale, the perturbed structure of a protective lipid layer of the gastric mucosa should manifest itself by an increased access of water into the layer interior. Indeed, our previous X-ray diffraction and molecular dynamics (MD) simulation study of a lipid bilayer containing three commercial NSAIDs with different gastric toxicity, aspirin, ketoprofen, and piroxicam, demonstrated that the most toxic drug has the ability to “pull” water molecules into the hydrophobic core of the bilayer modelling the outermost lipid layer of the gastric mucosa [35]. In the research presented in this paper, we also use simulation and experimental approaches to assess the possible toxic effect of three newly synthesized xanthone derivatives [36] 2-methyl-2[2(methyl)-6-xanthonoxy]-propionic acid (KS1), 2-methyl-2[4(methyl)-6-xanthonoxy]-propionic acid (KS2), and racemic (RS)2-[2-(methyl)-6-xanthonoxy]-propionic acid (KS3) (Fig. 1) on the gastrointestinal track. Investigated xanthone derivatives administered orally to rats showed varied anti-inflammatory activity in the carrageenan induced edema test, isomers KS1 and KS3 (Fig. 1) (symbol: MH-38 and MH-40, respectively, in [37]) have a strong and isomer KS2 (Fig. 1) (symbol: MH-44 in [37,38]) has a moderate anti-inflammatory activity as compared to aspirin and ketoprofen [37]. Unfortunately, of the three xanthones, only KS2 toxicity has been examined in pharmacological studies – the compound did not show any ulcerogenic effect in rats [38]. Our aim is to elucidate whether and how these potential drugs interact with the bilayer lipids and how these interactions affect the dynamic structure and other properties of a lipid bilayer. Primarily, we are interested in their interactions with water and ions at the bilayer interface as these interactions are the key factor affecting the bilayer hydrophobic barrier. Comparison of the effects of the three xanthones on the model membrane will be used to assess potential toxicity of two of them, KS1 and KS3, for which there are no experimental results concerning toxicity. As a model of the outer lipid layer of the gastric mucosa as well as of gastric mucosa cell membranes a palmitoyloleoylphosphatidylcholine-cholesterol (POPC-Chol) bilayer is used. Such a bilayer was chosen because POPC is the main phospholipid species and cholesterol is common constituent of human and pig mucus, e.g. [39,40].

Fig. 1. Chemical structures of (a) xanthone derivatives, 2-methyl-2[2-(methyl)-6-xanthonoxy]-propionic acid (KS1), 2-methyl-2[4-(methyl)-6-xanthonoxy]-propionic acid (KS2), and racemic (RS)-2-[2-(methyl)-6-xanthonoxy]-propionic acid (KS3) – hydrogen atoms in the red frame are not present in the ionized form; (b) POPC, and (c) Chol with atom numbering. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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2. Materials and methods As the model of a hydrophobic layer of phospholipids protecting gastric mucosa a POPC-Chol bilayer with POPC:Chol in a 7:2 molar ratio (22 mol% Chol) was used. In the bilayer containing xanthone molecules, the molar ratio of POPC:Chol:xanthone was 7:2:1. The content of Chol in the gastric mucus of the human stomach has not been precisely determined even though it is rich in cholesterol [41]; also intestinal mucus in rats is rich in cholesterol [42]. However, much less cholesterol has been found in human colonic mucus [40]. As the content of Chol in gastric mucosa has not been quantitatively determined and it is not known, we used the same POPC:Chol molar ratio (22 mol%) as in our previous study [43]. 2.1. X-ray diffraction X-ray measurements were carried out on multilamellar liposomes consisting of (1) only POPC, (2) POPC and cholesterol (Chol), and (3) POPC, Chol and xanthone molecules. POPC was purchased from Avanti Polar Lipids, and Chol from Sigma-Aldrich. Xanthone derivatives were synthetized at the Department of Chemical Technology of Drugs of the Jagiellonian University. To prepare multilamellar liposomes, POPC, Chol and xanthones in the required proportion were dissolved in chloroform. The solvent was evaporated under nitrogen, followed by drying under a vacuum pump for 12 h. Redistilled water was added to each sample at lipid/H2O 1:10, 1:15, 1:25, and 1:30 molar ratios. The mixtures were centrifuged until full homogenization was achieved. A few hours before the experiment, the samples were placed into sample holders. Small angle X-ray diffraction measurements were carried out at beamline A2 in HASYLAB at DESY using monochromatic X-ray radiation emitted by the DORIS synchrotron with wavelength of 0.15 nm (fixed energy of 8 keV). SAXS data collection was carried out with linear position-sensitive gas-filled detector calibrated using rat-tail tendon; more experimental details are given in Supporting information (SI). The temperatures and pressure of 1 atm were controlled independently during the measurements. The measurements were carried out at temperatures from 20 to 40 °C (at 2 degree step) and four hydrations (see above) to avoid artefacts connected to the lattice defects and to minimize the error in estimating the bilayer width at full-hydration; this error arises from the fact that some of the hydrating water occupies the inter-liposome space [44,45]. Positions of the lamellar peaks in the diffractograms were determined by nonlinear least squares fitting to the Gaussian function. The positions changed, as hydration increased (cf. animation, SI). The repeat period, d, at full bilayer hydration (lipid/H2O 1:37.9) was obtained by extrapolating of the linear dependence of d on sample hydration [45] to full hydration. To estimate the bilayer width (DB) and the average cross-sectional area per phospholipid (A/PC) for bilayers at full hydration, the Luzzati method [46] was used; details concerning calculations of these quantities are given in SI. 2.2. MD simulation 2.2.1. Simulation systems The reference POPC-Chol bilayer used in this study consisted of 132 POPC, 36 Chol (~22 mol% Chol) and 5000 water molecules. Six additional systems were created by adding to the POPC-Chol bilayer sixteen xanthone molecules of each of the three chemical types in two protonation states, neutral and anionic. Additionally, the chiral group of KS3 was in two racemic configurations. The initial structures of the xanthone molecules were constructed using the Cerius2 program [47], as in our previous study on commercially available NSAID molecules [35]. Details concerning generation of low energy conformations are given in SI. Eight conformationally diverse molecules of each xanthone were placed in each leaflet of the POPC-

Chol bilayer. To the bilayers containing xanthone molecules in the anionic state, 22 Na+ and 6 Cl− ions, and to the bilayers containing neutral xanthone molecules, 22 Na+ and 22 Cl− ions, were added to compensate the charges of anionic xanthones and to mimic physiological NaCl concentration. Due to the comparative character of this study, the numbers of PC, cholesterol, xanthone and water molecules in all systems were the same, except for the reference POPC-Chol bilayer where there were no xanthone molecules. In total, seven systems were constructed: (1) the reference POPC-Chol bilayer with the POPC:Chol molar ratio of ~ 7:2, and six POPC-Chol bilayers containing: (2) KS1 in the neutral protonation state (POPC-KS1-n); (3) KS1 in the anionic form (POPC-KS1-i); (4) neutral KS2 (POPC-KS2-n); (5) anionic KS2 (POPC-KS2-i); (6) racemic mixture (RS) of KS3 in the neutral form (POPC-KS3-n); and (7) racemic mixture of KS3 in the anionic form (POPC-KS3-i), with the POPC:Chol:xanthone molar ratio of 7:2:1. As the preferential location of xanthones in phospholipid bilayers is not known, when building the initial structures of the bilayer, we assumed the same probability of their location in the water and in the lipid phases. Thus, in each bilayer, eight xanthone molecules were placed in the hydrophobic bilayer core, parallel to the POPC chains and the other eight were placed in the water phase, near the interface, parallel to the membrane surface (Fig. 2a). Such orientations of molecules additionally diversified their initial locations in the bilayer. Each of the systems was MD simulated for 500 ns, using Gromacs package [48]. 2.2.2. Simulation parameters For all solute molecules and ions, the all-atom optimized potentials for liquid simulations (OPLS-AA) parameters [49], and for water, the transferable intermolecular potential three point model (TIP3P) [50] were used. The atomic charges of xanthones in neutral and anionic states were obtained using the Restrained Electrostatic Potential (RESP) method [51]. Charges were calculated for eight structurally diverse conformers of each xanthone to minimize conformational dependence of the charges. Details concerning calculations of atomic charges on xanthones are given in SI. 2.2.3. Simulation conditions The LINCS algorithm was applied to all CH, OH and NH bonds of the POPC, xanthone, Chol, and water molecules and the time step was set at 2 fs. The long-range electrostatic interactions were evaluated using the particle-mesh Ewald (PME) summation method with the β-spline interpolation order of 5, and a direct sum tolerance of 10−6 [52]. For the real space, three-dimensional periodic boundary conditions with the usual minimum image convention and a cut-off of 10 Å were used. The van der Waals interactions were cut off at 10 Å. The list of nonbonded pairs was updated every 10 fs. Simulations were carried out at a constant temperature of 310 K = 37 °C, which is above the main phase transition temperature for a pure POPC bilayer (−5 °C [53]), and a constant pressure (1 atm). The temperature was controlled using NoséHoover thermostat [54,55], separately for the solute and solvent, and the pressure was controlled by the Parrinello-Rahman method [56]. The relaxation times for temperatures and pressure were set at 0.6 and 1 ps, respectively. Applied pressure was controlled anisotropically, where each direction was treated independently and the trace of the pressure tensor was kept constant (1 atm). 2.2.4. Structural bilayer parameters The average cross-sectional area per POPC (A/PC) in the computer simulated POPC-Chol and POPC-Chol-xanthone bilayers was calculated at a given time step (every 1 ps) in two steps: (1) from the total surface area of the bilayer, the area occupied by the Chol molecules of (18 × 38 Å2) 684 Å2 was subtracted (18 is the number of Chol molecules in one leaflet; 38 Å2 is the approximate average surface area of a Chol molecule obtained in the Chol monolayer [57–59]; Chol crystal [60] and in MD simulated pure Chol bilayer [61]); (2) the resulting area

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Fig. 2. The (a) initial locations of KS3 molecules (red) within the POPC-Chol bilayer after energy minimization and (b) their final locations after 500 ns of the MD simulation. POPC (grey) and Chol (yellow) molecules are the transparent background; for clarity, the water is not shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

was divided by the number of POPC molecules in one bilayer leaflet, of 66. The bilayer width, DHH, was estimated as the distance between the maxima in the electron density profile across the bilayer—the maxima correspond to the average positions of the phosphate groups in the two bilayer leaflets. The value of DHH approximates the measure of the bilayer width as a head-to-head separation that can, in principle, be obtained directly in diffraction experiment but was not in this case (the bilayer width was obtained using the Luzzati method [46]).

2.2.5. Mechanical properties The mechanical properties of the MD simulated bilayer are characterised here by the area and volume compressibility moduli and the bending rigidity modulus [62]. To calculate the area and volume compressibility moduli, the 400-ns productive trajectory (see below) of each bilayer was divided into eight statistically independent 50-ns blocks. The number of blocks was determined by calculating the correlation function of fluctuations of the calculated quantity [63]; details are given in SI. The mean values (bA N and bV N) and mean squared

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fluctuations (σ2(A) and σ2(V)) of the simulation box area and volume were calculated from the block averages. The area (KA) and volume (KV) compressibility moduli were calculated from bA N and b V N and σ2(A) and σ2(V) according to the formulas [62]: K A ¼ kT hAi=σ 2 ðAÞ;

ð1Þ

and K V ¼ kT hV i=σ 2 ðV Þ:

ð2Þ

From KA and the effective bilayer width (dt), the bilayer bending rigidity modulus, κb, was calculated [62]: 2

κb ¼

K A dt ; 24

ð3Þ

where dt = (DHH – 1), and DHH is defined above. 2.3. Averages and errors The reported average values of quantities obtained in MD simulations are time averages calculated from the final 400-ns fragment (see below) of each trajectory sampled by 1-ps time steps, and the ensemble of molecules, unless stated otherwise. Errors in the average values are standard errors except for the entries in Table 1, where they are standard deviation estimates. For the lateral self-diffusion coefficient, D||, errors were estimated using the variance analysis [64]. Errors in experimental values of DB and A/PC were only approximately assessed based on the error estimates of the partial molecular volumes of POPC, Chol, and water [65]. 3. Results 3.1. Equilibration of the MD simulated systems In order to estimate the equilibration time of the simulated systems, time profiles of the potential energy, EPOT, average surface area per PC, A/PC, (see above) and vertical location of the centreof-masses of the xanthone molecules in the bilayer were recorded from the onset of simulation until 500 ns, that is, over the total simulation time, for each bilayer system. The exemplary EPOT and A/PC profiles for the POPC-KS1-n bilayer are shown in Fig. 3a and b, and the vertical location profiles for the neutral and negatively charged KS3 molecules in the POPC-KS3-n and -i bilayers, respectively, are shown in Fig. 4. The potential energy of each system reached a stable average value after 20 ns of simulation, while the average surface area per lipid stabilized within 20–100 ns of MD simulation. The time when most of the xanthone molecules reached their stable

location in their bilayers varied from 5 to 25 ns. Exchange of the initial environment from water to lipids and vice versa by some xanthones in each bilayer justified the assumption that after that time, the distribution of the xanthone molecules between the water and lipid phases represented the equilibrium state. However, in one or two instances, xanthone molecules intercalated from water into their bilayers after only a relatively long time between 250 and 450 ns (cf. Fig. 4b). As these were rare cases, the time of achieving thermal equilibrium by each of the simulated systems was estimated as 100 ns. In all MD simulated systems, most, but not all (Fig. 2b), of the xanthone molecules located in the bilayer interfacial region, and mainly from the side of the nonpolar core (Figs. 4 and 2b). Of those which intercalated, some located relatively deeply in the bilayer core but during the whole simulation time of 500 ns none of them diffused into the region of the lowest density or translocated to the opposite leaflet. In general, xanthones in the ionized state penetrated less deeply the core than those in the neutral state (see below). To check the effect of slowly penetrating molecules, all analyses were carried out in two steps; in the first, all xanthone molecules that intercalated the bilayer were included; in the second, only those molecules that intercalated the bilayer during the first 100 ns of MD simulation were included. The results of the analyses indicated that slowly penetrating molecules contribute negligibly to the calculated average values. Thus, all xanthone molecules that intercalated into the bilayer (regardless of when) were included in analyses and the first 100-ns fragment of each 500-ns trajectory, as well as the xanthone molecules that did not intercalate into the bilayer core were excluded from the analyses. An example of the final location of xanthone (KS3) molecules in the bilayer is shown in Fig. 2b. 3.2. Effects of xanthones on the bilayer 3.2.1. Spatial properties Among the membrane parameters, which provide information about the spatial organization of the lipid bilayer, are the average cross-sectional area per lipid, A/PC, and the membrane width, D (DHH or DB, see above). A/PC and D belong to a group of membrane parameters that can be obtained both by X-ray diffraction and MD simulation. A/PC is one of the main structural parameters of a lipid bilayer and correlates strongly with several other membrane parameters [66]. The values of DHH and A/PC, and DB and A/PC obtained for the fully hydrated bilayers from MD simulation and X-ray diffraction, respectively (cf. Methods and SI), are given in Table 1. Since in the experimental samples both protonation forms of xanthone molecules are present, the measured values of DB and A/PC are averages over both forms. For the MD simulated bilayers, the values of DHH and A/PC can be calculated for each protonation form separately so the values for both the average over the two protonation states and each protonation form separately are given in Table 1. The calculated values of A/PC for bilayers containing

Table 1 Bulk membrane parameters. Area per lipid [Å2]

Membrane width [Å] Bilayer

X-ray (DB)

MD (DHH) (-n/-i)

X-ray

MD (-n/-i)

POPC POPC-Chol POPC-KS1

35.9 ± 1.5 38.3 ± 1.5 37.1 ± 1.5

70.0 ± 1.5 65.5 ± 1.5 66.9 ± 1.5

POPC-KS2

38.5 ± 1.5

POPC-KS3

37.6 ± 1.5

35.5 ± 0.1* 38 ± 1.1 37.7 ± 1.4 (38.0/37.3) 37.3 ± 1.1 (37.5/37.1) 37.9 ± 1.1 (38.2/37.6)

63.5 ± 0.5* 56.0 ± 1.2 58.6 ± 1.4 (57.6/59.6) 59.2 ± 1.1 (58.6/59.9) 57.5 ± 1.2 (56.8/58.1)

64.5 ± 1.5 66.8 ± 1.5

The membrane width (DB and DHH) and average surface area per PC (A/PC) obtained using X-ray diffraction method (X-ray) and MD simulations (MD trajectory 100–500 ns), for all studied systems. The errors are standard deviation estimates. *Reference [43]. In parentheses, the values of DHH and A/PC calculated separately for MD simulated bilayers with the neutral (-n) and ionized (-i) forms of the xanthone molecules.

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Fig. 3. Time profiles of the (a) bilayer potential energy and (b) average surface area per PC (A/PC) for the POPC-KS1-n bilayer.

only deprotonated xanthones are systematically larger (of 1.3–2 Å2) than those containing protonated xanthones; the differences in DHH for both forms are less significant (Table 1) but, as expected, a larger area corresponds to a smaller width. The experimental values of A/PC derived using the Luzzati method [46] are overestimated as this method assumes that entire water added to the lipid sample goes between stacks of bilayers within a liposome and affects the measured lamellar repeat period, d. Actually, some of the water occupies available inter-liposome space and has no effect on d. Therefore, the values for A/PC in Table 1 that were obtained experimentally are systematically greater than those obtained from MD simulations. Larger values of A/PC obtained experimentally might be possibly caused also by a larger number of

xanthones in the deprotonated than protonated forms in the samples. The proportion of both forms in the samples is not possible to determine. However, the values of xanthones pKα predicted using the ACD/ I-Lab 2.0 Prediction Modules [67] are between 3.3 and 3.7, so in liposomes dissolved in distilled water of pH ~ 7 one might expect more deprotonated than protonated forms. As will be shown below, anionic xanthones attract more water molecules than neutral ones so the interface of the bilayer containing anionic xanthones is more hydrated thus, A/PC is larger, e.g. [68,69]. In the case of the bilayer width averaged over both protonation forms, the values obtained experimentally are in good agreement with those obtained from MD simulations. The effect of KS1, KS2 and KS3 on the bilayer width and A/PC is rather small. 3.2.2. Chain order A parameter which is related to A/PC is the molecular order parameter, Smol, of the phospholipid acyl chains in the bilayer. The profile of this parameter along the bilayer depth reports on the orientational disorder (fluctuations) of the acyl chains. For the nth segment of an acyl chain, Smol is defined through [70]:

 Smol ¼ 0:5  3cos2 θn −1

ð4Þ

where: θn is the instantaneous angle between the nth segmental vector, i.e., the (Cn − 1, Cn + 1) vector linking n − 1 and n + 1 carbon (C) atoms in the acyl chain and the bilayer normal with corrections for sp2 hybridization of the carbon atoms linked by the double bond [71]; and b…N denotes both the ensemble and the time average. Smol profiles along the β- and γ-chain averaged over both bilayer leaflets, for the simulated bilayers are shown in Fig. 5. The profiles and mean values (averaged over the segmental vectors ≥ 5) of Smol (Table 2), are similar for all simulated systems and their differences do not exceed the limits of errors. Nevertheless, it can be seen that PC chains in bilayers with neutral xanthones are systematically more ordered than those in bilayers with ionized xanthones (Fig. 5) and also, that, in general, KS2 slightly decreases the chain order, whereas KS3 slightly increases. The results correlate well with the discussed below location of the xanthones in the bilayers. However, the net effect of xanthones on the POPC acyl chain order is small.

Fig. 4. Time profiles of the location of the centre-of-mass of neutral (a) and negatively (b) charged KS3 molecules along the bilayer normal (z-axis). Black lines indicate the average vertical location of P atoms in each of the bilayer leaflets. The periodic boundary conditions allow the translocation of drug molecules through the water phase between the two water layers. Trajectories of individual xanthone molecules are in different colours. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

3.2.3. Lateral diffusion of POPC, Chol and xanthones Another property of the bilayer lipids related to A/PC is their lateral self-diffusion. To check whether xanthone molecules have an effect on the mobility of POPC and Chol in the bilayer, approximate values of the diffusion coefficients for their lateral self-diffusion, D||, in each bilayer were determined from the linear parts of the mean square displacement (MSD) curves and are given in Table 2. This is a very rough estimation of D|| as lipids in the bilayer undergo anomalous diffusion [72] but here only the relative values are of interest. As can be seen

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Fig. 5. Molecular order parameter (Smol) profiles calculated for the POPC β-chain (a, c) and γ-chain (b, d) for the bilayers containing neutral (a, b) and charged (c, d) KS1, KS2, KS3 as well as for the reference POPC-Chol bilayer (REF). The standard errors are less than the size of the symbols. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

from Table 2, the differences among the values of D|| are within the estimated error of 0.02 × 10−7 cm2/s. This means that in the bilayer, xanthones are kinetically transparent i.e., the drugs do not affect significantly mobility of the lipids. The lateral diffusion coefficient for POPC in the POPC-Chol bilayer of 1.03 × 10−7 cm2/s is, surprisingly, very close to the value obtained with 1 H NMR spectroscopy for the hydrated POPC-Chol bilayer in similar conditions (POPC:H2O = 30, 37 mol% Chol, 313 K) of 1 × 10−7 cm2/s [73]. 3.2.4. Lateral pressure profiles The lateral pressure across the bilayer is nonuniform due to the bilayer inhomogeneity [74]. The depth-dependent intra-membrane pressures, that is, the lateral pressure profile can be calculated from MD trajectory by dividing the bilayer into thin slices parallel to the bilayer surface and for each slice, calculating the pressure tensor [75]. The shape and the values of the minima and maxima of the lateral pressure profile depend on the lipid composition of the bilayer [76], their packing in the bilayer and their dynamics, which on the other hand are related to Table 2 Lateral diffusion and molecular order parameter. D × 10−7 ± 0.02 × 10−7 cm2/s

bSmol N

Bilayer

POPC

Chol

Xanthone

β-Chain

γ-Chain

REF KS1-n KS1-i KS2-n KS2-i KS3-n KS3-i

1.03 1.00 1.03 1.15 1.07 0.94 1.04

1.27 1.23 1.27 1.34 1.25 1.15 1.25

– 1.33 1.31 1.36 1.32 1.37 1.35

0.34 0.35 0.32 0.34 0.32 0.37 0.34

0.46 0.47 0.43 0.45 0.43 0.48 0.45

± ± ± ± ± ± ±

0.03 0.03 0.03 0.03 0.03 0.03 0.03

± ± ± ± ± ± ±

0.04 0.04 0.04 0.04 0.04 0.04 0.04

Diffusion coefficients, D × 10−7 cm2/s, for the translational self-diffusion in the bilayer plane (lateral) of POPC, cholesterol and xanthone molecules, obtained from fits to the linear fragments of the corresponding MSD curves; average (over the chain segments ≥ 5) values of Smol (bSmol N) for the POPC β- and γ-chains, for the reference POPC-Chol bilayer (REF) and POPC-Chol bilayers containing xanthone molecules, KS1, KS2, and KS3 in the neutral (-n) and ionic (-i) protonation states. Errors in the bSmol N values are standard error estimates.

Fig. 6. Lateral pressure profiles for bilayers containing protonated (neutral) (a) and deprotonated (charged) (b) xanthone molecules. The bilayer centre is at z = 0 nm, and the bilayer width (with the water phase) is ~ 7.7 nm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

A/PC. For each of the studied bilayers, the lateral pressure profile was calculated as described in Ref. [77] and is shown in Fig. 6. The profiles are characteristic for the PC bilayer containing cholesterol, cf. Ref. [78]. There is no significant difference between the profiles for bilayers containing xanthones and that for the reference bilayer. However, the smallest difference is for the bilayers containing KS3. KS1 and KS2, particularly those in the ionic form, slightly reduce the stresses within the bilayer (Fig. 6b). But the overall effect of xanthones on the lateral pressure profile is small. 3.2.5. Mechanical properties The values of the area, KA, and volume, KV compressibility moduli and bending rigidity modulus, κb, calculated (see Methods section) for all systems are given in Table 3. For the reference POPC-Chol bilayer at 310 K, the value of KA is 284.2 ± 43.4 dyn/cm and is not much different from those obtained in other MD simulations for the DOPC bilayer at 295 K of 325.0 ± 51.1 dyn/cm [77] and for the POPC bilayer at 303 K of 240 ± 10 dyn/cm [63]. The experimentally determined values of KA for PC bilayers are in the range between 230 and 340 dyn/cm [79–82]. The effect of Chol on KA is not firmly established; in Refs [81–83] such an effect was not observed, whereas data in Ref. [80] indicated that with increasing Chol content KA increases. Thus, the value of KA for the POPC-Chol bilayer rationally agrees with those obtained experimentally. The value of KV of 20.5 ± 0.17 kbar obtained here for the POPC-Chol bilayer is within the experimentally determined values of 20–40 kbar for various lipid bilayer also those containing Chol [84]. The value of κb of 21.7 ± 3.3 kT for the reference bilayer is within the values obtained experimentally for PC-Chol bilayers containing 20 mol% Chol that are 17.9 ± 1.9 kT [80] and 27.7 ± 2.9 kT [85]. As can be seen from Table 3, KS1, KS2, and KS3 affect the area compressibility modulus, KA, and bending rigidity modulus, κb, but practically do not affect the volume compressibility modulus, KV of the POPC-Chol bilayer. The values of KA for bilayers containing xanthone molecules are higher than for the bilayer without them (Table 3). Based on Eq. (1) and data in Table 3 one can conclude that the increased values of KA result not as much from the differences in the average simulation box surface areas, b Abox N, that vary b 6% among the bilayers but from significantly smaller variance (up to 74%) of Abox for bilayers containing xanthones compared to the reference POPC-Chol bilayer (Table 3). Explanation of very similar values of KV in all systems can also be based on Eq. (2) and data in Table 3 indicating that in each system the values of bVbox N and variance of Vbox are similar. Small differences in b Vbox N arise from different numbers of atoms in different systems; as in the POPC-Chol bilayer there are ~ 600 fewer atoms than the POPC-Chol-xanthone bilayers, the value of b Vbox N is the smallest. The bending rigidity modulus, κb is calculated directly from KA and the bilayer width (eq. 3). As the widths of the bilayers are similar (Table 1), the values of κb are proportional to those of KA. The results concerning volume compressibility are in line with the results of previous studies indicating that volume compressibility of a lipid bilayer is low e.g. Refs [86–89] and similar to that of most “incompressible” fluids [90], and also that dimensional changes are much

27

larger in the bilayer plane than in the overall volume [91]. It is estimated that the bilayer is at least 10-fold more compressible in area than in volume [88]. 3.3. Intermolecular interactions at the bilayer interface and bilayer hydration In the analyses below the following geometric criteria of a hydrogen bond (H-bond), water bridge (WB), and charge pair (CP) were used. A H-bond donor (D) and acceptor (A) form a H-bond when the D–A distance is ≤ 3.5 Å and the angle between the A–D vector, and the D\\H bond is ≤30° [92]. It has been shown that these criteria give a correct average number of H-bonds in MD simulated pure water [93]. A water bridge is made by a water molecule that is simultaneously H-bonded to two or more polar groups of different molecules (here only intermolecular water bridges are analysed) [94]. A charge pair is formed between a positively charged choline methyl group (N-CH3) of POPC and a negatively charged oxygen atom of another molecule when they are located within 4.6 Å (the position of the first minimum in the radial distribution function, cf. sec. 3.3.1.4) from each other (here only intermolecular charge pairs are analysed) [95]. To assess the effect of xanthone molecules on the bilayer hydration, in addition to determining the number of intermolecular interactions among lipid, xanthone, and water molecules at the bilayer interface, an analysis of the solvent accessible surface area (SASA) [96] was carried out with the radius of the solvent probe of 1.4 Å. 3.3.1. Interactions of xanthones with water, POPC, cholesterol, ions and other xanthones 3.3.1.1. Xanthone-water hydrogen bonds. To examine whether xanthone molecules interact with the bilayer water, the radial distribution functions (RDF) of the water oxygen atoms relative to oxygen atoms of the xanthone molecule in the neutral and anionic forms (O-O RDF) were calculated (Fig. 7). The RDFs shown in Fig. 7 indicate that water molecules make H-bonds with xanthones. The average numbers of xanthone⋯water H-bonds per xanthone of each type calculated directly based on the geometric criteria (see above) are given in Table 4. The RDFs in Fig. 7 and the entries in Table 4 demonstrate that irrespective of their chemical types, the deprotonated (ionic) xanthones make over two times more H-bonds with water than the protonated (neutral) ones. Nevertheless, for each protonation form, the numbers of H-bonds with water are for all xanthones practically the same within the limits of estimated errors (Table 4) as are the RFDs in Fig. 7. To assess possible gastric toxicity of xanthones, the RDFs for the neutral and anionic xanthones are compared in Fig. 7 with those for ketoprofen obtained in Ref. [35]. Ketoprofen was shown to be NSAID with high gastric toxicity [97]. As for both neutral and anionic NSAIDs, RDFs indicate much smaller attraction of water by xanthones than by ketoprofen, one can conclude that gastric toxicity of xanthones is less than that of ketoprofen.

Table 3 Membrane mechanical parameters. Bilayer

KA [dyn/cm]

bAbox N [nm2] (var) [nm4]

κb [kT]

REF KS1-n KS1-i KS2-n KS2-i KS3-n KS3-i

284.2 475.2 411.6 513.3 476.7 376.6 487.0

43.178 (0.650) 44.277 (0.398) 45.591 (0.474) 44.932 (0.374) 45.751 (0.410) 43.758 (0.497) 44.602 (0.391)

21.7 35.5 30.7 37.2 34.6 28.5 36.9

± ± ± ± ± ± ±

43.4 48.8 96.6 67.2 51.7 112.0 100.8

± ± ± ± ± ± ±

bVbox N [nm3] (var) [nm6]

KV [kbar] 3.3 3.6 7.2 4.9 3.8 8.5 7.6

20.51 20.74 20.91 20.86 20.83 20.82 20.94

± ± ± ± ± ± ±

0.17 0.08 0.13 0.10 0.15 0.13 0.13

337.30 (0.703) 343.63 (0.709) 342.85 (0.701) 343.80 (0.705) 342.92 (0.704) 343.14 (0.705) 342.31 (0.699)

Mean values of the area (KA) compressibility modulus, surface area of the simulation box bAbox N and its variance (var), bending rigidity modulus, κb, and volume (KV) compressibility modulus, volume of the simulation box bVbox N and its variance (var), for the reference POPC-Chol bilayer (REF) and POPC-Chol bilayers containing xanthone molecules, KS1, KS2, and KS3 in the neutral (-n) and ionic (-i) protonation states.

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M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

into the water phase (Fig. 9c), make significantly more H-bonds with water than those of the neutral xanthones (Table 4). The carboxylic group is terminal in all xanthones but in KS1 and KS2 it is flanked by two CH3 groups, whereas in KS3 by only one (Fig. 1). These two CH3 groups in KS1 and KS2 only slightly limit access of water molecules to their anionic carboxylic groups. The profiles in Fig. 9a indicate that even though some carbonyl groups of the neutral xanthone molecules reach quite deeply into the bilayer core, they do not attract detectable amount of water inside the core (cf. inset in Fig. 9a). However, the mostly distributed are the locations of the carbonyl groups of KS2 and they are the deepest reaching of those of other neutral xanthones; as such, they slightly increase water access below the POPC carbonyl groups (Fig. 9b, inset in Fig. 9a) nevertheless, differences among the neutral xanthones are very small. Carbonyl groups of the anionic xanthone molecules reach less deeply into the bilayer core than those of the neutral ones (Fig. 9c). The mostly distributed are the locations of the carbonyl groups of KS1 which slightly increase water access below the POPC carbonyl groups compared with other anionic xanthones (Fig. 9d, inset in Fig. 9c). But there are no apparent differences in water penetration between the reference bilayer and those containing xanthones either neutral or charged (cf. Table S1, SI).

Fig. 7. Xanthone-water radial distribution functions (O-O RDF) of the water oxygen atoms relative to all oxygen atoms of the xanthone molecule in the neutral (a) and anionic (b) forms. The RDFs are compared with those for ketoprofen (dotted lines) [35] – a reference NSAID with high toxicity [97]. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

To get a better insight into xanthone⋯water H-bonding, in each bilayer, RDFs of the water oxygen atoms were calculated relative to each oxygen atom of the xanthone molecules in the neutral and anionic states (Fig. 8). The RDFs clearly indicate that practically the only Hbond acceptors of both neutral and anionic xanthones are oxygen atoms of their carboxylic and carbonyl groups. The number density profiles of the groups along the bilayer normal (z-axis) are shown in Fig. 9. As the xanthone carbonyl groups are more deeply immersed into the bilayer core than the carboxyl ones (Fig. 9), it is not surprising that, on average, the carboxylic groups make at least twice as many H-bonds with water as the carbonyl groups do (Fig. 8 and Table 4). And also, that the carboxylic groups of the ionic xanthones, as charged and located further

3.3.1.2. Xanthone-POPC hydrogen bonds. Only xanthone in the neutral form can make xanthone⋯PC H-bond, because, in contrast to ionic xanthone and PC, it has an H-bond donor (OH) group. Calculated xanthonePC O-O RDFs (not shown) indicate that such H-bonds do form indeed. The H-bond acceptors are exclusively the POPC non-esterified phosphate oxygen atoms, O13 and O14 (Fig. 1b), collectively called as Op. As KS3 has only one CH3 group flanking the carboxylic moiety, one would expect that it makes more H-bonds with Op than KS1 and KS2. Indeed, the numbers of xanthone⋯Op H-bonds calculated for each xanthone directly based on geometrical criteria (Table 4) indicate that KS3 makes more H-bonds with Op than the other two xanthones, particularly KS2 which is located more deeply in the bilayer core. However, the differences in the numbers (Table 4) are mainly within estimated errors. 3.3.1.3. Xanthone-POPC water bridges. On average, each xanthone in the ionic form is linked with a POPC molecule via a water bridge, while only a half of the neutral xanthones make water bridges with POPC (Table 4). 3.3.1.4. Xanthone-POPC charge pairs. Only xanthone in the anionic form can make xanthone-POPC charge pairs. The calculated RDFs of the choline methyl groups of POPC (CH3) relative to negatively charged oxygen atoms of the xanthone molecule (COO−\\CH3 RDF) (not shown)

Table 4 Number of hydrogen bonds. Xanthone-H2O

Xanthone-POPC

Bilayer

#HB/XANT

#HB/XANT

#WB/XANT

KS1-n

1.48 ± 0.34 (0.36/0.74 ± 4.56 ± 0.29 (0.75/3.88 ± 2.06 ± 0.26 (0.56/0.90 ± 4.96 ± 0.38 (0.86/4.01 ± 1.62 ± 0.22 (0.33/0.85 ± 4.97 ± 0.39 (0.69/3.07 ±

0.56 ± 0.12

0.49 ± 0.18

KS1-i KS2-n KS2-i KS3-n KS1-i

#CP/XANT

0.2) 1.07 ± 0.27

0.55 ± 0.11

0.3) 0.38 ± 0.10

0.46 ± 0.18

0.2) 0.99 ± 0.26

0.50 ± 0.11

0.3) 0.59 ± 0.09

0.33 ± 0.22

0.2) 1.01 ± 0.38

0.49 ± 0.11

0.3)

Average numbers of xanthone⋯H2O H-bonds per xanthone (#HB/XANT) and in the parentheses per xanthone carbonyl and carboxyl groups; also, average numbers of xanthone-POPC Hbonds (#HB), water bridges (#WB), and charge pairs (#CP) per xanthone molecule (/XANT) in the POPC-Chol bilayers containing xanthone molecules KS1, KS2, and KS3 in the neutral (-n) and ionic (-i) protonation states.

M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

29

Fig. 8. Xanthone-water radial distribution functions (O-O RDF) of the water oxygen atoms relative to each oxygen atom of the xanthone molecules in the neutral and anionic forms. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

indicate that such interactions take place indeed. Average numbers of charge pairs per xanthone of each chemical type calculated directly are given in Table 4. The numbers of charge pairs between POPC and ionic xanthones are very similar to those of H-bonds between POPC and neutral xanthones and are practically the same for all ionic xanthones.

3.3.1.5. Xanthone-Chol hydrogen bonds. Cholesterol can act both as an Hbond acceptor, and donor, therefore xanthone ··· Chol can be formed with xanthones in both neutral and anionic forms. The average numbers of xanthone ··· Chol H-bonds are given in Table 5. A detailed analysis reveals that in the case of the ionic xanthones, the exclusive H-bond donor is the Chol hydroxyl group and the acceptors are the oxygen atoms of the ionized xanthone carboxylic group. In the case of the neutral xanthones, the H-bond donor is the xanthone carboxylic OH group and the acceptor is the oxygen atom of the Chol hydroxyl group. Chol preferably makes H-bonds with the ionic xanthones than with the neutral ones.

3.3.1.6. Xanthone-Chol water bridges. As in the case of H-bonds, in its interactions with xanthones via water bridges, Chol favours the ionic xanthones over the neutral ones, but these interactions are not numerous (Table 5).

3.3.1.7. Xanthone-ions interactions. Direct calculation of the number of xanthone-Na+ interactions per xanthone (the number of Na+ not further than 3.0 Å from a xanthone oxygen atom) indicates that even though deprotonated xanthones bind significantly more Na+ ions than protonated ones, for both protonation states the number of bound Na+ ions is very small (Table 5). The charged xanthones bind Na+ by the oxygen atoms of the ionized carboxyl group, and the neutral ones predominantly by the carboxyl C_O group.

3.3.1.8. Xanthone-xanthone interactions. Direct H-bonds between xanthone molecules are not detected, however, in a few cases, xanthonexanthone water bridges are observed (Table 5).

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M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

Fig. 9. Number density profiles along the bilayer normal (z-axis) of the xanthone carboxyl (closer to the water phase) and carbonyl (closer to the bilayer centre) groups (a, c) as well as POPC phosphate and carbonyl groups (b, d) in the lower bilayer leaflet of bilayers containing neutral (a, b) and charged (c, d) xanthone molecules, relative to number density profiles of the lipid and water molecules in the bilayers. The insets in panels (a) and (c) show the profiles of the number density of water molecules below the POPC carboxyl groups (multiplied by 100) in the systems with neutral (a) and anionic (c) xanthones.

3.3.2. Xanthone effect on lipid-lipid, lipid-water, and lipid-ion interactions 3.3.2.1. POPC-Chol interactions. The numbers of POPC-Chol interactions via H-bonds and water bridges determined for all bilayers are given in Table 6. The numbers indicate that the presence of xanthones slightly weakens these interactions, and the effect is more apparent for the ionic xanthones. This may be because the charged xanthones interact more effectively with POPC and Chol than the neutral ones thus, slightly blocking POPC-Chol interactions. 3.3.2.2. POPC-water interactions. The entries in Table 6 indicate that there is practically no effect of xanthones in either protonation state on the number of POPC⋯water H-bonds. This result is in accord with the calculated SASA's of POPC molecules and of the POPC acyl chains (Table S2, SI) that are practically the same for all the systems. This indicates that the access of water to POPC is not affected by the presence of

Xanthone-Xanthone

#HB/XANT

#WB/XANT

#Na+/XANT

#WB/XANT

KS1-n KS1-i KS2-n KS2-i KS3-n KS3-i

0.11 0.39 0.07 0.22 0.12 0.34

0.05 0.17 0.04 0.18 0.05 0.22

0.004 0.051 0.006 0.067 0.009 0.079

0.13 0.04 0.04 0.14 0.02 0.04

0.06 0.11 0.06 0.09 0.08 0.13

± ± ± ± ± ±

0.06 0.11 0.05 0.11 0.09 0.17

3.3.2.4. Chol-water interactions. Xanthones only slightly decrease the average number of Chol ⋯ water H-bonds (Table 6) and the decrease is more apparent for the bilayers containing anionic xanthones. This is

#Na+/POPC

#HB/POPC Xanthone-Na+

Bilayer

± ± ± ± ± ±

3.3.2.3. POPC-Na+ interactions. Average numbers of Na+ ions bound by POPC via both Op and Oc oxygen atoms in all bilayers are given in Table 6. The neutral xanthones do not affect Na+ binding by POPC and ionic xanthones slightly increase it. The increase is most likely due to Na+ binding by the ionic xanthones themselves (Table 5). However, the numbers of Na+ ions bound by POPC are small and the differences among them are within estimated errors.

Table 6 Number of intermolecular interactions.

Table 5 Number of intermolecular interactions. Xanthone-Chol

xanthones. The lack of the effect may result from a large number of the POPC H-bond acceptor groups and a relatively large number of POPC-xanthone water bridges.

± ± ± ± ± ±

0.016 0.056 0.021 0.061 0.023 0.062

± ± ± ± ± ±

0.09 0.05 0.05 0.08 0.05 0.09

Average numbers of xanthone-Chol H-bonds (#HB), and water bridges (#WB) as well as xanthone-Na + contacts and xanthone-xanthone water bridges (#WB) per xanthone (/XANT) molecule, in the POPC-Chol bilayers containing xanthone molecules KS1, KS2, and KS3 in the neutral (-n) and ionic (-i) protonation state.

Bilayer

POPC-Chol

POPC-water

POPC-Na

+

REF KS1-n KS1-i KS2-n KS2-i KS3-n KS3-i

0.19 0.18 0.16 0.18 0.16 0.18 0.13

6.97 6.84 7.10 7.01 7.02 6.88 6.91

0.027 0.025 0.038 0.025 0.043 0.025 0.044

0.013 0.013 0.015 0.012 0.016 0.012 0.015

± ± ± ± ± ± ±

0.04 0.04 0.03 0.04 0.03 0.04 0.03

± ± ± ± ± ± ±

0.14 0.14 0.12 0.14 0.14 0.14 0.13

± ± ± ± ± ± ±

#HB/Chol Chol-water 1.51 1.39 1.40 1.48 1.44 1.38 1.30

± ± ± ± ± ± ±

0.12 0.13 0.11 0.14 0.11 0.14 0.11

Average number of POPC-Chol, POPC-water, and Chol-water interactions per lipid molecule (#HB/lipid) and bound Na+ ions by POPC (#Na+/POPC), the reference POPC-Chol (REF) bilayer and in POPC-Chol bilayers containing xanthone molecules KS1, KS2, and KS3 in the neutral (-n) and ionic (-i) protonation state.

M. Markiewicz et al. / Biophysical Chemistry 220 (2017) 20–33

most likely because interactions of the anionic xanthones with Chol are more effective than those of the neutral ones. However, due to relatively large errors, the effect of the xanthone protonation state on the Chol⋯water H-bonds is not very evident. 3.3.2.5. Chol-Na+ interactions. Cholesterol does not interact with Na+ ions in any of the studied systems. 4. Discussion Naturally occurring xanthone derivatives exhibit a broad spectrum of therapeutic actions and have been used as drugs in traditional folk medicine for a long time [98]. Recently, synthetic derivatives of xanthones have become a group of very promising potential drugs e.g. [99]. The three xanthone derivatives investigated in this research, exhibit properties of nonsteroidal anti-inflammatory drugs [38]. As in many cases, long-term use of NSAIDs is associated with adverse effects from the gastrointestinal tract, search for new and low-toxic NSAIDs and their evaluation is a fully justified task. The mechanism of NSAIDs' gastric toxicity may be linked to their direct action on the outermost lipid layer of the gastric mucosa, which lowers its hydrophobicity, thus resistance to luminal acid e.g. [32,100]. The intention of the present study was to examine the possibility of assessing potential adverse effects of the three xanthone derivatives (KS1, KS2, and KS3, Fig. 1) on the outer lipid layer of the gastric mucosa using the POPC-Chol bilayer as the model and computer modelling as the method. Computer modelling methodology enabled us to determine the xanthones' effect on the bilayer bulk properties, and on the lipid-lipid, lipid-water, and lipid-ion interactions at the bilayer interface, but most importantly, their direct interactions with the bilayer water and ions. The computer models generated in this study were partially validated based on the results of X-ray diffraction measurements which confirmed that between 20 and 40 °C and under atmospheric pressure the studied bilayers are lamellar and at full hydration they are in the liquid-crystalline phase [36]. The values of the parameters derived by the two methods are either in good agreement or their differences can be explained by a systematic error. They are also in good agreement with experimental and other computer simulations studies where NSAIDs and anaesthetics in the lipid bilayer were found to locate in the bilayer interfacial region with their terminal polar groups strongly interacting with the phospholipids head groups [101–110]. Both X-ray diffraction measurements and MD simulations reveal that the effects of the xanthones on the bulk properties of the POPCChol bilayer are at most moderate. The bilayer width, acyl chain order, volume compressibility modulus, lateral pressure profiles, lateral diffusion of lipid molecules, and solvent accessible area of lipids, are practically unaffected by the presence of xanthone molecules in the bilayer. However, xanthones have evident effects on the bilayer area compressibility (KA) and the bending rigidity (κb) moduli. The higher values of these parameters for the bilayers containing xanthone molecules in both protonation states relative to the reference bilayer indicate that xanthones suppress lateral fluctuations of the bilayer and make it stiffer against bending deformation. Suppressed fluctuations might result from quite numerous direct xanthone-POPC and xanthone-Chol interactions at the bilayer interface. However, these interactions apparently have no strong effect on the lateral pressure profile across the bilayer. As has been already stressed, gastric toxicity of NSAIDs is connected with their disruption of the hydrophobic barrier of the protective lipid layer of the gastric mucosa. On the atomic level, such a disruption will be manifested by higher access of water and ions into the hydrophobic core of the model membrane. Thus, to assess potential adverse effects of the three xanthone derivatives, KS1, KS2, KS3, their interactions with water and Na+ in the bilayer modelling the protective lipid layer of the gastric mucosa are analysed in details. As it has been observed in previous studies of compounds of similar activity e.g. [35,102,104–108], the protonated xanthones penetrate the

31

bilayer deeper than their deprotonated counterparts. This deeper penetration results in significantly fewer interactions of the neutral xanthones with water and Na+ as well as with POPC and Chol compared to those of the charged xanthones. The distributions of the xanthone carbonyl groups along the bilayer normal reach much deeper into the bilayer core than the distribution of the POPC carbonyl groups. These groups bind some water molecules, nevertheless, in the bilayers containing neutral xanthones and in the reference bilayer, the normalised number densities of water molecules in the bilayer region below the POPC carbonyl groups (between 10 and 0 Å) are practically the same and their very small values differ in an unsystematic way (cf. Table S1, SI). The neutral xanthones have no effect on the number of POPCwater and POPC-Na+ interactions; neither on water access to POPC molecules (cf. Table S2, SI). Thus, their effect on the bilayer hydrophobic barrier may be considered negligible. The anionic xanthones interact much more effectively with water and Na+ ions as well as with POPC and Chol than the neutral ones. These interactions are mainly through the deprotonated carboxyl group. The distributions of these groups along the bilayer normal overlap with those of the POPC phosphate and carbonyl groups. Thus, interactions of water and Na+ with the deprotonated carboxyl groups do not increase their access to the bilayer core. Similarly as in the case of the neutral xanthones, the carbonyl groups of the charge xanthones reach below the POPC carbonyl groups but less deeply than those of the neutral xanthones, and they do not change the normalised number densities of water molecules in the bilayer region below the POPC carbonyl groups (cf. Table S1, SI). Moreover, the charged xanthones have insignificant effect on the number of POPC-water and POPC-Na+ interactions as well as on water access to POPC molecules (cf. Table S2, SI). So, similarly as in the case of the neutral xanthones, one can state that the effect of charge xanthones on the bilayer hydrophobic barrier may be considered negligible. In experimental studies, gastric toxicity of KS2 was established as low [38]. Its interactions with water, Na+, and with lipids in either protonation state are practically the same as those of the other xanthones, so based on this study it is justified to say that gastric toxicity of the other two xanthones, irrespectively of their protonation state is also low. Comparison of the xanthone-water RDFs with the ketoprofenwater RDFs [35] suggests that gastric toxicity of the studied xanthones is certainly lower than that of ketoprofen. Acknowledgment This work was supported by the Ministry of Science and Higher Education Republic of Poland (IP2011 033871). We are grateful to Prof. Pavol Balgavy and Dr. Daniela Uhrikova for their substantial help and advice. We thank HASYLAB at DESY, Hamburg, Germany, for beam time and support (Hasylab project I-05-089EC). Faculty of Biochemistry, Biophysics and Biotechnology of Jagiellonian University is a partner of the Leading National Research Center (KNOW) supported by the Ministry of Science and Higher Education. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bpc.2016.10.007. References [1] K.S. Masters, S. Brase, Xanthones from fungi, lichens, and bacteria: the natural products and their synthesis, Chem. Rev. 112 (2012) 3717–3776. [2] V. Kuete, T. Efferth, Cameroonian medicinal plants: pharmacology and derived natural products, Front. Pharmacol. 1 (2010). [3] I. Salmoiraghi, M. Rossi, P. Valenti, P. Da Re, Allylamine type xanthone antimycotics, Arch. Pharm. 331 (1998) 225–227. [4] C.K. Ho, Y.L. Huang, C.C. Chen, Garcinone E, a xanthone derivative, has potent cytotoxic effect against hepatocellular carcinoma cell lines, Planta Med. 68 (2002) 975–979.

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