Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes

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1 MIIREVIEW Molecular dynamics simulations of the interactions of medicinal plant extracts and drugs with lipid bilayer membranes Wojciech Kopec, Jelena Telenius and imanshu Khandelia MEMPYS Center for Biomembrane Physics, University of Southern Denmark, dense, Denmark Keywords anesthetics; cholesterol; curcumin; drugs; drug-membrane interactions; molecular dynamics, lateral pressure profile; lipid membrane; plant extracts; terpenes Correspondence. Khandelia, MEMPYS Center for Biomembrane Physics, University of Southern Denmark, dense, Denmark Fax: Tel: (Received 4 March 2013, accepted 10 April 2013) doi: /febs Several small drugs and medicinal plant extracts, such as the Indian spice extract curcumin, have a wide range of useful pharmacological properties that cannot be ascribed to binding to a single protein target alone. The lipid bilayer membrane is thought to mediate the effects of many such molecules directly via perturbation of the plasma membrane structure and dynamics, or indirectly by modulating transmembrane protein conformational equilibria. Furthermore, for bioavailability, drugs must interact with and eventually permeate the lipid bilayer barrier on the surface of cells. Biophysical studies of the interactions of drugs and plant extracts are therefore of interest. Molecular dynamics simulations, which can access time and length scales that are not simultaneously accessible by other experimental methods, are often used to obtain quantitative molecular and thermodynamic descriptions of these interactions, often with complementary biophysical measurements. This review considers recent molecular dynamics simulations of small drug-like molecules with membranes, and provides a biophysical description of possible routes of membrane-mediated pharmacological effects of drugs. The review is not exhaustive, and we focus on molecules containing aromatic ring-like structures to develop our hypotheses. We also show that some drugs and anesthetics may have an effect on the lipid bilayer analogous to that of cholesterol. Introduction Drugs interact with membranes. In conventional pharmacology, a drug s interaction with lipid bilayer membranes is recognized and optimized in the context of its ability to reach intracellular targets. Therefore, biophysical and biochemical investigations of drug membrane interactions have always been of interest for drug design. owever, recently, it has been recognized that some drugs, and natural plant extracts that operate as drugs, may exert their effects via interactions with the lipid bilayer membrane, by either perturbing membrane integrity itself, chaperoning co-administered drugs, increasing the bilayer permeability, binding to Abbreviations CARMM, Chemistry at arvard Macromolecular Mechanics; CL, Cholesterol; DLPC, 1,2-dilauroyl-sn-glycero-3-phosphocholine; DMPC, 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine; DMPE, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine; DPC, 1,2-dioleoyl-sn-glycero-3- phosphocholine; DPPC, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine; DPPG, 1,2-dipalmitoyl-sn-glycero-3-phospho-(1 -rac-glycerol); DPPS, 1,2- dipalmitoyl-sn-glycero-3-phospho-l-serine; DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; DSPE, 1,2-distearoyl-sn-glycero-3- phosphoethanolamine; GAFF, General AMBER Force Field; PLS, optimized potentials for liquid simulations; PPA, 1-palmitoyl-2-oleoyl-snglycero-3-phosphate; PPC, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; PPE, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine; PSM, palmitoylsphingomyelin; SM, sphingomyelin. FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2785

2 Simulations of small molecules with lipid bilayers W. Kopec et al. A F B G C D I E K L M Fig. 1. Possible effects of drug binding to the lipid membrane and membrane proteins. Green, phospholipid; blue, drug; red, (membrane) protein; orange, solvent molecule (water or ion) that does not spontaneously penetrate the lipid membrane. (A E) Effect of drug binding on membrane properties. Drug binding may affect membrane curvature (A) via asymmetric binding. Bound molecules may also affect membrane thickness (B, C), bilayer area (C) and phospholipid chain ordering (C). Bilayer-bound molecules may create defects in and pores through the membrane (D), and cause or prevent lipid phase separation (E). (F K) Effect of drug binding to membrane proteins and membrane permeation. Changes in membrane curvature (F) and membrane thickness (G, ) may cause hydrophobic mismatch and other local changes in the membrane environment of a membrane protein. This may cause a subsequent protein conformation change, e.g. ion channel opening or closing. Membrane permeation of ions or other molecules may also increase via channels and defects formed by membrane-bound drug molecules (I). If the drug molecules alter the phase separation properties of the membrane lipids, this may cause differential packing of the membrane proteins on the membrane (K). (L ) Binding of a drug molecule to a protein receptor. The drug may bind to a membrane-bound protein on either side of the membrane (L, M). Binding of this effector molecule causes activation of the protein, and hence ion channel opening or activation of a signaling cascade, for example. The drug molecule may also bind to a cytoplasmic protein after penetrating the membrane () FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

3 W. Kopec et al. Simulations of small molecules with lipid bilayers Fig. 2. Schematic illustration of the impact of the membrane lateral pressure profile on the position of a drug molecule in the lipid bilayer. Adapted from Bagatolli LA, Ipsen, J, Simonsen, AC & Mouritsen G (2010) An outlook on organization of lipids in membranes: searching for a realistic connection with the organization of biological membrane. Prog Lipid Res 49, , with permission from Elsevier. lipid signaling molecules, binding to transmembrane protein targets upon dissolving in the membrane, or altering membrane protein conformation by altering the biophysical properties of the lipid bilayer, without any specific protein binding (Fig. 1). The possible modes of action are not necessarily exclusive, and it is likely that some drugs have multiple concomitant effects. Some of these natural plant extracts and drugs have a wide range of activities, including anti-cancer, anti-depressant, anti-inflammatory and anti-microbial properties. These diverse effects suggest that inhibition or activation of cellular processes by specific protein binding may not be the only mechanism of drug action. In particular, drugs that are active in the nervous system often tend to have strong subsidiary anticarcinogenic or anti-bacterial effects, but it is not understood why. There is thus a renewed interest in studying the interactions of these small molecules with model and real lipid bilayer membranes. Among the various techniques used to probe small molecule membrane interactions, molecular dynamics (MD) simulations have been widely used to study drug membrane systems: both to capture interaction details on the molecular scale, and to extract thermodynamic properties that are directly comparable to complementary analytical measurements. MD simulations are particularly suited to study such systems owing to the ability of the method to quantify non-covalent interactions of the magnitude of thermal energy, which often determine the evolution and assembly of soft systems such as lipid bilayers. In this review, we survey the recent literature regarding interactions between natural plant extracts and chemically synthesized drugs with lipid bilayers, focusing particularly on those drugs that have a wide range of biological activities. Additionally, due to space limitations, we focus on a few drugs that have aromatic or cyclic groups, because the interactions of these molecules have a strong analogy to that of the quintessential membrane modulator cholesterol. The review is by no means comprehensive, and the emphasis is on the biophysics of the effects of drugs on membranes, rather than on pharmacological properties such as improved bioavailability in the cell. A general idea of our view of how drugs may alter the conformations of proteins via lipids is shown in Fig. 1. The review is organized as follows. Interactions of lipid bilayers with chemical drugs that are not isolated from natural sources are summarized first. The influence of natural plant extracts on lipid bilayers is reviewed next. A list of all the compounds reviewed here is provided in Table 1. Some general ideas and hypotheses about the biophysical impact of the molecules on lipid membranes and the role of the lipid bilayer in mediating the activity of these molecules are presented in the Discussion. An excellent review of older simulations of small molecules with lipid bilayers has been published previously [1]. Typically, MD simulations compare well with complementary experimental measurements. The few exceptions are discussed below, and may arise from either insufficient sampling, questionable force fields in simulations, or difficulties in extracting molecular-level details in analytical measurements. We also discuss a few experiments where the interpretation is unambiguous, and the simulations agree remarkably well with the analytical measurements. Interactions of drugs with bilayers Many drugs contain aromatic or, more generally, cyclic chemical groups that may be considered flat in 3D space [2,3]. The classes of drugs discussed in this section are: general and local anesthetics (GAs and LAs, respectively), non-steroidal anti-inflammatory drugs (SA- IDs), and drugs with a very diverse pharmacological profile, e.g. anti-psychotic drugs, which also exhibit anti-bacterial and anti-cancer activities [4]. The following statements are true for almost all drugs and small molecules discussed here: (a) amphipathic drugs parti- FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2787

4 Simulations of small molecules with lipid bilayers W. Kopec et al. Table 1. verview of described simulations. ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field Lidocaine Local anesthetic DMPC 100 Berger lipids S Articaine Local anesthetic (a) DMPC (b) PPC (a) 200 (b) 20 (a) Berger lipids (b) CARMM Prilocaine Local anesthetic PPC 20 CARMM Benzocaine 2 Local anesthetic (a) DPPC (b) Asymmetrical DPPC/DPPS (a) 50 (b) 40 (a) Berger lipids (b) Berger lipids Propofol General anesthetic DPPC 500 Berger lipids Cl Ketamine General anesthetic PPC 100 PLS 1-alkanols n General anesthetic DMPC Up to 70 Berger lipids Cl F alothane Br F F General anesthetic (a) DMPC (b) PPC (c) DMPC (a) 2 (b) ~ 30 (c) 16 Xenon Xe General anesthetic PPE Up to 150 GAFF (a) CARMM (b) CARMM (c) CARMM 2788 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

5 W. Kopec et al. Simulations of small molecules with lipid bilayers Table 1. (continued) ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field Dopamine 2 eurotransmitter, antioxidant DLPC, DLPC/ SM/CL Up to 210 PLS Aspirin Anti-inflammatory, anti-pyretic, analgesic, anti-cancer DPPC 100 Berger lipids Ibuprofen Anti-inflammatory, anti-pyretic, analgesic, anti-cancer DPPC 100 Berger lipids aproxen Anti-inflammatory, anti-pyretic, analgesic, anti-cancer DPPC 100 Berger lipids Salicylate Anti-inflammatory, may cause hearing loss (a) DPPC (b) DMPC (a) 30 (b) Up to 220 (a) Berger lipids (b) Berger lipids Chlorpromazine S Cl Anti-psychotic, anti-cancer, anti-tuberculosis (a) DPPC, DPPG monolayers (b) PPC (a) 8 (b) Up to 450 (a) CARMM (b) Berger lipids F Siramesine Anti-cancer PPC/PPA 90 Berger lipids 2 Amantadine Anti-viral, anti-parkinsonian PPC 15 PLS FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2789

6 Simulations of small molecules with lipid bilayers W. Kopec et al. Table 1. (continued) ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field 2 Rimantadine Anti-viral, anti-parkinsonian PPC 15 PLS Memantine 2 Cl Anti-viral, anti-parkinsonian PPC 15 PLS Fusidic acid Bacteriostatic antibiotic DPPC 100 Berger lipids Alprenolol b-blocker DMPC 300 Coarse-grained for lipids, GAFF for drugs 2 Atenolol b-blocker DMPC 300 Coarse-grained for lipids, GAFF for drugs Pindolol b-blocker DMPC 300 Coarse-grained for lipids, GAFF for drugs Progesterone Sex hormone DMPC 300 Coarse-grained for lipids, GAFF for drugs Testosterone Sex hormone DMPC 300 Coarse-grained for lipids, 2790 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

7 W. Kopec et al. Simulations of small molecules with lipid bilayers Table 1. (continued) ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field GAFF for drugs Cholesterol Essential component ofthe mammalian cell membrane (a) DPPC (b) DPPC, DPC (a) 100 (b) 100 (a) Berger lipids (b) Berger lipids Ketosterol (a) DPPC (b) DPPC, DPC (a) 200 (b) 100 (a) Berger lipids (b) Berger lipids Sitosterol Component of the plant cell membrane, anti-cholesterol DPPC 80 Berger lipids Stigmasterol Component of the plant cell membrane, anti-cholesterol, anti-cancer, antioxidant DPPC 80 Berger lipids Dioscin igh hemolytic activity DPPC/PPC/ PSM/CL 400 MARTII Catechins Isoflavones Anti-allergic, anti-cancer, anti-cholesterol, anti-diabetic, anti-inflammatory, anti-hypertensive, anti-microbial Antioxidant, anti-cancer (a) PPC/PPE (b) PPC (a) 100 (b) 200 (a) PLS (b) PLS DPC Up to 80 CARMM FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2791

8 Simulations of small molecules with lipid bilayers W. Kopec et al. Table 1. (continued) ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field Flavonols Antioxidant (a) DPC (b) DPPC (a) Up to 300 (b) Up to 20 (a) Berger lipids (b) Berger lipids ypericin Antiviral, anti-tumor (a) DPPC (b) DPPC, DPPC/CL (a) 60 (b) 300 (a) Berger lipids (b) Berger lipids Curcumin Anti-fungal, anti-bacterial, anti-tumor PPC 200 PLS Stilbenoids Antioxidant, anti-cancer, anti-inflammatory DPPC 2.5 GRMS 96 Lutein Antioxidant, pigment in human eye DPPC Monte Carlo simulation a-tocopherol (form of vitamin E) Antioxidant DMPC, DMPE, DSPC, DSPE o data o data Resorcinolic lipids Antioxidant, stabilizer for liposomal drug delivery systems DMPC Up to 360 GRMS 96 Perillyl alcohol Anti-bacterial, anti-cancer DMPC Up to 220 Berger lipids 2792 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

9 W. Kopec et al. Simulations of small molecules with lipid bilayers Table 1. (continued) ame Chemical structure Pharmacological properties Bilayer composition Length of each simulation (ns) Force field Perillyl acid Anti-bacterial, anti-cancer DMPC Up to 220 Berger lipids Perillyl aldehyde Anti-bacterial, anti-cancer DMPC Up to 220 Berger lipids Limonene Anti-bacterial, anti-cancer DMPC Up to 220 Berger lipids tion at the bilayer interface, while completely hydrophobic molecules may partition at the bilayer center, and (b) charged versions of a drug containing possible protonation or de-protonation sites always localize closer to the lipid water interface than the uncharged versions. Anesthetics Anesthetics work either by altering the conformational transition of proteins such as ion channels, or by modulation of properties of membrane such as fluidity and lateral pressure profile [5,6] (Fig. 2). The latter hypothesis was developed due to a strong correlation between potency and solubility in non-polar media [7,8]. A combination of both models of action is also possible, as lipids strongly influence proteins embedded in the plasma membrane and vice versa [9]. MD simulations of anesthetic molecules with lipid membranes have been widely performed, with the preliminary hypothesis that the partitioning properties of an anesthetic in the bilayer indicate its potency [5]. Local anesthetics Although LAs are a diverse group, they share similarities in chemical structure. Usually they contain an aromatic ring and an ionizable amino group. Thus, the coulombic charge on the molecule may vary depending on the p and/or the local environment near the LA molecule. Charged lidocaine molecules partition closer to the bilayer interface than their uncharged versions [10]. The aromatic ring of charged lidocaine is oriented parallel to the bilayer normal. Uncharged lidocaine molecules possess translational degrees of freedom both in the membrane lateral plane as well as across membrane leaflets ( flip-flop ). The uncharged form of lidocaine may thus be important for drug delivery, due to its higher mobility, while the charged form may be responsible for the anesthetic effect. Such localization of anesthetic molecules may influence the electrostatic potential across the bilayer, which in turn may affect electrogenic membrane proteins such as ion channels. Although both forms of the drug affect the electrostatic potential inside the membrane to a similar extent [11], the mechanism of influence is very different. Charged lidocaine molecules, together along with neutralizing Cl counter-ions, disturb the charge distribution across the bilayer, but the individual contributions cancel each other out, thus increasing the electrostatic potential only slightly. Insertion of uncharged lidocaine preserves the membrane charge structure, and the higher electrostatic potential inside the membrane is contributed by dipoles present in the drug molecules. Irrespective of the actual mechanism, the direction of the electrostatic potential change, as well as its absolute value, suggest that lidocaine and related LAs may FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2793

10 Simulations of small molecules with lipid bilayers W. Kopec et al. modulate voltage-sensitive ion channels by modulating the electrostatic potential profile of the lipid bilayer. Although articaine has some structural properties that distinguish it from lidocaine, namely a more lipophilic thiophene aromatic ring instead of a benzene ring and an additional ester group, its behavior in the lipid membrane is similar to that of lidocaine [12]. The uncharged form of articaine orders lipid tails, and the effect is more pronounced for carbon atoms located near the glycerol backbone of lipids, i.e. in the region occupied by LA molecules. This effect is qualitatively similar to the well-known ordering effect of cholesterol in membranes [13]. Uncharged articaine increases the lateral diffusion constant of lipids. owever, addition of the charged form of the drug decreases lipid order parameters. Interaction of uncharged articaine with PPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) bilayers was also studied using the CARMM force field [14]. Although only one drug molecule was simulated, for a relatively short time (20 ns), its partitioning into the glycerol region of the membrane is in agreement with results obtained using the Berger force field [12]. Unlike lidocaine and articaine, the presence of uncharged prilocaine reduced lipid order parameters when simulated using the CARMM force field [15]. The lower order parameters may be a consequence of the relatively high drug concentration used (drug : lipid ratio of 1 : 3), or the fact that prilocaine molecules are initially placed in the core of the membrane. Benzocaine, unlike other LAs, is found only in the uncharged form under physiological conditions. The amino group in benzocaine has a low pk a because it is bonded to an electron-withdrawing benzene ring. In terms of orientation, depth of insertion and effects on a model bilayer, the effects of uncharged benzocaine are similar to the effects of the charged versions of other LAs [16]. Porasso et al. [17] and Cascales et al. [18] studied interactions of two benzocaine molecules with symmetric and asymmetric mixed bilayers containing DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine) and DPPS (1,2-dipalmitoyl-sn-glycero-3-phospho-Lserine) lipids in different leaflets. In both cases, benzocaine decreased the lipid tail order parameters, and, although benzocaine is uncharged, its effect is more pronounced for negatively charged DPPS lipids. This supports previous observations that benzocaine s effect on lipid bilayers is similar to that of other charged LAs. General anesthetics Like LAs, GAs are of diverse types: inhaled GAs include inorganic gases such as nitrous oxide or xenon, and small, highly fluorinated organic molecules such as halothane, desflurane or methoxyflurane, while intravenous GAs are usually cyclic organic molecules with amphiphilic properties. Among intravenous GAs, the most common are barbiturates, benzodiazepines, ketamine and propofol. Several other classes of compounds also act as GAs, such as 1-alkanols (alcohols) [19]. Propofol binds pentameric ligand-gated ion channels with high affinity [20] in the transmembrane region of the protein. The depth of the drug molecules inside the bilayer calculated from MD simulations coincides with the depth of the protein s binding sites (ansen A, Khandelia, Sørensen KT, Duelund L, Simonsen AC, ansen PL, Serer A & Richard, M, unpublished results). The presence of propofol molecules in the membrane also orders lipid acyl chains. Propofol is significantly smaller than cholesterol, and its ordering effect is thus observable only for carbon atoms near the interface, and is negligible near the bilayer center. The simulation data are also in accordance with isothermal titration calorimetry and Langmuir monolayer surface pressure area isotherms. Ketamine, which, like propofol, is an inhibitor of ligand-gated ion channels, structurally resembles propofol to some degree, i.e. it has a benzene ring decorated with hydrophilic functional groups. The presence of ketamine leads to a net shift of lateral pressure toward the center of the membrane [21]. Using two cartoon membrane protein models, it was shown that such changes of the lateral pressure profile may influence the conformational equilibrium of transmembrane proteins. Remarkably, cholesterol induces a very similar quantitative change in the lateral pressure in the region of the DPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) bilayer where the rigid steroid structure is located [22]. Ethanol replaces water molecules at the interface between the lipid tails and the head groups, decreases lipid chains repulsion and slightly decreases order parameters [19]. Long-chain 1-alkanols, such as tetradecanol, align themselves along the bilayer normal, order the lipid chains, particularly near the bilayer center, and amplify the local pressure extremes in that region of the bilayer. The effect is again reminiscent of that of cholesterol. alothane, a potent inhaled GA, disorders lipid tails [23,24], like ethanol and higher alkanols do. Seeger et al. [25] used pressure perturbation calorimetry to characterize the melting point (phase-transition temperature) of vesicles with a variety of molecules including halothane and 1-octanol. In agreement with simulations of these anesthetics, they found that both molecules depressed the melting point by fluidizing the 2794 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

11 W. Kopec et al. Simulations of small molecules with lipid bilayers bilayer. General anesthesia caused by drugs may be reversed by applied ambient pressure, within the range from 80 [26] to 200 atm [27]. For halothane [24,28] and xenon [29], high external pressure leads to aggregation of GA molecules in the bilayer center. owever, it remains to be seen whether pressure reversal affects protein binding by the GA. In one of the only studies of the effects of small neuroactive molecules on cholesterol-containing membranes, rowski et al. [30] performed simulations of the neurotransmitter dopamine and its precursor L-dopa using pure and cholesterol-containing DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) bilayers. Both molecules occur in ionic forms in physiological solutions. owever, dopamine is positively charged, while L-dopa is zwitterionic. It has been postulated that neurotransmitters may act as anesthetics, due to their similar chemical structure [31]. Dopamine penetrates the bilayer more deeply than L-dopa does. The presence of charged molecules inside the cholesterol-free membranes slightly decreases the order parameters near the glycerol backbone, consistent with results obtained for charged LAs. owever, such an effect is not observed for cholesterol-containing membranes. This is expected, as cholesterol protects the membrane from disruption caused by other agents in the fluid phase [32 34]. Further studies of drugs and small molecules with membranes containing cholesterol are required to resolve the effects of drugs and cholesterol on membranes. on-steroidal anti-inflammatory drugs (SAIDs) SAIDs are a widespread class of drugs that exhibit anti-pyretic, analgesic and anti-inflammatory effects [35], but are also considered as potent agents against various cancers, Alzheimer s disease and heart disease [36 38]. It is believed that direct interactions of SA- IDs with phospholipids present in the gastrointestinal tract are responsible for their gastrointestinal toxicity [39,40]. The therapeutic effect of SAIDs may be partially induced by lipid drug interactions. The most common SAIDs are aspirin, ibuprofen and naproxen. All contain aromatic rings and are weak carboxylic acids. Thus, in aqueous media, they occur in both uncharged and anionic forms. In simulations with an umbrella sampling, charged forms of aspirin and ibuprofen penetrated the bilayer with their polar groups fully hydrated, creating local water defects in the bilayer similar to those reviewed elsewhere [1], while uncharged forms penetrate the membrane in dehydrated form [41]. The location of ibuprofen in the lipid bilayer is concentration-independent [41]. Similar results were also obtained for naproxen [42]. The presence of charged ibuprofen near the water/ membrane interface slightly decreases the hydration of lipid head groups. owever, these drug molecules do not induce any serious changes in the membrane properties such as membrane thickness, while small-angle neutron scattering measurements revealed that incorporation of ibuprofen significantly decreases the bilayer thickness [43]. Further investigation is required to reconcile these simulations and experiments. In MD simulations, salicylate, which is closely related to aspirin and is a widely used anti-acne agent, slightly decreases the area per lipid in the bilayer, in contrast with experimental results [44,45]. owever, in more extensive simulations, the ordering effect of salicylate was attributed to a force-field artifact caused by the presence of small, monovalent sodium ions that condense lipids by binding to their glycerol backbone [46]. Use of larger counter-ions such as potassium or tetramethylammonium unmasks the disordering effect of the drug, and a decrease in the order parameters and an increase in area per lipid is observed, in agreement with the experimental data. We therefore suggest use of larger counter-ions in conjunction with the Berger force field to avoid these problems in future simulations. verall, the effect of negatively charged SAIDs on zwitterionic membrane properties is similar to that of positively charged LAs. As expected, charged molecules are found much closer to the water/membrane interface than their uncharged analogs, and cause minor disruption of the bilayer structure as a result of hydration of their charged groups. Drugs with a wide pharmacological profile Anti-psychotic drugs used widely in the treatment of psychiatric diseases also exhibit potent anti-cancer activity [47]. Some anti-psychotic drugs are also known to be effective anti-microbial agents and excellent codrugs in anti-tuberculosis formulations [48,49]. Like anesthetics, anti-psychotics may operate via direct interactions with the lipid membrane [49]. Chlorpromazine is a phenothiazine derivative that was previously used against schizophrenia. It possesses a rigid, tricyclic, hydrophobic skeleton, and may be present in either neutral or cationic forms in aqueous media. Together with other phenothiazines, such as trifluoperazine and thioridazine, chlorpromazine may cause reversal of multiple drug resistance, by suppressing bacterial efflux pumps, which has proven promising in treatment of tuberculosis [49,50]. Both the cationic and uncharged versions of chlorpromazine order lipid acyls very slightly, in the region FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2795

12 Simulations of small molecules with lipid bilayers W. Kopec et al. occupied by drug molecules, and the effect is more pronounced for the uncharged drug, which penetrates deeper into the membrane [51]. We have also observed an ordering effect on the bilayer, caused by charged thioridazine at high drug concentrations (drug:lipid ratios of 1 : 4 and 1 : 2), when using the Berger force field for lipids (Kopec W, Duelund L & Khandelia, unpublished results). For the phenothiazines, data from simulations typically complement experimental measurements that used magic-angle spinning solidstate 13 C MR, 31 P MR and calorimetry to estimate the localization of phenothiazines in the bilayer [52]. The solidification of the membrane in the liquid crystalline phase as reported by fluorescence spectroscopy, electron spin resonance and microcalorimetry agrees well with simulation results [53]. Another drug with a wide spectrum of activity is siramesine, which failed drug trials as an anti-anxiety and anti-depressant, but was later shown to have anticancer properties [54]. In simulations with mixed PPA (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate)/ PPC lipids, siramesine forms robust complexes with PPA, a phospholipid that is an important second messenger, promoting cell survival [55]. Cancer cells are known to produce PA (Phosphatidic acid) to avoid apoptosis [56]. The presence of PA in bilayers induces aggregation of siramesine molecules at the membrane surface, due to strong electrostatic interactions between the cationic drugs and exposed negatively charged phosphorous groups of PA lipids. By binding to PA, the drug prevents it from cascading signaling processes that delay cancer cell death [54]. Simulation results also substantiate the high affinity of siramesine for PA lipids in preference to other anionic lipids, as revealed by fluorescence spectroscopy and Langmuir isotherms. Amantadine, rimantadine and memantine are tricycle-decane derivatives often used in the treatment of influenza, and also as agents against Parkinson s disease. The molecules possess an amino group that, when protonated, confers a positive charge to the molecules. Although the drugs have cyclic rings, their three-dimensional shapes are more spherical than flat. The amino groups of the drugs remain solvated while interacting with lipid head groups, leading to considerable deformation of the membrane [57]. wing to their more incompatible shape in terms of lipid packing, the molecules disrupt membranes more than charged LAs or SAIDs do. A drug that is very closely related to cholesterol is the steroid-based antibiotic drug, fusidic acid. Uncharged fusidic acid orders a DPPC bilayer just like cholesterol does, while the ordering effect for the charged version of the drug is significantly weaker [58]. The rigidifying effect of fusidic acid is even stronger than that of cholesterol. It was thus postulated, in line with differential scanning calorimetry and fluorescence spectroscopy experiments [58], that fusidic acid is organized into microdomains, and the domain boundaries promote water permeation through the membrane. Compared to cholesterol, fusidic acid is more tilted in the membrane, and its presence creates additional free volume inside the membrane. Thus, the structural shape and smoothness of the molecules play an important role in their interactions with phospholipids, and may be crucial in inducing the ordering effect and/or in domain formation. The results for anti-psychotics and fusidic acid suggest that, for drugs with large hydrophobic backbones, e.g. phenothiazines with three fused rings or steroids with a rigid cyclic skeleton, the presence of charges in the hydrophilic component does not affect interactions with lipid membranes as drastically as in the case of smaller molecules such as SAIDs. The large hydrophobic frame of these compounds allows them to penetrate the bilayer more deeply, resulting in ordering of the fluid phase. An increase in the number of aromatic rings in azo-aromatic fluorescent probes correlated with the increasing depth of immersion of these molecules in a DPPC bilayer [59]. rsi and Essex [60] used a dual-resolution method to describe interactions of three b-blockers and human sex hormones (progesterone and testosterone) with DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) bilayers. The lipid and water molecules are modeled using a coarse-grained approach, while the drug and hormone molecules are described in full atomistic detail. The b- blockers partition near the lipid head groups, while the steroid-based hormones are located more deeply in the membrane. The authors propose that the two interfaces the water/membrane interface and the lipid head group/lipid tails interface are the preferred binding sites for most amphipathic membrane-binding molecules. The attractive forces acting at the interfaces, arising from the lateral pressure profile of the membrane, stabilize most molecules partitioned in the polar/ apolar interface, while repulsive forces acting above and below this region prevent movement of molecules. Interactions of plant extracts with bilayers Plants and plant extracts have been used in traditional medicine in various parts of the world for centuries. Plant extracts have played and still play a major role in modern medicine [61,62]. The uses of plant extracts range from serious heart diseases (digitalis extract) to 2796 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

13 W. Kopec et al. Simulations of small molecules with lipid bilayers breath freshener (eucalyptus oil). The chemical composition of many of these plant extracts has been thoroughly investigated [63], and the active substances have been isolated. Most of these active molecules are rather small and volatile, and are also relatively easy to synthesize in laboratory. Due to their small size and volatility, the olfactory and taste receptors recognize these substances, and, being aromatic, the molecules often have a distinctive taste and smell. Many of these active molecules contain substituted aromatic rings or diene chains, making the compounds autofluorescent and brightly colored. For example, anthocyanins, which are the common colorants of berries and fruits, also serve as potent antioxidants in the body [64]. In some cases, theories and treatments parallel to modern medicine have attracted recent attention due to apparently lower side-effects and the aesthetic pleasure of using natural compounds in preference to synthetic ones for treating disorders that are not life-threatening. Modern medicine has tested many such plant extracts that have a remarkably wide range of beneficial properties, prime examples being the Indian root extract curcumin [65] and terpene extracts from plants commonly used in East Asian cuisine [66]. Many plant extracts have been shown to be membrane-active, and here we review simulations of some plant extracts with lipid membranes, with particular emphasis on similarities or dissimilarities with cholesterol. Plant sterols The interactions of plant sterols and cholesterol (the only sterol molecule of the animal plasma membrane) with lipid membranes have been widely investigated. It is well-known that cholesterol orders fluid lipid bilayers, and introduces the liquid ordered phase [67] observed by MR [68], X-ray diffraction [69] and calorimetry [70]. Plant sterols and cholesterol align with the lipid bilayer and increase lipid tail ordering and membrane thickness [13,22]. Experimentally, each plant sterol interacts differently with the membrane, and the subtle differences in the structure of the sterol molecules may cause significant differences in the membrane properties [71]. Animal cells are fine-tuned to maintain a stable cell membrane fluidity via cholesterol, and replacing cholesterol with plant sterols may alter the balance of the membrane significantly [72] by altering the pressure profile along the membrane normal [71]. In MD simulations, replacing cholesterol with plant sterols affects the thickness of the layer, the sterol orientation, membrane fluidity and elastic properties of membranes [22,73]. A membrane containing ketosterol instead of cholesterol is thinner and more fluid. Ketosterol cannot form all the hydrogen bonds with membrane lipids that cholesterol forms. The effects are strongest when the lipids of the membrane are saturated [22,73]. MD simulations also show significant differences between plant sterols; for example, sitosterol orders the membrane more than stigmasterol does [74]. MD simulations will most probably continue to play a key role in investigating the effect of cholesterol and plant sterols on the membrane properties, as simulation studies may be easily used to trace major differences in membrane properties back to minor differences in the structure of the sterols. Saponins ther sterol-like molecules extracted from plants may also interact with lipid membranes, and the result may be more dramatic, such as leaking or disruption of the cell membrane. This is often seen with saponins, which have sugar moieties attached to a cholesterol-like core [75]. Saponins are soapy substances that are abundant in many plants and marine organisms. Before animal fat-based or synthetic detergents became popular, the plant-derived saponins were widely used for washing clothes. owadays, saponins are used in cosmetics for their soapiness, and in agriculture pesticides for their anti-microbial properties. owever, many saponins are cytotoxic [76]. ne saponin molecule, dioscin, has been investigated via MD simulations [77]. Dioscin molecules adopt a cholesterol-like orientation in the membrane, diffuse towards cholesterol-rich parts of the membrane, and bind cholesterol. The dioscin cholesterol binding is more favorable than cholesterol cholesterol binding. The biggest contributions to this strong pairwise interaction are favorable electrostatic interactions and a reduction of the solvation energy. The molecules bind head-to-head, i.e. the sterol moieties bind along each other with their principal axis pointing in the same direction. The accumulation of dioscin molecules in the membrane and the binding to cholesterol molecules bends the lipid membrane drastically, and the authors propose this to be the underlying cause of hemolysis of red blood cells by saponins. This suggests that the saponin cholesterol aggregation seen in many experiments [76,78] also has biological significance. Saponins thus disintegrate membranes by binding to the sterol molecules in the membrane [77], affecting the delicate effect of sterols as membrane stabilizers. Flavonoids Flavonoids are ring structure-containing plant extracts that possess antioxidant, anti-bacterial, anti-cancer, FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2797

14 Simulations of small molecules with lipid bilayers W. Kopec et al. and other health-promoting properties [79,80]. These are responsible for many of the scents and tastes in aromatic plants, such as those of fruits, berries and tea leaves. They contain two aromatic rings, which are usually connected by a heterocycle with one oxygen atom, and one of the three rings is able to rotate in relation to the other two rings. The aromatic rings of flavonoids contain many hydroxyl substituents, and some of them even contain phenyl substituents. All plant flavonoids investigated by MD simulations (catechins [81,82], isoflavones [83] and flavonols [84,85]) penetrate readily into lipid membranes, and this may be responsible for their health-promoting actions [79,80]. The flavonoids form multiple hydrogen bonds with the membrane lipid head groups when they first bind to the surface, and form double the number of hydrogen bonds with the lipids when they penetrate to the plane of the glycerol region of the lipids [81,82]. Many of the bulkiest flavonoids containing phenyl substituents absorb into the membrane as aggregates, and remain as aggregates in the membrane, causing significant membrane distortion [82]. When absorbed, the flavonoids thin the membrane [83] due to creation of excess free volume in the acyl tail region. The membrane fluidity is correlated with its bending modulus, which, in simulations, is significantly smaller when flavonoids absorb to the membrane [83]. n the other hand, flavonoids tend to decrease the area occupied by each lipid molecule in the plane of the lipid glycerol groups [83]. This tightly packed region in the bilayer coincides with the high lateral pressure peak of the lipid bilayers. The number of hydroxyl groups that the flavonoid possesses determines how deep into the membrane it can penetrate, and also the magnitude of change in membrane fluidity due to flavonoid penetration [83]. The more hydroxyl groups the flavonoid has, the more it interacts with the lipid head groups only, and the smaller the change in membrane fluidity. In general, isoflavones soften the membrane, increase membrane elasticity, and decrease both the area per lipid molecule and membrane thickness [83]. Catechins decrease the area per lipid, as they bind closely to the lipid molecules in the membrane [81,82]. Quercetin increases water permeability of the membrane, as the polar groups of the molecule reside deep in the membrane [84,85]. In the body, the liver removes excess flavonoids by adding functional groups to the molecules. Glucuronided, methylated and sulfated flavonoids all have an impaired ability to absorb into the membrane in MD simulations [84]. ypericin The largest of the plant-derived polyphenolic substances investigated by MD simulations is the eight ring-containing planar molecule hypericin, extracted from St John s wort (ypericum perforatum). ypericin releases reactive oxygen species in response to light, and may be used in laser-assisted treatment of lipid capsules containing viruses (such as IV), and many cancers, such as skin cancer. In MD simulations, the native form of hypericin adsorbs to the interface of lipid membranes [86,87]. The molecule forms multiple hydrogen bonds with the water molecules even when membrane-bound. The hypericins aggregate in the water phase, and an adsorbed aggregate does not disintegrate in the membrane. Brominated hypericin settles deeper into the membrane and penetrates the whole membrane, enabling hypericin to possibly reach intracellular organelles [86,87]. This effect is more pronounced in membranes containing a physiological amount (23%) of cholesterol than in systems with lower cholesterol content. In the future, hypericin may thus serve as a model compound for cancer therapy drugs synthesized for laser radiation therapy. Curcumin and stilbenoids Stilbenoids and curcumin have two aromatic rings substituted with hydroxyl groups and other substituents, one at each end of the molecule, just like flavonoids. owever, unlike flavonoids, they have a diene chain like an elastic stick connecting the two aromatic rings, instead of a third ring structure. In the case of curcumin, the diene chain is also substituted. Curcumin is an anti-fungal, anti-bacterial and antitumor molecule extracted from the plant turmeric [88]. In MD simulations, curcumin does not penetrate membranes when aggregated, similarly to the bulkiest flavonoids, but this may be a meta-stable state. Free curcumin molecules penetrate the lipid membrane (Telenius J, Kongsted J & Khandelia, unpublished results). Most molecules settle at the glycerol region of the membrane, with their principal axis parallel to the membrane. Such localization is also observed experimentally by fluorescence quenching [89]. owever, in simulations, some molecules settle parallel to the lipid tails. When there are multiple molecules organized parallel to lipid tails, they form bundles, which span the entire membrane (Telenius J, Kongsted J & Khandelia, unpublished results). This behavior resembles the cytolytic mechanism of action of antibacterial peptides. The curcumin bundles probably cannot transfer ions, but, as the molecules are conjugated dienes and thus readily 2798 FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS

15 W. Kopec et al. Simulations of small molecules with lipid bilayers conduct electrons, the bundles may just short-circuit the membrane, and thus depolarize the membranes. Remarkably, results obtained by Ramamoorthy et al. suggest that curcumin is incorporated into membranes in a similar way to cholesterol [90]. Also, a strong ordering effect induced by curcumin was measured using ion currents induced by dimerization of gramicidin [91]. Stilbenoids, which resemble curcumin structurally, are known to function as potent antioxidants [92]. In MD simulations, the methylated and ethylated forms of stilbenoids (which are less polar than native stilbenoids) settle deep in the membrane, tilted but almost parallel with the membrane lipids [93]. owever, the duration of the simulation was only 2 ns. If this observation holds over longer time scales, the spatial organization of the stilbenoid aromatic head parallel with unsaturated lipid double bonds may explain its antioxidant properties, as the substituted aromatic head may act as a catalyst in the reduction reaction of the lipid double bonds, and the diene chain may act either as an electron donor or an electron sink in the reaction due to its high conductivity. Carotenoids Carotenoids, which are known antioxidants and health-promoting agents that are abundant in many plants such as carrot and berries such as blueberry, contain a long polyterpene chain, which is a long conjugated diene, having methyl substituents on regular intervals. The diene is longer than in stilbenoids, and the ring structures on the ends of the diene are not aromatic. The rings are not necessarily highly substituted with polar groups either. The length and hydrophobicity of b-carotenes enables them to span biological membranes and settle parallel to the lipid tails of the membrane. This effect has yet to be investigated via long time scale MD simulations, and so far the only simulations for carotenoids have been Monte Carlo simulations for lutein [94]. The simulations show that b-carotenes may influence the liquid gel transition of the lipid layer, and that their orientation in the membrane (parallel to the membrane or parallel to the lipid hydrocarbon chains) depends on the lipid bilayer phase state. Locally, carotenoids tend to decrease lipid tail order parameters. Molecules with a hydrocarbon tail substituent There are numerous plant extracts that consist of only one ring structure and have a long hydrocarbon chain as one of the substituents of the ring. The ring may be either aromatic or non-aromatic, and the long hydrocarbon tail of the molecule often has methyl substituents. In addition to the hydrocarbon chain, the ring structure is usually rich in hydroxyl group substituents. The polar ring may position itself near the lipid head groups, and the non-polar tail may penetrate between lipid tails into the membrane. ne of these molecules is the most active form of vitamin E: a-tocopherol, a potent antioxidant that maintains lipid bilayers in a homogenous phase even in the liquid liquid phase separation region of phospholipid membranes [95]. In MD simulations, a-tocopherol achieves this by inhibiting coalescence of the microdomains of each phase to a large phase-separated unit, by binding to the phase boundaries of these microphase-separated systems [95]. owever, vitamin E has no effect on lipid tail order. In MD simulations, a-tocopherol may also flip-flop in the membrane [96]. It is possible that the polar ring of a-tocopherol may reduce the oxidized lipids, flip to the water phase to be reduced itself, and flop back to continue to another cycle. The molecule also shows very high diffusion rates at high temperatures [96], making scanning for oxidized lipids efficient just when it is needed, i.e. when the membrane is the most fluid and thus most penetrable by oxidizing agents due the high temperature. Another family of this kind of molecules are the resorcinolic lipids that are abundant in plant membranes. The hydrocarbon tails of resorcinolic lipids penetrate one leaflet of the membrane, and the longest of them may penetrate the whole membrane [97]. They exhibit various configurations in the membrane: the tail may bend back to the leaflet it came from, or lie in the middle of the membrane, or span both leaflets. Resorcinolic lipids increase lipid tail order parameters, and decrease transmembrane water permeation. owever, when they are absorbed into an intact phospholipid bilayer, they may cause phase transition to hexagonal phase, phase separation by generation of gel domains, or water pore formation during absorption [97]. Small cyclic molecules The final group of membrane-active plant extracts is a group of small ring-structured molecules with only one ring and small substituents. ne of these is salicylate, which has been discussed above. Small cyclic terpenes, consisting of one non-aromatic ring and only a few polar substituents, also have a wide range of reported activities [66]. Perillyl derivatives (perillyl alcohol, acid and aldehyde) [46,98] and limonene [98] have been investigated using a combination of MD and isothermal titration calorimetry. Many small terpenes have favorable smells (limonene FEBS Journal 280 (2013) ª 2013 The Authors Journal compilation ª 2013 FEBS 2799