How Does the P7C3-Series of Neuroprotective Small Molecules

Article pubs.acs.org/jcim

How Does the P7C3-Series of Neuroprotective Small Molecules Prevent Membrane Disruption? Amin Reza Zolghadr* and Maryam Heydari Dokoohaki Department of Chemistry, Shiraz University, Shiraz 71946-84795, Iran S Supporting Information *

ABSTRACT: Molecular dynamics (MD) simulations are conducted to suggest a mechanism of action for the aminopropyl dibromocarbazole derivative (P7C3) small molecule, which protects neurons from apoptotic cell death. At first, the influence of embedded Aβ42 stacks on the structure of membrane is studied. Then, the effect of P7C3 molecules on the Aβ42 fibril enriched membrane and Aβ42 fibril depleted membrane (when Aβ42 fibrils are originally dissolved in the aqueous phase) are evaluated. Also, the formation of an amyloid ion channel in the Aβ42 enriched membrane is examined by calculating deuterium order parameter, density profile, and surface thickness. For Aβ42 in the fully inserted state, ion channel-like structures are formed. The presence of P7C3 molecules in this case just postpones membrane destruction but could not prevent pore formation. In contrast, when both Aβ42 and P7C3 molecules are embedded in the aqueous solution, the P7C3 molecules are self-assembled at membrane/ionic aqueous solution interface and prevent the precipitation and deposition of Aβ42 fibrils into the membrane.

1. INTRODUCTION

underlying P7C3 efficacy is not clear and still needs subsequent work. In spite of the fact that the molecular root of AD is not fully clarified, past investigations have represented that a key pathogenic pathway in AD is amyloid-beta (Aβ) peptide.4 Aβ proteins have a potent tendency to form protofibrils with βstrands that are perpendicular to the fiber direction.5,6 β- and γSecretase, two membrane-bound proteases, cleave the amyloid precursor protein (APP) sequentially and commonly produce amyloid-beta fragments with 40- and 42-residue.7 A critical factor in the pathogenesis of Alzheimer’s diseases is selfaggregation of amyloid-beta peptides into toxic soluble protofibrils or oligomers.8,9 Accordingly, both Aβ40 and Aβ42 have been the subject of extensive studies of peptide aggregation.10,11 The molecular basis of neurotoxicity of Aβ has been proposed by a great variety of hypotheses. Amyloid hypothesis indicates that Aβ oligomers may directly damage cell membranes, alter neuronal ion homeostasis, and eventually cause cell death.12 There has been a recent shift in focus from mature fibers to soluble prefibrillar oligomers as the main neurotoxic species in amyloid disease.13,14 Recently, the initial steps of Aβ aggregation were probed by high-resolution atomic force microscopy.15 The effect of fibril fragmentation on the cell membrane injury studied by Xue et al. in a systematic manner.16 They indicated that this fragmentation, induced by the mechanical stress, thermal motion, and chaperone activity,17 increases the interaction of Aβ fibrils with cell membranes.

Alzheimer’s disease (AD) is a common cause of dementia that is associated with daily living through anxiety, depression, apathy, psychosis, memory loss, and cognitive impairment. The number of individuals that would be affected by AD is predicted to nearly triple over the next 40 years, which emphasizes the need for reconsideration of either the treatment or the approach to drug development in order to decelerate the future burden of disease on individuals, society, and the economy.1 Target-based approaches have been pursued by some researchers to discover new treatments for Alzheimer’s diseases. Recent evidence proposes small drug molecules that enhance neurogenesis and retard neuronal death. To find chemicals capable of protecting neurons, Pieper et al. searched a collection of 2000 small molecules of different origins to identify neuroprotective agents.2 Their assay revealed the P7C3 class of aminopropyl dibromocarbazole compounds (see Scheme 1) that elevated the number of surviving cells by blocking apoptosis.3 The introduction of the P7C3 class of neuroprotective drugs opens up a new avenue for design and screening of potent Alzheimer’s drug. However, the mechanism Scheme 1. Structure of P7C3

Received: March 15, 2017 Published: July 10, 2017 © 2017 American Chemical Society

2009

DOI: 10.1021/acs.jcim.7b00151 J. Chem. Inf. Model. 2017, 57, 2009−2019

Article

Journal of Chemical Information and Modeling

resolution necessary for describing membrane disruption in AD pathogenesis. Also, the aggregation of a carbazolimum-based drug candidate, P7C3, at phospholipid membranes/ionic aqueous solution is presented. Altogether, a new molecular mechanism is provided for P7C3 that opens up a new avenue to develop a novel Alzheimer’s drug.

It has been shown that Aβ proteins can insert into the lipid bilayer and form ion channels, permeable to physiological ions which might lead to depolarization of cell membrane, uncontrolled calcium influx, and ultimately cell death.18 According to several studies, Aβ toxicity is caused by the formation of pores or channels in membranes.19,20 Kayed et al.21 have indicated that just oligomeric Aβ is capable of directly injuring brain neurons. However, Ambroggio et al.22 have corroborated that monomeric Aβ species could absorb into the membrane and perturb the bilayer. Arispe et al.23,24 have shown that Aβ40 was incorporated into the artificial membranes and formed ion channels. McLaurin and Chakrabartty indicated that Aβ disrupts membranes containing acidic phospholipids,25 and Rhee et al. showed that Aβ42 forms calcium-permeable channels.26 With the help of molecular dynamics (MD) simulations impressive advancement is being made in the understanding of peptide−lipid interactions.27,28 The success of applying MD to capture the conformational dynamics of proteins inserted in lipid bilayers,29,30 and protein−lipid interactions,31 has been addressed in the aforementioned research. The advances made in the simulations of interface between model membrane and aqueous solution that require the close cooperation with experiments well reviewed by Berkowitz and Vácha.32 Distinct experimental techniques, such as vibrational sum frequency generation spectroscopy,33 infrared (IR) spectroscopy,34 small angle neutron scattering,35 low-temperature magic angle nuclear magnetic resonance (NMR),36 and X-ray scattering,37 were used to study the interface between lipid bilayer and aqueous solution. Also, more advanced surface-specific spectroscopic techniques are employed to acquire a comprehensive picture of water structure at the surfaces of biomembranes.38,39 MD simulation of bee venom peptide, melittin,40,41 in dipalmitoylphosphatidylcholine (DPPC) and dimyristoylphosphatidylcholine (DMPC) lipid bilayers has been conducted in order to relate its toxic effect with cell membrane perturbation.42,43 Water molecules can pierce into the membrane due to the interaction of melittin with leaflets of the phospholipid bilayer. Based on the same characteristic of Aβ and melittin, such as their α-helical conformations that interact asymmetrically with membrane leaflets, simulations of both are assumed to cause some degree of perturbation on the bilayer. Lemkul and Bevan have indicated that disordering of lipids is observed due to the electrostatic interactions between the Aβ40 and the hydrophilic backbone of membrane phospholipids.44 In fact, amyloid beta oligomers are capable of interacting with cell membranes,45−48 forming specific ion channels across the membrane, and disrupting intracellular ion homeostasis.49,50 A molecular level understanding of interactions between Aβ oligomer and membrane that could lead to the formation of ion channels is missing. Central questions remain, especially regarding the chemical and structural characteristics of Aβ oligomer that enable it to perturb the membrane. Comprehensive investigations of the binding mechanism of peptide and lipid membrane are needed to understand this phenomenon. In this study, the primary goal is to understand in detail the mechanism of cell membrane damage and cell death in AD. To this aim, the effect of the interactions between Aβ oligomers and the phospholipid bilayer on the membrane structure are studied thoroughly. Simulations of amyloid fibrils in a model DPPC membrane can provide the atomistic

2. DETAILS OF SIMULATIONS The simulations consisted of four different systems: (I) a protein-free control system (lipid membrane/water), (II) an Aβ stack interacting with membrane/aqueous ionic solutions, (III) an Aβ stack interacting with membrane/P7C3 enriched aqueous ionic solutions (this system is similar to system II, while 24 molecules of P7C3 are inserted in the aqueous phase); (IV) membrane/aqueous ionic solutions enriched with 48 molecules of P7C3 and an Aβ stack. We used the 3D structure of Alzheimer’s Aβ42 fibril (PDB code 2BEG)51 which was determined using solid-state NMR.52 The net charge of each protein stack was −5e which was neutralized by the addition of the same amount of positive ions. The stability of this structure was also tested in our previous simulations.53 Also, Lemkul and Bevan have simulated this structure to reveal key aspects that are essential to the stability of the Aβ42 fibril.54,55 They have shown from backbone rootmean-square deviations that the wild-type protofibril did not differ significantly from the initial NMR structure. The pentameric structures they acquired throughout the six independent 100 ns simulations using the GROMOS 53A6 force field were judged stable. Masman et al. have shown that the core region of the fibril, consisting of residues 17−42, is substantially responsible for its stability.56 We have used the pre-equilibrated coordinates and force field parameters of the DPPC bilayer which comprised 64 lipids per membrane leaflet.57 Initial structures for the DPPC membranes were taken from the Tieleman group website.58 The initial coordinates of P7C3 were obtained by an ab initio calculation at the MP2/6-311++G** level of theory. The structure and topology of P7C3 were produced by the small molecule topology generator PRODRG.59 The partial atomic charges are calculated by using natural population analysis as implemented in Gaussian 09 program.60 Simulations were performed with the GROMACS 4.5.4 program using the GROMOS96 53A6 force field61 under constant number, pressure, and temperature (NPT) conditions for all our systems. All simulations were carried out in the presence of explicit water by using simple point charge (SPC) model. The net charge on the protofibrils was neutralized by addition of sodium ions. Periodic boundary conditions applied in three dimensions were employed for the whole ensembles. The equilibration was performed in three stages: (1) Initial energy minimization using the steepest descent algorithm has been conducted to remove close contacts and find a minimum on the potential energy surface. (2) An equilibration NVT run has been carried out over 5 ns while restraining the position of the fibril by force constant of 1000 kJ mol−1 nm−2 to their initial position. (3) The system has been equilibrated for 5 ns using an NPT ensemble. The final configurations from the above-mentioned procedures were taken as the initial structures for the 400 ns production runs at 310 K. The first 50 ns of each trajectory was set aside for equilibration and was discarded during structural analysis. The 350 ns of sampling was sufficient to observe the convergence of measurements. 2010


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Figure 1. Snapshots of the Aβ stack interacting with membrane/aqueous ionic solutions (system II) at different simulation time intervals. The Aβ stack is colored green (using a cartoon representation), and for the lipid bilayer, the red, blue, and yellow points represent the oxygen, nitrogen, and phosphorus atoms of DPPC headgroups, respectively. Pink lines are the acyl chains. Water and ions are shown with blue lines and purple spheres, respectively.

Analysis. Density Profile. The transbilayer, or z-dependent, number density profiles were calculated using the g_density utility from the GROMACS package. The density profiles of the DPPC polar headgroups, the DPPC tails, water, and protein were obtained over the simulation time for each system. Deuterium Order Parameters. The deuterium order parameter SCD, which can be determined experimentally by NMR spectroscopy, is evaluated to explore the impact of Aβ42 fibrils on the membrane structure. SCD is calculated as 1/ 2⟨(3cos2 θ − 1)⟩ where θ is the angle formed by the C−H bond vector of the nth carbon atom of sn-1 and sn-2 chains with

The equations of motion have been solved by the leapfrog integrator with a time step of 2 fs. The temperature was maintained at 310 K using Berendsen thermostat with a coupling constant of 0.1 ps.62 The pressure was kept at 1 bar by coupling semiisotropically to a barostat with a coupling constant of 1 ps. Both the electrostatic and van der Waals interactions used a short-range cutoff of 1.2 nm and the longrange electrostatics were evaluated using the particle mesh Ewald (PME) method.63 Visualization of results was accomplished using the VMD software.64 2011


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Figure 2. Density profiles of the headgroups, the lipid tail, and the Aβ-fibril obtained from the trajectories of (A) the control system and system II after (B) 100, (C) 300, and (D) 400 ns.

systems.67 The g_membed tool works by placing the Aβ fibril at the desired position in the middle of the bilayer, removing eight lipids, and then allowing surrounding lipids to relax. After this procedure, additional water molecules were added to the system to resize the box to 10.5 nm along the z-axis (with the bilayer in the x−y plane), leading to approximately 3.5 nm of space between periodic images of the fibril in the z-dimension to eliminate interactions between periodic images. We also performed simulations using an ensemble of larger lipids, with 256 molecules (see Figure S1). Because no appreciable difference due to the ensemble size was noted, we continued the simulation using the smaller one. Figure 1 shows the snapshots of system II as time progressed. The position of Aβ oligomers in this simulation is in line with experimental findings that indicated Aβ fibrils across the hydrophobic core of a lipid bilayer.25 The simulated root-mean-square distances (RMSDs) to the corresponding initial conformation show that Aβ fibrils remain stable over the course of the 400 ns simulation (see Figure S2). Also, the time evolution of radius of gyration (Rg) of fibrils in system II is examined and depicted in Figure S3. The mean gyration radius does not vary over the simulation time. At t = 0 ns, Aβ fibril oriented almost parallel to the membrane plane (xy). Whereas during the simulation time, the Aβ fibril oriented almost tilted to the membrane plane but retained a β-sheet structure. Deformation of the membrane in the center of the bilayer is significant after 50 ns of simulation. Due to the fibril−lipid interactions, the hydrophilic backbone of membrane phospholipids tilts significantly around the fibrils,

the bilayer normal. The averages are over simulation trajectory. SCD quantifies the order of the phospholipid tails in the membrane with regard to the bilayer normal. The −SCD(n) values for lipid bilayers are commonly between 0 and 0.5. A −SCD value of 0.5 represents the perfect alignment of the lipid tail to the bilayer normal and a value close to the zero indicates a random orientation. Deuterium order parameters were determined using the GROMACS program g_order. Surface Area per Lipid. The average surface area per lipid (APL) was obtained by dividing the simulation cell area in the dimension parallel to the plane of the bilayer (x−y plane) by the number of lipids in one of the leaflets and averaging over all frames. The GridMAT-MD code was used to map the lipid atomic positions onto a 2D lattice.65 Average Bilayer Thickness. The effect of the Aβ42 stack on the average thickness of the lipid bilayer is evaluated by measuring the phosphorus atom distances between each leaflet of the bilayer. The GridMAT-MD tool was used to calculate this descriptor of membrane disruption. It has been shown that membrane thickness decreases when monomeric Aβ40 inserts into the hydrophobic core of membranes.66

3. RESULTS Disruption of Membrane in the Presence of Aβ Stacks. This system was considered to simulate the structure of the neuronal cell membrane when Aβ stacks are inserted into the cell membrane. In this simulation the fibril was intercalated into a pre-equilibrated bilayer using g_membed, a Gromacs tool typically used for the preparation of membrane protein 2012


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Journal of Chemical Information and Modeling causing the hydrocarbon chains of the phospholipids to orient toward the bilayer interface. As clearly seen at 20 ns, water and ions diffused into the bilayer due to the membrane deformation. The water permeation is assisted by hydrogen bonding between water and the β-amyloid protein (see Figure S4). Aβ fibrils adopted a near-transmembrane structure, pulling water and ions into the membrane such as a channel. This leakage of ions would have serious consequences for cellular homeostasis.19 After 400 ns of simulation, the Aβ oligomer associated strongly with the bilayer and the structure of the membrane changed substantially as a result of electrostatic and vdW interactions (see Figure 1). Another perspective where acyl chains are removed for clarity is depicted in Figure S5. Obviously, various tilt angles have been adopted by the Aβ protein during the course of the simulations. The tilt angle is defined as the angle between the β-sheet axis and the bilayer normal. A 90° tilt angle is in accordance with a parallel to the membrane plane orientation. The β-sheet axis of the Aβ protein is made by two points, one near the top and the other near the bottom of the β-sheet. The results indicated that the tilt angle increased over the simulation time and the protein became increasingly tilted relative to the bilayer normal in accordance with Tieleman et al. findings.68 We also performed simulations using an ensemble of POPC lipids, consisting of 256 1palmitoyl-2-oleoylphosphatidylcholine (POPC) molecules. Both DPPC and POPC lipids predict ion and water flux through the membrane. As the results were found to be unaffected by the structure of phospholipids, we continued the simulations using the DPPC lipids (see Figure S6). Figure 2A depicts the density profile of DPPC headgroups and tails for the control system. The density profile of lipid membrane is divided into two regions, a headgroup region and a hydrocarbon chain region. The headgroups in control simulations tend to aggregate, and hence, dense regions of polar parts are formed in the surface layers. On the other hand, the segregation of nonpolar groups is seen in the subsurface regions. The presence of headgroups favorably in the interface pushes the tails into the subsurface region. Thus, an enrichment of tail density in the subsurface region is substantial. The density profiles of the control system confirm the usual picture of bilayer structure. Figure 2B−D illustrates the density profiles of system II during the simulation times of 100, 300, and 400 ns, respectively. The extent of membrane disruption by Aβ42 is clearly seen at different simulation times by diminishing the characteristic peaks of lipid density profile. It shows that the average density of DPPC headgroups decreases from 0.706 ± 0.031 g cm−3 for the control system to 0.563 ± 0.037 g cm−3 for the disrupted system after 400 ns (Figure 2D). The density peaks of DPPC hydrocarbon chains decrease substantially in comparison with pure DPPC and finally reach a plateau. In this state, a significant change in the ordering of hydrocarbon chain can be identified. The interactions of Aβ42 proteins and lipids have a significant effect on the membrane structure and function. The lipids could respond to the presence of Aβ42 fibrils by changing their hydrophobic tails order. A higher value of the order parameter corresponds to more ordered orientation and a lower value indicates a less ordered structure. The order parameters −SCD are plotted as a function of the carbon atom along the lipid tails for DPPC in Figure 3 and provide a quantitative measure of the degree of order along the lipid acyl chains. The chemical formula of the DPPC molecule is shown on the right side of Figure 3 in which the carbon tails are numbered as follow: sn-1

Figure 3. Average deuterium order parameters obtained for the sn-1 and sn-2 chains of the DPPC over the 400 ns of the simulation. The calculated values of the control system and experimental71 deuterium order parameters of DPPC at 310 K (pink open triangles) are also shown. The chemical formula of a DPPC molecule is shown on the right side.

chain consists of C34 and C36−C50 and sn-2 is C15 and C17− C31 with the positions of 2−15, respectively. The trend of order parameters obtained for pure DPPC system is likely the same for both sn-1 and sn-2 chains. Our result of pure membrane lies within an acceptable range when compared to the previous experimental and simulation results.69,70 The control system is identified by the first region of relatively high acyl chain order near the interface which resembles a plateau (except deviation in the first data point), followed by a second region that diminishes monotonically toward the lower −SCD in the core of the lipid bilayer. The order parameter of the lipid is locally disturbed by the interactions with Aβ42. The presence of Aβ42 stacks leads to a decrease in the order of first region and an increase in the second part. The decrease in the order of the first region can be described mainly by electrostatic interactions between the Aβ42 and the lipid headgroups. The more ordered structure of lipid tails is attributed to the higher vdW interactions between alkyl chains in the presence of fibrils. For all simulated systems, the APL headgroup for the top and bottom leaflets of the DPPC bilayer was calculated at the start of production runs (t = 0 ns) and also at the end of the simulation (t = 400 ns) and are shown in Table 1. The values of APL for the control system are within 3% of experimental estimates of the area per lipid of DPPC which was obtained at 323 K.72 The results of simulated pure DPPC bilayers give an average APL headgroup of 0.614 ± 0.012 nm2, in agreement with previous simulation and experimental results.73,74 For system II, APL values in each leaflet exhibit an asymmetric arrangement of lipids around the Aβ oligomers. The average APL headgroup for the top and bottom leaflets of the DPPC bilayer was 0.484 ± 0.016 and 0.599 ± 0.016 nm2, respectively. The substantial decrease in the APL values is due to the tilting of the lipid monolayers. The APL for top leaflet is decreased significantly from the control system, whereas the APL for bottom leaflet is only slightly less than that of the control system. This is attributed to the different electrostatic and vdW interactions of each leaflet with amyloid fibrils. The thickness of membrane around the embedded Aβ protein is depicted in Figure S7. As shown in Table 2, the 2013


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neural cells that are attacked by Aβ fibrils (system III). Figure 4 depicts the snapshots of this system at different simulation time intervals. Initially, the drug molecules were dissolved in the aqueous phase and separated from each other by about 2 nm. As simulation started, the P7C3 molecules very quickly moved toward the membrane phase and reached the interface within 50 ns (close snapshots are shown). Throughout this process, the P7C3 molecules are accumulated right where the protein is located in the membrane. These snapshots confirm that although small amounts of water pass through the membrane, the Aβ protein tilting and ion channel formation are postponed. One of the main consequences of the accumulation of P7C3 is membrane shrinkage. It can be described regarding the decrease of the surface area per lipid molecule, which is shown in Table 1. Alteration of the membrane thickness in system III is expected to be governed by two different factors. First, the thickness of monolayers changes as Aβ fibrils disrupt the membrane. Second, the change in the averaged size of lipid molecule in the direction normal to the bilayer plane due to the presence of P7C3 molecules. The sign of these effects depends on the ordering of the lipid chains. Prevention of Aβ Penetration into the Membrane by Enrichment of Aqueous Phase with P7C3. The possibility of spontaneous incorporation of soluble Aβ molecules into phospholipid membrane was evaluated by Arispe et al.23 Their experimental results reveal that when Aβ molecules are dissolved in an aqueous solution, a structure is attained which allows direct incorporation of the peptide molecules into the lipid bilayer. Also, we have performed a simulation of Aβ peptide permeation by starting from a structure with fibrils embedded in the aqueous phase. The results of 400 ns simulation (see Figure S10) shows that the Aβ peptide deposited spontaneously in the membrane. Once within the membrane, the amyloid channels direct cations in a way quite similar to ion channels. Therefore, the observed channel activity results from the insertion of Aβ molecules into the bilayer. In this part, we have performed simulations to propose a mechanism for the action of P7C3 anti-Alzheimer’s disease activity. To this aim, both P7C3 molecules and Aβ fibrils are placed in the water phase (system IV). As we have shown elsewhere, the P7C3 molecules assembled at lipid/water

Table 1. Area per Lipid of Simulated Systems at the Start of Production Runs and at the End of Simulationsa name of system exp system II t = 0 ns system III t = 0 ns system IV t = 0 ns

area per lipid (nm2)

name of system

0.630 ± 0.01 (T = 323 K) top: 0.596 ± 0.048 bottom: 0.646 ± 0.001 top: 0.607 ± 0.090 bottom: 0.646 ± 0.075 top: 0.646 ± 0.061 bottom: 0.646 ± 0.061

control (system I) system II t = 400 ns system III t = 400 ns system IV t = 400 ns

area per lipid (nm2) 0.614 ± 0.012 top: 0.484 ± 0.016 bottom: 0.599 ± 0.016 top: 0.456 ± 0.012 bottom: 0.527 ± 0.017 top: 0.450 ± 0.018 bottom: 0.404 ± 0.031

a

The experimental value for the unperturbed membrane is included for comparison.

Table 2. Bilayer Thickness of Simulated Systems at the Start of Production Runs and at the End of Simulations thickness of bilayer (nm) control (system I) system II system III system IV

t = 0 ns 3.770 3.983 3.932 3.857

± ± ± ±

0.040 0.057 0.210 0.030

t = 400 ns 3.770 0.460 0.906 4.188

± ± ± ±

0.040 0.085−5.060 ± 0.024 0.033−5.000 ± 0.048 0.040

thickness of control system (at 310 K) is 3.770 ± 0.040 nm in fairly good accordance with the experimental result of 3.78 nm at 323 K.75 It should be noted that, in system II, Aβ oligomers can lead to a magnitude of bilayer thinning about 0.46 nm in regions where the phospholipids became more tilted over time. As shown in Figure S8, the interaction of membrane with Aβ fibrils diminished the number of backbone hydrogen bonds between peptide chains. However, each chain largely retains its secondary structure throughout the simulation. The main interactions of lipids with Aβ fibril are shown in Figure S9. The interactions of Ala42, Leu17, Gly33, Phe19, Val18, Asn27, Phe20, and Asp23 residues with lipid headgroups are dominant. Treatment of Aβ Affected Membrane with P7C3. The aim of this part is the study of the influence of P7C3 on the

Figure 4. Snapshots of the Aβ stack interacting with membrane/P7C3 enriched aqueous ionic solutions (system III) at different simulation time intervals. The color codes are the same as Figure 1. The atoms of P7C3 molecules are shown as cyan spheres. 2014


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Figure 5. Snapshots of the membrane/aqueous ionic solutions enriched with 48 molecules of P7C3 and Aβ stack (system IV) at different simulation time intervals. The color codes are the same as Figure 1. The atoms of P7C3 molecules are shown as cyan spheres.

Figure 6. (A) Density profiles and (B) order parameters of system IV.

interface such as a surface active molecule.76 P7C3 molecules accumulate near the boundary between the hydrophilic parts of the lipid bilayer and water, where their polar groups interact with polar lipid head groups and water. The membrane thickness increases slightly, which can be attributed to the increase in lipid ordering due to the accumulation of P7C3 molecules at the membrane interface. Figure 5 shows that at t = 50 ns, the P7C3 molecules self-aggregated at the interface of water and membrane. This assembly prevents precipitation of Aβ oligomers into the cell membranes. In this way, P7C3 molecules protect newborn neurons from apoptotic cell death as MacMillan et al. discovered experimentally.3 To present a quantitative estimate of this finding, the density profiles and order parameters of system IV are depicted in Figure 6. Density profiles of Figure 6A retain the characteristic structure of a lipid. The mass density of lipid tails has a well near the center of the bilayer. Equilibrium distribution of P7C3 and Aβ fibrils outside the lipid bilayer can be described by the mass density profiles. As shown in Figure 6B, the values of −SCD for both sn-1 and sn-2 chains are slightly higher in the system IV than in the control system confirming that Aβ42

stacks could not disturb the order of lipid bilayer in the presence of P7C3 molecules as a neuroprotective agent. The interaction of P7C3 and Aβ with water and DPPC for systems III and IV are investigated with special attention paid to the formation of hydrogen bonds between these constituents (see Figures S11 and S12). The number of hydrogen bonds between water and Aβ for system IV is increased substantially in comparison with system II (see Figure S12). The analysis of binding preferences between the polar groups of P7C3 and atoms of both lipids and water (see Figures S11 and S12 in the Supporting Information) revealed that P7C3 forms hydrogen bonds predominantly through its hydroxyl and amine groups. The average number of hydrogen bonds per time frame between P7C3 and DPPC are 9.80 ± 2.25 and 23.63 ± 3.01 for systems III and IV, respectively. The increase in the P7C3··· DPPC number of hydrogen bonds in system IV represents the higher accumulation of P7C3 at the membrane interfacial region when the Aβ fibrils are dissolved in the aqueous phase. The number of hydrogen bonds between Aβ and membrane are 19.12 ± 3.73 and 4.83 ± 1.81 for systems III and IV, respectively. In this way, localization of P7C3 at the interfacial 2015


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and increases bilayer thickness. On the other hand, polar regions interact with the lipid headgroup and affect lipid order, leading to a reduction in bilayer thickness. Studies of amyloid fibril interactions with lipid membrane are critical for unraveling the molecular mechanisms underlying anti-AD activity. P7C3 was discovered through an in vivo assay planned to recognize drug-like chemicals that elevate newborn neurons by protecting neural precursor cells from programmed cell death.81,82 The analysis of toxicity profiles indicated that P7C3 is not toxic. Also, the P7C3 class of aminopropyl carbazoles have been found to be effective in blocking apoptosis of neurons in other parts of the central nervous system including amyotrophic lateral sclerosis,83 Parkinson’s disease,84,85 stress-associated depression,86 and traumatic brain injury.87,88 Accordingly, the P7C3 series of compounds may facilitate the generation of a new category of neuroprotective small molecules.89 Good blood−brain barrier permeability makes P7C3 derivatives an outstanding stage for the future advances of neuroprotective drugs. Little is known about the molecular mechanism of P7C3 compounds. Though the exact mechanism of P7C3 action is still under scrutiny, our recent report suggests that the inhibition of β-secretase could be considered as one possible mechanism of action. 53 The interactions are dominantly through hydrogen bonding between the hydroxyl group of P7C3 and the aspartic acid residue Asp228. Also, Manetsch and co-workers have considered 1,3-disubstituted 2propanol derivative as β-secretase inhibitor.90 Also, Bertini et al. proposed carbazole-containing aryl carboxamides as β-secretase inhibitors.91 In addition a class of hydroxyethylamine molecules were studied using high-throughput screening (HTS) fluorescence assay.92 This assay recommend a dibromo-substituted carbazole containing an alpha-naphthylaminopropan-2-ol derivative, as a potent inhibitor of β-secretase. We have also given some consideration to the question of how P7C3 might protect neurons. Our simulations were designed to examine whether P7C3 interacts directly with the lipid membrane. Moreover, we have recently identified that the surface activity of P7C3 is critical for the structural characteristics of being a drug.76 These findings encouraged us to evaluate whether P7C3, which is considered as an “interface active molecule”, could assemble at the membrane surface and thus function as a neuroprotective agent by inhibition of Aβ deposition into the membrane. To answer this question, we studied the interactions of Aβ fibrils with a model membrane in the presence of P7C3. In this investigation, a mixture of P7C3 and Aβ oligomers was added in the aqueous phase of the membrane−water interface. With these simulations set (system IV), we showed that P7C3 can self-assemble at the membrane− water interface and efficiently prevent the insertion of the peptide within the membrane. As shown in Figure 5, Aβ channel activity was almost completely blocked by P7C3. The effect of P7C3 on the Aβ peptide−membrane interactions was then quantified by measuring density profiles and order parameters. Moreover, these results indicate that the density profiles of the phospholipid membrane and the bilayer thickness were not disturbed when the drug molecules are added to the aqueous phase. We have shown previously that P7C3 tightly interacts with the Asp23-Lys28 salt bridges in the Aβ peptide fibrils, through its hydroxyl group.53 On the other hand, Fantini and coworkers indicated that Lys-28 appeared to be common to the cholesterol-binding site of these peptides.93 They have shown

region was shown to influence the interactions of Aβ with the membrane.

4. DISCUSSION A significant body of data has proposed that polymerization of Aβ peptide into amyloid fibrils is an essential stage in the pathogenesis of AD. As amyloidogenic peptides are believed to be toxic, much work has been performed to inhibit β-secretase and γ-secretase enzymes that are responsible for the generation of Aβ fragments.77 However, all of these efforts were unsuccessful in either preclinical or clinical phases. Some studies show that β-secretase and γ-secretase inhibitors may be incapable of ameliorating AD as nonamyloidogenic peptides seem to have a capability of disrupting membrane as well as amyloidogenic peptides.78 Amyloid ion-channels would provide a root for pathophysiological effects of amyloidogenic peptides on the cell membranes; the Aβ peptides that form ion pores in the membrane disturb ionic balance which leads to cellular degeneration.48 In the present work, we have taken this into account and concentrated on a novel paradigm through which P7C3 could protect neurons directly by self-assembly at the membrane interface and hinder ion channel formation. Our simulations indicate that the ion channel-like activity of Aβ fibrils in the membrane disorder structure which can lead to neurodegeneration and cell death in Alzheimer’s disease. The present results demonstrate that Aβ fibrils disrupt lipid bilayers (see Figure S5) in line with previous experimental findings. Arispe et al. have demonstrated using discrete conductance changes that synthetic Aβ peptides can form channels that permit the ions to enter the lipid bilayer.20 Quist et al. have shown that different amyloid molecules form channel-like structures in membrane using atomic force microscopy.49,79 To our knowledge, no simulation studies have investigated the Aβ fibril channel formation process yet. Previous studies are focused on the interaction of Aβ monomers with lipid membrane.22 Xu et al. performed MD to study Aβ40 in DPPC lipid bilayers.80 The results obtained by these researchers indicate that the Aβ40 can leave the DPPC bilayer within 100 ns of MD simulation while keeping much of its helical secondary structure. Their results suggested that Aβ could interact with lipids and cause nearby lipids to become more tilted relative to control system. We have focused primarily on the interactions of Aβ protofibrils with the membrane. The results presented above indicate that the tilting of lipids are occurred when the Aβ fibril is embedded into lipids. Due to the electrostatic and vdW interactions between lipid bilayer and protofibrils, a pore is formed in the membrane and water molecules penetrate into the lipid bilayer. The binding between lipid headgroups and protofibril decreases the number of backbone hydrogen bonds between peptides. However, these interactions could not affect the folding of toxic β-sheet fibrils. The simulation of control system revealed that the structure of the membrane is generally in agreement with the available experimental and simulated results. However, there are significant perturbations in the structure of the lipid when Aβ embedded into the membrane. One of the substantial influences of Aβ on the bilayer structure is that of membrane thinning in some regions and thickening in other parts. The impact of amyloid fibrils on the membrane thickness is adjusted by their structure. Amyloid fibrils are large, anisotropic, and rigid molecules which adopt a tilt orientation within the bilayer. Hydrophobic parts of amyloid partitions to the nonpolar region 2016



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that the amino group of Lys-28 can form a hydrogen bond with the OH group of cholesterol. In fact, cholesterol interacts with Aβ peptides, facilitates the path of their insertion in the membrane, and motivates their tendency to form ion channellike structures.94,95 As shown in Figure S12 the average number of hydrogen bonds between Aβ and P7C3 is 7.78 ± 1.19 over the last 200 ns of simulations for system IV. Therefore, we propose that P7C3, through its hydrophilic and hydrophobic groups, could block the cholesterol binding site of Aβ peptides at the lipid membrane interface. Taken together, we showed that Aβ42 fibrils could form ion channels in the membrane and that P7C3 could block the formation of these channels by inhibiting the cholesterol binding site of Aβ fibrils.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.7b00151. Snapshots of the Aβ stack interacting with a larger membrane with 256 molecules, snapshots of the Aβ stack interacting with POPC membrane, another perspective of simulated systems where acyl chains are removed for clarity, RMSD and Rg of fibrils over 400 ns, bilayer thickness map, and number of hydrogen bonds between different components (PDF)

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REFERENCES

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5. CONCLUSIONS In the present study, we described a new mechanism of action of P7C3 small molecules which have been recently reported to protect developing neurons in a mouse model of hippocampal neurogenesis. To this aim we investigate two distinct but converging fields of study. On one hand, the effect of Aβ42 fibrils on the membrane was evaluated by insertion of the Aβ42 fibrils in the center of the DPPC bilayer. The density profiles indicate that the membrane sustains substantial structural changes when Aβ42 peptides are embedded. The hydrophobic mismatch between fibrils and peptides leads to the tilting of the Aβ42 stack and formation of ion channel-like structures. The overall trends in the order parameters were considerably altered in the presence of the Aβ42 stack. On the other hand, when P7C3 is dissolved in the aqueous phase, the precipitation of toxic Aβ42 stacks into the membrane is inhibited. An important result from our simulations is the self-assembly of P7C3 molecules at the water−membrane interface and prevention of Aβ42 precipitation into the membrane by inhibiting the cholesterol binding site of the peptide. The crucial role of P7C3 is to block amyloid channel formation.

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Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: +98 713 613 7157. ORCID

Amin Reza Zolghadr: 0000-0002-6289-3794 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are indebted to the research council of the Shiraz University for the financial supports. We would like to thank Prof. Kenneth S. Suslick for helpful discussions. 2017


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