Inhibition Mechanisms of a Pyridazine-Based Amyloid Inhibitor: As a

Article pubs.acs.org/JPCB

Inhibition Mechanisms of a Pyridazine-Based Amyloid Inhibitor: As a β‑Sheet Destabilizer and a Helix Bridge Maker Hamid R. Kalhor* and M. Parsa Jabbari Biochemistry Research Laboratory, Department of Chemistry, Sharif University of Technology, PO Box: 11365-11155, Tehran, Iran S Supporting Information *

ABSTRACT: Conformational diseases have been investigated extensively in recent years; as a result, a number of drug candidates have been introduced as amyloid inhibitors; however, no effective therapies have been put forward. RS-0406 with pyridazine as its core chemical structure has so far shown promising results in inhibiting amyloid formation. In the present work, using molecular dynamics, we undertook the investigation of RS-0406 interactions with U-shaped Aβ1−42 and Aβ1−40 pentamers, Aβ1−42 monomers, and double-horseshoe-like Aβ1−42. To set better parameters for the small molecule, experimental and computational log P values were obtained. In addition, an analogue of RS-0406 was also simulated for comparison. The results indicate that RS-0406 may inhibit amyloid formation exploiting two different mechanisms. One mechanism includes stabilizing the α helix, in the monomer peptide, by binding to the flanking sites of the amyloidogenic region. The second mechanism mediates through interaction of the small molecules near the amyloidogenic regions, leading to destabilization of the β-sheets in both the U-shaped and the S-shaped fibril. Notably, a persistent interaction between the imidazole ring of His14 from an S-shaped structure and the pyridazine ring of RS-0406 was observed. The unique structural properties of RS-0406, including aromaticity, H-bonding capability, flexibility, and symmetry, allow the small molecule to noticeably affect amyloid formation.

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sizes and turn into insoluble fibrils in the third stage of amyloid formation.11,12 Indeed, a number of debilitating human diseases, including Alzheimer’s and Parkinson’s diseases, are known as amyloidrelated diseases in which one peptide or protein tends to form insoluble amyloid, leading to severe pathological effects.13,14 In the pathology of Alzheimer’s disease,13 amyloid precursor protein, a neuronal membrane integral protein, is erroneously cleaved by β-secretase and the presenilin−γ-secretase complex protein, generating Aβ peptides. These peptides have been shown to be assembled into soluble oligomers and bring about damage to the synaptic function.13 Although Aβ1−42 and Aβ1−40 fragments have been shown to be the most amyloidogenic fragments,15 Aβ1−42 has displayed much faster oligomerization and more neurotoxicity.16,17 The Aβ peptide contains at least two amyloidogenic regions, including hydrophobic-rich sequences (K16LVFFA) and the second region encompassing residues 25−35, which has been shown to be highly amyloidogenic and has displayed an elevated neurotoxicity.18 Over the last few years, a number of Aβ amyloid structures have been resolved by NMR.19−21 The three-dimensional (3D) structure of fibrils of Aβ1−42 with U-shaped pentameric structures had been the main structural model for explaining

INTRODUCTION

The most fundamental self-assembly process that a functioning cell must carry on in all given situations pertains to protein folding.1,2 Protein folding not only happens spontaneously but also gives each protein its unique structure and ultimately its function. To safeguard such a delicate and complex process as protein folding, cells have evolved a number of ways to ascertain proper folding of their proteome. For instance, genetically encoded chaperons have been used by cells for proper folding of their proteins in adverse conditions, such as elevated temperature.3,4 In addition, small organic osmolytes also have been shown as effective factors in influencing protein folding in various organisms.5,6 In spite of all of the aforementioned mechanisms, proteins may still become partially unfolded and subsequently undergo conformational changes, leading to misfolded products.7 The major pathway of protein misfolding is better known as amyloid formation, leading to products which are insoluble fibrils.7 Over the last few years, a number of studies have shed lights on the molecular mechanism of amyloid formation.8−10 The kinetic studies have revealed that a protein/peptide destined for amyloid formation undergoes three distinct phases; in the first phase, called lag phase, the peptide/protein monomer is assembled into a nucleus upon which the larger oligomer fragments are formed. In the second phase, soluble oligomers are exponentially created; these oligomers are heterogeneous in © XXXX American Chemical Society

Received: May 29, 2017 Revised: July 19, 2017

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DOI: 10.1021/acs.jpcb.7b05189 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B

Figure 1. Structures of peptides and small molecules used in this study. (A) Initial structures of the U-shaped Aβ17−42 pentamer model (PDB ID: 2BEG), Aβ1−42 monomer model (PDB ID:1Z0Q), and Aβ1−42 double-horseshoe model (PDB ID: 2NAO). In the U-shaped structure, chains A−E are colored as blue, red, gray, orange, and yellow, respectively. In the double-horseshoe structure, chains A−F are colored as blue, red, gray, orange, yellow, and tan, respectively. Images were generated by visual molecular dynamics (VMD). (B) Molecular structures of RS-0406 (top) and the analogue lacking hydroxyl groups (bottom).

simulations, including atomic-scale movement calculations, total energy calculation (Hamiltonian), and underscoring classical statistics. RS-0406, a small organic molecule, which was identified through combinatorial screening, has shown promising results in inhibiting amyloid formation both in vitro and in animal studies,34,35 and currently, the small molecule is under investigation in phase II clinical trial.36 However, to the best of our knowledge, there has not been any report on the details of molecular interactions of RS-0406 delineating its mechanism of amyloid inhibition. As shown in Figure 1B, RS-0406 contains three aromatic rings with pyridazine as its central ring. Although the molecule is flat, it possesses a number of single bonds upon which the aromatic rings can rotate and the entire molecule may possess a bent geometry. Pyridazine with its unique chemical and electronic structures has found its way in a number of medicinal drugs.37 Therefore, RS-0406, having a symmetrical and flexible structure, may present additional unique properties that could be crucial in interacting with an amyloid structure.

the biological activity of Aβ. In the U-shaped structure (Figure 1A, upper panel), residues 1−17 were found to be disordered and residues 18−42 formed two β-sheets, β1 and β2.19 A more recent 3D structure of Aβ1−42, forming a double-horseshoe-like (or an inverted letter S), was elucidated by solid-state NMR. This double-horseshoe structure was composed of three layers of dimeric S-shaped strands with two-fold symmetry (Figure 1A, the lowest panel); this structure has been suggested to be a more disease-relevant amyloid fibril.21,22 Both natural and synthetic products have been shown as effective approaches to reduce amyloid formation.23−28 Besides providing the therapeutic value that natural and synthetic compounds may have in future, these types of studies have helped in obtaining valuable information on the molecular mechanism of amyloid formation.23,24 However, because of the complexity of amyloid formation, it has been difficult to investigate the details of the misfolding process. Molecular dynamics (MD) simulation has played a pivotal role in studying such complex processes as amyloid formation.29−33 There exist a number of advantages in studying macromolecules using MD B


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neutral. To be certain about validation of the topology file, free energy change of transferring RS-0406 in both water and 1octanol solvents from a fully interactive to a noninteractive state was calculated (ΔG1 and ΔG2, respectively). According to eq 1, the difference between the mentioned free energy changes is equal to free energy change of transferring the molecule from water to 1-octanol,40 which is related to the corresponding log P value, as shown in eq 2.

The goals of the present work pertain to the mechanisms through which small molecule RS-0406 can affect amyloid formation. Molecular dynamics was used to investigate the details of the interaction of the small molecule and different forms of amyloid β structures. Our immediate goal has not been focused on determining equilibrated structures in the simulations but rather to investigate the interactions of the small molecule with different forms of Aβ structures throughout the simulations. To better parameterize the small molecule, log P (lipophilicity index) was calculated for RS-0406. To make a comprehensive study, the interaction of the small molecule was investigated with the Aβ monomer, U-shaped pentameric oligomer, and double-horseshoe-like (S-shape) structure, summing of more than 2 μs simulations. The simulation results revealed two major mechanisms that may be exploited by RS-0406 in the inhibition of amyloid fibrillation: these include mediation through stabilizing the Aβ α helix and the other is used to destabilize the β-sheet structure either by backbone interaction or residue-specific binding (in S-shape structure). The interaction of an analogue of RS-0406, lacking phenolic hydroxyl groups, with amyloid was also investigated. The results underscore the importance of phenolic hydroxyl in addition to symmetry, aromaticity, and flexibility of pyridazinederived compounds in affecting amyloid formation. Because our assessments of RS-0406 interactions with different forms of Aβ are mostly based on local and entire secondary structural variations, we performed our simulations using OPLSaa force field. In addition, a recent work on Aβ1−42 dimers has revealed that OPLSaa can be considered a reliable force field for secondary structural contents.38

ΔtrsG = ΔG1 − ΔG2

(1)

ΔtrsG = −RT ln(P) = −2.303RT log P

(2)

where P is the equilibrium constant for transferring the molecule from water to 1-octanol. To prepare a system containing RS-0406 and water, the molecule was centered in a cubic box with faces at least 1.0 nm apart from the centered molecule and solvated with SPC water. For RS-0406 in an octanol system, a cubic box with faces at least 2.0 nm apart from the centered molecule was prepared and filled with equilibrated 1-octanol molecules; because the octanol phase is a mixture of 1-octanol and water with 0.26 mole fraction of water,41 water molecules were added to obtain this mixture. Several simulations have been done with different coupling parameters, λ, ranging from 0 (i.e., fully interactive state) to 1 (i.e., noninteractive state) in each system, and the free energy change was calculated from integration of the system Hamiltonian obtained in each λ value.40 Eleven λ values for both Coulombic and van der Waals interactions (0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.0, 1.0, 1.0, 1.0, and 1.0 for Coulombic and 0.0, 0.0, 0.0, 0.0, 0.0, 0.0, 0.2, 0.4, 0.6, 0.8, and 1.0 for van der Waals interactions) were settled. In each simulation, the whole system was energy-minimized using a steep method and further minimized using a l-bfgs method, equilibrated for 100 ps in an NVT (canonical) ensemble and for 100 ps in an NPT (isothermal isobaric) ensemble and simulated for 1.0 ns in an NPT ensemble. All of the equilibration and MD simulations ware conducted using a Langevin dynamics integrator with an integration step of 2 fs. Temperature coupling at 300 K handled by the Langevin dynamics integrator and pressure coupling at 1.0 bar in NPT ensembles were carried out by a Parrinello− Rahman integrator. Neighbor searching and short-range nonbonded interactions were applied with a verlet cutoff scheme and updated every 20 steps (40 fs). The particle mesh Ewald (PME) method was used for electrostatic interactions with a cutoff value of 1.2 nm, and van der Waals forces were switched to zero between rvdw-switch = 1.0 and 1.2 nm by potential-switch. Simulation of Different Forms of Aβ. In this study, we assembled the following systems using GROMACS 5.2 modules: (1) Aβ17−42 pentamer in water (control system), (2) Aβ17−42 pentamer in the presence of four randomly positioned RS-0406 molecules, (3) Aβ17−42 pentamer docked with one molecule of RS-0406, (4) Aβ1−42 monomer in water (control system), (5) Aβ1−42 monomer in the presence of four randomly positioned RS-0406 molecules, (6) Aβ17−40 pentamer in water (control system), (7) Aβ17−40 pentamer in the presence of four randomly positioned RS-0406 molecules, (8) Aβ17−42 pentamer in the presence of four randomly positioned RS-0406 analogues lacking hydroxyl moieties, (9) doublehorseshoe Aβ1−42 in water (control), (10) double-horseshoe Aβ1−42 in the presence of four randomly positioned RS-0406 molecules, and (11) double-horseshoe Aβ1−42 docked with one

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MATERIALS AND METHODS Chemicals. RS-0406 was a kind gift provided by A. Nazari, at Sharif University of Technology. 1-Octanol and Tris base were purchased from Merck Co., Germany. Experimental Measurement of log P Value. Measurements of log P were performed using the shake-flask method.39 Briefly, 100 mL of RS-0406 solution in water (39 mg/100 mL), 25 mL of RS-0406 solution in 1-octanol (83 mg/L), and 0.5 mol/L solution of Tris buffer at pH 8.25 were prepared as stock solutions. The stock solution in water and the buffer solution were mixed to obtain standard solutions for absorption calibration curves. The absorptions of the wavelength range between 200 and 400 nm were scanned, and calibration curves were plotted at 212 and 304 nm, which are absorption peaks of the resulting spectra. The 304 nm wavelength was chosen due to its better linear response to variation in concentration. Stock solutions of RS-0406 in water and 1-octanol were diluted to total volumes of 5.00 and 1.00 mL, respectively. The resulting octanol and water phases were mixed and shook overnight, and the absorption of the aqueous aliquot at 304 nm was measured using an ultraviole/visible spectrophotometer (PerkinElmer Lambda 25). Simulation of Small Molecules with Proper Parameters. The GROMCS 5.2 package was chosen for the simulation. The RS-0406 structure file (pdb file) was obtained using the PRODRG 2.x online server, and the topology file was prepared manually, in which OPLSaa force field was chosen. Atomic OPLSaa parameters of the molecules similar to RS0406 fragments (e.g., ammonia, pyridazine, and phenol) were assigned to the corresponding atoms in the molecule. Partial charges of the atoms contributing in bonds between the mentioned fragments were adjusted to make the molecule C


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The Journal of Physical Chemistry B molecule of RS-0406. PDB files of the U-shaped pentamer (PDB ID: 2BEG), double-horseshoe structure (PDB ID: 2NAO), and the monomer peptide (PDB ID: 1ZOQ) were obtained from the RSCB server,42 and the first model was chosen in each case. Each system was energy-minimized, equilibrated, and simulated, as described in detail in the following paragraph. MD Simulation Details. All of the simulations have been done under OPLSaa force field. In each protein, hydrogen atoms were added and titrable residues were protonated corresponding to physiological pH. Each protein was centered in a cubic box with faces at least 1.0 nm apart from the closest atom of the protein. For systems 2, 5,7, and 10, four molecules of RS-0406 were added to the box; for system 8, four randomly positioned RS-0406 analogues were added; and for systems 3 and 11, the oligomers were docked with one molecule of RS0406 using Auto Dock Tools 1.5.6 software and no further RS0406 molecules were added. The resulting systems were solvated by SPC216 water and neutralized by adding sufficient number of ions. Each system was energy-minimized by the steepest descent integrator with emtol value of 100.0 kJ/mol nm. After minimization, 100 ps equilibration under an NVT ensemble was carried out by the leap-frog integrator and the temperature was set at 300 K. Furthermore, 100 ps equilibration under an NPT ensemble has been done using the leap-frog integrator, in which the temperature and pressure were 300 K and 1.0 bar, respectively. In each equilibration step, position restraints on protein atoms were applied. In the next step, the systems were simulated under an NPT ensemble with no position restraints and the simulation time was 400 ns for the systems containing U-shaped Aβ17−42 and 100 ns for those containing monomer peptides, U-shaped Aβ17−40, and doublehorseshoe structures. In all equilibration and MD simulation steps, the integration time was set to be 2 fs; for equilibrations, velocities, coordinates, and energies were saved every 1.0 ps, and for MD simulation, energies were saved every 10.0 ps. All bonds were constrained using the Lincs algorithm. Short-range electrostatic and van der Waals interactions were updated every 20 fs (10 × 2 fs) with a verlet cutoff scheme, and the cutoff value was 1.0 nm for equilibrations and 1.4 nm for MD simulation. Long-range electrostatic interactions were calculated using the PME method with the Fourier spacing value of 0.16. Temperature couplings have been performed by the Vrescale method with a 0.1 ns time constant, and pressure couplings have been performed by the Parrinello−Rahman method with a 2 ps time constant. Simulations of systems 2 and 5 were repeated at least twice.

explanation). The value computed for log P turned to be 2.38, which closely agreed with the experimental value. RS-0406 Acting as a β Breaker, Destabilizing the βSheet Structure. To investigate the interaction of RS-0406 with U-shaped Aβ1−42 pentamers, the simulation was carried out for up to 400 ns. However, after 200 ns simulations, no significant changes were observed. In the presence of the small molecule, the change in the root-mean-square deviation (RMSD) value of chain A was significant compared to that of the other chains (Figure 2A,B). In addition, in the presence of RS-0406, RMSD values for chains A and E were significantly increased during the simulation compared to those for the control (Figure S1). To compare a less amyloidogenic structure, U-shaped Aβ40 pentamer interactions with the small molecule were investigated; the simulation was carried out for up to 100 ns; the RMSD variation for Aβ40 was not as significant as that for Aβ42 (Figure S2). The secondary structural analysis of the amyloid pentamers in the presence and absence of RS-0406 illustrated that during the simulation the β-sheet content of the pentamer was markedly reduced (Figure 2C). In fact, examining the time interval of 75−120 ns, it was observed that the number of βsheet residues was significantly reduced in the presence of RS0406. Remarkably, the number of H-bonds of the main chains in β-sheet strands during the simulation was substantially reduced in the presence of small molecules (Figure S3). As shown in Figure 3, a snapshot at 175 ns, RS-0406 molecules were found to insert themselves between different peptide chains, destabilizing interstrand interactions of β-sheets. The RS-0406 interactions include side-chain hydrophobic packing and backbone hydrogen bonding (Figure 3B,C). Indeed, the interaction sites of all RS-0406 molecules were mapped near one of the amyloidogenic regions of the Aβ peptide (residues 25−35); remarkably, both hydroxyl groups of one of the RS0406 molecules formed H-bonds with backbones of Val36 from chain B and Val36 from chain D, respectively (Figure 3C); additionally, the phenolic ring of the small molecule was in hydrophobic contact with the side chain of Val39 from chain E. Another small molecule was found to form a hydrophobic interaction with Ile41 from chain A and Ile31 from chain E (Figure 3D). Interactions of other small molecules are shown in Figure S4A,B. Consequently, these interactions may lead to the disruption of β-sheet interstrand hydrogen bonds and prevent these strands from further aggregation. Additionally, the importance of symmetrical interactions of small molecules is underscored. All of the secondary structural changes allude to the point that RS-0406 destabilizes the peptide backbone interactions and replaces them with its own H-bond interactions. To better understand how the structural moieties of the small molecule are able to affect the oligomeric structure, the number of H-bonds between the pentameric peptide and the specific moiety of RS-0406 was investigated. The number of Hbonds of phenolic hydroxyl group with the U-shaped structure reached at the maximum value of seven (Figure S5A). The number of H-bonds of the amine moiety with the peptide reached a maximum of four during the simulation (Figure S5B). The nitrogen atoms of pyridazine reached a maximum number of six H-bonding interactions with the peptide (Figure S5C). A comparison of the patterns of H-bonding of different moieties of the small molecules suggested that the phenolic moiety displayed a higher number of H-bonds during simulation.

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RESULTS Assignment of Parameters for Small Molecules. Our initial approach was to identify proper OPLSaa parameters for the small molecule. One of the reliable methods to validate the parameters for small molecules has been determining the partitioning coefficient between octanol and water, known as log P.40 To better obtain log P for small molecules studied in this work, we performed the shake-flask method, as described in detail in Materials and Methods.39 The experimental log P value for RS-0406 was measured to be 2.30. To calculate log P computationally for the small molecule, the free energy of transfer of small molecules from a fully interactive state to a fully noninteractive state in both water and 1-octanol solvents was computed (see Materials and Methods for more D


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H-bonds with the N-terminus of chain C. These interactions most likely disrupt interstrand backbone H-bonds (Figure 4B). In addition, a number of aromatic and aliphatic amino acids were close enough for hydrophobic interactions (Figure 4C). Overall, a single molecule of the small molecule was capable of deforming the pentameric structure globally (Figure 4A). RS-0406 Acting as an Inhibitor through CrossBridging the Helices in the Aβ Monomer. To investigate how RS-0406 can inhibit the Aβ monomer to form amyloid aggregates, the monomeric Aβ1−42, as shown in its initial structure in Figure 1A (middle panel), was simulated in the presence and absence of RS-0406. The purpose of this simulation is to obtain the effect of the small molecule on the equilibrated structure in an aqueous environment. As shown in Figure 5A, RMSD for monomeric peptides was much less increased in the presence of the small molecule. Because the monomeric form is α helical, the number of H-bonds between residues n and n + 4 was measured. In the presence of small molecules, the number of H-bonds (n−(n + 4)) was less decreased during the simulation in the presence of RS-0406 (Figure 5A). A snapshot of the final stage of simulation suggested a possible mechanism for stabilizing the amyloidogenic region (KLVFFA) of the Aβ peptide (Figure 5B). The small molecule (RS-0406) may stabilize the Aβ monomer in two ways: one is H-bonding with backbone residues Asp1 and Glu22, flanking the amyloidogenic region (Figure 5C). The second approach that RS-0406 could stabilize the helical conformation of the amyloidogenic region is mediated through hydrophobic contacts between Leu17 and Phe20 with RS-0406 aromatic rings (Figure 5D). Indeed, Leu17 seemed to be caged in by all three aromatic rings of RS-0406. When the number of hydrogen bonds of the fragment containing the amyloidogenic region (KLVFFAEDVGS) of the Aβ peptide was calculated during the simulation, it showed a decrease in the absence of small molecule significantly, whereas the number of H-bonds stayed constant in the presence of RS-0406 (Figure S6). This result emphasizes the role of the small molecule in interaction with the Aβ backbone, in preventing possible conformational change of the amyloidogenic region through partial unfolding, leading to a coil or a β strand. RS-0406 Analogue Lacking Hydroxyl Moieties, Revealing the Importance of Hydroxyl Groups. To investigate the role of hydroxyl groups of the small molecule, its analogue lacking the hydroxyl groups was investigated (Figure 1B, the lower panel); the corresponding force field parameters were adjusted starting from RS-0406 parameters, so that a calculated log P value was obtained. When the analogue was simulated with the Aβ pentamer, the result showed imperceptible changes as compared with the control (in the absence of any small molecule); that is, the RMSD value in the presence of the analogue and in the control for chains A and E became very similar, whereas RMSD in the presence of RS0406 displayed a much higher value (Figure 6A). To probe into the stabilization of β-sheets of the Aβ pentamer in the presence of the analogue, interstrand hydrogen bonds of the pentamer were calculated (Figure 6B); the result demonstrated any significant changes relative to the control. A snapshot at the final stage of the simulation (100 ns) did not show much conformational change of the pentamer (Figure 6C). Interactions of RS-0406 with the Double-Horseshoe Structure. To better explore the effects of the small molecule on the amyloid structure, a more disease-relevant amyloid fibril was exploited. The simulation of RS-0406 with the dimeric

Figure 2. RMSD and secondary structure analysis of the U-shaped Aβ17−42 pentamer in the absence of RS-0406 and in the presence of four randomly positioned RS-0406 molecules. (A) Chains A−E: Cα RMSD values of the U-shaped structure in the presence of RS-0406 molecules. (B) Chains A−E: Cα RMSD values of the U-shaped structure in the absence of RS-0406 molecules. (C) Comparison of number of residues with β-sheet conformation in the presence and absence of small molecules.

To better assess the interactions of RS-0406, molecular docking simulation was used to insert the small molecule into the Aβ pentameric peptide. A single molecule of RS-0406, which was trapped inside the peptide, displayed multiple interactions at the final stage of the simulation with strands B− E of the pentameric peptide (Figure 4A). These interactions include H-bonding with the peptide backbone and hydrophobic interactions; the H-bonding interactions with the phenolic and pyridazine rings of RS-0406 include the backbones of two Leu17 from chains C and E, and also, pyridazine nitrogen forms E


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Figure 3. Snapshot at 175 ns of simulation for the U-shaped Aβ pentamer with four randomly positioned small molecules. (A) U-shaped Aβ pentamer and RS-0406. The strands are colored in the same manner as in Figure 1A. Four RS-0406 molecules numbered as 1−4 are colored gray, light gray, green, and dark green, respectively. The image is generated by VMD. (B) First RS-0406 molecule, forming H-bonds with the backbones of Val36 in chains B and D; generated by PyMol. (C) Second view of first RS-0406, revealing insertion of the small molecule among chains B, D, and E; generated by PyMol. (D) Hydrophobic interactions of second RS-0406 with side chains of Ile41 from chain A and Ile31 from chain B; generated by PyMol.

double-horseshoe (similar to letter S) Aβ1−42 was performed in the presence of four small molecules that were randomly positioned (Figure 7A,B). The Cα RMSD of the six chains revealed moderate changes with respect to those of control. The RMSD change for chains A−C is more pronounced than that of the other three chains (Figure 7A). When the secondary structural analysis was performed for double-horseshoe during the simulation, no significant differences in the entire structure were revealed in the presence of the small molecule (Figure S7A). To look for more regional secondary structural changes, the interstrand H-bonds and the number of β-sheet residues for the major amyloidogenic region of Aβ (K16LVFFA) were analyzed. The results demonstrated a lack of significant changes in the amyloidogenic region of Aβ as compared to those in the control (Figure S7B,C). However, the trajectory of the simulation revealed that two of the small molecules were

bound to the double-horseshoe structure form 20 ns to the end of the simulation. The interactions of the small molecules were mapped near N-terminus residues 1−7 of chains D and E (Figure 7B); the types of interactions that were observed include hydrophobic packing and H-bonding with the backbone (Figure S7D). An alternative approach was to dock a single molecule of RS0406 into the double-horseshoe structure. Notably, the docked molecule was mapped near the major amyloidogenic region of chains A−C. Meaningful secondary structure changes in some local regions were revealed due to the interaction of the docked molecule (Figure 7C). Particularly, regions 29−34 in second amyloidogenic regions of chains A−C showed significant changes; that is, the numbers of β-sheet residues and interstrand H-bonds were smaller with respect to those in the control (Figure 7C). One notable interaction between the F


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Figure 4. Snapshot at the final stage of simulation of the U-shaped Aβ17−42 pentamer docked with one molecule of RS-0406, presenting hydrophobic interactions with the side chains of residues and backbone H-bonding. (A) Structures of the U-shaped structure and the small molecule; generated by VMD. (B) H-bond formation with backbones of Leu17 from chains C and E and N-terminus from chain C with the pyridazine moiety of RS0406. In addition, separation of Leu17−Val18 peptide bonds of chains C−E is shown; generated by PyMol. (C) Hydrophobic packing of the RS0406 phenolic moiety with aromatic and aliphatic side chains of chains B−D; generated by PyMol.

hydroxyl moieties was utilized as a comparison. To fully understand the mechanism of action of RS-0406, a number of simulations with different forms of Aβ, including the Aβ42 monomer, U-shaped Aβ42 pentamer, U-shaped Aβ40 pentamer, and double-horseshoe structure, were performed. The results illustrate that RS-0406 may inhibit amyloid fibrillation in both the oligomerization phase and monomeric state. Indeed, it was shown that RS-0406 may interfere with the α-to-β conformational transition of the Aβ1−42 monomer in aqueous solution. As previously, it has been shown that this conversion was reversible and the α conformation (in membrane environment) would fully shift to a β-sheet structure

docked molecule and the double-horseshoe structure is the noncovalent interaction of His14 of chain C with the pyridazine region of RS-0406 (Figure 7D). Measurement of the distances between the imidazole ring of His and the pyridazine ring demonstrated that this noncovalent interaction persists during the simulation (Figure S8).

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DISCUSSION In the present work, to better parameterize RS-0406 in silico, log P of the compound was experimentally measured, and it agreed well with the computationally calculated log P values. Furthermore, the analogue of RS-0406 lacking external G


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Figure 5. Analysis of the Aβ1−42 monomer in presence and absence of RS-0406. (A) Protein Cα RMSD values (left) and number of backbone n−(n + 4) H-bonds (right) during 100 ns simulation vs time. (B) Entire structure of the monomer and three bound RS-0406 molecules; first amyloidogenic region (KLVFFA) is shown in blue, generated by VMD. (C) H-bond formation with backbones of Asp1 and Glu22, generated by PyMol. (D) Hydrophobic interactions with Leu17 and Phe20 side chains in the amyloidogenic region, generated by PyMol.

in aqueous environment.43 How could a small organic molecule destabilize a β-sheet structure (in the oligomerization state) and also stabilize an α-helical structure in the Aβ monomer? RS0406 interacts with β-sheet backbones of the pentamer through H-bonds (Figure 3); these interactions could possibly compete with the β-sheet interstrand H-bonds existing in the U-shaped

and S-shaped structures. As a result, this competition may prevent β-sheet stacking and consequently further oligomerization would be hampered. Remarkably, when the small molecule was docked onto the U-shaped (pentameric) structure (Figure 4), similar hydrogen bond interactions between the small molecule and backbone were formed. Notably, hydrophobic H


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Figure 6. Analysis of the U-shaped Aβ pentamer in presence of the RS-0406 analogue lacking hydroxyl groups. (A) Cα RMSD values in the presence of the analogue (black line), in the presence of RS-0406 (red line), and in the control (green line). The graph on the right side corresponds to chain A, and the left graph corresponds to chain E. (B) Number of interstrand hydrogen bonds between the main chains of adjacent strands in the presence of the analogue (black line), in the presence of RS-0406 (red line), and in the control (green line). (C) Snapshot at 100 ns of simulation, revealing the relative stability of the β-sheet structure in the presence of the analogue, generated by VMD. I


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Figure 7. Structural analysis of the Aβ1−42 double-horseshoe model in the presence and absence of RS-0406. (A) Cα RMSD values of chains A−F. The graph on the right corresponds to the Aβ oligomer in the presence of four RS-0406 molecules, and the left graph corresponds to the control system. (B) Snapshot at 40 ns of the Aβ oligomer simulation with four small molecules. Chains are colored in the same manner as in Figure 1A. The small molecules are bound to N-terminal residues 1−7 from chains D−F, generated by VMD. (C) Secondary structural analysis of residues 29−34, from chains A−C in double-horseshoe Aβ when docked with a single small molecule. The right graph monitors the number of interstrand H-bonds in residues 29−34 from chains A to C, whereas the left graph shows the number of β-sheet residues in this region. (D) Snapshot at 53 ns of J


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The Journal of Physical Chemistry B Figure 7. continued

simulation of the docked system. Representing the entire structure of the double-horseshoe structure docked with RS-0406 (left), and noncovalent interaction of RS-0406 with His14 from chain C (right). This interaction was persistent during the simulation. The left image was generated by VMD, and the right image was generated by PyMol.

The importance of RS-0406 as an inhibitor mainly relies on its unique structural elements. These elements include the pyridazine ring, aromatic moieties, hydroxyl and amino groups, and four single bonds, causing molecular flexibility. The pyridazine moiety has been shown to easily form H-bonds with proteins, as well as reducing log P for the molecule.37 The importance of aromatic moieties and hydrogen bonding has been previously demonstrated in inhibition of amyloids, such as polyphenol compounds.24 The molecular flexibility renders the molecule capable of probing an optimal interaction, which is lacking in most previous novel inhibitors. To better investigate the role of RS-0406 hydroxyl groups, the pentamer structure was simulated with an RS-0406 analogue (lacking hydroxyl moieties). The results showed the importance of hydroxyl groups (Figure 6). The hydroxyl group involvement in amyloid inhibitions has been previously reported using other molecules such as polyphenols.24 To attain a more comprehensive insight into the mechanism of amyloid inhibition, various amyloid inhibitors that were investigated previously using molecular dynamics are compared (Table S1). Previously, the inhibitory effects of these molecules were investigated experimentally.49−53 Although these small molecules share similar mechanisms for inhibition of amyloids, RS-0406 may seem to be a more promising drug candidate for amyloid inhibition due to not only its flexibility and symmetry, which lead the molecule to insert itself into the Aβ oligomeric structure and cross-bridge into the Aβ monomer, but also its small size and its log P value (2.3), making it easier to the pass blood−brain barrier. To better explore the effects of RS-0406 on large oligomeric structures, such as a double-horseshoe structure, one of our ongoing projects is examining the role of both singly docked and a few randomly positioned small molecules (in a single simulation) with the Aβ structure. Our future goals are to take advantage of replica-exchange molecular dynamics simulations of RS-0406 and different forms of Aβ; such approaches have been applied to a number of inhibitors to get a better mechanistic insight into Aβ inhibition.54,55

interactions between aromatic moieties of the ligand and amyloidogenic side chains of the Aβ peptide were abundant. These interactions emphasize the importance of aromatic moieties in amyloid inhibition, as revealed previously.24 In exploring the mechanism of Aβ monomer inhibition, interactions of RS-0406 with the peptide account for how this ligand may prevent the amyloidogenic helical structure from the partial unfolding process leading to oligomerization (Figure 5). Because the RS-0406 molecule is symmetrical and relatively flexible, as shown in Figure 5C,D, three molecules of RS-0406 cooperatively interact with both sides of the helical segment encompassing the amyloidogenic region. These interactions included backbone hydrogen bonding between one small molecule and Asp1 of the peptide and between the other small molecule and Leu22. Furthermore, the third small molecules interact with amyloidogenic side chains (KLVFFA), which have remained in an α-helical structure. Notably, the three small molecules were in contact with each other through hydrophobic/π−π interactions. All of these interactions may suggest a unique mechanism of stabilizing the amyloidogenic helical structure during the Aβ monomer partial unfolding steps. The interactions of randomly positioned RS-0406 molecules with the double-horseshoe structure did not reveal significant effects on the secondary structure of the fibril. These results might be partly due to the limited time of the simulation that was performed or due to the lack of capability of RS-0406 to inhibit the more disease-relevant structure of Aβ fibrils. Notably, the structure of double-horseshoe is considered more stable by optimally burying the hydrophobic side chains of Aβ.21 However, when a single molecule of RS-0406 was docked into the S-shape structure, meaningful changes in secondary structure were obtained (Figure 7C). The pyridazine ring of the docked molecule was found to interact persistently during the entire simulation with the imidazole ring of His14 from chain C (Figure S8). Notably, His14 of Aβ has been previously shown to be important in neurotoxicity and membrane binding of amyloid fibrils.44,45 Additionally, the interaction of the pyridazine containing small molecule with His14 may induce conformational changes in amyloidogenic regions of the protofibril structure. It must be mentioned that the U-shaped structure of Aβ lacks 1−17 residues that might not be as important for drug interactions due to their disordered structure. Additionally, in the S-shaped model, all of the residues are present although residues 1−15 were found to be partially disordered.21 However, certain studies have shed lights on the importance of position two; particularly, an A2V mutation was shown to be protective in a heterozygous form in Alzheimer disease and the same mutation in its homozygous form was shown to be promoting an early type of dementia in Alzheimer disease.46 Furthermore, recent studies have shed light on how the mutation in the heterozygous form resulted in slow forming pathways and leading to different conformational distributions by increasing intramolecular interactions.47,48 Interestingly, RS0406 in the simulation with the double-horseshoe model was found to be closely associated with residues 1−7 during the most of the trajectory.

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CONCLUSIONS In this work, we have used all-atom molecular dynamics to study in detail the interactions of RS-0406 with amyloid β monomer, U-shaped pentamer, and double-horseshoe structures. Initially, to set the correct parameters, log P for the small molecule was measured, and it agreed well with its computed value. Our results indicate that the small molecule may inhibit amyloid formation mainly through two different mechanisms. Using backbone and side-chain interactions, the small molecule interferes with the β-sheet structure, as observed in the simulation of U-shaped and S-shaped structures. One noticeable interaction in the simulation of a double-horseshoe structure, docked with a single small molecule, pertained to His14 and pyridazine ring of RS-0406. In addition, the small molecule exploits a number of noncovalent interactions with the side chains of the amyloidogenic region in the monomer. Simulation of the RS-0406 analogue lacking hydroxyl groups K


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showed markedly reduced interactions and effects in inhibition of amyloid formation.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b05189. Table S1 categorizes some amyloid inhibitors, Figures S1 and S2 demonstrate Cα RMSD values for each chain for the U-shaped Aβ42 and Aβ40 pentamers in the presence of small molecules compared to those of control systems, Figure S3 illustrates a comparison of the number of interstrand H-bonds in U-shaped Aβ42/RS-0406 with that in the control system, Figure S4 shows a snapshot of the interactions of two other RS-0406 molecules with Ushaped Aβ42 at 175 ns, Figure S5 shows the number of H-bonds formed by different moieties of RS-0406 with the U-shaped model, Figure S6 shows the number of n− (n + 4) H-bonds in the Aβ monomer plotted versus time. Figure S7 shows structural analysis and some snapshots of the simulation with the double-horseshoe structure, and Figure S8 indicates the distance between His14 from chain C of the double-horseshoe structure and the pyridazine ring of RS-0406 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Email: [email protected]. Tel:+982166165315. Fax: +982166165315. ORCID

Hamid R. Kalhor: 0000-0002-0654-0513 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We would like to thank Arash Firouzbakht for his technical assistance and the center for High Performance computing (HPC) at Sharif University of Technology for the usage of their facilities. This work was supported partially by Sharif University of Technology grant (G930614).

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Inhibition Mechanisms of a Pyridazine-Based Amyloid Inhibitor: As a

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