Conserved water mediated H-bonding dynamics of Ser117 and Thr119 residues in human transthyretin–thyroxin complexation: Inhibitor modeling study through docking and molecular dynamics simulation

Transthyretin (TTR) is a protein whose aggregation and deposition causes amyloid diseases in human beings. Amyloid fibril formation is prevented by binding of thyroxin (T4) or its analogs to TTR. The MD simulation study of several solvated X-ray structures of apo and holo TTR has indicated the role of a conserved water molecule and its interaction with T4 binding residues Ser117 and Thr119. Geometrical and electronic consequences of those interactions have been exploited to design a series of thyroxin analogs (Mod1–4) by modifying 5′ or 3′ or both the iodine atoms of thyroxin. Binding energy of the designed ligands has been calculated by docking the molecules in tetrameric structure of the protein. Theoretically investigated pharmacological parameters along with the binding energy data indicate the potentiality of 3′,5′-diacetyl-3,5-dichloro-l-thyronine (Mod4) to act as a better inhibitor for TTR-related amyloid diseases.     

Oligomerization preceding amyloid fibril formation: a process in common to intrinsically disordered and globular proteins

Neurodegenerative diseases present a big burden to society. At the molecular level many of them - if not all - show protein aggregation (as an epiphenomenon or as a cause). The knowledge on details of thermodynamics and kinetics as well as structure of the protein aggregates, especially the early and soluble oligomers, may help in designing inhibitors for early stages of such diseases. Here, a possible outlook on more general mechanism for their formation is discussed. The oligomers of amyloid forming proteins, which are present prior and during nucleation and amyloid fibril formation, are claimed to be toxic to cells. Oligomers of the globular proteins and the intrinsically disordered proteins (IDPs), form in?vitro upon partial denaturation and renaturation, respectively. Often they form if the sample is heated or freeze-thawed for a few cycles. A question is asked if this does not highlight one important property in common to globular proteins and IDPs, namely, a high energetic barrier dividing such oligomers from the monomers. This also would imply existence of two populations of states, one, the monomer - being metastable - at least under the conditions, which promote fibril formation.     

Inhibition of peptide aggregation by lipids: insights from coarse-grained molecular simulations

The amyloidogenic peptide apolipoprotein C-II(60-70) is known to exhibit lipid-dependent aggregation behaviour. While the peptide rapidly forms amyloid fibrils in solution, fibrillization is completely inhibited in the presence of lipids. In order to obtain molecular-level insights into the mechanism of lipid-dependent fibril inhibition, we have employed molecular dynamics simulations in conjunction with a coarse-grained model to study the aggregation of an amyloidogenic peptide, apoC-II(60-70), in the absence and presence of a short-chained lipid, dihexanoylphosphatidylcholine (DHPC). Simulation of a solution of initially dispersed peptides predicts the rapid formation of an elongated aggregate with an internal hydrophobic core, while charged sidechains and termini are solvent-exposed. Inter-peptide interactions between aromatic residues serve as the principal driving force for aggregation. In contrast, simulation of a mixed peptide-DHPC solution predicts markedly reduced peptide aggregation kinetics, with subsequent formation of a suspension of aggregates composed of smaller peptide oligomers partially inserted into lipid micelles. Both effects are caused by strong interactions between the aromatic residues of the peptide with the lipid hydrophobic tails. This suggests that lipid-induced aggregate inhibition is partly due to the preferential binding of peptide aromatic sidechains with lipid hydrophobic tails, reducing inter-peptide hydrophobic interactions. Furthermore, our simulations suggest that the morphology of peptide aggregates is strongly dependent on their local lipid environment, with greater contacts with lipids resulting in the formation of more elongated aggregates. Finally, we find that peptides disrupt lipid self-assembly, which has possible implications for explaining the cytotoxicity of peptide oligomers.     

Conformational changes of Aβ (1–42) monomers in different solvents

Amyloid proteins are known to be the main cause of numerous degenerative and neurodegenerative diseases. In general, amyloids are misfolded from monomers and they tend to have β-strand formations. These misfolded monomers are then transformed into oligomers, fibrils, and plaques. It is important to understand the forming mechanism of amyloids in order to prevent degenerative diseases to occur. Aβ protein is a highly noticeable protein which causes Alzheimer’s disease. It is reported that solvents affect the forming mechanism of Aβ amyloids. In this research, Aβ_1–42 was analyzed using an all-atom MD simulation with the consideration of effects induced by two disparate solvents: water and DMSO. As a result, two different conformation changes of Aβ_1–42 were exhibited in each solvent. It was found that salt-bridge of Asp23 and Lys28 in Aβ_1–42 was the key for amyloid folding based on the various analysis including hydrogen bond, electrostatic interaction energy and salt-bridge distance. Since this salt-bridge region plays a crucial role in initiating the misfolding of Aβ_1–42, this research may shed a light for studies related in amyloid folding and misfolding.     

The molecular dynamics of assembly of the ubiquitous aortic medial amyloidal medin fragment

In recent years there is an increased understanding of the molecular conformation of amyloid fibrils. However, much less is known about the early events that lead to the formation of these medically important assemblies. The clarification of these very important mechanistic details on the process may indicate directions towards the inhibition of the early stages of the assembly, where harmful species are most likely to form. Here, we study the dynamics of assembly of short amyloidogenic peptide fragments from the medin polypeptide. This polypeptide is of unique interest since amyloid deposits composed of medin are found almost in all the population above the age of 50. Twelve independent 50 ns long molecular dynamics simulations in explicit water have been run on peptide NH₂–NFGSVQFV–COOH, the minimal recognition hexapeptide element, NH₂–NFGSVQ–COOH, and several single-point mutants. In all cases a three-stranded polymeric β-sheet was used as the basic unit from which fibrils can be formed. Our results clearly indicate the need of well-defined sequence and stereochemical constraints to allow the formation of stable well-ordered aggregates. One of the key findings is the need for the presence of a phenylalanine residue, but not other hydrophobic amino acids, in specific positions within the peptide. Taken together, the results are consistent with recent high-resolution structures of amyloid assemblies and provide unique insights into the dynamics of these structures.     

Protocol for fast screening of multi-target drug candidates: Application to Alzheimer’s disease

The treatment of many diseases may require drugs that are capable to attack multiple targets simultaneously. Obviously, the virtual screening of multi-target drug candidates is much more time consuming compared to the single-target case. This, in particular, concerns the last step of virtual screening where the binding free energy is computed by conventional molecular dynamics simulation. To overcome this difficulty we propose a simple protocol which is relied on the fast steered molecular dynamics simulation and on available experimental data on binding affinity of reference ligand to a given target. Namely, first we compute non-equilibrium works generated during pulling ligands from the binding site using the steered molecular dynamics method. Then as top leads we choose only those compounds that have the non-equilibrium work larger than that of a reference compound for which the binding free energy has been already known from experiment.Despite many efforts no cures for AD (Alzheimer’s disease) have been found. One of possible reasons for this failure is that drug candidates were developed for a single target, while there are exist many possible pathways to AD. Applying our new protocol to five targets including amyloid beta fibril, peroxisome proliferator-activated receptor γ, retinoic X receptor α, β- and γ-secretases, we have found two potential drugs (CID 16040294 and CID 9998128) for AD from the large PubChem database. We have also shown that these two ligands can interfere with the activity of popular Acetylcholinesterase target through strong binding towards it.     

Identification of potential bivalent inhibitors from natural compounds for acetylcholinesterase through in silico screening using multiple pharmacophores

The symptomatic cure observed in the treatment of Alzheimer's disease (AD) by FDA approved drugs could possibly be due to their specificity against the active site of acetylcholinesterase (AChE) and not by targeting its pathogenicity. The AD pathogenicity involved in AChE protein is mainly due to amyloid beta peptide aggregation, which is triggered specifically by peripheral anionic site (PAS) of AChE. In the present study, a workflow has been developed for the identification and prioritization of potential compounds that could interact not only with the catalytic site but also with the PAS of AChE. To elucidate the essential structural elements of such inhibitors, pharmacophore models were constructed using PHASE, based on a set of fifteen best known AChE inhibitors. All these models on validation were further restricted to the best seven. These were transferred to PHASE database screening platform for screening 89,425 molecules deposited at the “ZINC natural product database”. Novel lead molecules retrieved were subsequently subjected to molecular docking and ADME profiling. A set of 12 compounds were identified with high pharmacophore fit values and good predicted biological activity scores. These compounds not only showed higher affinity for catalytic residues, but also for Trp86 and Trp286, which are important, at PAS of AChE. The knowledge gained from this study, could lead to the discovery of potential AChE inhibitors that are highly specific for AD treatment as they are bivalent lead molecules endowed with dual binding ability for both catalytic site and PAS of AChE.     

Effect of D23N mutation on the dimer conformation of amyloid β-proteins: Ab initio molecular simulations in water

The molecular pathogenesis of Alzheimer's disease (AD) is deeply involved in aggregations of amyloid β-proteins (Aβ) in a diseased brain. The recent experimental studies indicated that the mutation of Asp23 by Asn (D23N) within the coding sequence of Aβ increases the risk for the pathogeny of cerebral amyloid angiopathy and early-onset familial ADs. Fibrils of the D23N mutated Aβs can form both parallel and antiparallel structures, and the parallel one is considered to be associated with the pathogeny. However, the structure and the aggregation mechanism of the mutated Aβ fibrils are not elucidated at atomic and electronic levels. We here investigated solvated structures of the two types of Aβ dimers, each of which is composed of the wild-type or the D23N mutated Aβ, using classical molecular mechanics and ab initio fragment molecular orbital (FMO) methods, in order to reveal the effect of the D23N mutation on the structure of Aβ dimer as well as the specific interactions between the Aβ monomers. The results elucidate that the effect of the D23N mutation is significant for the parallel structure of Aβ dimer and that the solvating water molecules around the Aβ dimer have significant contribution to the stability of Aβ dimer.     

The molecular dynamics of assembly of the ubiquitous aortic medial amyloidal medin fragment

In recent years there is an increased understanding of the molecular conformation of amyloid fibrils. However, much less is known about the early events that lead to the formation of these medically important assemblies. The clarification of these very important mechanistic details on the process may indicate directions towards the inhibition of the early stages of the assembly, where harmful species are most likely to form. Here, we study the dynamics of assembly of short amyloidogenic peptide fragments from the medin polypeptide. This polypeptide is of unique interest since amyloid deposits composed of medin are found almost in all the population above the age of 50. Twelve independent 50 ns long molecular dynamics simulations in explicit water have been run on peptide NH₂–NFGSVQFV–COOH, the minimal recognition hexapeptide element, NH₂–NFGSVQ–COOH, and several single-point mutants. In all cases a three-stranded polymeric β-sheet was used as the basic unit from which fibrils can be formed. Our results clearly indicate the need of well-defined sequence and stereochemical constraints to allow the formation of stable well-ordered aggregates. One of the key findings is the need for the presence of a phenylalanine residue, but not other hydrophobic amino acids, in specific positions within the peptide. Taken together, the results are consistent with recent high-resolution structures of amyloid assemblies and provide unique insights into the dynamics of these structures.     

Effect of graphene oxide on the conformational transitions of amyloid beta peptide: A molecular dynamics simulation study

The interactions between nanomaterials (NMs) and amyloid proteins are central to the nanotechnology-based diagnostics and therapy in neurodegenerative disorders such as Alzheimer's and Parkinson's. Graphene oxide (GO) and its derivatives have shown to modulate the aggregation pattern of disease causing amyloid beta (Aβ) peptide. However, the mechanism is still not well understood. Using molecular dynamics simulations, the effect of graphene oxide (GO) and reduced graphene oxide (rGO) having carbon:oxygen ratio of 4:1 and 10:1, respectively, on the conformational transitions (alpha-helix to beta-sheet) and the dynamics of the peptide was investigated. GO and rGO decreased the beta-strand propensity of amino acid residues in Aβ. The peptide displayed different modes of adsorption on GO and rGO. The adsorption on GO was dominated by electrostatic interactions, whereas on rGO, both van der Waals and electrostatic interactions contributed in the adsorption of the peptide. Our study revealed that the slight increase in the hydrophobic patches on rGO made it more effective inhibitor of conformational transitions in the peptide. Alpha helix-beta sheet transition in Aβ peptide could be one of the plausible mechanism by which graphene oxide may inhibit amyloid fibrillation.     

Antiviral drug acyclovir exhibits antitumor activity via targeting βTrCP1: Molecular docking and dynamics simulation study

The critical role of βTrCP1 in cancer development makes it a discerning target for the development of small drug like molecules. Currently, no inhibitor exists that is able to target its substrate binding site. Through molecular docking and dynamics simulation assays, we explored the comparative binding pattern of βTrCP1-WD40 domain with ACV and its phospho-derivatives (ACVMP, ACVDP and ACVTP). Consequently, through principal component analysis, βTrCP1-ACVTP was found to be more stable complex by obscuring a reduced conformational space than other systems. Thus based on the residual contribution and hydrogen bonding pattern, ACVTP was considered as a noteworthy inhibitor which demarcated binding in the cleft formed by βTrCP1-WD40 specific β-propeller. The outcomes of this study may provide a platform for rational design of specific and potent inhibitor against βTrCP1, with special emphasis on anticancer activity.     

Structure-based design of hERG-neutral antihypertensive oxazalone and imidazolone derivatives

Angiotensin II receptor type 1 (AT1) antagonists are the most recent drug class against hypertension. Recently first crystal structure of AT1 receptor is deposited to the protein data bank (PDB ID: 4YAY). In this work, several molecular screening methods such as molecular docking and de novo design studies were performed and it is found that oxazolone and imidazolone derivatives reveal similar/better interaction energy profiles compared to the FDA approved sartan molecules at the binding site of the AT1 receptor. A database consisting of 3500-fragments were used to enumerate de novo designed imidazolone and oxazolone derivatives and hereby more than 50000 novel small molecules were generated. These derivatives were then used in high throughput virtual screening simulations (Glide/HTVS) to find potent hit molecules. In addition, virtual screening of around 18 million small drug-like compounds from ZINC database were screened at the binding pocket of the AT1 receptor via Glide/HTVS method. Filtered structures were then used in more sophisticated molecular docking simulations protocols (i.e., Glide/SP; Glide/XP; Glide/IFD; Glide/QPLD, and GOLD). However, the K⁺ ion channel/drug interactions should also be considered in studies implemented in molecular level against their cardiovascular risks. Thus, selected compounds with high docking scores via all diverse docking algorithms are also screened at the pore domain regions of human ether-a-go-go-related gene (hERG1) K⁺ channel to remove the high affinity hERG1 blocking compounds. High docking scored compounds at the AT1 with low hERG1 affinity is considered for long molecular dynamics (MD) simulations. Post-processing analysis of MD simulations assisted for better understanding of molecular mechanism of studied compounds at the binding cavity of AT1 receptor. Results of this study can be useful for designing of novel and safe AT1 inhibitors.     

Discovery of high affinity ligands for β2-adrenergic receptor through pharmacophore-based high-throughput virtual screening and docking

Novel high affinity compounds for human β₂-adrenergic receptor (β₂-AR) were searched among the clean drug-like subset of ZINC database consisting of 9,928,465 molecules that satisfy the Lipinski's rule of five. The screening protocol consisted of a high-throughput pharmacophore screening followed by an extensive amount of docking and rescoring. The pharmacophore model was composed of key features shared by all five inactive states of β₂-AR in complex with inverse agonists and antagonists. To test the discriminatory power of the pharmacophore model, a small-scale screening was initially performed on a database consisting of 117 compounds of which 53 antagonists were taken as active inhibitors and 64 agonists as inactive inhibitors. Accordingly, 7.3% of the ZINC database subset (729,413 compounds) satisfied the pharmacophore requirements, along with 44 antagonists and 17 agonists. Afterwards, all these hit compounds were docked to the inactive apo form of the receptor using various docking and scoring protocols. Following each docking experiment, the best pose was further evaluated based on the existence of key residues for antagonist binding in its vicinity. After final evaluations based on the human intestinal absorption (HIA) and the blood brain barrier (BBB) penetration properties, 62 hit compounds have been clustered based on their structural similarity and as a result four scaffolds were revealed. Two of these scaffolds were also observed in three high affinity compounds with experimentally known K_i values. Moreover, novel chemical compounds with distinct structures have been determined as potential β₂-AR drug candidates.     

An investigation of structurally diverse carbamates for acetylcholinesterase (AChE) inhibition using 3D-QSAR analysis

In order to identify the essential structural features and physicochemical properties for acetylcholinesterase (AChE) inhibitory activity in some carbamate derivatives, the systematic QSAR (Quantitative Structure Activity Relationship) studies (CoMFA, advance CoMFA and CoMSIA) have been carried out on a series of (total 78 molecules) taking 52 and 26 molecules in training and test set, respectively. Statistically significant 3D-QSAR (three-dimensional Quantitative Structure Activity Relationship) models were developed on training set molecules using CoMFA and CoMSIA and validated against test set compounds. The highly predictive models (CoMFA q(2)=0.733, r(2)=0.967, predictive r(2)=0.732, CoMSIA q(2)=0.641, r(2)=0.936, predictive r(2)=0.812) well explained the variance in binding affinities both for the training and the test set compounds. The generated models suggest that steric, electrostatic and hydrophobic interactions play an important role in describing the variation in binding affinity. In particular the carbamoyl nitrogen should be more electropositive; substitutions on this nitrogen should have high steric bulk and hydrophobicity while the amino nitrogen should be electronegative in order to have better activity. These studies may provide important insights into structural variations leading to the development of novel AChE inhibitors which may be useful in the development of novel molecules for the treatment of Alzheimer's disease.     

Identification of potential inhibitors against the Zika virus using consensus scoring

The Zika virus (ZIKV) is a life threatening pathogen of zoonotic importance with prevalence in some parts of Africa and America. Unfortunately, there is yet to be a single approved vaccine or antiviral drug to treat the diseases and deformations being caused by the Zika virus infection. In this study, about 36 million compounds from MCULE database were virtually screened against a real matured ZIKV protein using a consensus scoring method to get improved hit rates. The consensus scoring method combined the result from the 25 top ranked molecules from both MCULE and Drug Score eXtended (DSX) docking programs which led to the selection of two hit compounds. The inhibition constant (Ki) values of 0.08 and 0.30 μm were obtained for the two selected compounds MCULE-8830369631-0-1 and MCULE-9236850811-0-1 respectively, to remark them as hit compounds. The molecular interactions of the two selected hit compounds with the amino acids (ALA 48, ILE 49, ILE 468 and LEU 472) present in the ZIKV protein indicated that they both have similar binding modes. The result of the computationally predicted physicochemical properties including ADMET for the selected compounds showed their great potential in becoming lead compounds upon optimization and thus could be used in treating the Zika virus diseases.     

Protein post-translational modifications and misfolding: New concepts in heart failure

A new concept in the field of heart-failure (HF) research points to a role of misfolded proteins, forming preamyloid oligomers (PAOs), in cardiac toxicity. This is largely based on few studies reporting the presence of PAOs, similar to those observed in neurodegenerative diseases, in experimental and human HF. As the majority of proteinopathies are sporadic in nature, protein post-translational modifications (PTMs) likely play a major role in this growing class of diseases. In fact, PTMs are known regulators of protein folding and of the formation of amyloid species in well-established proteinopathies. Proteomics has been instrumental in identifying both chemical and enzymatic PTMs, with a potential impact on protein mis-/folding. Here we provide the basics on how proteins fold along with a few examples of PTMs known to modulate protein misfolding and aggregation, with particular focus on the heart. Due to its innovative content and the growing awareness of the toxicity of misfolded proteins, an “Alzheimer's theory of HF” is timely. Moreover, the continuous innovations in proteomic technologies will help pinpoint PTMs that could contribute to the process. This nuptial between biology and technology could greatly assist in identifying biomarkers with increased specificity as well as more effective therapies.     

Relaxed complex scheme suggests novel inhibitors for the lyase activity of DNA polymerase beta

DNA polymerase beta (pol β), the error-prone polymerase of base excision repair, plays a significant role in chemotherapeutic agent resistance. Its over expression reduces the efficacy of anticancer drug therapies including ionizing radiation, bleomycin, monofunctional alkylating agents and cisplatin. Small-scale studies on different types of cancer showed that pol β is mutated in approximately 30% of tumors. These mutations further lower pol β fidelity in DNA synthesis exposing the genome to serious mutations. These findings suggested pol β as a promising therapeutic target for cancer treatment. More than 60 pol β-inhibitors have been identified so far, however, most of them are either not potent or specific enough to become a drug. Here, we applied the relaxed complex scheme virtual screening (RCSVS) to allow for the full receptor flexibility in filtering the NCI diversity set, DrugBank compounds and a library of ～ 9000 fragmental compounds for novel pol β inhibitors. In this procedure we screened the set of ～ 12,500 compounds against an ensemble of 11 dominant-receptor structures representing the essential backbone dynamics of the 8 kDa domain of pol β. Our results predicted new compounds that can bind with higher affinity to the lyase active site compared to pamoic acid (PA), a well-known inhibitor of DNA pol β.     

A systematic methodology for large scale compound screening: A case study on the discovery of novel S1PL inhibitors

Decrease in sphingosine 1-phosphate (S1P) concentration induces migration of pathogenic T cells to the blood stream, disrupts the CNS and it is implicated in multiple sclerosis (MS), a progressive inflammatory disorder of the central nervous system (CNS), and Alzheimer’s disease (AD). A promising treatment alternative for MS and AD is inhibition of the activity of the microsomal enzyme sphingosine 1-phosphate lyase (S1PL), which degrades intracellular S1P. This report describes an integrated systematic approach comprising virtual screening, molecular docking, substructure search and molecular dynamics simulation to discover novel S1PL inhibitors. Virtual screening of the ZINC database via ligand-based and structure-based pharmacophore models yielded 10000 hits. After molecular docking, common substructures of the top ranking hits were identified. The ligand binding poses were optimized by induced fit docking. MD simulations were performed on the complex structures to determine the stability of the S1PL-ligand complex and to calculate the binding free energy. Selectivity of the selected molecules was examined by docking them to hERG and cytochrome P450 receptors. As a final outcome, 15 compounds from different chemotypes were proposed as potential S1PL inhibitors. These molecules may guide future medicinal chemistry efforts in the discovery of new compounds against the destructive action of pathogenic T cells.     

Ultra-sensitive, label-free probing of the conformational characteristics of amyloid beta aggregates with a SERS active nanofluidic device

One of the primary pathological hallmarks of Alzheimer’s disease is the formation of neutric plaques in the brain. The aggregation of amyloid beta peptide (Aβ) is central to the formation of these plaques and thus trace detection and characterization of these aggregates can have significant implications for understanding and diagnosing diseases. Here we have demonstrated a label-free surface enhanced Raman scattering technique combined with nanofluidics that is able to sensitively detect Aβ aggregates and to characterize their structural and surface properties at concentrations that are much lower than the limit of detection of existing instrumentation. With our device we have successfully detected Aβ aggregates formed at a very low concentration range of 10?fM to 1?μM and shown that the extent of protein aggregation and its resulting conformational characteristics are dependent on the initial Aβ concentration. The ability to observe the early stages of the aggregation process with analytical techniques, like that demonstrated here, could help to develop a better understanding of the conditions which lead to conformational disease, such as neurodegenerative diseases.