Josephine W. Wu

The discovery of potential acetylcholinesterase inhibitors: A combination of pharmacophore modeling, virtual screening, and molecular docking studies

BackgroundAlzheimers disease (AD) is the most common cause of dementia characterized by progressive cognitive impairment in the elderly people. The most dramatic abnormalities are those of the cholinergic system. Acetylcholinesterase (AChE) plays a key role in the regulation of the cholinergic system, and hence, inhibition of AChE has emerged as one of the most promising strategies for the treatment of AD.MethodsIn this study, we suggest a workflow for the identification and prioritization of potential compounds targeted against AChE. In order to elucidate the essential structural features for AChE, three-dimensional pharmacophore models were constructed using Discovery Studio 2.5.5 (DS 2.5.5) program based on a set of known AChE inhibitors.ResultsThe best five-features pharmacophore model, which includes one hydrogen bond donor and four hydrophobic features, was generated from a training set of 62 compounds that yielded a correlation coefficient of R = 0.851 and a high prediction of fit values for a set of 26 test molecules with a correlation of R2 = 0.830. Our pharmacophore model also has a high Güner-Henry score and enrichment factor. Virtual screening performed on the NCI database obtained new inhibitors which have the potential to inhibit AChE and to protect neurons from Aβ toxicity. The hit compounds were subsequently subjected to molecular docking and evaluated by consensus scoring function, which resulted in 9 compounds with high pharmacophore fit values and predicted biological activity scores. These compounds showed interactions with important residues at the active site.ConclusionsThe information gained from this study may assist in the discovery of potential AChE inhibitors that are highly selective for its dual binding sites.

Inhibition of Amyloid Fibrillization of Hen Egg-White Lysozymes by Rifampicin and p-Benzoquinone

It has been reported that more than 20 different human proteins can fold abnormally, resulting in the formation of pathological deposits and several lethal degenerative diseases. Despite extensive investigations on amyloid fibril formation, the detailed molecular mechanism remained rather elusive. The current research, utilizing hen egg‐white lysozymes as a model system, is aimed at exploring inhibitory activities of two potential molecules against lysozyme fibril formation. We first demonstrated that the formation of lysozyme amyloid fibrils at pH 2.0 was markedly enhanced by the presence of agitation in comparison with its quiescent counterpart. Next, via numerous spectroscopic techniques and transmission electron microscopy, our results revealed that the inhibition of lysozyme amyloid formation by either rifampicin or its analogue p‐benzoquinone followed a concentration‐dependent fashion. Furthermore, while both inhibitors were shown to acquire an anti‐aggregating and a disaggregating activity, rifampicin, in comparison with p‐benzoquinone, served as a more effective inhibitor against in vitro amyloid fibrillogenesis of lysozyme. It is our belief that the data reported in this work will not only reinforce the findings validated by others that rifampicin and p‐benzoquinone serve as two promising preventive molecules against amyloid fibrillogenesis, but also shed light on a rational design of effective therapeutics for amyloidogenic diseases.

The Importance of Steric Zipper on the Aggregation of the MVGGVV Peptide Derived from the Amyloid β Peptide

Abstract Amyloid-like fibrils are found in many fatal diseases, such as Alzheimers disease, Parkinsons disease, type II diabetes mellitus, and prion diseases. Recently, the structural characterization of the MVGGVV peptide from the C-terminal hydrophobic segment of the amyloid-β (Aβ) peptide has revealed a general feature of amyloid-like fibrils, termed as “steric zipper”, which is constituted by a tight side-chain complementation of the opposing β-sheet layers. In this study, several all-atom molecular dynamics simulations with explicit water were conducted to investigate the importance of steric zipper on the aggregation of the MVGGVV peptide. Our results show that the structural stability of the MVGGVV oligomers increases with increasing the number of β-strands. We further proposed that the octameric structure (the SH2-ST4 model in this study) is the possible nucleus seed for MVGGVV protofibril formation. Our results also demonstrated that hydrophobic interaction is the principle driving force to stabilize the adjacent β-strands while the steric zipper involved M1, V2, V5 and V6 is responsible for holding the neighboring β-sheet layers together. Finally, a twisted model of the MVGGVV assembly (SH2-ST50), based on the averaged twisted angle of ∼ 11.5° between the adjacent β-strands of the SH2-ST4 model, was proposed. Our results gain insights into the aggregation of the MVGGVV peptide in atomic details and may provide a hint for designing new inhibitors able to prevent the fibril formation of the Aβ peptide.

RING domains functioning as E3 ligases reveal distinct structural features: a molecular dynamics simulation study.

Abstract RING domain, a cysteine-rich motif that chelates two zinc ions, has been shown to regulate many biological processes such as mediating a crucial step in the ubiquitinylation pathway In order to investigate the distinct structural features for the RING domains functioning as E3 ligases, several molecular dynamics simulations involving the c-Cbl, CNOT4 (with E3 ligase function), and p44 (no E3 ligase function) RING domains were conducted in this study. Our results reveal that the structural stability of the recognition site is a basic requirement for the RING domains functioning as E3 ligases. The structural stability of the recognition site is maintained by the hydrophobic core and hydrogen bonding network. Another important structural feature of the RING domains functioning as E3 ligases is the stable distances between the recognition site and the zinc ion binding sites S1 and S2. Moreover, the RING domains functioning as E3 ligases seem to exhibit lower β stability due to the higher proportion of proline residues in their sequences. However, no significant difference of the other secondary (α and turn) and the tertiary structural stabilities can be observed among these three RING domains.

Carnosine's Effect on Amyloid Fibril Formation and Induced Cytotoxicity of Lysozyme

Carnosine, a common dipeptide in mammals, has previously been shown to dissemble alpha-crystallin amyloid fibrils. To date, the dipeptides anti-fibrillogensis effect has not been thoroughly characterized in other proteins. For a more complete understanding of carnosines mechanism of action in amyloid fibril inhibition, we have investigated the effect of the dipeptide on lysozyme fibril formation and induced cytotoxicity in human neuroblastoma SH-SY5Y cells. Our study demonstrates a positive correlation between the concentration and inhibitory effect of carnosine against lysozyme fibril formation. Molecular docking results show carnosines mechanism of fibrillogenesis inhibition may be initiated by binding with the aggregation-prone region of the protein. The dipeptide attenuates the amyloid fibril-induced cytotoxicity of human neuronal cells by reducing both apoptotic and necrotic cell deaths. Our study provides solid support for carnosines amyloid fibril inhibitory property and its effect against fibril-induced cytotoxicity in SH-SY5Y cells. The additional insights gained herein may pave way to the discovery of other small molecules that may exert similar effects against amyloid fibril formation and its associated neurodegenerative diseases.

Molecular dynamics simulation of the induced‐fit binding process of DNA aptamer and L‐argininamide

Aptamers are rare functional nucleic acids with binding affinity to and specificity for target ligands. Recent experiments have lead to the proposal of an induced-fit binding mechanism for L-argininamide (Arm) and its binding aptamer. However, at the molecular level, this mechanism between the aptamer and its coupled ligand is still poorly understood. The present study used explicit solvent molecular dynamics (MD) simulations to examine the critical bases involved in aptamer-Arm binding and the induced-fit binding process at atomic resolution. The simulation results revealed that the Watson-Crick pair (G10-C16), C9, A12, and C17 bases play important roles in aptamer-Arm binding, and that binding of Arm results in an aptamer conformation optimized through a general induced-fit process. In an aqueous solution, the mechanism has the following characteristic stages: (a) adsorption stage, the Arm anchors to the binding site of aptamer with strong electrostatic interaction; (b) binding stage, the Arm fits into the binding site of aptamer by hydrogen-bond formation; and (c) complex stabilization stage, the hydrogen bonding and electrostatic interactions cooperatively stabilize the complex structure. This study provides dynamics information on the aptamer-ligand induced-fit binding mechanism. The critical bases in aptamer-ligand binding may provide a guideline in aptamer design for molecular recognition engineering.

The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract Huntingtons disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.

Comparative analysis of human γD-crystallin aggregation under physiological and low pH conditions.

Cataract, a major cause of visual impairment worldwide, is the opacification of the eye’s crystalline lens due to aggregation of the crystallin proteins. The research reported here is aimed at investigating the aggregating behavior of γ-crystallin proteins in various incubation conditions. Thioflavin T binding assay, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, intrinsic (tryptophan) fluorescence spectroscopy, light scattering, and electron microscopy were used for structural characterization. Molecular dynamics simulations and bioinformatics prediction were performed to gain insights into the γD-crystallin mechanisms of fibrillogenesis. We first demonstrated that, except at pH 7.0 and 37°C, the aggregation of γD-crystallin was observed to be augmented upon incubation, as revealed by turbidity measurements. Next, the types of aggregates (fibrillar or non-fibrillar aggregates) formed under different incubation conditions were identified. We found that, while a variety of non-fibrillar, granular species were detected in the sample incubated under pH 7.0, the fibrillogenesis of human γD-crystallin could be induced by acidic pH (pH 2.0). In addition, circular dichroism spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and intrinsic fluorescence spectroscopy were used to characterize the structural and conformational features in different incubation conditions. Our results suggested that incubation under acidic condition led to a considerable change in the secondary structure and an enhancement in solvent-exposure of the hydrophobic regions of human γD-crystallin. Finally, molecular dynamics simulations and bioinformatics prediction were performed to better explain the differences between the structures and/or conformations of the human γD-crystallin samples and to reveal potential key protein region involved in the varied aggregation behavior. Bioinformatics analyses revealed that the initiation of amyloid formation of human γD-crystallin may be associated with a region within the C-terminal domain. We believe the results from this research may contribute to a better understanding of the possible mechanisms underlying the pathogenesis of senile nuclear cataract.

Combining Structure-Based Pharmacophore and In Silico Approaches to Discover Novel Selective Serotonin Reuptake Inhibitors

Inhibition of human serotonin transporter (hSERT) has been reported to be a potent strategy for the treatment for depression. To discover novel selective serotonin reuptake inhibitors (SSRIs), a structure‐based pharmacophore model (SBPM) was developed using the docked conformations of six highly active SSRIs. The best SBPM, consisting of four chemical features: two ring aromatics (RAs), one hydrophobic (HY), and one positive ionizable (PI), was further validated using Gunner‐Henry (GH) scoring and receiver operating characteristic (ROC) curve methods. This well‐validated SBPM was then used as a 3D‐query in virtual screening to identify potential hits from National Cancer Institute (NCI) database. These hits were subsequently filtered by absorption, distribution, metabolism, excretion, and toxicity (ADMET) prediction and molecular docking, and their binding stabilities were validated by 20‐ns MD simulations. Finally, only two compounds (NSC175176 and NSC705841) were identified as potential leads, which exhibited higher binding affinities in comparison with the paroxetine. Our results also suggest that cation–π interaction plays a crucial role in stabilizing the hSERT‐inhibitor complex. To our knowledge, the present work is the first structure‐based virtual screening study for new SSRI discovery, which should be a useful guide for the rapid identification of novel therapeutic agents from chemical database.

Investigation of the early stages of human γD-crystallin aggregation process

Cataract, a major cause of visual impairment worldwide, is a common disease of the eye lens related to protein aggregation. Several factors including the exposure of ultraviolet irradiation and possibly acidic condition may induce the unfolding and subsequent aggregation of the crystallin proteins leading to crystalline lens opacification. Human γD-crystallin (HγDC), a 173 residue monomeric protein, abundant in the nucleus of the human eye lens, has been shown to aggregate and form amyloid fibrils under acidic conditions and that this aggregation route is thought to be a potential initiation pathway for the onset of age-related nuclear cataract. However, the underlying mechanism of fibril formation remains elusive. This report is aimed at examining the structural changes and possible amyloid fibril formation pathway of HγDC using molecular dynamics and molecular docking simulations. Our findings demonstrated that incubation of HγDC under the acidic condition redistributes the protein surface charges and affects the protein interaction with its surrounding solvent environment. This brings about a twist motion in the overall tertiary structure that gives rise to newly formed anti-parallel β-strands in the C-terminal flexible loop regions. The change in protein structural conformation also involves an alteration in specific salt-bridge interactions. Altogether, these findings revealed a plausible mechanism for amyloid fibril formation of HγDC that is important to the early stages of HγDC aggregation involved in cataractogenesis.