Using ΔK280 TauRD Folding Reporter Cells to Screen TRKB Agonists as Alzheimer's Disease Treatment Strategy.

Misfolded aggregation of the hyperphosphorylated microtubule binding protein Tau in the brain is a pathological hallmark of Alzheimer's disease (AD). Tau aggregation downregulates brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB) signaling and leads to neurotoxicity. Therefore, enhancement of BDNF/TRKB signaling could be a strategy to alleviate Tau neurotoxicity. In this study, eight compounds were evaluated for the potential of inhibiting Tau misfolding in human neuroblastoma SH-SY5Y cells expressing the pro-aggregator Tau folding reporter (ΔK280 TauRD-DsRed). Among them, coumarin derivative ZN-015 and quinoline derivatives VB-030 and VB-037 displayed chemical chaperone activity to reduce ΔK280 TauRD aggregation and promote neurite outgrowth. Studies of TRKB signaling revealed that ZN-015, VB-030 and VB-037 treatments significantly increased phosphorylation of TRKB and downstream Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase 1/2 (ERK) and AKT serine/threonine kinase (AKT), to activate ribosomal S6 kinase (RSK) and cAMP response element-binding protein (CREB). Subsequently, p-CREB enhanced the transcription of pro-survival BDNF and BCL2 apoptosis regulator (BCL2), accompanied with reduced expression of anti-survival BCL2-associated X protein (BAX) in ΔK280 TauRD-DsRed-expressing cells. The neurite outgrowth promotion effect of ZN-015, VB-030 and VB-037 was counteracted by a RNA interference-mediated knockdown of TRKB, suggesting the role of these compounds acting as TRKB agonists. Tryptophan fluorescence quenching analysis showed that ZN-015, VB-030 and VB-037 interacted directly with a Pichia pastoris-expressed TRKB extracellular domain, indirectly supporting the role through TRKB signaling. The results of up-regulation in TRKB signaling open up the therapeutic potentials of ZN-015, VB-030 and VB-037 for AD.

A Neuroprotective Action of Quercetin and Apigenin through Inhibiting Aggregation of Aß and Activation of TRKB Signaling in a Cellular Experiment.

Alzheimer's disease (AD) is a neurodegenerative disease with progressive memory loss and the cognitive decline. AD is mainly caused by abnormal accumulation of misfolded amyloid ß (Aß), which leads to neurodegeneration via a number of possible mechanisms such as down-regulation of brain-derived neurotrophic factor-tropomyosin-related kinase B (BDNF-TRKB) signaling pathway. 7 ,8-Dihydroxyflavone (7,8-DHF), a TRKB agonist, has demonstrated potential to enhance BDNF-TRKB pathway in various neurodegenerative diseases. To expand the capacity of flavones as TRKB agonists, two natural flavones quercetin and apigenin, were evaluated. With tryptophan fluorescence quenching assay, we illustrated the direct interaction between quercetin/apigenin and TRKB extracellular domain. Employing Aß folding reporter SH-SY5Y cells, we showed that quercetin and apigenin reduced Aß-aggregation, oxidative stress, caspase-1 and acetylcholinesterase activities, as well as improved the neurite outgrowth. Treatments with quercetin and apigenin increased TRKB Tyr516 and Tyr817 and downstream cAMP-response-element binding protein (CREB) Ser133 to activate transcription of BDNF and BCL2 apoptosis regulator (BCL2), as well as reduced the expression of pro-apoptotic BCL2 associated X protein (BAX). Knockdown of TRKB counteracted the improvement of neurite outgrowth by quercetin and apigenin. Our results demonstrate that quercetin and apigenin are to work likely as a direct agonist on TRKB for their neuroprotective action, strengthening the therapeutic potential of quercetin and apigenin in treating AD.

Virtual Screening and Testing of GSK-3 Inhibitors Using Human SH-SY5Y Cells Expressing Tau Folding Reporter and Mouse Hippocampal Primary Culture under Tau Cytotoxicity.

Glycogen synthase kinase-3ß (GSK-3ß) is an important serine/threonine kinase that implicates in multiple cellular processes and links with the neurodegenerative diseases including Alzheimer's disease (AD). In this study, structure-based virtual screening was performed to search database for compounds targeting GSK-3ß from Enamine's screening collection. Of the top-ranked compounds, 7 primary hits underwent a luminescent kinase assay and a cell assay using human neuroblastoma SH-SY5Y cells expressing Tau repeat domain (TauRD) with pro-aggregant mutation ΔK280. In the kinase assay for these 7 compounds, residual GSK-3ß activities ranged from 36.1% to 90.0% were detected at the IC50 of SB-216763. In the cell assay, only compounds VB-030 and VB-037 reduced Tau aggregation in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. In SH-SY5Y cells expressing ΔK280 TauRD, neither VB-030 nor VB-037 increased expression of GSK-3α Ser21 or GSK-3ß Ser9. Among extracellular signal-regulated kinase (ERK), AKT serine/threonine kinase 1 (AKT), mitogen-activated protein kinase 14 (P38) and mitogen-activated protein kinase 8 (JNK) which modulate Tau phosphorylation, VB-037 attenuated active phosphorylation of P38 Thr180/Tyr182, whereas VB-030 had no effect on the phosphorylation status of ERK, AKT, P38 or JNK. However, both VB-030 and VB-037 reduced endogenous Tau phosphorylation at Ser202, Thr231, Ser396 and Ser404 in neuronally differentiated SH-SY5Y expressing ΔK280 TauRD. In addition, VB-030 and VB-037 further improved neuronal survival and/or neurite length and branch in mouse hippocampal primary culture under Tau cytotoxicity. Overall, through inhibiting GSK-3ß kinase activity and/or p-P38 (Thr180/Tyr182), both compounds may serve as promising candidates to reduce Tau aggregation/cytotoxicity for AD treatment.

Neuroprotective Action of Coumarin Derivatives through Activation of TRKB-CREB-BDNF Pathway and Reduction of Caspase Activity in Neuronal Cells Expressing Pro-Aggregated Tau Protein.

Hyperphosphorylation and aggregation of the microtubule binding protein tau is a neuropathological hallmark of Alzheimer's disease/tauopathies. Tau neurotoxicity provokes alterations in brain-derived neurotrophic factor (BDNF)/tropomycin receptor kinase B (TRKB)/cAMP-response-element binding protein (CREB) signaling to contribute to neurodegeneration. Compounds activating TRKB may therefore provide beneficial effects in tauopathies. LM-031, a coumarin derivative, has demonstrated the potential to improve BDNF signaling in neuronal cells expressing pro-aggregated ΔK280 tau mutant. In this study, we investigated if LM-031 analogous compounds provide neuroprotection effects through interaction with TRKB in SH-SY5Y cells expressing ΔK280 tauRD-DsRed folding reporter. All four LMDS compounds reduced tau aggregation and reactive oxygen species. Among them, LMDS-1 and -2 reduced caspase-1, caspase-6 and caspase-3 activities and promoted neurite outgrowth, and the effect was significantly reversed by knockdown of TRKB. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in these cells, implying that the neuroprotective effects of LMDS-1/2 are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. Furthermore, LMDS-1/2 demonstrated their ability to quench the intrinsic fluorescence of tryptophan residues within the extracellular domain of TRKB, thereby consolidating their interaction with TRKB. Our results suggest that LMDS-1/2 exert neuroprotection through activating TRKB signaling, and shed light on their potential application in therapeutics of Alzheimer's disease/tauopathies.

Novel TRKB agonists activate TRKB and downstream ERK and AKT signaling to protect Aß-GFP SH-SY5Y cells against Aß toxicity.

Decreased BDNF and impaired TRKB signaling contribute to neurodegeneration in Alzheimer's disease (AD). We have shown previously that coumarin derivative LM-031 enhanced CREB/BDNF/BCL2 pathway. In this study we explored if LM-031 analogs LMDS-1 to -4 may act as TRKB agonists to protect SH-SY5Y cells against Aß toxicity. By docking computation for binding with TRKB using 7,8-DHF as a control, all four LMDS compounds displayed potential of binding to domain d5 of TRKB. In addition, all four LMDS compounds exhibited anti-aggregation and neuroprotective efficacy on SH-SY5Y cells with induced Aß-GFP expression. Knock-down of TRKB significantly attenuated TRKB downstream signaling and the neurite outgrowth-promoting effects of these LMDS compounds. Among them, LMDS-1 and -2 were further examined for TRKB signaling. Treatment of ERK inhibitor U0126 or PI3K inhibitor wortmannin decreased p-CREB, BDNF and BCL2 in Aß-GFP cells, implicating the neuroprotective effects are via activating TRKB downstream ERK, PI3K-AKT and CREB signaling. LMDS-1 and -2 are blood-brain barrier permeable as shown by parallel artificial membrane permeability assay. Our results demonstrate how LMDS-1 and -2 are likely to work as TRKB agonists to exert neuroprotection in Aß cells, which may shed light on the potential application in therapeutics of AD.

Novel Synthetic Coumarin-Chalcone Derivative (E)-3-(3-(4-(Dimethylamino)Phenyl)Acryloyl)-4-Hydroxy-2H-Chromen-2-One Activates CREB-Mediated Neuroprotection in Aß and Tau Cell Models of Alzheimer's Disease.

Abnormal accumulations of misfolded Aß and tau proteins are major components of the hallmark plaques and neurofibrillary tangles in the brains of Alzheimer's disease (AD) patients. These abnormal protein deposits cause neurodegeneration through a number of proposed mechanisms, including downregulation of the cAMP-response-element (CRE) binding protein 1 (CREB) signaling pathway. Using CRE-GFP reporter cells, we investigated the effects of three coumarin-chalcone derivatives synthesized in our lab on CREB-mediated gene expression. Aß-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells were used to evaluate these agents for possible antiaggregative, antioxidative, and neuroprotective effects. Blood-brain barrier (BBB) penetration was assessed by pharmacokinetic studies in mice. Of the three tested compounds, (E)-3-(3-(4-(dimethylamino)phenyl)acryloyl)-4-hydroxy-2H-chromen-2-one (LM-021) was observed to increase CREB-mediated gene expression through protein kinase A (PKA), Ca2+/calmodulin-dependent protein kinase II (CaMKII), and extracellular signal-regulated kinase (ERK) in CRE-GFP reporter cells. LM-021 exhibited antiaggregative, antioxidative, and neuroprotective effects mediated by the upregulation of CREB phosphorylation and its downstream brain-derived neurotrophic factor and BCL2 apoptosis regulator genes in Aß-GFP- and ΔK280 tauRD-DsRed-expressing SH-SY5Y cells. Blockage of the PKA, CaMKII, or ERK pathway counteracted the beneficial effects of LM-021. LM-021 also exhibited good BBB penetration ability, with brain to plasma ratio of 5.3%, in in vivo pharmacokinetic assessment. Our results indicate that LM-021 works as a CREB enhancer to reduce Aß and tau aggregation and provide neuroprotection. These findings suggest the therapeutic potential of LM-021 in treating AD.

Multi-Target Effects of Novel Synthetic Coumarin Derivatives Protecting Aß-GFP SH-SY5Y Cells against Aß Toxicity.

Alzheimer's disease (AD) is a common neurodegenerative disease presenting with progressive memory and cognitive impairments. One of the pathogenic mechanisms of AD is attributed to the aggregation of misfolded amyloid ß (Aß), which induces neurotoxicity by reducing the expression of brain-derived neurotrophic factor (BDNF) and its high-affinity receptor tropomyosin-related kinase B (TRKB) and increasing oxidative stress, caspase-1, and acetylcholinesterase (AChE) activities. Here, we have found the potential of two novel synthetic coumarin derivatives, ZN014 and ZN015, for the inhibition of Aß and neuroprotection in SH-SY5Y neuroblastoma cell models for AD. In SH-SY5Y cells expressing the GFP-tagged Aß-folding reporter, both ZN compounds reduced Aß aggregation, oxidative stress, activities of caspase-1 and AChE, as well as increased neurite outgrowth. By activating TRKB-mediated extracellular signal-regulated kinase (ERK) and AKT serine/threonine kinase 1 (AKT) signaling, these two ZN compounds also upregulated the cAMP-response-element binding protein (CREB) and its downstream BDNF and anti-apoptotic B-cell lymphoma 2 (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of ZN014 and ZN015. A parallel artificial membrane permeability assay showed that ZN014 and ZN015 could be characterized as blood-brain barrier permeable. Our results suggest ZN014 and ZN015 as novel therapeutic candidates for AD and demonstrate that ZN014 and ZN015 reduce Aß neurotoxicity via pleiotropic mechanisms.

Flavones 7,8-DHF, Quercetin, and Apigenin Against Tau Toxicity via Activation of TRKB Signaling in ΔK280 TauRD-DsRed SH-SY5Y Cells.

Alzheimer's disease (AD) is a progressive neurodegenerative disease with memory loss and cognitive decline. Neurofibrillary tangles (NFTs) formed by hyperphosphorylated Tau protein are one of the pathological hallmarks of several neurodegenerative diseases including AD. Heat shock protein family B (small) member 1 (HSPB1) is a molecular chaperone that promotes the correct folding of other proteins in response to environmental stress. Nuclear factor erythroid 2-like 2 (NRF2), a redox-regulated transcription factor, is the master regulator of the cellular response to excess reactive oxygen species. Tropomyosin-related kinase B (TRKB) is a membrane-bound receptor that, upon binding brain-derived neurotrophic factor (BDNF), phosphorylates itself to initiate downstream signaling for neuronal survival and axonal growth. In this study, four natural flavones such as 7,8-dihydroxyflavone (7,8-DHF), wogonin, quercetin, and apigenin were evaluated for Tau aggregation inhibitory activity and neuroprotection in SH-SY5Y neuroblastoma. Among the tested flavones, 7,8-DHF, quercetin, and apigenin reduced Tau aggregation, oxidative stress, and caspase-1 activity as well as improved neurite outgrowth in SH-SY5Y cells expressing ΔK280 TauRD-DsRed folding reporter. Treatments with 7,8-DHF, quercetin, and apigenin rescued the reduced HSPB1 and NRF2 and activated TRKB-mediated extracellular signal-regulated kinase (ERK) signaling to upregulate cAMP-response element binding protein (CREB) and its downstream antiapoptotic BCL2 apoptosis regulator (BCL2). Knockdown of TRKB attenuated the neuroprotective effects of these three flavones. Our results suggest 7,8-DHF, quercetin, and apigenin targeting HSPB1, NRF2, and TRKB to reduce Tau aggregation and protect cells against Tau neurotoxicity and may provide new treatment strategies for AD.

LMDS-1, a potential TrkB receptor agonist provides a safe and neurotrophic effect for early-phase Alzheimer's disease.

RATIONALE: The signaling pathways of tropomyosin-related kinase B (TrkB) receptor play a pivotal role in axonal sprouting, proliferation of dendritic arbor, synaptic plasticity, and neuronal differentiation. The levels of BDNF and TrkB receptor were reduced in patients with Alzheimer's disease (AD). OBJECTIVES: The activation of TrkB signaling pathways is a potential strategy for AD therapies. We intended to identify potential TrkB agonists to activate the neuroprotective signaling to alleviate the pathological features of AD mice. RESULTS: Both of the Aß-deteriorated hippocampal primary neurons and mouse models were generated and showed AD characteristics. We first investigated 12 potential TrkB agonists with primary hippocampal neurons of mice. Both 7,8-DHF and LMDS-1 were identified to have better effect than the other compounds on dendritic arborization of the neurons and were further applied to the Aß-injected mouse model. The short-term cognitive behavior and pathology in the mice were improved by LMDS-1. Further investigation indicated that LMDS-1 activated the TrkB through phosphorylation at Y516 rather than Y816. In addition, the ERK but not CaMKII or Akt was activated in the mouse hippocampus with LMDS-1 administration. LMDS-1 treatment also upregulated CREB and BDNF while downregulated the GSK3ß active form and tau phosphorylation. CONCLUSIONS: This study suggests that LMDS-1 upregulates the expression of BDNF and ameliorates the early-phase phenotypes of the AD-like mice through the pTrkB (Y516)-ERK-CREB pathway. In addition, LMDS-1 has better effect than 7,8-DHF in ameliorating the behavioral and pathological features of AD-like mice.

Targeting Inflammation, PHA-767491 Shows a Broad Spectrum in Protein Aggregation Diseases.

Many protein aggregation diseases (PAD) affect the nervous system. Deposits of aggregated disease-specific proteins are found within or around the neuronal cells of neurodegenerative diseases. Although the main protein component is disease-specific, oligomeric aggregates are presumed to be the key agents causing the neurotoxicity. Evidence has shown that protein aggregates cause a chronic inflammatory reaction in the brain, resulting in neurodegeneration. Therefore, strategies targeting anti-inflammation could be beneficial to the therapeutics of PAD. PHA-767491 was originally identified as an inhibitor of CDC7/CDK9 and was found to reduce TDP-43 phosphorylation and prevent neurodegeneration in TDP-43 transgenic animals. We recently identified PHA-767491 as a GSK-3ß inhibitor. In this study, we established mouse hippocampal primary culture with tau-hyperphosphorylation through the activation of GSK-3ß using Wortmannin and GF109203X. We found that PHA-767491 significantly improved the neurite outgrowth of hippocampal primary neurons against the neurotoxicity induced by GSK-3ß. We further showed that PHA-767491 had neuroprotective ability in hippocampal primary culture under oligomeric Aß treatment. In addition, PHA-767491 attenuated the neuroinflammation in mouse cerebellar slice culture with human TBP-109Q agitation. Further study of SCA17 transgenic mice carrying human TBP-109Q showed that PHA-767491 ameliorated the gait ataxia and the inflammatory response both centrally and peripherally. Our findings suggest that PHA-767491 has a broad spectrum of activity in the treatment of different PAD and that this activity could be based on the anti-inflammation mechanism.

Effective methane conversion to methanol on bi-functional graphene-oxide-supported platinum nanoclusters (Pt5) - a DFT study.

Nowadays identifying a high-performance catalyst for converting methane to methanol is crucial because methanol serves as an excellent energy source and has wide chemical applications. In the present study, we used DFT, a computational chemistry method, to investigate the reaction mechanism of methanol production by conversion of methane on Pt5 nanoparticles supported on graphene oxide (GO) substrates. Computational results predicted that the Pt5/GO system exhibits excellent catalysis efficiency, compared with those of the previously examined Pt2/GO and Pt2O2/GO systems. Energetics of examined molecular species and the reaction mechanism showed that the Pt5/GO system exhibits high stability in this catalysis reaction and catalyzes the reaction efficiently. Moreover, between the two investigated surfaces GO and UGO, GO performed better and should be a promising catalyst support to convert methane into methanol.

Novel compound VB-037 inhibits Aß aggregation and promotes neurite outgrowth through enhancement of HSP27 and reduction of P38 and JNK-mediated inflammation in cell models for Alzheimer's disease.

The pathogenesis of Alzheimer's disease (AD) is involved in the aggregation of misfolded amyloid ß (Aß), which upregulates the activity of acetylcholinesterase (AChE), increases the production of reactive oxygen species (ROS), enhances neuroinflammation, and eventually leads to neuronal death. Therefore, compounds targeting these mechanisms may be candidates for multitarget drugs in AD treatment. We found that two quinoline derivatives, VB-030 and VB-037, markedly reduced Aß aggregation and ROS levels in the thioflavin T biochemical assay and Tet-On Aß-green fluorescent protein (GFP) 293 AD cell model. These compounds further improved neurite outgrowth, reduced AChE activity and upregulated the molecular chaperone heat shock protein family B [small] member 1 (HSP27), whereas knockdown of HSP27 counteracted the compounds' neuroprotective effects on the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Furthermore, VB-037 attenuated lipopolysaccharide (LPS)/interferon (IFN)-γ-induced activation of BV-2 microglial cells. In addition, VB-037 demonstrated its potential to diminish LPS/IFN-γ-induced upregulation of caspase 1 activity, expression of interleukin (IL)-1ß, and active phosphorylation of mitogen-activated protein kinase 14 (P38), mitogen-activated protein kinase 8 (JNK), and Jun proto-oncogene, AP-1 transcription factor subunit (JUN) signalings, as well as improve cell viability in the Tet-On Aß-GFP SH-SY5Y AD neuronal model. Our findings strongly indicate the potential of VB-037 for modifying AD progression by targeting multiple mechanisms, thereby offering a new drug development avenue for AD treatment.

Chronic low dose of AM404 ameliorates the cognitive impairment and pathological features in hyperglycemic 3xTg-AD mice.

RATIONALE: Hyperglycemia accelerates the progression of Alzheimer's disease (AD), and GSK3ß plays a potential link between AD and hyperglycemia. Therefore, a direct or indirect GSK3ß inhibition may have potential to delay the progression of AD. Our previous biochemical assay identified AM404 as a GSK3ß inhibitor at high dose (IC50 = 5.353 µM); however, other study suggests that AM404 impaired synaptic plasticity of hippocampus at high dose (10 mg/kg; i.p.). Therefore, the dose and duration of treatment are crucial for the effects of AM404. OBJECTIVE: The effects and molecular mechanisms of AM404 at low dose were evaluated from in vitro to in vivo models. METHODS: AM404 (0.1-0.5 µM) was tested on tau hyperphosphorylated mouse hippocampal primary cultures treated with Wortmannin (WT) and GF109203X (GFX). Hyperglycemic triple transgenic AD (3×Tg-AD) mice at 6 months old were intraperitoneally injected with AM404 (0.25 mg/kg) for 4 weeks. The spatial learning and memory of mice were measured using the Morris water maze. Mouse brain and serum samples were collected for pathological analyses. RESULTS: AM404 (0.5 µM) exhibited significantly augmented neuroprotection toward tau hyperphosphorylation in primary cultures. The chronic systemic administration of AM404 (0.25 mg/kg) attenuated cognitive deficits in hyperglycemic 3×Tg-AD mice. Moreover, chronic low dose of AM404 significantly attenuated Aß production, tau protein phosphorylation, and inflammation associated with an increase of pS473Akt and pS9-GSK3ß. Therefore, AM404 at low dose, not only increased neuroprotection, but also ameliorated cognitive deficit, could be partly by regulating the Akt/GSK3ß signaling, which may contribute to downregulation of Aß, tau hyperphosphorylation, and inflammation in hyperglycemic 3×Tg-AD mice. CONCLUSIONS: These results highlight that chronic administration of AM404 at low dose may be through the Akt/GSK3ß pathway to ameliorate the impairment in hyperglycemic 3×Tg-AD mice.

Synthesis of (3S,4S,5S)-trihydroxylpiperidine derivatives as enzyme stabilizers to improve therapeutic enzyme activity in Fabry patient cell lines.

A series of 3S,4S,5S-trihydroxylated piperidines bearing structural diversity at C-2 or C-6 positions has been synthesized and tested to determine their ability to stabilize the activity of recombinant human α-Galactosidase A (rh-α-Gal A). Hit molecules were identified by rapid inhibitory activity screening, and then further investigated for their ability to protect this enzyme from thermo-induced denaturation and enhance its activity in Fabry patient cell lines. Our study resulted in the identification of a new class of small molecules as enzyme stabilizers for the potential treatment of Fabry disease. Of these, stabilizer 21 was the most effective, showing a 12-fold increase in rh-α-Gal A activity in Fabry disease cell lines.

Investigation of the bindings of a class of inhibitors with GSK3ß kinase using thermodynamic integration MD simulation and kinase assay.

GSK3ß kinase is a noteworthy target for discovery of the drugs that will be used to treat several diseases. In the effort to identify a new inhibitor lead compound, we utilized thermodynamic integration (TI)-molecular dynamics (MD) simulation and kinase assay to investigate the bindings between GSK3ß kinase and five compounds that were analogous to a known inhibitor with an available crystal structure. TI-MD simulations of the first two compounds (analogs 1 and 2) were used for calibration. The computed binding affinities of analogs 1 and 2 agreed well with the experimental results. The rest three compounds (analogs 3-5) were newly obtained from a database search, and their affinity data were newly measured in our labs. TI-MD simulations predicted the binding modes and the computed ΔΔG values have a reasonably good correlation with the experimental affinity data. These newly identified inhibitors appear to be new leads according to our survey of GSK3ß inhibitors listed in recent review articles. The predicted binding modes of these compounds should aid in designing new derivatives of these compounds in the future.

Identifying GSK-3ß kinase inhibitors of Alzheimer's disease: Virtual screening, enzyme, and cell assays.

Glycogen synthase kinase 3ß (GSK-3ß) is widely known as a critical target protein for treating Alzheimer's disease (AD). We utilized virtual screening to search databases for compounds with the potential to be used in drugs targeting GSK-3ß kinase, and kinase as well as cell assays to investigate top-scored, selected compounds. Virtual screening of >1.1 million compounds in the ZINC and in-house databases was conducted using an optimized computational protocol in the docking program GOLD. Of the top-ranked compounds, 16 underwent a luminescent kinase assay and a cell assay using HEK293 cells expressing DsRed-tagged ΔK280 in the repeat domain of tau (tauRD). The compounds VB-003 (a potent GSK-3ß inhibitor) and VB-008 (AM404, an anandamide transport inhibitor), with determined IC50 values of 0.25 and 5.4µM, respectively, were identified as reducing tau aggregation. Both compounds increased expression of phospho-GSK-3ß (Ser9) and reduced endogenous tau phosphorylation at the sites of Ser202, Thr231, and Ser396. In the ∆K280 tauRD-DsRed SH-SY5Y cells, VB-008, but not VB-003, enhanced HSPB1 and GRP78 expression, increased ∆K280 tauRD-DsRed solubility, and promoted neurite outgrowth. Thus VB-008 performed best to the end of the present study. The identified compound VB-008 may guide the identification and synthesis of potential inhibitors analogous to this compound.

Investigation of differences in the binding affinities of two analogous ligands for untagged and tagged p38 kinase using thermodynamic integration MD simulation.

Thermodynamic integration (TI) molecular dynamics (MD) simulations for the binding of a pair of a reference ("ref") ligand and an analogous ("analog") ligand to either tagged (with six extra residues at the N-terminus) or untagged p38 kinase proteins were carried out in order to probe how the binding affinity is influenced by the presence or absence of the peptide tag in p38 kinase. This possible effect of protein length on the binding affinity of a ligand-which is seldom addressed in the literature-is important because, even when two labs claim to have performed experiments with the same protein, they may actually have studied variants of the same protein with different lengths because they applied different protein expression conditions/procedures. Thus, if we wanted to compare ligand binding affinities measured in the two labs, it would be necessary to account for any variation in ligand binding affinity with protein length. The pair of ligand-p38 kinase complexes examined in this work (pdb codes: 3d7z and 3lhj, respectively) were ideal for investigating this effect. The experimentally determined binding energy for the ref ligand with the untagged p38 kinase was -10.9 kcal mol(-1), while that for the analog ligand with the tagged p38 kinase was -11.9 kcal mol(-1). The present TI-MD simulation of the mutation of the ref ligand into the analog ligand while the ligand is bound to the untagged p38 kinase predicted that the binding affinity of the analog ligand is 2.0 kcal mol(-1) greater than that of the ref ligand. A similar simulation also indicated that the same was true for ligand binding to the tagged protein, but in this case the binding affinity for the analog ligand is 2.5 kcal mol(-1) larger than that for the ref ligand. These results therefore suggest that the presence of the peptide tag on p38 kinase increased the difference in the binding energies of the ligands by a small amount of 0.5 kcal mol(-1). This result supports the assumption that the presence of a peptide tag has only a minor effect on ΔG values. The error bars in the computed ΔG values were then estimated via confidence interval analysis and a time autocorrelation function for the quantity dV/dλ. The estimated correlation time was ~0.5 ps and the error bar in the ΔG values estimated using nanosecond-scale simulations was ±0.3 kcal mol(-1) at a confidence level of 95%. These predicted results can be verified in future experiments and should prove useful in subsequent similar studies. Graphical Abstract Thermodynamic cycles for binding of two analogous ligands with untagged and tagged p38 kinases and associated Gibbs free energy.

Using thermodynamic integration MD simulation to compute relative protein-ligand binding free energy of a GSK3ß kinase inhibitor and its analogs.

Thermodynamic integration molecular dynamics simulation was used to investigate how TI-MD simulation preforms in reproducing relative protein-ligand binding free energy of a pair of analogous GSK3ß kinase inhibitors of available experimental data (see Fig. 1), and to predict the affinity for other analogs. The computation for the pair gave a ΔΔG of 1.0 kcal/mol, which was in reasonably good agreement with the experimental value of -0.1 kcal/mol. The error bar was estimated at 0.5 kcal/mol. Subsequently, we employed the same protocol to proceed with simulations to find analogous inhibitors with a stronger affinity. Four analogs with a substitution at one site inside the binding pocket were the first to be tried, but no significant enhancement in affinity was found. Subsequent simulations for another 7 analogs was focused on substitutions at the benzene ring of another site, which gave two analogs (analogs 9 and 10) with ΔΔG values of -0.6 and -0.8 kcal/mol, respectively. Both analogs had a OH group at the meta position and another OH group at the ortho position at the other side of the benzene ring, as shown in Table 3. To explore further, another 4 analogs with this characteristic were investigated. Three analogs with ΔΔG values of -2.2, -1.7 and -1.2 kcal/mol, respectively, were found. Hydrogen bond analysis suggested that the additional hydrogen bonds of the added OH groups with Gln185 and/or Asn64, which did not appear in the reference inhibitor or as an analog with one substitution only in the examined cases, were the main contributors to an enhanced affinity. A prediction for better inhibitors should interest experimentalists of enzyme and/or cell assays. Analysis of the interactions between GSK3ß kinase and the investigated analogs will be useful in the design of GSK3ß kinase inhibitors for compounds of this class.

Computation of relative binding free energy for an inhibitor and its analogs binding with Erk kinase using thermodynamic integration MD simulation.

In the present study, we carried out thermodynamic integration molecular dynamics simulation for a pair of analogous inhibitors binding with Erk kinase to investigate how computation performs in reproducing the relative binding free energy. The computation with BCC-AM1 charges for ligands gave -1.1 kcal/mol, deviated from experimental value of -2.3 kcal/mol by 1.2 kcal/mol, in good agreement with experimental result. The error of computed value was estimated to be 0.5 kcal/mol. To obtain convergence, switching vdw interaction on and off required approximately 10 times more CPU time than switching charges. Residue-based contributions and hydrogen bonding were analyzed and discussed. Furthermore, subsequent simulation using RESP charge for ligand gave ΔΔG of -1.6 kcal/mol. The computed results are better than the result of -5.6 kcal/mol estimated using PBSA method in a previous study. Based on these results, we further carried out computations to predict ΔΔG for five new analogs, focusing on placing polar and nonpolar functional groups at the meta site of benzene ring shown in the Fig. 1, to see if these ligands have better binding affinity than the above ligands. The computations resulted that a ligand with polar -OH group has better binding affinity than the previous examined ligand by ~2.0 kcal/mol and two other ligands have better affinity by ~1.0 kcal/mol. The predicted better inhibitors of this kind should be of interest to experimentalist for future experimental enzyme and/or cell assays.

Prediction of the binding mode between GSK3ß and a peptide derived from GSKIP using molecular dynamics simulation.

GSK3ß plays an important role in many physiological functions; dysregulated GSK3ß is involved in human diseases such as diabetes, cancer, and Alzheimer's disease. This study uses MD simulations to determine the interaction between GSK3ß and a peptide derived from GSKIP, a novel GSK3ß interacting protein. Results show that GSKIPtide is inlaid in a binding pocket consisting of an α-helix and an extended loop near the carboxy-terminal end. This binding pocket is hydrophobic, and is responsible for the protein-protein interaction of two other GSK3ß interacting proteins: FRAT and Axin. The GSKIPtide binding mode is closer to that of AxinGID (in the Axin-GSK3-interacting domain). The single-point mutations of V267G and Y288F in GSK3ß differentiate the binding modes between GSK3 and GSKIPtide, AxinGID, and FRATide. The V2677G mutation of GSK3ß reduces the GSKIPtide binding affinity by 70% and abolishes the binding affinity with AxinGID, but has no effect on FRATide. However, GSK3ß Y288F completely abolishes the FRATide binding without affecting GSKIPtide or AxinGID binding. An analysis of the GSK3ß-GSKIPtide complex structure and the X-ray crystal structures of GSK3ß-FRATide and GSK3ß-AxinGID complexes suggests that the hydroxyl group of Y288 is crucial to maintaining a hydrogen bond network in GSK3ß-FRATide. The hydrophobic side chain of V267 maintains the integrity of helix-helix ridge-groove hydrophobic interaction for GSK3ß-GSKIPtide and GSK3ß-AxinGID. This study simulates these two mutant systems to provide atomic-level evidence of the aforementioned experimental results and validate the wild-type complex structure prediction.