Structural determinants for activation of the Tau kinase CDK5 by the serotonin receptor 5-HT7R.

BACKGROUND: Multiple neurodegenerative diseases are induced by the formation and deposition of protein aggregates. In particular, the microtubule-associated protein Tau leads to the development of so-called tauopathies characterized by the aggregation of hyperphosphorylated Tau within neurons. We recently showed that the constitutive activity of the serotonin receptor 7 (5-HT7R) is required for Tau hyperphosphorylation and aggregation through activation of the cyclin-dependent kinase 5 (CDK5). We also demonstrated physical interaction between 5-HT7R and CDK5 at the plasma membrane suggesting that the 5-HT7R/CDK5 complex is an integral part of the signaling network involved in Tau-mediated pathology. METHODS: Using biochemical, microscopic, molecular biological, computational and AI-based approaches, we investigated structural requirements for the formation of 5-HT7R/CDK5 complex. RESULTS: We demonstrated that 5-HT7R domains responsible for coupling to Gs proteins are not involved in receptor interaction with CDK5. We also created a structural model of the 5-HT7R/CDK5 complex and refined the interaction interface. The model predicted two conserved phenylalanine residues, F278 and F281, within the third intracellular loop of 5-HT7R to be potentially important for complex formation. While site-directed mutagenesis of these residues did not influence Gs protein-mediated receptor signaling, replacement of both phenylalanines by alanine residues significantly reduced 5-HT7R/CDK5 interaction and receptor-mediated CDK5 activation, leading to reduced Tau hyperphosphorylation and aggregation. Molecular dynamics simulations of 5-HT7R/CDK5 complex for wild-type and receptor mutants confirmed binding interface stability of the initial model. CONCLUSIONS: Our results provide a structural basis for the development of novel drugs targeting the 5-HT7R/CDK5 interaction interface for the selective treatment of Tau-related disorders, including frontotemporal dementia and Alzheimer's disease.

NeuM: A Neuron-Selective Probe Incorporates into Live Neuronal Membranes via Enhanced Clathrin-Mediated Endocytosis in Primary Neurons.

The development of a small-molecule probe designed to selectively target neurons would enhance the exploration of intricate neuronal structures and functions. Among such probes, NeuO stands out as the pioneer and has gained significant traction in the field of research. Nevertheless, neither the mechanism behind neuron-selectivity nor the cellular localization has been determined. Here, we introduce NeuM, a derivative of NeuO, designed to target neuronal cell membranes. Furthermore, we elucidate the mechanism behind the selective neuronal membrane trafficking that distinguishes neurons. In an aqueous buffer, NeuM autonomously assembles into micellar structures, leading to the quenching of its fluorescence (Φ=0.001). Upon exposure to neurons, NeuM micelles were selectively internalized into neuronal endosomes via clathrin-mediated endocytosis. Through the endocytic recycling pathway, NeuM micelles integrate into neuronal membrane, dispersing fluorescent NeuM molecules in the membrane (Φ=0.61). Molecular dynamics simulations demonstrated that NeuM, in comparison to NeuO, possesses optimal lipophilicity and molecular length, facilitating its stable incorporation into phospholipid layers. The stable integration of NeuM within neuronal membrane allows the prolonged monitoring of neurons, as well as the visualization of intricate neuronal structures.

Exploration of Tetrahydroisoquinoline- and Benzo[c]azepine-Based Sphingosine 1-Phosphate Receptor 1 Agonists for the Treatment of Multiple Sclerosis.

Because of the wide use of Fingolimod for the treatment of multiple sclerosis (MS) and its cardiovascular side effects such as bradycardia, second-generation sphingosine 1-phosphate receptor 1 (S1P1) agonist drugs for MS have been developed and approved by FDA. The issue of bradycardia is still present with the new drugs, however, which necessitates further exploration of S1P1 agonists with improved safety profiles for next-generation MS drugs. Herein, we report a tetrahydroisoquinoline or a benzo[c]azepine core-based S1P1 agonists such as 32 and 60 after systematic examination of hydrophilic groups and cores. We investigated the binding modes of our representative compounds and their molecular interactions with S1P1 employing recent S1P1 cryo-EM structures. Also, favorable ADME properties of our compounds were shown. Furthermore, in vivo efficacy of our compounds was clearly demonstrated with PLC and EAE studies. Also, the preliminary in vitro cardiovascular safety of our compound was verified with human iPSC-derived cardiomyocytes.

Novel potent blockers for TWIK-1/TREK-1 heterodimers as potential antidepressants.

TREK-1 (TWIK-related potassium channel-1) is a subunit of the two-pore domain potassium (K2p) channel and is widely expressed in the brain. TREK-1 knockout mice were shown to have antidepressant-like effects, providing evidence for the channel's potential as a therapeutic target. However, currently there is no good pharmacological inhibitor specifically targeting TREK-1 containing K2p channels that also displays similar antidepressant-like effects. Here, we sought to find selective and potent inhibitors for TREK-1 related dimers both in vitro and in vivo. We synthesized and evaluated 2-hydroxy-3-phenoxypropyl piperidine derivatives yielding a library from which many TREK-1 targeting candidates emerged. Among these, hydroxyl-phenyl- (2a), piperidino- (2g), and pyrrolidino- (2h) piperidinyl substituted compounds showed high potencies to TREK-1 homodimers with significant antidepressant-like effects in forced swim test and tail suspension test. Interestingly, these compounds were found to have high potencies to TWIK-1/TREK-1 heterodimers. Contrastingly, difluoropiperidinyl-4-fluorophenoxy (3e) and 4-hydroxyphenyl-piperidinyl-4-fluorophenoxy (3j) compounds had high potencies to TREK-1 homodimer but lower potency to TWIK-1/TREK-1 heterodimers without significant antidepressant-like effects. We observed positive correlation between inhibition potency to TWIK-1/TREK-1 and immobility time, and no correlation between inhibition potency to TREK-1 homodimer and immobility time. This was consistent with molecular docking simulations of selected compounds to TREK-1 homodimeric and TWIK-1/TREK-1 heterodimeric models. Existing antidepressant fluoxetine was also found to potently inhibit TWIK-1/TREK-1 heterodimers. Our study reveals novel potent TWIK-1/TREK-1 inhibitors 2a, 2g, and 2h as potential antidepressants and suggest that the TWIK-1/TREK-1 heterodimer could be a potential novel molecular therapeutic target for antidepressants.

Levosimendan inhibits disulfide tau oligomerization and ameliorates tau pathology in TauP301L-BiFC mice.

Tau oligomers play critical roles in tau pathology and are responsible for neuronal cell death and transmitting the disease in the brain. Accordingly, preventing tau oligomerization has become an important therapeutic strategy to treat tauopathies, including Alzheimer's disease. However, progress has been slow because detecting tau oligomers in the cellular context is difficult. Working toward tau-targeted drug discovery, our group has developed a tau-BiFC platform to monitor and quantify tau oligomerization. By using the tau-BiFC platform, we screened libraries with FDA-approved and passed phase I drugs and identified levosimendan as a potent anti-tau agent that inhibits tau oligomerization. 14C-isotope labeling of levosimendan revealed that levosimendan covalently bound to tau cysteines, directly inhibiting disulfide-linked tau oligomerization. In addition, levosimendan disassembles tau oligomers into monomers, rescuing neurons from aggregation states. In comparison, the well-known anti-tau agents methylene blue and LMTM failed to protect neurons from tau-mediated toxicity, generating high-molecular-weight tau oligomers. Levosimendan displayed robust potency against tau oligomerization and rescued cognitive declines induced by tauopathy in the TauP301L-BiFC mouse model. Our data present the potential of levosimendan as a disease-modifying drug for tauopathies.

Neuroprotective Effects of Ethanol Extract of Polyscias fruticosa (EEPF) against Glutamate-Mediated Neuronal Toxicity in HT22 Cells.

In traditional herbal medicine, the Polyscias fruticosa has been frequently used for the treatment of ischemia and inflammation. Oxidative stress mediated by elevated glutamate levels cause neuronal cell death in ischemia and various neurodegenerative diseases. However, so far, the neuroprotective effects of this plant extract against glutamate-mediated cell death have not been investigated in cell models. The current study investigates the neuroprotective effects of ethanol extracts of Polyscias fruticosa (EEPF) and elucidates the underlying molecular mechanisms of EEPFs relevant to neuroprotection against glutamate-mediated cell death. The oxidative stress-mediated cell death was induced by 5 mM glutamate treatment in HT22 cells. The cell viability was measured by a tetrazolium-based EZ-Cytox reagent and Calcein-AM fluorescent dye. Intracellular Ca2+ and ROS levels were measured by fluorescent dyes, fluo-3 AM and 2',7'-dichlorodihydrofluorescein diacetate (DCF-DA), respectively. Protein expressions of p-AKT, BDNF, p-CREB, Bax, Bcl-2, and apoptosis-inducing factor (AIF) were determined by western blot analysis. The apoptotic cell death was measured by flow cytometry. The in vivo efficacy of EEPF was evaluated using the Mongolian gerbil mouse by surgery-induced brain ischemia. EEPF treatment showed a neuroprotective effect against glutamate-induced cell death. The EEPF co-treatment reduced the intracellular Ca2+ and ROS and apoptotic cell death. Furthermore, it recovered the p-AKT, p-CREB, BDNF, and Bcl-2 levels decreased by glutamate. The EEPF co-treatment suppressed the activation of apoptotic Bax, the nuclear translocation of AIF, and mitogen-activated protein kinase (MAPK) pathway proteins (ERK1/2, p38, JNK). Further, EEPF treatment significantly rescued the degenerative neurons in the ischemia-induced Mongolian gerbil in vivo model. EEPF exhibited neuroprotective properties that suppress glutamate-mediated neurotoxicity. The underlying mechanism of EEPF is increasing the level of p-AKT, p-CREB, BDNF, and Bcl-2 associated with cell survival. It has therapeutic potential for the treatment of glutamate-mediated neuropathology.

Glutamate Permeability of Chicken Best1.

Bestrophin-1 (Best1) is a calcium (Ca2+)-activated chloride (Cl-) channel which has a phylogenetically conserved channel structure with an aperture and neck in the ion-conducting pathway. Mammalian mouse Best1 (mBest1) has been known to have a permeability for large organic anions including gluconate, glutamate, and D-serine, in addition to several small monovalent anions, such as Cl­, bromine (Br-), iodine (I-), and thiocyanate (SCN-). However, it is still unclear whether non-mammalian Best1 has a glutamate permeability through the ion-conducting pathway. Here, we report that chicken Best1 (cBest1) is permeable to glutamate in a Ca2+-dependent manner. The molecular docking and molecular dynamics simulation showed a glutamate binding at the aperture and neck of cBest1 and a glutamate permeation through the ion-conducting pore, respectively. Moreover, through electrophysiological recordings, we calculated the permeability ratio of glutamate to Cl- (PGlutamate/PCl) as 0.28 based on the reversal potential shift by ion substitution from Cl- to glutamate in the internal solution. Finally, we directly detected the Ca2+-dependent glutamate release through cBest1 using the ultrasensitive two-cell sniffer patch technique. Our results propose that Best1 homologs from non-mammalian (cBest1) to mammalian (mBest1) have a conserved permeability for glutamate.

Identification of novel discoidin domain receptor 1 (DDR1) inhibitors using E-pharmacophore modeling, structure-based virtual screening, molecular dynamics simulation and MM-GBSA approaches.

Dysregulation of the discoidin domain receptor (DDR1), a collagen-activated receptor tyrosine kinase, has been linked to several human cancer diseases including non-small cell lung carcinoma (NSCLC), ovarian cancer, glioblastoma, and breast cancer, in addition to several inflammatory and neurological conditions. Although there are some selective DDR1 inhibitors that have been discovered during the last two decades, a combination of elevated cytotoxicity, kinome selectivity and/or poor DMPK profile has prevented more in-depth studies from being performed. As such, no DDR1 inhibitor has reached clinical investigation to date, forming an urgent need to develop specific DDR1 inhibitor(s) using various drug discovery means. However, the recent discovery of VU6015929, a potent and selective DDR1 kinase inhibitor, with enhanced physiochemical and DMPK properties in addition to its clean kinome profile marked a milestone in the development of DDR1 inhibitors. Herein, VU6015929 was used to construct a 3D e-pharmacophore model which was validated via calculating the difference of score between the active compounds and decoys. The validated e-pharmacophore model was then utilized to screen 20 million drug-like compounds obtained from the freely accessible Zinc database. The generated hits were ranked using high throughput virtual screening technique (HTVS), and the top 8 small molecules were subjected to a molecular docking study and MM-GBSA calculations. Protein-ligand complexes of compounds 1, 2, 3 and the standard compound (VU6015929) were performed for 100 ns and compared with the DDR1 unbound protein state and the DDR1 bound to a co-crystallized ligand. The molecular docking, MD and MM-GBSA outputs revealed compounds 1-3 as potential DDR1 inhibitors, with compound 2 displaying superior binding affinity, comparable binding stability and average binding free energy for the ligand-enzyme complex compared to VU6015929.

Highly Potent, Selective, and Competitive Indole-Based MAO-B Inhibitors Protect PC12 Cells against 6-Hydroxydopamine- and Rotenone-Induced Oxidative Stress.

Monoamine oxidase B (MAO-B) is responsible for dopamine metabolism and plays a key role in oxidative stress by changing the redox state of neuronal and glial cells. To date, no disease-modifying therapy for Parkinson's disease (PD) has been identified. However, MAO-B inhibitors have emerged as a viable therapeutic strategy for PD patients. Herein, a novel series of indole-based small molecules was synthesized as new MAO-B inhibitors with the potential to counteract the induced oxidative stress in PC12 cells. At a single dose concentration of 10 µM, 10 compounds out of 30 were able to inhibit MAO-B with more than 50%. Among them, compounds 7b, 8a, 8b, and 8e showed 84.1, 99.3, 99.4, and 89.6% inhibition over MAO-B and IC50 values of 0.33, 0.02, 0.03, and 0.45 µM, respectively. When compared to the modest selectivity index of rasagiline (II, a well-known MAO-B inhibitor, SI > 50), compounds 7b, 8a, 8b and 8e showed remarkable selectivity indices (SI > 305, 3649, 3278, and 220, respectively). A further kinetic study displayed a competitive mode of action for 8a and 8b over MAO-B with Ki values of 10.34 and 6.63 nM. Molecular docking studies of the enzyme-inhibitor binding complexes in MAO-B revealed that free NH and substituted indole derivatives share a common favorable binding mode: H-bonding with a crucial water "anchor" and Tyr326. Whereas in MAO-A the compounds failed to form favorable interactions, which explained their high selectivity. In addition, compounds 7b, 8a, 8b, and 8e exhibited safe neurotoxicity profiles in PC12 cells and partially reversed 6-hydroxydopamine- and rotenone-induced cell death. Accordingly, we report compounds 7b, 8a, 8b, and 8e as novel promising leads that could be further exploited for their multi-targeted role in the development of a new oxidative stress-related PD therapy.

Pharmacophore-Based Virtual Screening of Novel Competitive Inhibitors of the Neurodegenerative Disease Target Kynurenine-3-Monooxygenase.

The pathogenesis of several neurodegenerative diseases such as Alzheimer's or Huntington's disease has been associated with metabolic dysfunctions caused by imbalances in the brain and cerebral spinal fluid levels of neuroactive metabolites. Kynurenine monooxygenase (KMO) is considered an ideal therapeutic target for the regulation of neuroactive tryptophan metabolites. Despite significant efforts, the known KMO inhibitors lack blood-brain barrier (BBB) permeability and upon the mimicking of the substrate binding mode, are subject to produce reactive oxygen species as a side reaction. The computational drug design is further complicated by the absence of complete crystal structure information for human KMO (hKMO). In the current work, we performed virtual screening of readily available compounds using several protein-ligand complex pharmacophores. Each of the pharmacophores accounts for one of three distinct reported KMO protein-inhibitor binding conformations. As a result, six novel KMO inhibitors were discovered based on an in vitro fluorescence assay. Compounds VS1 and VS6 were predicted to be BBB permeable and avoid the hydrogen peroxide production dilemma, making them valuable, novel hit compounds for further drug property optimization and advancement in the drug design pipeline.

Platycodin D, a natural component of Platycodon grandiflorum, prevents both lysosome- and TMPRSS2-driven SARS-CoV-2 infection by hindering membrane fusion.

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann-Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.

Modulation of SETDB1 activity by APQ ameliorates heterochromatin condensation, motor function, and neuropathology in a Huntington's disease mouse model.

The present study describes evaluation of epigenetic regulation by a small molecule as the therapeutic potential for treatment of Huntington's disease (HD). We identified 5-allyloxy-2-(pyrrolidin-1-yl)quinoline (APQ) as a novel SETDB1/ESET inhibitor using a combined in silico and in vitro cell based screening system. APQ reduced SETDB1 activity and H3K9me3 levels in a HD cell line model. In particular, not only APQ reduced H3K9me3 levels in the striatum but it also improved motor function and neuropathological symptoms such as neuronal size and activity in HD transgenic (YAC128) mice with minimal toxicity. Using H3K9me3-ChIP and genome-wide sequencing, we also confirmed that APQ modulates H3K9me3-landscaped epigenomes in YAC128 mice. These data provide that APQ, a novel small molecule SETDB1 inhibitor, coordinates H3K9me-dependent heterochromatin remodelling and can be an epigenetic drug for treating HD, leading with hope in clinical trials of HD.

Molecular Characterization of Hepatitis C Virus for Developed Antiviral Agents Resistance Mutations and New Insights into in-silico Prediction Studies.

BACKGROUND: Identification and characterization of developed antiviral drug resistance mutations are key to the success of antiviral therapies against hepatitis C virus (HCV), which remains a worldwide highly prevalent pathogenic disease. Although most studies focus on HCV genotypes 1, 2 or 3, the investigation of drug resistance in HCV genotype 4, predominant in North Africa, is especially significant in Egypt. METHODS: We performed mutational and genotypic analysis of the untranslated region (UTR) and nonstructural protein 5B (NS5B) drug resistance-associated regions of HCV for patients in the surrounding villages of Mansoura city, who were not responding to different antiviral treatments (sofosbuvir (SOF), ribavirin, and interferon). Furthermore, molecular modelling approaches (homology modelling and docking studies) were used to investigate the significance of the identified NS5B mutations for SOF and ribavirin binding in the HCV genotype 4a NS5B active site. RESULTS: Genotypic analysis confirmed all samples to have genotype 4 with sub-genotype 4a predominant. Partial sequencing of the UTR and NS5B resistance-associated regions identified D258E, T282S and A307G mutations in all isolates of NS5B. The UTR mutation site at position 243 was associated with interferon resistance, whereas the NS5B T282S mutation was considered as significant for SOF and ribavirin resistance. Docking studies in the HCV genotype 4a homology model predict SOF and ribavirin to accommodate a nucleotide-like binding mode, in which the T282 residue does interfere with the binding as it would in HCV genotypes 1 and 2. Mutation energy calculations predict T282S to moderately destabilize the binding of SOF and ribavirin by 0.57 and 0.47 kcal/mol, respectively. CONCLUSION: The performed study identified and characterized several antiviral drug resistance mutations of HCV genotype 4a and proposed a mechanism by which the T282S mutation may contribute to SOF and ribavirin resistance.

Discovery of N-(1-(3-fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide: a novel, selective, and competitive indole-based lead inhibitor for human monoamine oxidase B.

Herein, two new series of N-substituted indole-based analogues were rationally designed, synthesized via microwave heating technology, and evaluated as noteworthy MAO-B potential inhibitors. Compared to the reported indazole-based hits VI and VII, compounds 4b and 4e exhibited higher inhibitory activities over MAO-B with IC50 values of 1.65 and 0.78 µM, respectively. When compared to the modest selectivity index of rasagiline (II, a well-known MAO-B inhibitor, SI > 50), both 4b and 4e also showed better selectivity indices (SI > 60 and 120, respectively). A further kinetic evaluation of the most potent derivative (4e) displayed a competitive mode of inhibition (inhibition constant (K i)/MAO-B = 94.52 nM). Reasonable explanations of the elicited biological activities were presented via SAR study and molecular docking simulation. Accordingly, the remarkable MAO-B inhibitory activity of 4e (N-(1-(3-fluorobenzoyl)-1H-indol-5-yl)pyrazine-2-carboxamide), with its selectivity and competitive inhibition, advocates its potential role as a promising lead worthy of further optimization.

PyrPeg, a Blood-Brain-Barrier-Penetrating Two-Photon Imaging Probe, Selectively Detects Neuritic Plaques, Not Tau Aggregates.

Amyloid-ß (Aß) tracers have made a significant contribution to the treatment of Alzheimer's disease (AD) by allowing a definitive diagnosis in living patients. Unfortunately, they also detect tau and other protein aggregates that compromise test accuracy. In AD research, there has been a growing need for in vivo Aß imaging by two-photon microscopy, which enables deep-brain-fluorescence imaging. There is no suitable neuritic Aß probe for two-photon microscopy. Here we report PyrPeg, a novel two-photon fluorescent probe that can selectively target insoluble Aß rather than tau and α-synuclein aggregates in the AD model brain and postmortem brain. When injected intravenously, PyrPeg detects the neuritic plaques in the brain and olfactory bulb of the AD model. PyrPeg may serve as a useful blood-brain-barrier-penetrating diagnostic tool for optical and functional monitoring of insoluble forms of Aß aggregates in the living AD brain.

Visualization of soluble tau oligomers in TauP301L-BiFC transgenic mice demonstrates the progression of tauopathy.

Accumulation of abnormal tau aggregates in the brain is a pathological hallmark of multiple neurodegenerative disorders including Alzheimer's disease. Increasing evidence suggests that soluble tau aggregates play a key role in tau pathology as neurotoxic species causing neuronal cell death and act as prion-like seeds mediating tau propagation. Despite the pathological relevance, there is a paucity of methods to monitor tau oligomerization in the brain. As a tool to monitor tau self-assembly in the brain, we generated a novel tau transgenic mouse, named TauP301L-BiFC. By introducing bimolecular fluorescence complementation technique to human tau containing a P301L mutation, we were able to monitor and quantify tau self-assembly, represented by BiFC fluorescence in the brains of transgenic TauP301L-BiFC mice. TauP301L-BiFC mice showed soluble tau oligomerization from 3 months, showing significantly enriched BiFC fluorescence in the brain. Then, massive tau fragmentation occured at 6 months showing dramatically decreased TauP301L-BiFC fluorescence. The fragmented tau species served as a seed for insoluble tau aggregation. In a result, insoluble TauP301L-BiFC aggregates coaggregated with endogenous mouse tau accumulated in the brain, showing subsequently increased BiFC fluorescence from 9 months. Neuronal degeneration and cognitive deficits were observed from 12 months of age. TauP301L-BiFC mouse model demonstrated that methylene blue reduced the amount of soluble tau oligomers in the brain, resulting in the prevention of cognitive impairments. We assure that TauP301L-BiFC mice are a bona-fide animal tool to monitor pathological tau oligomerization in AD and other tauopathies.

Nrf2 activator via interference of Nrf2-Keap1 interaction has antioxidant and anti-inflammatory properties in Parkinson's disease animal model.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by abnormal movement, including slowed movements, shuffling gait, lack of balance, and tremor. Oxidative stress has been shown to play a decisive role in dopaminergic neuronal cell death in PD. The nuclear factor E2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) signaling pathway provides the main defense system against oxidative stress by inducing the expression of antioxidant enzyme genes. Direct interference in the Keap1-Nrf2 protein-protein interaction (PPI) has emerged as an effective strategy for Nrf2 activation. Therefore, we searched for novel Nrf2 activators that can disrupt Nrf2-Keap1 interaction by using a virtual screening approach and identified a potent Nrf2 activator, KKPA4026. KKPA4026 was confirmed to induce the expression of the Nrf2-dependent antioxidant enzymes heme oxygenase-1, glutamate-cysteine ligase catalytic subunit, glutamate-cysteine ligase regulatory subunit, and NAD(P)H:quinone oxidoreductase 1 in BV-2 cells. Furthermore, KKPA4026 showed anti-inflammatory effects in an Nrf2-dependent manner. In a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model of PD, KKPA4026 effectively attenuated PD-associated behavioral deficits and protected dopaminergic neurons. In summary, we identified KKPA4026 as a novel Nrf2 activator and suggested that Nrf2 activation through interference with the Nrf2-Keap1 interaction may be effective for PD treatment.

CRISPR-mediated gene correction links the ATP7A M1311V mutations with amyotrophic lateral sclerosis pathogenesis in one individual.

Amyotrophic lateral sclerosis (ALS) is a severe disease causing motor neuron death, but a complete cure has not been developed and related genes have not been defined in more than 80% of cases. Here we compared whole genome sequencing results from a male ALS patient and his healthy parents to identify relevant variants, and chose one variant in the X-linked ATP7A gene, M1311V, as a strong disease-linked candidate after profound examination. Although this variant is not rare in the Ashkenazi Jewish population according to results in the genome aggregation database (gnomAD), CRISPR-mediated gene correction of this mutation in patient-derived and re-differentiated motor neurons drastically rescued neuronal activities and functions. These results suggest that the ATP7A M1311V mutation has a potential responsibility for ALS in this patient and might be a potential therapeutic target, revealed here by a personalized medicine strategy.

Evaluation of anti-depressant effects of phthalazinone-based triple-acting small molecules against 5-HT2A, 5-HT2C, and the serotonin transporter.

Development of highly effective, safe, and fast-acting anti-depressants is urgently required for the treatment of major depressive disorder. It has been suggested that targeting 5-HT2A and 5-HT2C in addition to inhibition of serotonin reuptake may be beneficial in generating anti-depressant agents with better pharmacology and less adverse effects. We have developed phthalazinone-based compounds that potently bind to 5-HT2A, 5-HT2C, and the serotonin transporter. The representative compounds 11j and 11l displayed strong binding affinities against these targets, and showed favorable toxicity profiles as determined by hERG binding and CYP inhibition assays. Furthermore, these compounds presented promising anti-depressant effects comparable to fluoxetine and also synergistic effects with fluoxetine in forced swimming test, which implicates these compounds can be developed to help the treatment of major depressive disorder.

Thiazolidine-2,4-dione-based irreversible allosteric IKK-ß kinase inhibitors: Optimization into in vivo active anti-inflammatory agents.

Selective kinase inhibitors development is a cumbersome task because of ATP binding sites similarities across kinases. On contrast, irreversible allosteric covalent inhibition offers opportunity to develop novel selective kinase inhibitors. Previously, we reported thiazolidine-2,4-dione lead compounds eliciting in vitro irreversible allosteric inhibition of IKK-ß. Herein, we address optimization into in vivo active anti-inflammatory agents. We successfully developed potent IKK-ß inhibitors with the most potent compound eliciting IC50 = 0.20 µM. Cellular assay of a set of active compounds using bacterial endotoxin lipopolysaccharide (LPS)-stimulated macrophages elucidated significant in vitro anti-inflammatory activity. In vitro evaluation of microsomal and plasma stabilities showed that the promising compound 7a is more stable than compound 7p. Finally, in vivo evaluation of 7a, which has been conducted in a model of LPS-induced septic shock in mice, showed its ability to protect mice against septic shock induced mortality. Accordingly, this study presents compound 7a as a novel potential irreversible allosteric covalent inhibitor of IKK-ß with verified in vitro and in vivo anti-inflammatory activity.