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.

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.

Discovery of 3,4-dichloro-N-(1H-indol-5-yl)benzamide: A highly potent, selective, and competitive hMAO-B inhibitor with high BBB permeability profile and neuroprotective action.

Since there is no disease-modifying treatment discovered yet for Parkinson's disease (PD), there is still a vital need to develop novel selective monoamine oxidase B (MAO-B) inhibitors as promising therapeutically active candidates for PD patients. Herein, we report the design, synthesis, and full characterization of new twenty-six indole derivatives as potential human MAO-B (hMAO-B) selective inhibitors. Six compounds (2i, 3b-e, and 5) exhibited low micromolar to nanomolar inhibitory activities over hMAO-B; compared to our recently reported N-substituted indole-based lead compound VIII (hMAO-B IC50 = 777 nM), compound 5 (3,4-dichloro-N-(1H-indol-5-yl)benzamide) exhibited 18-fold increase in potency (IC50 = 42 nM). A selectivity study over hMAO-A revealed an excellent selectivity index of compound 5 (SI > 2375) with a 47-fold increase compared to rasagiline (II, a well-known MAO-B inhibitor, SI > 50). A further kinetic evaluation of compound 5 over hMAO-B showed a reversible and competitive mode of inhibition with Ki value of 7 nM. Highly effective permeability and high CNS bioavailability of compound 5 with Pe = 54.49 × 10-6 cm/s were demonstrated. Compound 5 also exhibited a low cytotoxicity profile and a promising neuroprotective effect against the 6-hydroxydopamine-induced neuronal cell damage in PC12 cells, which was more effective than that of rasagiline. Docking simulations on both hMAO-B and hMAO-A supported the in vitro data and served as further molecular evidence. Accordingly, we report the discovery of compound 5 as one of the most potent indole-based MAO-B inhibitors to date which is noteworthy to be further evaluated as a promising agent for PD treatment.

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.

Design, Synthesis and Biological Evaluation of New HDAC1 and HDAC2 Inhibitors Endowed with Ligustrazine as a Novel Cap Moiety.

INTRODUCTION: Histone deacetylases (HDACs) represent one of the most validated cancer targets. The inhibition of HDACs has been proven to be a successful strategy for the development of novel anticancer candidates. METHODS: This work describes design and synthesis of a new set of HDAC inhibitors (7a-c and 8a, b) utilizing ligustrazine as a novel cap moiety, and achieving the pharmacophoric features required to induce the desired inhibition. RESULTS: The newly synthesized derivatives were evaluated for their potential inhibitory activity toward two class I histone deacetylases, namely HDAC1 and HDAC2. The tested ligustrazine-based compounds were more potent toward HDAC2 (IC50 range: 53.7-205.4 nM) than HDAC1 (IC50 range: 114.3-2434.7 nM). Furthermore, the antiproliferative activities against two HDAC-expressing cancer cell lines; HT-29 and SH-SY5Y were examined by the MTT assay. Moreover, a molecular docking study of the designed HDAC inhibitors (7a-c and 8a,b) was carried out to investigate their binding pattern within their prospective targets; HDAC1 (PDB-ID: 4BKX) and HDAC2 (PDB-ID: 6G3O). DISCUSSION: Compound 7a was found to be the most potent analog in this study toward HDAC1 and HDAC2 with IC50 values equal 114.3 and 53.7 nM, respectively. Moreover, it was the most effective counterpart (IC50 = 1.60 µM), with 4.7-fold enhanced efficiency than reference drug Gefitinib (IC50 = 7.63 µM) against SH-SY5Y cells. Whereas, compound 8a (IC50 = 1.96 µM) was the most active member toward HT-29 cells, being 2.5-times more potent than Gefitinib (IC50 = 4.99 µM). Collectively, these results suggest that 7a merits further optimization and development as an effective new HDACI lead compound.