Psychoplastogenic DYRK1A Inhibitors with Therapeutic Effects Relevant to Alzheimer's Disease.

Tauopathy, neuronal atrophy, and psychological impairments are hallmarks of neurodegenerative diseases, such as Alzheimer's disease, that currently lack efficacious clinical treatments capable of rectifying these issues. To address these unmet needs, we used rational drug design to combine the pharmacophores of DYRK1A inhibitors and isoDMTs to develop psychoplastogenic DYRK1A inhibitors. Using this approach, we discovered a nonhallucinogenic compound capable of promoting cortical neuron growth and suppressing tau hyperphosphorylation while also having the potential to mitigate the biological and psychological symptoms of dementia. Together, our results suggest that hybridization of the DYRK1A and psychoplastogen pharmacophores represents a promising strategy for identifying compounds that might address the cognitive as well as the behavioral and psychological symptoms of dementia.

Functional Selection of Tau Oligomerization-Inhibiting Aptamers.

Pathological hyperphosphorylation and aggregation of microtubule-associated Tau protein contribute to Alzheimer's Disease (AD) and other related tauopathies. Currently, no cure exists for Alzheimer's Disease. Aptamers offer significant potential as next-generation therapeutics in biotechnology and the treatment of neurological disorders. Traditional aptamer selection methods for Tau protein focus on binding affinity rather than interference with pathological Tau. In this study, we developed a new selection strategy to enrich DNA aptamers that bind to surviving monomeric Tau protein under conditions that would typically promote Tau aggregation. Employing this approach, we identified a set of aptamer candidates. Notably, BW1c demonstrates a high binding affinity (Kd=6.6 nM) to Tau protein and effectively inhibits arachidonic acid (AA)-induced Tau protein oligomerization and aggregation. Additionally, it inhibits GSK3ß-mediated Tau hyperphosphorylation in cell-free systems and okadaic acid-mediated Tau hyperphosphorylation in cellular milieu. Lastly, retro-orbital injection of BW1c tau aptamer shows the ability to cross the blood brain barrier and gain access to neuronal cell body. Through further refinement and development, these Tau aptamers may pave the way for a first-in-class neurotherapeutic to mitigate tauopathy-associated neurodegenerative disorders.

Small molecule inhibitor of tau self-association in a mouse model of tauopathy: A preventive study in P301L tau JNPL3 mice.

Advances in tau biology and the difficulties of amyloid-directed immunotherapeutics have heightened interest in tau as a target for small molecule drug discovery for neurodegenerative diseases. Here, we evaluated OLX-07010, a small molecule inhibitor of tau self-association, for the prevention of tau aggregation. The primary endpoint of the study was statistically significant reduction of insoluble tau aggregates in treated JNPL3 mice compared with Vehicle-control mice. Secondary endpoints were dose-dependent reduction of insoluble tau aggregates, reduction of phosphorylated tau, and reduction of soluble tau. This study was performed in JNPL3 mice, which are representative of inherited forms of 4-repeat tauopathies with the P301L tau mutation (e.g., progressive supranuclear palsy and frontotemporal dementia). The P301L mutation makes tau prone to aggregation; therefore, JNPL3 mice present a more challenging target than mouse models of human tau without mutations. JNPL3 mice were treated from 3 to 7 months of age with Vehicle, 30 mg/kg compound dose, or 40 mg/kg compound dose. Biochemical methods were used to evaluate self-associated tau, insoluble tau aggregates, total tau, and phosphorylated tau in the hindbrain, cortex, and hippocampus. The Vehicle group had higher levels of insoluble tau in the hindbrain than the Baseline group; treatment with 40 mg/kg compound dose prevented this increase. In the cortex, the levels of insoluble tau were similar in the Baseline and Vehicle groups, indicating that the pathological phenotype of these mice was beginning to emerge at the study endpoint and that there was a delay in the development of the phenotype of the model as originally characterized. No drug-related adverse effects were observed during the 4-month treatment period.

Effective immunotherapy occurs in neurons.

Neuronal clearance of proteins in the brain proves to be important for immunotherapy.

De novo designed protein inhibitors of amyloid aggregation and seeding.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-ß (Aß) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aß, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Metformin in Alzheimer's disease: An overview of potential mechanisms, preclinical and clinical findings.

Alzheimer's disease (AD) is the most common neurodegenerative disorder worldwide. The association between AD and other diseases such as diabetes is well-studied. In parallel, potential disease-modifying effects of therapeutic agents used for diabetes have been investigated in the context AD of. Metformin is a biguanide and the most commonly prescribed medication for type 2 diabetes Due to its pleiotropic properties, metformin's potential disease-modifying effects are widely studied on different pathophysiological plyers of AD such as amyloid-ß (Aß) production and clearance, tau phosphorylation, and neuroinflammation, in relevant in vitro and in vivo models. In this review, we summarize the relevant scientific literature on the effects of metformin on various aspects of AD pathophysiology.

In Silico Analysis of Metabolites from Peruvian Native Plants as Potential Therapeutics against Alzheimer's Disease.

BACKGROUND: Despite research on the molecular bases of Alzheimer's disease (AD), effective therapies against its progression are still needed. Recent studies have shown direct links between AD progression and neurovascular dysfunction, highlighting it as a potential target for new therapeutics development. In this work, we screened and evaluated the inhibitory effect of natural compounds from native Peruvian plants against tau protein, amyloid beta, and angiotensin II type 1 receptor (AT1R) pathologic AD markers. METHODS: We applied in silico analysis, such as virtual screening, molecular docking, molecular dynamics simulation (MD), and MM/GBSA estimation, to identify metabolites from Peruvian plants with inhibitory properties, and compared them to nicotinamide, telmisartan, and grapeseed extract drugs in clinical trials. RESULTS: Our results demonstrated the increased bioactivity of three plants' metabolites against tau protein, amyloid beta, and AT1R. The MD simulations indicated the stability of the AT1R:floribundic acid, amyloid beta:rutin, and tau:brassicasterol systems. A polypharmaceutical potential was observed for rutin due to its high affinity to AT1R, amyloid beta, and tau. The metabolite floribundic acid showed bioactivity against the AT1R and tau, and the metabolite brassicasterol showed bioactivity against the amyloid beta and tau. CONCLUSIONS: This study has identified molecules from native Peruvian plants that have the potential to bind three pathologic markers of AD.

Therapeutic potential of quinazoline derivatives for Alzheimer's disease: A comprehensive review.

Quinazolines are considered as a promising class of bioactive heterocyclic compounds with broad properties. Particularly, the quinazoline scaffold has an impressive role in the design and synthesis of new CNS-active drugs. The drug-like properties and pharmacological characteristics of quinazoline could lead to different drugs with various targets. Among CNS disorders, Alzheimer's disease (AD) is a progressive neurodegenerative disorder with memory loss, cognitive decline and language dysfunction. AD is a complex and multifactorial disease therefore, the need for finding multi-target drugs against this devastative disease is urgent. A literature survey revealed that quinazoline derivatives have diverse therapeutic potential for AD as modulators/inhibitors of ß-amyloid, tau protein, cholinesterases, monoamine oxidases, and phosphodiesterases as well as other protective effects. Thus, we describe here the most relevant and recent studies about anti-AD agents with quinazoline structure which can further aid the development and discovery of new anti-AD agents.

Cannabidiol Inhibits Tau Aggregation In Vitro.

A hallmark of Alzheimer's disease (AD) is the accumulation of tau protein in the brain. Compelling evidence indicates that the presence of tau aggregates causes irreversible neuronal destruction, eventually leading to synaptic loss. So far, the inhibition of tau aggregation has been recognized as one of the most effective therapeutic strategies. Cannabidiol (CBD), a major component found in Cannabis sativa L., has antioxidant activities as well as numerous neuroprotective features. Therefore, we hypothesize that CBD may serve as a potent substance to hamper tau aggregation in AD. In this study, we aim to investigate the CBD effect on the aggregation of recombinant human tau protein 1N/4R isoform using biochemical methods in vitro and in silico. Using Thioflavin T (ThT) assay, circular dichroism (CD), and atomic force microscopy (AFM), we demonstrated that CBD can suppress tau fibrils formation. Moreover, by quenching assay, docking, and job's plot, we further demonstrated that one molecule of CBD interacts with one molecule of tau protein through a spontaneous binding. Experiments performed by quenching assay, docking, and Thioflavin T assay further established that the main forces are hydrogen Van der Waals and some non-negligible hydrophobic forces, affecting the lag phase of tau protein kinetics. Taken together, this study provides new insights about a natural substance, CBD, for tau therapy which may offer new hope for the treatment of AD.

Structure-activity relationship around PI-2620 highlights the importance of the nitrogen atom position in the tricyclic core.

Tau aggregates represent a critical pathology in Alzheimer's disease (AD) and other forms of dementia. The extent of Tau neurofibrillary tangles across defined brain regions corresponds well to the observed level of cognitive decline in AD. Compound 1 (PI-2620) was recently identified as a promising Tau positron emission tomography tracer for AD and non-AD tauopathies. To evaluate the impact of the N-atom position with respect to Tau- and off-target binding, tricyclic core analogs of PI-2620 with nitrogen atoms at different positions were prepared. Affinity to aggregated Tau was evaluated using human AD brain homogenates, and their off-target binding was evaluated in a monoamine oxidase A (MAO-A) competition assay. The novel tricyclic core derivatives all displayed inferior Tau binding or MAO-A off-target selectivity, indicating PI-2620 to be the optimal design for high affinity binding to Tau and high MAO-A selectivity.

Impact of Sterilization Methods on the Seeding Ability of Human Tau Proteopathic Seeds.

The protein tau, when misfolded in neurodegenerative diseases, has several prion-like properties including being able to spread by cell-to-cell transfer, induce templated seeding, and exist in distinct conformational strains. These properties of transmission may present health hazards when lesion-containing biospecimens are used in research and neuropathology laboratories. We evaluated the impact standard sterilization and cleaning methods have on the capacity of tau seeds to induce aggregation. We employed a previously developed, highly sensitive FRET-based biosensor assay to assess remnant tau seeding after exposure to these procedures. For tau species derived from human Alzheimer disease tissue (brain homogenate and sarkosyl-insoluble fibrils), both autoclaving and incubation in 90.6% formic acid were sufficient to reduce tau bioactivity. By contrast, boiling was not always effective in completely blocking bioactivity in the seeding assay. Notably, only formic acid incubation was able to produce a similar reduction in tissue from a P301L mutant tau mouse model of tauopathy. Our study highlights nuances in methods for inactivation of tau seeding which may support adapted tissue processing procedures, especially in research settings. These findings also highlight the importance of universal precautions when handling human neuropathological and research laboratory materials.

Dual targeting of acetylcholinesterase and tau aggregation: Design, synthesis and evaluation of multifunctional deoxyvasicinone analogues for Alzheimer's disease.

Development of multitargeted ligands have demonstrated remarkable efficiency as potential therapeutics for Alzheimer's disease (AD). Herein, we reported a new series of deoxyvasicinone analogues as dual inhibitor of acetylcholinesterase (AChE) and tau aggregation that function as multitargeted ligands for AD. All the multitargeted ligands 11(a-j) and 15(a-g) were designed, synthesized, and validated by 1HNMR, 13CNMR and mass spectrometry. All the synthesized compounds 11(a-j) and 15(a-g) were screened for their ability to inhibit AChE, BACE1, amyloid fibrillation, α-syn aggregation, and tau aggregation. All the screened compounds possessed weak inhibition of BACE-1, Aß42 and α-syn aggregation. However, several compounds were identified as potential hits in the AChE inhibitory screening assay and cellular tau aggregation screening. Among all compounds, 11f remarkably inhibited AChE activity and cellular tau oligomerization at single-dose screening (10 µM). Moreover, 11f displayed a half-maximal inhibitory concentration (IC50) value of 0.91 ± 0.05 µM and half-maximal effective concentration (EC50) value of 3.83 ± 0.51 µM for the inhibition of AChE and cellular tau oligomerization, respectively. In addition, the neuroprotective effect of 11f was determined in tau-expressing SH-SY5Y cells incubated with Aß oligomers. These findings highlighted the potential of 11f to function as a multifunctional ligand for the development of promising anti-AD drugs.

Thiophene-Based Dual Modulators of Aß and Tau Aggregation.

Alzheimer's disease is characterized by the accumulation of amyloid beta (Aß) and Tau aggregates in the brain, which induces various pathological events resulting in neurodegeneration. There have been continuous efforts to develop modulators of the Aß and Tau aggregation process to halt or modify disease progression. A few small-molecule-based inhibitors that target both Aß and Tau pathology have been reported. Here, we report the screening of a targeted library of small molecules to modulate Aß and Tau aggregation together with their in vitro, in silico and cellular studies. In vitro ThT fluorescence assay, dot blot assay, gel electrophoresis and transmission electron microscopy (TEM) results have shown that thiophene-based lead molecules effectively modulate Aß aggregation and inhibit Tau aggregation. In silico studies performed by employing molecular docking, molecular dynamics and binding-free energy calculations have helped in understanding the mechanism of interaction of the lead thiophene compounds with Aß and Tau fibril targets. In cellulo studies revealed that the lead candidate is biocompatible and effectively ameliorates neuronal cells from Aß and Tau-mediated amyloid toxicity.

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display.

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau ß-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.

Multifaceted Alzheimer's Disease: Building a Roadmap for Advancement of Novel Therapies.

Alzheimer's disease (AD) is one of the most prevailing neurodegenerative disorders of elderly humans associated with cognitive damage. Biochemical, epigenetic, and pathophysiological factors all consider a critical role of extracellular amyloid-beta (Aß) plaques and intracellular neurofibrillary tangles (NFTs) as pathological hallmarks of AD. In an endeavor to describe the intricacy and multifaceted nature of AD, several hypotheses based on the roles of Aß accumulation, tau hyperphosphorylation, impaired cholinergic signaling, neuroinflammation, and autophagy during the initiation and advancement of the disease have been suggested. However, in no way do these theories have the potential of autonomously describing the pathophysiological alterations located in AD. The complex pathological nature of AD has hindered the recognition and authentication of successful biomarkers for the progression of its diagnosis and therapeutic strategies. There has been a significant research effort to design multi-target-directed ligands for the treatment of AD, an approach which is developed by the knowledge that AD is a composite and multifaceted disease linked with several separate but integrated molecular pathways.

Phosphorylated TAR DNA-binding protein-43: Aggregation and antibody-based inhibition.

TAR DNA-binding protein-43 (TDP-43) pathology, including fibrillar aggregates and mutations, develops in amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). Hyperphosphorylation and aggregation of TDP-43 contribute to pathology and are viable therapeutic targets for ALS. In vivo inhibition of TDP-43 aggregation was evaluated using anti-TDP-43 antibodies with promising outcomes. However, the exact mechanism of antibody-based inhibition targeting TDP-43 is not well understood but may lead to the identification of viable immunotherapies. Herein, the mechanism of in vitro aggregation of phosphorylated TDP-43 was explored, and the anti-TDP-43 antibodies tested for their inhibitor efficacies. Specifically, the aggregation of phosphorylated full-length TDP-43 protein (pS410) was monitored by transmission electron microscopy (TEM), turbidity absorbance, and thioflavin (ThT) spectroscopy. The protein aggregates were insoluble, ThT-positive and characterized with heterogeneous morphologies (fibers, amorphous structures). Antibodies specific to epitopes 178-393 and 256-269, within the RRM2-CTD domain, reduced the formation of ß-sheets and insoluble aggregates, at low antibody loading (antibody: protein ratio = 1 µg/mL: 45 µg/mL). Inhibition outcomes were highly dependent on the type and loading of antibodies, indicating dual functionality of such inhibitors, as aggregation inhibitors or aggregation promoters. Anti-SOD1 and anti-tau antibodies were not effective inhibitors against TDP-43 aggregation, indicating selective inhibition.

Targeting both BDNF/TrkB pathway and delta-secretase for treating Alzheimer's disease.

Alzheimer's disease (AD) is the most common dementia, and no disease-modifying therapeutic agents are currently available. BDNF/TrkB signaling is impaired in AD and is associated with prominent delta-secretase (δ-secretase, also known as asparaginyl endopeptidase or legumain) activation, which simultaneously cleaves both APP and Tau and promotes Aß production and neurofibrillary tangles (NFT) pathologies. Here we show that the optimized δ-secretase inhibitor (#11a) or TrkB receptor agonist (CF3CN) robustly blocks δ-secretase activity separately, and their combination synergistically blunts δ-secretase, exhibiting promising therapeutic efficacy in 3xTg AD mouse model. The optimal δ-secretase inhibitor reveals demonstrable brain exposure and oral bioavailability, suppressing APP N585 and Tau N368 cleavage by δ-secretase. Strikingly, CF3CN treatment evidently escalates BDNF levels. Both #11a and CF3CN display strong in vivo PK/PD properties and ability to suppress δ-secretase activity in the brain. Orally administrated CF3CN strongly activates TrkB that triggers active Akt to phosphorylate δ-secretase T322, preventing its proteolytic activation and mitigating AD pathologies. #11a or CF3CN significantly diminishes AD pathogenesis and improves cognitive functions with the combination exhibiting the maximal effect. Thus, our data support that these derivatives are strong pharmaceutical candidates for the treatment of AD.

Discovery of 1-(phenylsulfonyl)-1H-indole-based multifunctional ligands targeting cholinesterases and 5-HT6 receptor with anti-aggregation properties against amyloid-beta and tau.

Multifunctional ligands as an essential variant of polypharmacology are promising candidates for the treatment of multi-factorial diseases like Alzheimer's disease. Based on clinical evidence and following the paradigm of multifunctional ligands we have rationally designed and synthesized a series of compounds targeting processes involved in the development of the disease. The biological evaluation led to the discovery of two compounds with favorable pharmacological characteristics and ADMET profile. Compounds 17 and 35 are 5-HT6R antagonists (Ki = 13 nM and Ki = 15 nM respectively) and cholinesterase inhibitors with distinct mechanisms of enzyme inhibition. Compound 17, a tacrine derivative is a reversible inhibitor of acetyl- and butyrylcholinesterase (IC50 = 8 nM and IC50 = 24 nM respectively), while compound 35 with rivastigmine-derived phenyl N-ethyl-N-methylcarbamate fragment is a selective, pseudo-irreversible inhibitor of butyrylcholinesterase (IC50 = 455 nM). Both compounds inhibit aggregation of amyloid ß in vitro (75% for compound 17 and 68% for 35 at 10 µM) moreover, compound 35 is a potent tau aggregation inhibitor in cellulo (79%). In ADMET in vitro studies both compounds showed acceptable metabolic stability on mouse liver microsomes (28% and 60% for compound 17 and 35 respectively), no or little effect on CYP3A4 and 2D6 up to a concentration of 10 µM and lack of toxicity on HepG2 cell line (IC50 values of 80 and 21 µM, for 17 and 35 respectively). Based on the pharmacological characteristics and favorable pharmacokinetic properties, we propose compounds 17 and 35 as an excellent starting point for further optimization and in-depth biological studies.

From virtual screening hits targeting a cryptic pocket in BACE-1 to a nontoxic brain permeable multitarget anti-Alzheimer lead with disease-modifying and cognition-enhancing effects.

Starting from six potential hits identified in a virtual screening campaign directed to a cryptic pocket of BACE-1, at the edge of the catalytic cleft, we have synthesized and evaluated six hybrid compounds, designed to simultaneously reach BACE-1 secondary and catalytic sites and to exert additional activities of interest for Alzheimer's disease (AD). We have identified a lead compound with potent in vitro activity towards human BACE-1 and cholinesterases, moderate Aß42 and tau antiaggregating activity, and brain permeability, which is nontoxic in neuronal cells and zebrafish embryos at concentrations above those required for the in vitro activities. This compound completely restored short- and long-term memory in a mouse model of AD (SAMP8) relative to healthy control strain SAMR1, shifted APP processing towards the non-amyloidogenic pathway, reduced tau phosphorylation, and increased the levels of synaptic proteins PSD95 and synaptophysin, thereby emerging as a promising disease-modifying, cognition-enhancing anti-AD lead.