Effective Inhibitor Of Aggregation Research Articles

Role of dopaminergic agonists on full‐length tau protein aggregation

AbstractBackgroundGlobally, a number of individuals who live with Alzheimer’s disease (AD), an incurable and debilitating disease, is increasing. Tau protein has a structural role, maintains integrity of microtubules and is one of the underlying causes of the tauopathies [1]. It is unfolded and highly soluble under physiological conditions. Dopamine receptor agonists have been used to target motor symptoms of AD [2] but there is a gap in knowledge on how these compounds play a role in tau protein aggregation.MethodHerein, we propose to evaluate the use of a unique class of multivalent inhibitors with various moieties (catechol, non‐catechol, biphenyl, piperazine, and thiazole) [3] as potential inhibitors for in vitro full‐length tau protein aggregation induced by heparin. Fluorescence aggregation assay, transmission electron microscopy and 4,4′‐Dianilino‐1,1′‐binaphthyl‐5,5′‐disulfonic acid dipotassium salt (Bis‐ANS) fluorescence spectroscopy were used to obtain IC50 values, characterize the aggregation morphologies and evaluate the hydrophobicity of tau aggregates, respectively.ResultThe catechol containing compounds, D‐519 and D‐520, prevented aggregation of tau whereas the non‐catechol and thiazole containing compounds (D‐264 and D‐636) were poor inhibitors. Based on the inhibitors’ density function theory calculations, effective aggregation inhibitors are characterized by their high polarity and polarizability.ConclusionData indicate that small molecule inhibitors may be useful therapeutics targeting tau aggregation related neurodegenerative diseases.

Inhibition of Tau Protein Aggregation Using Small Molecule Inhibitors and Immunotherapies

AbstractBackgroundTau protein aggregation is one of the biomarkers of neurodegenerative diseases and is a viable drug target. Small molecules and immunotherapies have been tested targeting tauopathies. Understanding of the inhibitor mode‐of‐action is needed in order to develop effective drugs against neurodegeneration.MethodThe full length tau protein (2N4R, 441) was used in vitro with aggregation inducers: heparin and arachidonic acid. We have used small molecule inhibitors based of dopamine receptor agonist as inhibitors of tau aggregation in vitro. The inhibition of tau aggregation was also conducted in vitro using anti‐tau antibodies. The biophysical methods including electron microscopy and fluorescence spectroscopy were used to measure inhibitor efficiency.ResultThe anti‐tau antibodies which target epitope in R1‐R4 repeat domain were most effective in inhibition aggregation. The small molecules containing catechol groups were effective aggregation inhibitors and were in micromolar range, which was similar to the efficacy of methylene blue.ConclusionThe tau aggregation was inhibited in vitro using dual approach, with small and large molecules, indicating that both strategies are viable against tauopathies. Future work will require design of multifunctional inhibitors to improve selectivity and potency.

The multifunctional dopamine D2/D3 receptor agonists also possess inhibitory activity against the full-length tau441 protein aggregation

Neurodegeneration leads to variety of diseases which are linked to aberrant protein or peptide aggregation, as a one possible mechanism. Hence, small drug molecules targeting aggregation are of interest. Tau protein aggregation is one of the biomarkers of neurodegenerative diseases and is a viable drug target. Toward multifunctional inhibitors, we aim to incorporate structural elements in a potential drug in order to preserve dopamine agonist activity, which elevates disease symptoms associated with motor skills, and promote inhibitory activity against aggregation of the full-length tau (2N4R, tau441) protein. In our design, we introduced various moieties (catechol, non-catechol, biphenyl, piperazine, and thiazole) to determine which functional group leads to the greatest aggregation inhibition of tau. In vitro, tau aggregation was induced by heparin and monitored by using fluorescence aggregation assay, transmission electron microscopy and 4,4′-Dianilino-1,1′-binaphthyl-5,5′-disulfonic acid dipotassium salt (Bis-ANS) fluorescence spectroscopy. The catechol containing compounds, D-519 and D-520, prevented aggregation of tau. By contrast, non-catechol and thiazole containing compounds (D-264 and D-636) were poor inhibitors. The Bis-ANS studies revealed that the potent inhibitors bound solvent-exposed hydrophobic sites. Based on the density functional theory calculations on inhibitors tested, the compounds characterized with the high polarity and polarizability were more effective aggregation inhibitors. These findings could lead to the development of small multifunctional drug inhibitors for the treatment of tau-associated neurodegeneration.

Potent α-Synuclein Aggregation Inhibitors, Identified by High-Throughput Screening, Mainly Target the Monomeric State

α-Synuclein (αSN) aggregation is central to the etiology of Parkinson's disease (PD). Large-scale screening of compounds to identify aggregation inhibitors is challenged by stochastic αSN aggregation and difficulties in detecting early-stage oligomers (αSOs). We developed a high-throughput screening assay combining SDS-stimulated αSN aggregation with FRET to reproducibly detect initial stages in αSN aggregation. We screened 746,000 compounds, leading to 58 hits that markedly inhibit αSN aggregation and reduce αSOs' membrane permeabilization activity. The most effective aggregation inhibitors were derivatives of (4-hydroxynaphthalen-1-yl)sulfonamide. They interacted strongly with the N-terminal part of monomeric αSN and reduced αSO-membrane interactions, possibly by affecting electrostatic interactions. Several compounds reduced αSO toxicity toward neuronal cell lines. The inhibitors introduced chemical modifications of αSN that were, however, not a prerequisite for inhibitory activity. We also identified several phenyl-benzoxazol compounds that promoted αSN aggregation (proaggregators). These compounds may be useful tools to modulate αSN aggregation in cellula.

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.

Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

BackgroundHuntington's Disease (HD) is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt) is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.ResultsAs such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG) as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP). Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene.ConclusionSingle-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

1035. Short G-Rich Oligonucleotides as a Therapeutic for Huntingtonss Disease

Huntington's Disease (HD) is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt) is produced by HD genes that contain an increased number of glutamine codons and this expansion leads to the production of a protein that misfolds. Recentstudies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions.

In Vitro Studies of the Interaction of Heparin, Low Molecular Weight Heparin and Heparinoids with Platelets

Heparin, low molecular weight heparins, and heparinoids were studied for their ability to inhibit the aggregation of platelets by various agonists and for their ability to adhere to collagen. Heparin was a very effective inhibitor of aggregation with collagen and with ristocetin as it was with adhesion to collagen. The heparinoids showed little effect on aggregation or adhesion. Heparan sulfate and pentosan polysulfate did show slight inhibitory activity against collagen aggregation and adhesion and both interacted with the antibody induced by heparin therapy. It is of interest that dermatan sulfate and the pentasaccharide were almost inert in these experiments, and are unlikely to induce bleeding by inhibition of platelet function. It is highly probable that interference with the interaction of von Willebrand factor with platelets and collagen is a major mechanism for bleeding in the heparinized patient.

Calcium-dependent stimulation of platelet aggregation by PGE2

THERE is considerable interest in the effects of prostaglandins on blood platelets, partly because the synthesis of prostaglandins by stimulated platelets is important in haemostasis, and partly because platelets can be used as models to investigate the actions of prostaglandins (PGs) at a cellular level1. The effects of different PGs on platelets are diverse: PGE1 is a potent inhibitor of platelet aggregation2 with a Ki value around 30 nM and PGD2 is an even more effective inhibitor of aggregation of human platelets, but not those of some other species3,4. PGE2 inhibits platelet aggregation at high concentrations (> 1 × 10−6 M), and there is some controversy as to whether lower concentrations enhance aggregation5. When human platelets are stimulated by collagen they synthesise PGE2 from arachidonic acid6 and the cyclic endoperoxide intermediates in the biosynthesis of PGE2 exert a direct aggregating effect7,8. Synthetic derivatives of PGE2 can also induce the aggregation of human platelets9, but PGE2 itself has never been found to induce aggregation directly.