Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of a-Synuclein Protect Against Diverse a-Synuclein Mediated Dysfunctions

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease

Targeting the Intrinsically Disordered Structural Ensemble of a-Synuclein by Small Molecules as a Potential Therapeutic Strategy for Parkinson's Disease

Abstract The misfolding of intrinsically disordered proteins such as a-synuclein, tau and the Ab peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets a-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of a-synuclein-mediated dysfunction, including rescue of a-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of a-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting a-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

Funder: Howard Hughes Medical Institute (HHMI); doi: https://doi.org/10.13039/100000011Abstract: The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease

Neuroprotective effects of polyphenols in cellular models of Parkinson\u27s disease

Parkinson’s disease (PD) is a neurodegenerative disorder which disrupts the lives of 5 million people worldwide. PD is characterized by a loss of dopaminergic neurons in the substantia nigra, resulting in muscle rigidity, tremors, loss of physical movement and eventually cognitive dysfunction in most patients. Current therapies against PD alleviate symptoms without slowing the progression of the disease. The death of dopaminergic neurons is mainly caused by excessive production of reactive oxygen species (ROS) and down-regulation of cellular cytoprotective mechanisms. Hence, current efforts in the field of Parkinson’s-related research are directed towards discovering drug candidates with ROS scavenging/antioxidant properties. Dietary polyphenols are excellent candidates for neuroprotection in PD because they combine potent antioxidant activities with the ability to up-regulate endogenous cytoprotective responses. Moreover, epidemiological evidence suggests that a high intake of flavonoids (a type of polyphenol) is associated with lower risk of PD. Hence we hypothesized that polyphenols are neuroprotective in PD models. A previous study done in our lab showed that botanical extracts rich in anthocyanins (ANC) and proanthocyanidins (PAC) protected against dopaminergic cell loss elicited by rotenone (a mitochondrial toxin epidemiologically linked to PD) in primary midbrain cultures obtained from embryonic rats. Based on this study, we aimed to determine whether the extracts were also protective against neurotoxicity elicited by paraquat (PQ), another environmental toxin associated with high PD risk. PQ is a pro-oxidant that triggers a buildup of ROS by engaging in a redox cycling mechanism in the cytosol. Given that rotenone and PQ have different mechanisms of action, we were motivated to compare the extent of protection conferred by the botanical extracts against the two PD-related insults. Our data showed that botanical extracts rich in ANC, PAC and stilbenes (a subclass of polyphenols) protected against PQ neurotoxicity. Interestingly, a subset of extracts showed differential neuroprotective activity against rotenone and PQ. We hypothesized that these extracts alleviated neurotoxicity elicited by the two different insults by activating different cytoprotective mechanisms. In partial support of this hypothesis, we found that a subset of the botanical extracts induced an increase in the activity of Nrf-2 (nuclear factor erythroid 2-related factor 2), a transcription factor involved in regulating the expression of cellular antioxidant enzymes, whereas some of the extracts alleviated mitochondrial dysfunction elicited by rotenone. Taken together, these data identify botanical extracts with neuroprotective activity against two PD-related insults, and they shed light on differences in neuroprotective mechanisms that can alleviate toxicity elicited by rotenone versus PQ. Because a blueberry (BB) extract rich in ANC protected against both rotenone- and PQ-mediated neurotoxicity in the study outlined above, we further assessed the neuroprotective activity of this extract against dopaminergic cell death triggerd by an additional PD-related insult, and we investigated the underlying neuroprotective mechanisms. The BB extract was found to alleviate dopaminergic cell death and neurite loss in primary midbrain cultures exposed to virus encoding a mutant form of alpha-synuclein (αSyn), a presynaptic protein that is thought to form neurotoxic aggregates involved in the pathogenesis of familial and sporadic PD. Additionally, we found that the BB extract suppressed glial activation elicited by the bacterial endotoxin lipopolysaccharide (LPS), a classic inflammatory agent, and rotenone. Additional experiments revealed that the BB extract and individual ANC stimulated the transcriptional activity of Nrf2 and upregulated DJ-1, a redox-sensitive chaperone protein implicated in PD. Furthermore, we showed that the pro-oxidant activity of the BB extract was necessary for Nrf-2 activation and the extract’s neuroprotective activity. Furthermore, we showed that although DJ-1 was not necessary for BB-mediated activation of Nrf-2, DJ-1 knockdown abrogated BB-mediated neuroprotection against rotenone toxicity. Together, the data from this study suggest that BB ANC exert neuroprotection via dual action of Nrf2 and DJ-1. Having shown that an ANC-rich BB extract alleviated neurotoxicity induced by various PD-related insults, we further tested a ‘wild’ variety of BB for neuroprotective activity against PQ toxicity. Because the wild BB extract had a slightly different polyphenol profile compared to the ‘cultivated’ variety tested earlier, we hypothesized that the two varieties might have different neuroprotective activities. In support of this idea, we found that although extracts prepared from both BB varieties protected against PQ neurotoxicity, they possessed different potencies. Additionally, we tested different fractions of the wild BB extract enriched in different types of polyphenols. We found that the ANC-rich fraction alleviated PQ neurotoxicity and that synergies among various polyphenols apparently contributed to the neuroprotective activity of the wild BB extract. Collectively, our data indicate that polyphenols are protective against different PD-related insults. Our results also suggest that ANC and ANC-rich extracts are excellent candidates for PD therapy. Finally, our findings provide insight into neuroprotective mechanisms that could reduce PD risk and/or slow disease progression and identify Nrf-2 and DJ-1 as key therapeutic targets in PD

A mutation map for human glycoside hydrolase genes

International audienceGlycoside hydrolases (GHs) are found in all domains of life, and at least 87 distinct genes encoding proteins related to GHs are found in the human genome. GHs serve diverse functions from digestion of dietary polysaccharides to breakdown of intracellular oligosaccharides, glycoproteins, proteoglycans and glycolipids. Congenital disorders of GHs (CDGHs) represent more than 30 rare diseases caused by mutations in one of the GH genes. We previously used whole-exome sequencing of a homogenous Danish population of almost 2000 individuals to probe the incidence of deleterious mutations in the human glycosyltransferases (GTs) and developed a mutation map of human GT genes (GlyMAP-I). While deleterious disease-causing mutations in the GT genes were very rare, and in many cases lethal, we predicted deleterious mutations in GH genes to be less rare and less severe given the higher incidence of CDGHs reported worldwide. To probe the incidence of GH mutations, we constructed a mutation map of human GH-related genes (GlyMAP-II) using the Danish WES data, and correlating this with reported disease-causing mutations confirmed the higher prevalence of disease-causing mutations in several GH genes compared to GT genes. We identified 76 novel nonsynonymous single-nucleotide variations (nsSNVs) in 32 GH genes that have not been associated with a CDGH phenotype, and we experimentally validated two novel potentially damaging nsSNVs in the congenital sucrase-isomaltase deficiency gene, SI. Our study provides a global view of human GH genes and disease-causing mutations and serves as a discovery tool for novel damaging nsSNVs in CDGHs

Yeast Reveal a ‘Druggable’ Rsp5/Nedd4 Network That Ameliorates α-Synuclein Toxicity in Neurons

α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson’s disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum–to-Golgi vesicle trafficking.JPB FoundationHoward Hughes Medical Institute (Collaborative Innovation Award)Eleanor Schwartz Charitable FoundationNational Institutes of Health (U.S.). Ruth L. Kirschstein National Research Service Award (Fellowship F32GM099817)National Institutes of Health (U.S.) (Grant GM58160

Targeting the intrinsically disordered structural ensemble of α-synuclein by small molecules as a potential therapeutic strategy for Parkinson's disease.

The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions

Funder: Howard Hughes Medical Institute (HHMI); doi: https://doi.org/10.13039/100000011Abstract: The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson’s disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson’s disease

Novel Small Molecules Targeting the Intrinsically Disordered Structural Ensemble of α-Synuclein Protect Against Diverse α-Synuclein Mediated Dysfunctions.

The over-expression and aggregation of α-synuclein (αSyn) are linked to the onset and pathology of Parkinson's disease. Native monomeric αSyn exists in an intrinsically disordered ensemble of interconverting conformations, which has made its therapeutic targeting by small molecules highly challenging. Nonetheless, here we successfully target the monomeric structural ensemble of αSyn and thereby identify novel drug-like small molecules that impact multiple pathogenic processes. Using a surface plasmon resonance high-throughput screen, in which monomeric αSyn is incubated with microchips arrayed with tethered compounds, we identified novel αSyn interacting drug-like compounds. Because these small molecules could impact a variety of αSyn forms present in the ensemble, we tested representative hits for impact on multiple αSyn malfunctions in vitro and in cells including aggregation and perturbation of vesicular dynamics. We thereby identified a compound that inhibits αSyn misfolding and is neuroprotective, multiple compounds that restore phagocytosis impaired by αSyn overexpression, and a compound blocking cellular transmission of αSyn. Our studies demonstrate that drug-like small molecules that interact with native αSyn can impact a variety of its pathological processes. Thus, targeting the intrinsically disordered ensemble of αSyn offers a unique approach to the development of small molecule research tools and therapeutics for Parkinson's disease

ELN484228 provides protection in cellular models of αSyn-mediated dysfunction.

<p><b>A.</b> ELN484228 alleviates αSyn-mediated impairment of vesicular dynamics. H4 neuroglioma cells over-expressing αSyn from a tetracycline inducible promoter were cultured for 24 hours in the absence or presence of 1 µg/ml tetracycline to induce αSyn and ELN484228 or control compound ELN484217 (compound number 38 in table S4 in Supporting Information text <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087133#pone.0087133.s001" target="_blank">file S1</a>). Open bars: without compound, black bars: with indicated amount of compound. Cells were assayed for phagocytic activity as a measure of αSyn-mediated impairment of vesicular function. 4 μ beads were added for 90 minutes and a phagocytic index was calculated by microscopic visualization. Each sample was run in triplicate and experiments were run three independent times. The phagocytic indices for each individual experiment were averaged and statistical analyses run on the final averages from the three experiments. T-test analysis of the combined averages of the three experiments revealed a significant difference in phagocytosis between Tet-induced samples with and without ELN484228 (n = 3+/− s.e.m *p≤0.001 versus no compound Tet-induced sample). <b>B.</b> Microglia isolated from postnatal day 1 to 3 pups from hSNCA^E46K transgenic (αSyn ) or non-transgenic littermates were incubated for 24 hours with 100 µM ELN484217 or ELN484228 followed by addition of 10 µm beads for 90 minutes. A phagocytic index was calculated by microscopic visualization (n = 3+/− s.e.m *p≤0.001). <b>C.</b> ELN484228 alleviates loss of dopaminergic neurons and neurite retraction induced by the A53T mutant of αSyn. Primary rat embryonic midbrain cultures were non-transduced (‘control’) or transduced with adenovirus encoding A53T αSyn, in the absence or presence of 10 µM ELN484228. The cells were then stained immunocytochemically for MAP2 and TH. Preferential dopaminergic cell death was assessed by evaluating the percentage of MAP2-positive cells that also stained positive for TH. The lengths of neurites staining positive for both MAP2 and TH were measured using the NIS-Elements software. Data are plotted as the mean ± s.e.m. n = 3 for neuron viability analysis; n = 160–206 for neurite length analysis. *p-value≤0.05, ***p-value≤0.001 versus aSyn A53T virus in the absence of compound; one-way ANOVA with Newman-Keuls post-test. <b>D.</b> ELN484228 reduces translocation of αSyn to the phagocytic cup<b>.</b> To assess αSyn translocation, H4 cells were treated with 100 µM ELN484228 and 1 µg/ml tetracycline for 24 hours; cells were then stimulated with 4 μ beads for 90 minutes. Samples were fixed and stained with 5C12 antibody to detect αSyn (red). Cells were counterstained with 488-phalloidin (green) and Hoechts (blue). A dotted circle indicates the position of the bead. <b>E.</b> ELN484228 reduces translocation of αSyn to synapses. Rat hippocampal neurons (∼21DIV) grown in serum-free conditions were treated for 24 hours with 1 µM ELN484228 or 0.01% DMSO vehicle. On the left side is a representative confocal microscopic image showing localization of αSyn (red) detected with 5C12 antibody, and localization of the presynaptic marker synaptophysin (green). Scale bar is 5 µm. Images were subjected to quantitative analysis and synaptic αSyn levels were determined as the amount of signal that colocalizes with the synaptic synaptophysin marker. Automated measurements were performed in Metamorph imaging analysis software to determine synaptic αSyn and synaptophysin levels by integrated intensity or pixel area, respectively. Values represent mean +/− SEM, n = 1000 terminals (αSyn) or 18 optical fields (synaptophysin) per condition, and derived from 2–3 independent cultures. Quantitation demonstrates that ELN484228 reduces the synaptic levels of αSyn in rat hippocampal neurons.</p