Novel 4-triazole phenyl amide (4-TPA) molecules: Potent promoters of α-synuclein fibril disassembly.

Parkinson's disease profoundly compromises patients' daily lives, and the disassembly of α-synuclein aggregates, a primary pathological factor, represents a promising therapeutic approach. In this study, we conducted a systematic screening and optimization process to identify the novel scaffold B37, a 4-triazolyl-phenylamine derivative, exhibiting a potent disassembly activity of 1.1 µM against α-synuclein preformed fibrils. Notably, B37 demonstrated significant neuroprotective effects, ameliorated autophagic dysfunction induced by preformed fibrils, mitigated oxidative stress, and restored the co-localization of preformed fibrils with lysosomes. Transmission electron microscopy corroborated its in vitro disassembly function. Pharmacokinetic profiling revealed favorable parameters with a receptible blood-brain barrier permeability. B37 emerges as a promising lead compound for further optimization, aiming to develop a highly effective agent targeting the disassembly of α-synuclein aggregates to treat neurodegenerative diseases like Parkinson's disease.

Discovery of potent inhibitors of α-synuclein aggregation using structure-based iterative learning.

Machine learning methods hold the promise to reduce the costs and the failure rates of conventional drug discovery pipelines. This issue is especially pressing for neurodegenerative diseases, where the development of disease-modifying drugs has been particularly challenging. To address this problem, we describe here a machine learning approach to identify small molecule inhibitors of α-synuclein aggregation, a process implicated in Parkinson's disease and other synucleinopathies. Because the proliferation of α-synuclein aggregates takes place through autocatalytic secondary nucleation, we aim to identify compounds that bind the catalytic sites on the surface of the aggregates. To achieve this goal, we use structure-based machine learning in an iterative manner to first identify and then progressively optimize secondary nucleation inhibitors. Our results demonstrate that this approach leads to the facile identification of compounds two orders of magnitude more potent than previously reported ones.

In vitro inhibition of α-Synuclein aggregation and disaggregation of preformed fibers by polyphenol hybrids with 2-conjugated benzothiazole.

The misfolding and aggregation of α-Syn play a pivotal role in connecting diverse pathological pathways in Parkinson's disease (PD). Preserving α-Syn proteostasis and functionality by inhibiting its aggregation or disaggregating existing aggregates using suitable inhibitors represents a promising strategy for PD prevention and treatment. In this study, a series of benzothiazole-polyphenol hybrids was designed and synthesized. Three identified compounds exhibited notable inhibitory activities against α-Syn aggregation in vitro, with IC50 values in the low micromolar range. These inhibitors demonstrated sustained inhibitory effects throughout the entire aggregation process, stabilizing α-Syn proteostasis conformation. Moreover, the compounds effectively disintegrated preformed α-Syn oligomers and fibers, potentially by binding to specific domains within the fibers, inducing fibril instability, collapse, and ultimately resulting in smaller-sized aggregates and monomers. These findings offer valuable insights into the therapeutic potential of polyphenol hybrids with 2-conjugated benzothiazole targeting α-Syn aggregation in the treatment of PD.

In silico study on graphene quantum dots modified with various functional groups inhibiting α­synuclein dimerization.

HYPOTHESIS: Graphene quantum dots (GQDs) with various functional groups are hypothesized to inhibit the α-synuclein (αS) dimerization, a crucial step in Parkinson's disease pathogenesis. The potential of differently functionalized GQDs is systematically explored. EXPERIMENTS: All-atom replica-exchange molecular dynamics simulations (accumulating to 75.6 µs) in explicit water were performed to study the dimerization of the αS non-amyloid component region and the influence of GQDs modified with various functional groups. Conformation ensemble, binding behavior, and free energy analysis were conducted. FINDINGS: All studied GQDs inhibit ß-sheet and backbone hydrogen bond formation in αS dimers, leading to looser oligomeric conformations. Charged GQDs severely impede the growth of extended ß-sheets by providing extra contact surface. GQD binding primarily disrupts αS inter-peptide interactions through π-π stacking, CH-π interactions, and for charged GQDs, additionally through salt-bridge and hydrogen bonding interactions. GQD-COO- showed the most optimal inhibitory effect, binding mode, and intensity, which holds promise for the development of nanomedicines targeting amyloid aggregation in neurodegenerative diseases.

C9-Functionalized Doxycycline Analogs as Drug Candidates to Prevent Pathological α-Synuclein Aggregation and Neuroinflammation in Parkinson's Disease Degeneration.

Doxycycline, a semi-synthetic tetracycline, is a widely used antibiotic for treating mild-to-moderate infections, including skin problems. However, its anti-inflammatory and antioxidant properties, combined with its ability to interfere with α-synuclein aggregation, make it an attractive candidate for repositioning in Parkinson's disease. Nevertheless, the antibiotic activity of doxycycline restricts its potential use for long-term treatment of Parkinsonian patients. In the search for non-antibiotic tetracyclines that could operate against Parkinson's disease pathomechanisms, eighteen novel doxycycline derivatives were designed. Specifically, the dimethyl-amino group at C4 was reduced, resulting in limited antimicrobial activity, and several coupling reactions were performed at position C9 of the aromatic D ring, this position being one of the most reactive for introducing substituents. Using the Thioflavin-T assay, we found seven compounds were more effective than doxycycline in inhibiting α-synuclein aggregation. Furthermore, two of these derivatives exhibited better anti-inflammatory effects than doxycycline in a culture system of microglial cells used to model Parkinson's disease neuroinflammatory processes. Overall, through structure-activity relationship studies, we identified two newly designed tetracyclines as promising drug candidates for Parkinson's disease treatment.

Who Ever Said It Would Be Easy? Reflecting on Two Clinical Trials Targeting α-Synuclein.

Two recent, high-profile manuscripts reported negative results with two parallel approaches of passive immunization targeting α-synuclein in a population of patients with early Parkinson's disease (PD). These phase II studies failed to show a bona fide disease-modifying neuroprotective effect on PD progression, despite the evidence that these antibodies effectively bind native α-synuclein in human serum. Here, we discuss the possible reasons that could help explain the lack of clinical efficacy. In particular, we highlight (1) the wealth of evidence supporting the notion of α-synuclein as a valid therapeutic target; (2) the lack of evidence of target engagement in the aforementioned studies, especially of the elusive oligomeric species, the likely culprits in disease pathogenesis and/or its propagation; (3) the limitations, especially in terms of timing passive immunization, of preclinical models, where the same α-synuclein antibodies succeeded in mitigating disease manifestations; (4) the consideration of possibly intervening at an even earlier stage of disease in future trials; and (5) the multitude of strategies beyond passive immunization that could be used to combat α-synuclein-mediated neurodegeneration, if in the end the current approach is not fruitful. Overall, our perception is that converging developments in the field, among them novel bioassays and biomarkers, improved cellular and animal models and objective measurements of motor activities integrated into clinical trials, if further optimized, will gradually move the momentum of the field forward. This, to better test the concept of whether α-synuclein-targeting therapies can indeed deliver the "holy grail" of neuroprotection to the benefit of the PD community. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Trial of Prasinezumab in Early-Stage Parkinson's Disease.

BACKGROUND: Aggregated α-synuclein plays an important role in the pathogenesis of Parkinson's disease. The monoclonal antibody prasinezumab, directed at aggregated α-synuclein, is being studied for its effect on Parkinson's disease. METHODS: In this phase 2 trial, we randomly assigned participants with early-stage Parkinson's disease in a 1:1:1 ratio to receive intravenous placebo or prasinezumab at a dose of 1500 mg or 4500 mg every 4 weeks for 52 weeks. The primary end point was the change from baseline to week 52 in the sum of scores on parts I, II, and III of the Movement Disorder Society-sponsored revision of the Unified Parkinson's Disease Rating Scale (MDS-UPDRS; range, 0 to 236, with higher scores indicating greater impairment). Secondary end points included the dopamine transporter levels in the putamen of the hemisphere ipsilateral to the clinically more affected side of the body, as measured by 123I-ioflupane single-photon-emission computed tomography (SPECT). RESULTS: A total of 316 participants were enrolled; 105 were assigned to receive placebo, 105 to receive 1500 mg of prasinezumab, and 106 to receive 4500 mg of prasinezumab. The baseline mean MDS-UPDRS scores were 32.0 in the placebo group, 31.5 in the 1500-mg group, and 30.8 in the 4500-mg group, and mean (±SE) changes from baseline to 52 weeks were 9.4±1.2 in the placebo group, 7.4±1.2 in the 1500-mg group (difference vs. placebo, -2.0; 80% confidence interval [CI], -4.2 to 0.2; P = 0.24), and 8.8±1.2 in the 4500-mg group (difference vs. placebo, -0.6; 80% CI, -2.8 to 1.6; P = 0.72). There was no substantial difference between the active-treatment groups and the placebo group in dopamine transporter levels on SPECT. The results for most clinical secondary end points were similar in the active-treatment groups and the placebo group. Serious adverse events occurred in 6.7% of the participants in the 1500-mg group and in 7.5% of those in the 4500-mg group; infusion reactions occurred in 19.0% and 34.0%, respectively. CONCLUSIONS: Prasinezumab therapy had no meaningful effect on global or imaging measures of Parkinson's disease progression as compared with placebo and was associated with infusion reactions. (Funded by F. Hoffmann-La Roche and Prothena Biosciences; PASADENA ClinicalTrials.gov number, NCT03100149.).

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.

Immunisation with UB-312 in the Thy1SNCA mouse prevents motor performance deficits and oligomeric α-synuclein accumulation in the brain and gut.

Alpha synuclein has a key role in the pathogenesis of Parkinson's disease (PD), Dementia with Lewy Bodies (LBD) and Multiple System Atrophy (MSA). Immunotherapies aiming at neutralising toxic αSyn species are being investigated in the clinic as potential disease modifying therapies for PD and other synucleinopathies. In this study, the effects of active immunisation against αSyn with the UB-312 vaccine were investigated in the Thy1SNCA/15 mouse model of PD. Young transgenic and wild-type mice received an immunisation regimen over a period of 6 weeks, then observed for an additional 9 weeks. Behavioural assessment was conducted before immunisation and at 15 weeks after the first dose. UB-312 immunisation prevented the development of motor impairment in the wire test and challenging beam test, which was associated with reduced levels of αSyn oligomers in the cerebral cortex, hippocampus and striatum of Thy1SNCA/15 mice. UB-312 immunotherapy resulted in a significant reduction of theαSyn load in the colon, accompanied by a reduction in enteric glial cell reactivity in the colonic ganglia. Our results demonstrate that immunisation with UB-312 prevents functional deficits and both central and peripheral pathology in Thy1SNCA/15 mice.

Discovery of novel 6-hydroxybenzothiazole urea derivatives as dual Dyrk1A/α-synuclein aggregation inhibitors with neuroprotective effects.

A role of Dyrk1A in the progression of Down syndrome-related Alzheimer's disease (AD) is well supported. However, the involvement of Dyrk1A in the pathogenesis of Parkinson's disease (PD) was much less studied, and it is not clear whether it would be promising to test Dyrk1A inhibitors in relevant PD models. Herein, we modified our previously published 1-(6-hydroxybenzo[d]thiazol-2-yl)-3-phenylurea scaffold of Dyrk1A inhibitors to obtain a new series of analogues with higher selectivity for Dyrk1A on the one hand, but also with a novel, additional activity as inhibitors of α-synuclein (α-syn) aggregation, a major pathogenic hallmark of PD. The phenyl acetamide derivative b27 displayed the highest potency against Dyrk1A with an IC50 of 20 nM and high selectivity over closely related kinases. Furthermore, b27 was shown to successfully target intracellular Dyrk1A and to inhibit SF3B1 phosphorylation in HeLa cells with an IC50 of 690 nM. In addition, two compounds among the Dyrk1A inhibitors, b1 and b20, also suppressed the aggregation of α-synuclein (α-syn) oligomers (with IC50 values of 10.5 µM and 7.8 µM, respectively). Both compounds but not the Dyrk1A reference inhibitor harmine protected SH-SY5Y neuroblastoma cells against α-syn-induced cytotoxicity, with b20 exhibiting a higher neuroprotective effect. Compound b1 and harmine were more efficient in protecting SH-SY5Y cells against 6-hydroxydopamine-induced cell death, an effect that was previously correlated to Dyrk1A inactivation in cells but not yet verified using chemical inhibitors. The presented dual inhibitors exhibited a novel activity profile encouraging for further testing in neurodegenerative disease models.

1-Indanone and 1,3-indandione Derivatives as Ligands for Misfolded α-Synuclein Aggregates.

The development of imaging agents for in vivo detection of alpha-synuclein (α-syn) pathologies faces several challenges. A major gap in the field is the lack of diverse molecular scaffolds with high affinity and selectivity to α-syn fibrils for in vitro screening assays. Better in vitro scaffolds can instruct the discovery of better in vivo agents. We report the rational design, synthesis, and in vitro evaluation of a series of novel 1-indanone and 1,3-indandione derivatives from a Structure-Activity Relationship (SAR) study centered on some existing α-syn fibril binding ligands. Our results from fibril saturation binding experiments show that two of the lead candidates compounds 8 and 32 bind α-syn fibrils with binding constants (Kd ) of 9.0 and 18.8 nM, respectively, and selectivity of greater than 10× for α-syn fibrils compared with amyloid-ß (Aß) and tau fibrils. Our results demonstrate that the lead ligands avidly label all forms of α-syn on PD brain tissue sections, but only the dense core of senile plaques in AD brain tissue, respectively. These results are corroborated by ligand-antibody colocalization data from Syn211, which shows immunoreactivity toward all forms of α-syn aggregates, and Syn303, which displays preferential reactivity toward mature Lewy pathology. Our results reveal that 1-indanone derivatives have desirable properties for the biological evaluation of α-synucleinopathies.

Vitamin D Inhibits the Early Aggregation of α-Synuclein and Modulates Exocytosis Revealed by Electrochemical Measurements.

Alpha-synuclein (α-Syn) localizes at presynaptic terminal and modulates synaptic functions. Increasing evidence demonstrate that α-Syn oligomers, forming at the early of aggregation, are cytotoxic and is thus related to brain neurodegenerative diseases. Herein, we find that vitamin D (VD) can reduce neurocytotoxicity. The reduced neurocytotoxicity might be attributed to the less amount of large-sized α-Syn oligomers inhibited by VD, measured by electrochemical collision at single particle level, which are not observable with traditionally ensembled method. Single-cell amperometry (SCA) results show that VD can recover the amount of neurotransmitter release during exocytosis induced by α-Syn oligomers, further verifying the neuroprotection of VD. Our study reveals the neuroprotective role of VD through inhibiting α-Syn aggregation, which is envisioned to be of great importance in treatment and prevention of the neurodegenerative diseases.

Chaperon-mediated autophagy can regulate diquat-induced apoptosis by inhibiting α-synuclein accumulation cooperatively with macroautophagy.

α-Synuclein, which is associated with Parkinson's disease, is cleared by the ubiquitin-proteasome system and autophagy lysosome system. Chaperon-mediated autophagy (CMA) and macroautophagy are major subtypes of autophagy and play a critical role in pesticide-induced α-synucleinopathy. In this study, we explored the role of CMA in diquat (DQ)-induced α-synucleinopathy and characterized the relationship between CMA and macroautophagy in the clearance of pathologic α-synuclein for the prevention of DQ neurotoxicity. DQ was cytotoxic to SH-SY5Y cells in a concentration-dependent manner, as shown by decreased cell viability and increased cytotoxicity. DQ treatment was also found to induce autophagy such as CMA and macroautophagy by monitoring the expression of Lamp2A and microtubule-associated protein 1A/1B light chain 3B (LC3-II) respectively. Following DQ treatment, SH-SY5Y cells were found to have induced phosphorylated and detergent-insoluble α-synuclein deposits, and MG132, a proteasome inhibitor, effectively potentiated both CMA and macroautophagy for preventing α-synuclein aggregation. Interestingly, CMA impairment by Lamp2A-knock down decreased the LC3II expression compared to in DQ-treated cells transfected with control siRNA. In Lamp2-knock down cells, pathologic α-synuclein was increased 12 h after DQ treatment, but there was no change observed at 24 h. In DQ-treated cells, macroautophagy by 3-methyladenine and bafilomycin inhibition increased Lamp2A expression, indicating an increase in CMA activity. In addition, CMA modulation affected apoptosis, and inhibiting lysosome activity by NH4Cl increased apoptosis in DQ-treated cells. An increase in autophagy was confirmed to compensate for the decrease in lysosome activity. Pretreatment with z-VAD-fmk, a pan-caspase inhibitor, significantly enhanced the macroautophagy response of DQ-exposed cells without alterations in Lamp2A expression. Our results suggest that CMA can regulate DQ-induced α-synucleinopathy cooperatively with macroautophagy, and crosstalk between macroautophagy and CMA plays an important role in DQ-induced cytotoxicity. Taken together, autophagy modulation may be a useful treatment strategy in pesticide-induced neurodegenerative disorders through preventing α-synucleinopathy.

Sycosterol A, an α-Synuclein Inhibitory Sterol from the Australian Ascidian Sycozoa cerebriformis.

During a recent biodiscovery study to identify new α-synuclein (α-syn) aggregation inhibitors, we screened 29 Australian marine sponge and ascidian extracts in an MS binding assay. This resulted in an extract from the ascidian Sycozoa cerebriformis showing activity toward α-syn. The bioassay and MS guided isolation process led to the identification of one new polyoxygenated sterol sulfate, sycosterol A (1). The structure of this low-yielding steroid was elucidated from HRMS and NMR analysis. Sycosterol A displayed moderate antiaggregation activity with 46.2% (±1.8) inhibition when screened against α-syn at a 5:1 (sycosterol A:α-syn) molar ratio. The α-syn antiaggregation activity displayed by 1 and the recent discovery of similar sterols with α-syn antiaggregation activity and potent antiprion activity suggest this unique class may be useful antineurodegenerative compounds.

Design, synthesis and structure-activity evaluation of novel 2-pyridone-based inhibitors of α-synuclein aggregation with potentially improved BBB permeability.

The treatment of Parkinson's disease (PD), the second most common neurodegenerative human disorder, continues to be symptomatic. Development of drugs able to stop or at least slowdown PD progression would benefit several million people worldwide. SynuClean-D is a low molecular weight 2-pyridone-based promising drug candidate that inhibits the aggregation of α-synuclein in human cultured cells and prevents degeneration of dopaminergic neurons in a Caenorhabditis elegans model of PD. Improving SynuClean-D pharmacokinetic/pharmacodynamic properties, performing structure/activity studies and testing its efficacy in mammalian models of PD requires the use of gr-amounts of the compound. However, not enough compound is on sale, and no synthetic route has been reported until now, which hampers the molecule progress towards clinical trials. To circumvent those problems, we describe here an efficient and economical route that enables the synthesis of SynuClean-D with good yields as well as the synthesis of SynuClean-D derivatives. Structure-activity comparison of the new compounds with SynuClean-D reveals the functional groups of the molecule that can be disposed of without activity loss and those that are crucial to interfere with α-synuclein aggregation. Several of the derivatives obtained retain the parent's compound excellent in vitro anti-aggregative activity, without compromising its low toxicity. Computational predictions and preliminary testing indicate that the blood brain barrier (BBB) permeability of SynuClean-D is low. Importantly, several of the newly designed and obtained active derivatives are predicted to display good BBB permeability. The synthetic route developed here will facilitate their synthesis for BBB permeability determination and for efficacy testing in mammalian models of PD.

(De)stabilization of Alpha-Synuclein Fibrillary Aggregation by Charged and Uncharged Surfactants.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. An important hallmark of PD involves the pathological aggregation of proteins in structures known as Lewy bodies. The major component of these proteinaceous inclusions is alpha (α)-synuclein. In different conditions, α-synuclein can assume conformations rich in either α-helix or ß-sheets. The mechanisms of α-synuclein misfolding, aggregation, and fibrillation remain unknown, but it is thought that ß-sheet conformation of α-synuclein is responsible for its associated toxic mechanisms. To gain fundamental insights into the process of α-synuclein misfolding and aggregation, the secondary structure of this protein in the presence of charged and non-charged surfactant solutions was characterized. The selected surfactants were (anionic) sodium dodecyl sulphate (SDS), (cationic) cetyltrimethylammonium chloride (CTAC), and (uncharged) octyl ß-D-glucopyranoside (OG). The effect of surfactants in α-synuclein misfolding was assessed by ultra-structural analyses, in vitro aggregation assays, and secondary structure analyses. The α-synuclein aggregation in the presence of negatively charged SDS suggests that SDS-monomer complexes stimulate the aggregation process. A reduction in the electrostatic repulsion between N- and C-terminal and in the hydrophobic interactions between the NAC (non-amyloid beta component) region and the C-terminal seems to be important to undergo aggregation. Fourier transform infrared spectroscopy (FTIR) measurements show that ß-sheet structures comprise the assembly of the fibrils.

A Downsized and Optimised Intracellular Library-Derived Peptide Prevents Alpha-Synuclein Primary Nucleation and Toxicity Without Impacting Upon Lipid Binding.

Misfolding and aggregation of alpha-synuclein (αS) within dopaminergic neurons is a key factor in the development and progression of a group of age-related neurodegenerative diseases, termed synucleinopathies, that include Parkinson's disease (PD). We previously derived a peptide inhibitor from a 209,952-member intracellular library screen by employing the preNAC region (45-54) as a design template. At least six single-point mutations firmly linked to early-onset Parkinson's disease (E46K, H50Q, G51D, A53T/E/V) are located within this region, strongly implicating a pathogenic role within αS that leads to increased cytotoxicity. A library-derived ten residue peptide, 4554W, was consequently shown to block αS aggregation at the point of primary nucleation via lipid induction, inhibiting its conversion into downstream cytotoxic species. Here we couple truncation with a full alanine scan analysis, to establish the effect upon the αS aggregation pathway relative to 4554W. This revealed the precise residues responsible for eliciting inhibitory interaction and function, as well as those potentially amenable to modification or functionalisation. We find that modification N6A combined with N-terminal truncation results in a peptide of significantly increased efficacy. Importantly, our data demonstrate that the peptide does not directly disrupt αS lipid-binding, a desirable trait since antagonists of αS aggregation and toxicity should not impede association with small synaptic neurotransmitter vesicles, and thus not disrupt dopaminergic vesicle fusion and recycling. This work paves the way toward the major aim of deriving a highly potent peptide antagonist of αS pathogenicity without impacting on native αS function.

FRET-based assay for intracellular evaluation of α-synuclein aggregation inhibitors.

Aggregation of small neuronal protein α-synuclein (αSyn) in amyloid fibrils is considered to be one of the main causes of Parkinson's disease. Inhibition of this aggregation is a promising approach for disease treatment. Dozens of compounds able to inhibit αSyn fibrillization in solution were developed during the last decade. However, the applicability of most of them in the cellular environment was not established because of the absence of a suitable cell-based assay. In this work, we developed an assay for testing αSyn aggregation inhibitors in cells that is based on fluorescence resonance energy transfer (FRET) between labeled αSyn molecules in fibrils. The assay directly reports the amount of fibrillized αSyn and is more reliable than the assays based on cell viability. Moreover, we showed that cell viability decline does not always correlate with the amount of misfolded αSyn. The developed FRET-based assay does not interfere with the aggregation process and is suitable for high-throughput testing of αSyn aggregation inhibitors. Its application can sort out non-specific inhibitors and thus significantly facilitate the development of drugs for Parkinson`s disease.

Discovery of styrylaniline derivatives as novel alpha-synuclein aggregates ligands.

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Early diagnosis is the key to treatment but is still a great challenge in the clinic now. The discovery of alpha-synuclein (α-syn) aggregates ligands has become an attractive strategy to meet the early diagnosis of PD. Herein, we designed and synthesized a series of styrylaniline derivatives as novel α-syn aggregates ligands. Several compounds displayed good potency to α-syn aggregates with Kd values less than 0.1 µM. The docking study revealed that the hydrogen bonds and cation-pi interaction between ligands and α-syn aggregates would be crucial for the activity. The representative compound 7-16 not only detected α-syn aggregates in both SH-SY5Y cells and brain tissues prepared from two kinds of α-syn preformed-fibrils-injected mice models but also showed good blood-brain barrier penetration characteristics in vivo with a brain/plasma ratio over 1.0, which demonstrates its potential as a lead compound for further development of in vivo imaging agents.

Tat-p27 Ameliorates Neuronal Damage Reducing α-Synuclein and Inflammatory Responses in Motor Neurons After Spinal Cord Ischemia.

p27Kip1 (p27) regulates the cell cycle by inhibiting G1 progression in cells. Several studies have shown conflicting results on the effects of p27 against cell death in various insults. In the present study, we examined the neuroprotective effects of p27 against H2O2-induced oxidative stress in NSC34 cells and against spinal cord ischemia-induced neuronal damage in rabbits. To promote delivery into NSC34 cells and motor neurons in the spinal cord, Tat-p27 fusion protein and its control protein (Control-p27) were synthesized with or without Tat peptide, respectively. Tat-p27, but not Control-27, was efficiently introduced into NSC34 cells in a concentration- and time-dependent manner, and the protein was detected in the cytoplasm. Tat-p27 showed neuroprotective effects against oxidative stress induced by H2O2 treatment and reduced the formation of reactive oxygen species, DNA fragmentation, and lipid peroxidation in NSC34 cells. Tat-p27, but not Control-p27, ameliorated ischemia-induced neurological deficits and cell damage in the rabbit spinal cord. In addition, Tat-p27 treatment reduced the expression of α-synuclein, activation of microglia, and release of pro-inflammatory cytokines such as interleukin-1ß and tumor necrosis factor-α in the spinal cord. Taken together, these results suggest that Tat-p27 inhibits neuronal damage by decreasing oxidative stress, α-synuclein expression, and inflammatory responses after ischemia.