Design, synthesis, structure-activity relationship studies, and evaluation of novel GLS1 inhibitors.

Glutaminase converts glutamine into glutamic acid and has two isoforms: glutaminase 1 (GLS1) and glutaminase 2 (GLS2). GLS1 is overexpressed in several tumors, and research to develop glutaminase inhibitors as antitumor drugs is currently underway. The present study examined candidate GLS1 inhibitors using in silico screening and attempted to synthesize novel GLS1 inhibitors and assess their GLS1 inhibitory activities in a mouse kidney extract and against recombinant mouse and human GLS1. Novel compounds were synthesized using compound C as the lead compound, and their GLS1 inhibitory activities were evaluated using the mouse kidney extract. Among the derivatives tested, the trans-4-hydroxycyclohexylamide derivative 2j exhibited the strongest inhibitory activity. We also assessed the GLS1 inhibitory activities of the derivatives 2j, 5i, and 8a against recombinant mouse and human GLS1. The derivatives 5i and 8a significantly decreased the production of glutamic acid at 10 mM. In conclusion, we herein identified two compounds that exhibited GLS1 inhibitory activities with equal potencies as known GLS1 inhibitors. These results will contribute to the development of effective novel GLS1 inhibitors with more potent inhibitory activity.

Design and structural optimization of thiadiazole derivatives with potent GLS1 inhibitory activity.

GLS1 is an attractive target not only as anticancer agents but also as candidates for various potential pharmaceutical applications such as anti-aging and anti-obesity treatments. We performed docking simulations based on the complex crystal structure of GLS1 and its inhibitor CB-839 and found that compound A bearing a thiadiazole skeleton exhibits GLS1 inhibition. Furthermore, we synthesized 27 thiadiazole derivatives in an effort to obtain a more potent GLS1 inhibitor. Among the synthesized derivatives, 4d showed more potent GLS1 inhibitory activity (IC50 of 46.7 µM) than known GLS1 inhibitor DON and A. Therefore, 4d is a very promising novel GLS1 inhibitor.

Benziodarone and 6-hydroxybenziodarone are potent and selective inhibitors of transthyretin amyloidogenesis.

Transthyretin amyloidosis is a progressive systemic disorder that is caused by the amyloid deposition of transthyretin in various organs. Stabilization of the native transthyretin is an effective strategy for the treatment of transthyretin amyloidosis. In this study we demonstrate that the clinically used uricosuric agent benziodarone is highly effective to stabilize the tetrameric structure of transthyretin. An acid-induced aggregation assay showed that benziodarone had strong inhibitory activity similar to that of tafamidis, which is currently used as a therapeutic agent for transthyretin amyloidosis. Moreover, a possible metabolite, 6-hydroxybenziodarone, retained the strong amyloid inhibitory activity of benziodarone. An ex vivo competitive binding assay using a fluorogenic probe showed that benziodarone and 6-hydroxybenziodarone were highly potent for selective binding to transthyretin in human plasma. An X-ray crystal structure analysis revealed that the halogenated hydroxyphenyl ring was located at the entrance of the thyroxine binding channel of transthyretin and that the benzofuran ring was located in the inner channel. These studies suggest that benziodarone and 6-hydroxybenziodarone would potentially be effective against transthyretin amyloidosis.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

The hydrophobic residue Leu73 is crucial for the high stability and low aggregation properties of murine transthyretin.

Destabilization of human transthyretin leads to its aggregation into amyloid fibrils, which causes a rare, progressive and fatal systemic disorder called ATTR amyloidosis. By contrast, murine transthyretin is known to be very stable and therefore does not aggregate into amyloid fibrils in vivo or in vitro. We examined the hydrophobic residues responsible for the high-stability and low-aggregation properties of murine transthyretin using site-directed mutagenesis. Urea-induced unfolding and thioflavin T fluorescence aggregation assay revealed that Leu73 of murine transthyretin largely contributes to its high stability and low aggregation properties: the I73L mutation stabilized human transthyretin, while the L73I mutation destabilized murine transthyretin. In addition, the I26V/I73L mutation stabilized the amyloidogenic V30M mutant of human transthyretin to the same degree as the suppressor mutation T119M, which protects transthyretin against amyloid fibril aggregation. The I73L mutation resulted in no significant differences in the overall structure of the transthyretin tetramer or the contacts of side-chains in the hydrophobic core of the monomer. We also found that Leu73 of murine transthyretin is conserved in many mammals, while Ile73 of human transthyretin is conserved in monkeys and cats. These studies will provide new insights into the stability and aggregation properties of transthyretin from various mammals.

Determination of Hardness of a Pharmaceutical Oral Jelly by Using T2 Relaxation Behavior Measured by Time-Domain NMR.

Hardness is a critical quality characteristic of pharmaceutical oral jelly. In this study, the hardness was determined by using the T2 relaxation curves measured by time-domain NMR. For sample preparation, kappa- and iota-carrageenans, and locust bean gum, were used as gel-forming agents. Ten test jellies with different gel-forming agent composition were prepared, and their hardness and T2 relaxation curves were measured by a texture analyzer and time-domain NMR (TD-NMR). A negative correlation between T2 relaxation time (T2) and hardness was observed; however, it was difficult to determine the hardness directly from the T2 value. That is probably because the T2 relaxation curve contains information about molecular states, not only of water but also of the solute, and T2 values calculated by single-exponential curve fitting only express one property of the test jelly. By considering this issue, partial least squares (PLS) regression analysis was performed on the T2 relaxation curves for hardness determination of the test jellies. According to the analysis, an accurate and reliable PLS model was created that enabled accurate assessment of the hardness of the test jellies. TD-NMR enables the measurement of samples nondestructively and rapidly with low cost, and so could be a promising method for evaluation of the hardness of pharmaceutical oral jellies.

A low amyloidogenic E61K transthyretin mutation may cause familial amyloid polyneuropathy.

Patients with transthyretin (TTR)-type familial amyloid polyneuropathy (FAP) typically exhibit sensory dominant polyneuropathy and autonomic neuropathy. However, the molecular pathogenesis of the neuropathy remains unclear. In this study, we characterize the features of FAP TTR the substitution of lysine for glutamic acid at position 61 (E61K). This FAP was late-onset, with sensory dominant polyneuropathy, autonomic neuropathy, and cardiac amyloidosis. Interestingly, no amyloid deposits were found in the endoneurium of the four nerve specimens examined. Therefore, we examined the amyloidogenic properties of E61K TTR in vitro. Recombinant wild-type TTR, the substitution of methionine for valine at position 30 (V30M) TTR, and E61K TTR proteins were incubated at 37°C for 72 hr, and amyloid fibril formation was assessed using the thioflavin-T binding assay. Amyloid fibril formation by E61K TTR was less than that by V30M TTR, and similar to that by wild-type TTR. E61K TTR did not have an inhibitory effect on neurite outgrowth from adult rat dorsal root ganglion (DRG) neurons, but V30M TTR did. Furthermore, we studied the sural nerve of our patient by terminal deoxynucleotidyl transferase dUTP nick end labeling and electron microscopy. A number of apoptotic cells were observed in the endoneurium of the nerve by transferase dUTP nick end labeling. Chromatin condensation was confirmed in the nucleus of non-myelinating Schwann cells by electron microscopy. These findings suggest that E61K TTR is low amyloidogenic, in vitro and in vivo. However, TTR aggregates and amyloid fibrils in the DRG may cause sensory impairments in FAP because the DRG has no blood-nerve barrier. Moreover, Schwann cell apoptosis may contribute to the neurodegeneration.

Expression of SOLOIST/MRTFB i4, a novel neuronal isoform of the mouse serum response factor coactivator myocardin-related transcription factor-B, negatively regulates dendritic complexity in cortical neurons.

Megakaryoblastic leukemia 2 (MKL2)/myocardin-related transcription factor-B (MRTFB), a serum response factor (SRF) coactivator, is an important regulator of gene expression and neuronal morphology. Here, we show that different mouse MRTFB splice isoforms, including a novel fourth MRTFB isoform named spliced neuronal long isoform of SRF transcriptional coactivator (SOLOIST)/MRTFB isoform 4 (MRTFB i4), play distinct roles in this process. SOLOIST/MRTFB i4 has a short exon that encodes 21 amino acid residues ahead of the first RPXXXEL (RPEL) motif in MRTFB isoform 3. Quantitative PCR revealed that SOLOIST/MRTFB i4 and isoform 1 were enriched in the forebrain and neurons, and up-regulated during brain development. Conversely, isoform 3 was detected in various tissues, including both neurons and astrocytes, and was down-regulated in the developing brain. Reporter assays supported the SRF-coactivator function of SOLOIST/MRTFB i4 as well as isoform 1. Acute expression of MRTFB isoform 1, but not isoform 3 or SOLOIST/MRTFB i4, in neuronal cells within 24 hr drastically increased endogenous immediate early gene [c-fos, egr1, and activity-regulated cytoskeleton-associated protein] expression, but not endogenous actinin α1, ß-actin, gelsolin, or srf gene expression measured by qPCR. Over-expression of SOLOIST/MRTFB i4 reduced the dendritic complexity of cortical neurons, whereas over-expression of isoform 1 increased this complexity. Co-expression of isoform 1 and SOLOIST/MRTFB i4 in cortical neurons revealed that isoform 1 competitively counteracted down-regulation by SOLOIST/MRTFB i4. Our findings indicate that MRTFB isoforms have unique expression patterns and differential effects on gene expression and dendritic complexity, which contribute to shaping neuronal circuits, at least in part.

Inhibitory activities of anthraquinone and xanthone derivatives against transthyretin amyloidogenesis.

Transthyretin is a tetrameric protein which functions as a transporter of thyroxine and retinol-binding protein. Misfolding and amyloid aggregation of transthyretin are known to cause wild-type and hereditary transthyretin amyloidosis. Stabilization of the transthyretin tetramer by low molecular weight compounds is an efficacious strategy to inhibit the aggregation pathway in the amyloidosis. Here, we investigated the inhibitory activities of anthraquinone and xanthone derivatives against amyloid aggregation, and found that xanthone-2-carboxylic acid with one chlorine or methyl group has strong inhibitory activity comparable with that of diflunisal, which is one of the best known stabilizers of transthyretin. X-ray crystallographic structures of transthyretin in complex with the compounds revealed that the introduction of chlorine, which is buried in a hydrophobic region, is important for the strong inhibitory effect of the stabilizer against amyloidogenesis. An in vitro absorption, distribution, metabolism and elimination (ADME) study and in vivo pharmacokinetic study demonstrated that the compounds have drug-like features, suggesting that they have potential as therapeutic agents to stabilize transthyretin.

A cell-based high-throughput screening method to directly examine transthyretin amyloid fibril formation at neutral pH.

Transthyretin (TTR) is a major amyloidogenic protein associated with hereditary (ATTRm) and nonhereditary (ATTRwt) intractable systemic transthyretin amyloidosis. The pathological mechanisms of ATTR-associated amyloid fibril formation are incompletely understood, and there is a need for identifying compounds that target ATTR. C-terminal TTR fragments are often present in amyloid-laden tissues of most patients with ATTR amyloidosis, and on the basis of in vitro studies, these fragments have been proposed to play important roles in amyloid formation. Here, we found that experimentally-formed aggregates of full-length TTR are cleaved into C-terminal fragments, which were also identified in patients' amyloid-laden tissues and in SH-SY5Y neuronal and U87MG glial cells. We observed that a 5-kDa C-terminal fragment of TTR, TTR81-127, is highly amyloidogenic in vitro, even at neutral pH. This fragment formed amyloid deposits and induced apoptosis and inflammatory gene expression also in cultured cells. Using the highly amyloidogenic TTR81-127 fragment, we developed a cell-based high-throughput screening method to discover compounds that disrupt TTR amyloid fibrils. Screening a library of 1280 off-patent drugs, we identified two candidate repositioning drugs, pyrvinium pamoate and apomorphine hydrochloride. Both drugs disrupted patient-derived TTR amyloid fibrils ex vivo, and pyrvinium pamoate also stabilized the tetrameric structure of TTR ex vivo in patient plasma. We conclude that our TTR81-127-based screening method is very useful for discovering therapeutic drugs that directly disrupt amyloid fibrils. We propose that repositioning pyrvinium pamoate and apomorphine hydrochloride as TTR amyloid-disrupting agents may enable evaluation of their clinical utility for managing ATTR amyloidosis.

The Accumulation of Heparan Sulfate S-Domains in Kidney Transthyretin Deposits Accelerates Fibril Formation and Promotes Cytotoxicity.

The highly sulfated domains of heparan sulfate (HS), alias HS S-domains, are made up of repeated trisulfated disaccharide units [iduronic acid (2S)-glucosamine (NS, 6S)] and are selectively remodeled by extracellular endoglucosamine 6-sulfatases (Sulfs). Although HS S-domains are critical for signal transduction of several growth factors, their roles in amyloidoses are not yet fully understood. Herein, we found HS S-domains in the kidney of a patient with transthyretin amyloidosis. In in vitro assays with cells stably expressing human Sulfs, heparin, a structural analog of HS S-domains, promoted aggregation of transthyretin in an HS S-domain-dependent manner. Interactions of cells with transthyretin fibrils and cytotoxicity of these fibrils also depended on HS S-domains at the cell surface. Furthermore, glypican-5, encoded by the susceptibility gene for nephrotic syndrome GPC5, was found to be accumulated in the transthyretin amyloidosis kidney. Our study, thus, provides a novel insight into the pathologic roles of HS S-domains in amyloidoses, and we propose that enzymatic remodeling of HS chains by Sulfs may offer an effective approach to inhibiting formation and cytotoxicity of amyloid fibrils.

Transthyretin Amyloid Fibril Disrupting Activities of Extracts and Fractions from Juglans mandshurica Maxim. var. cordiformis (Makino) Kitam.

Transthyretin-related amyloidosis is a slowly progressive disorder caused by deposition of insoluble amyloid plaques formed by fibrillization of mutant or defective transthyretin (TTR) monomers that leads to neurodegeneration and organ failure. Thus, any compound exhibiting TTR amyloid formation inhibitory activity or TTR amyloid fibril disrupting activity might be a potential candidate for the development of therapies for these disorders. Our aim in this study was the evaluation of the TTR amyloid fibril disrupting potential of extracts of leaves and immature fruits of two Juglans plants, i.e., Juglans mandshurica var. sachalinensis and Juglans mandshurica var. cordiformis. The TTR amyloid fibril disrupting activity was measured by Thioflavin-T (ThT) assay and PROTEOSTAT® Protein aggregation assay methods. A fifty percent acetone extract of the fruits of Juglans mandshurica var. cordiformis showed strong amyloid fibril disrupting activity, and was further fractionated using different solvents. Ethyl acetate and n-butanol fractions showed significant activity in both assays. Syringic acid was isolated and identified as main compound in both of these fractions; however, it did not show any activity. Furthermore, some of the previously reported compounds from Juglans plants including naphthoquinone derivatives and phenolic compounds were evaluated to identify the potential bioactive compounds. Among them, juglone, a naphthoquinone derivative showed promising activity. However, juglone also showed strong cytotoxicity in HEK293 cells. Thus, future studies should focus on the isolation and identification of naphthoquinone derivatives or other compounds from Juglans plan ts with potent bioactivity and low cytotoxicity.

Design, synthesis, and evaluation of novel inhibitors for wild-type human serine racemase.

Most of the endogenous free d-serine (about 90%) in the brain is produced by serine racemase (SR). d-Serine in the brain is involved in neurodegenerative disorders and epileptic states as an endogenous co-agonist of the NMDA-type glutamate receptor. Thus, SR inhibitors are expected to be novel therapeutic candidates for the treatment of these disorders. In this study, we solved the crystal structure of wild-type SR, and tried to identify a new inhibitor of SR by in silico screening using the structural information. As a result, we identified two hit compounds by their in vitro evaluations using wild-type SR. Based on the structure of the more potent hit compound 1, we synthesized 15 derivatives and evaluated their inhibitory activities against wild-type SR. Among them, the compound 9C showed relatively high inhibitory potency for wild-type SR. Compound 9C was a more potent inhibitor than compound 24, which was synthesized by our group based upon the structural information of the mutant-type SR.

Inhibition of the Amyloidogenesis of Transthyretin by Natural Products and Synthetic Compounds.

Hereditary transthyretin (TTR)-related amyloidosis is caused by mutations in the TTR gene. The mutations destabilize the tetramer and/or monomer of TTR, and thus the stabilization of TTR is a key strategy for the treatment of TTR-related amyloidosis. In this review, we summarized the natural products and synthetic compounds that have been shown to inhibit the amyloidogenesis of TTR. The stabilizers and/or the amyloid fibril disrupters isolated from natural sources may become lead compounds for the treatment of TTR-related amyloidosis.

A novel serine racemase inhibitor suppresses neuronal over-activation in vivo.

Serine racemase (SRR) is an enzyme that produces d-serine from l-serine. d-Serine acts as an endogenous coagonist of NMDA-type glutamate receptors (NMDARs), which regulate many physiological functions. Over-activation of NMDARs induces excitotoxicity, which is observed in many neurodegenerative disorders and epilepsy states. In our previous works on the generation of SRR gene knockout (Srr-KO) mice and its protective effects against NMDA- and Aß peptide-induced neurodegeneration, we hypothesized that the regulation of NMDARs' over-activation by inhibition of SRR activity is one such therapeutic strategy to combat these disease states. In the previous study, we performed in silico screening to identify four compounds with inhibitory activities against recombinant SRR. Here, we synthesized 21 derivatives of candidate 1, one of four hit compounds, and performed screening by in vitro evaluations. The derivative 13J showed a significantly lower IC50 value in vitro, and suppressed neuronal over-activation in vivo.

Rifampicin is a candidate preventive medicine against amyloid-ß and tau oligomers.

Amyloid-ß, tau, and α-synuclein, or more specifically their soluble oligomers, are the aetiologic molecules in Alzheimer's disease, tauopathies, and α-synucleinopathies, respectively. These proteins have been shown to interact to accelerate each other's pathology. Clinical studies of amyloid-ß-targeting therapies in Alzheimer's disease have revealed that the treatments after disease onset have little benefit on patient cognition. These findings prompted us to explore a preventive medicine which is orally available, has few adverse effects, and is effective at reducing neurotoxic oligomers with a broad spectrum. We initially tested five candidate compounds: rifampicin, curcumin, epigallocatechin-3-gallate, myricetin, and scyllo-inositol, in cells expressing amyloid precursor protein (APP) with the Osaka (E693Δ) mutation, which promotes amyloid-ß oligomerization. Among these compounds, rifampicin, a well-known antibiotic, showed the strongest activities against the accumulation and toxicity (i.e. cytochrome c release from mitochondria) of intracellular amyloid-ß oligomers. Under cell-free conditions, rifampicin inhibited oligomer formation of amyloid-ß, tau, and α-synuclein, indicating its broad spectrum. The inhibitory effects of rifampicin against amyloid-ß and tau oligomers were evaluated in APPOSK mice (amyloid-ß oligomer model), Tg2576 mice (Alzheimer's disease model), and tau609 mice (tauopathy model). When orally administered to 17-month-old APPOSK mice at 0.5 and 1 mg/day for 1 month, rifampicin reduced the accumulation of amyloid-ß oligomers as well as tau hyperphosphorylation, synapse loss, and microglial activation in a dose-dependent manner. In the Morris water maze, rifampicin at 1 mg/day improved memory of the mice to a level similar to that in non-transgenic littermates. Rifampicin also inhibited cytochrome c release from the mitochondria and caspase 3 activation in the hippocampus. In 13-month-old Tg2576 mice, oral rifampicin at 0.5 mg/day for 1 month decreased amyloid-ß oligomer accumulation, tau hyperphosphorylation, synapse loss, and microglial activation, but not amyloid deposition. Rifampicin treatment to 14-15-month-old tau609 mice at 0.5 and 1 mg/day for 1 month also reduced tau oligomer accumulation, tau hyperphosphorylation, synapse loss, and microglial activation in a dose-dependent fashion, and improved the memory almost completely at 1 mg/day. In addition, rifampicin decreased the level of p62/sequestosome-1 in the brain without affecting the increased levels of LC3 (microtubule-associated protein light chain 3) conversion, suggesting the restoration of autophagy-lysosomal function. Considering its prescribed dose and safety in humans, these results indicate that rifampicin could be a promising, ready-to-use medicine for the prevention of Alzheimer's disease and other neurodegenerative diseases.

Exogenous amyloidogenic proteins function as seeds in amyloid ß-protein aggregation.

Amyloid ß-protein (Aß) aggregation is considered to be a critical step in the neurodegeneration of Alzheimer's disease (AD). In addition to Aß, many proteins aggregate into the amyloid state, in which they form elongated fibers with spines comprising stranded ß-sheets. However, the cross-seeding effects of other protein aggregates on Aß aggregation pathways are not completely clear. To investigate the cross-seeding effects of exogenous and human non-CNS amyloidogenic proteins on Aß aggregation pathways, we examined whether and how sonicated fibrils of casein, fibroin, sericin, actin, and islet amyloid polypeptide affected Aß40 and Aß42 aggregation pathways using the thioflavin T assay and electron microscopy. Interestingly, the fibrillar seeds of all amyloidogenic proteins functioned as seeds. The cross-seeding effect of actin was stronger but that of fibroin was weaker than that of other proteins. Furthermore, our nuclear magnetic resonance spectroscopic studies identified the binding sites of Aß with the amyloidogenic proteins. Our results indicate that the amyloidogenic proteins, including those contained in foods and cosmetics, contribute to Aß aggregation by binding to Aß, suggesting their possible roles in the propagation of Aß amyloidosis.

Interaction between tachyplesin I, an antimicrobial peptide derived from horseshoe crab, and lipopolysaccharide.

Lipopolysaccharide (LPS) is a major constituent of the outer membrane of Gram-negative bacteria and is the very first site of interactions with antimicrobial peptides (AMPs). In order to gain better insight into the interaction between LPS and AMPs, we determined the structure of tachyplesin I (TP I), an antimicrobial peptide derived from horseshoe crab, in its bound state with LPS and proposed the complex structure of TP I and LPS using a docking program. CD and NMR measurements revealed that binding to LPS slightly extends the two ß-strands of TP I and stabilizes the whole structure of TP I. The fluorescence wavelength of an intrinsic tryptophan of TP I and fluorescence quenching in the presence or absence of LPS indicated that a tryptophan residue is incorporated into the hydrophobic environment of LPS. Finally, we succeeded in proposing a structural model for the complex of TP I and LPS by using a docking program. The calculated model structure suggested that the cationic residues of TP I interact with phosphate groups and saccharides of LPS, whereas hydrophobic residues interact with the acyl chains of LPS.

Phenolic compounds prevent the oligomerization of α-synuclein and reduce synaptic toxicity.

Lewy bodies, mainly composed of α-synuclein (αS), are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies. Epidemiological studies showed that green tea consumption or habitual intake of phenolic compounds reduced Parkinson's disease risk. We previously reported that phenolic compounds inhibited αS fibrillation and destabilized preformed αS fibrils. Cumulative evidence suggests that low-order αS oligomers are neurotoxic and critical species in the pathogenesis of α-synucleinopathies. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds (myricetin (Myr), curcumin, rosmarinic acid (RA), nordihydroguaiaretic acid, and ferulic acid) on αS oligomerization. Using methods such as photo-induced cross-linking of unmodified proteins, circular dichroism spectroscopy, the electron microscope, and the atomic force microscope, we showed that Myr and RA inhibited αS oligomerization and secondary structure conversion. The nuclear magnetic resonance analysis revealed that Myr directly bound to the N-terminal region of αS, whereas direct binding of RA to monomeric αS was not detected. Electrophysiological assays for long-term potentiation in mouse hippocampal slices revealed that Myr and RA ameliorated αS synaptic toxicity by inhibition of αS oligomerization. These results suggest that Myr and RA prevent the αS aggregation process, reducing the neurotoxicity of αS oligomers. To develop disease modifying therapies for α-synucleinopathies, we examined effects of phenolic compounds on α-synuclein (αS) oligomerization. Phenolic compounds, especially Myricetin (Myr) and Rosmarinic acid (RA), inhibited αS oligomerization and secondary structure conversion. Myr and RA ameliorated αS synaptic toxicity on the experiment of long-term potentiation. Our results suggest that Myr and RA prevent αS aggregation process and reduce the neurotoxicity of αS oligomers. Phenolic compounds are good candidates of disease modifying drugs for α-synucleinopathies.

Structural stabilization of transthyretin by a new compound, 6-benzoyl-2-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione.

Familial amyloid polyneuropathy (FAP) is a genetic, adult-onset, neurodegenerative disorder caused by amyloid formation of transthyretin (TTR), a thyroxine-binding protein. Mutation in TTR causes a propensity of TTR tetramer to dissociate to monomer, which is the first step to amyloidosis. Thus, a drug that can stabilize the tetramer structure will have therapeutic benefit. Here, by virtual screening and biochemical assays, we identified small molecule 6-benzoyl-2-hydroxy-1H-benzo[de]isoquinoline-1,3(2H)-dione (L6) that can prevent the dissociation of TTR to monomer. X-ray crystallography reveals that L6 binds to the T4 binding pocket of TTR. These findings show that L6 is a candidate TTR stabilizer.