A RaPID Macrocyclic Peptide That Inhibits the Formation of α-Synuclein Amyloid Fibrils

There is considerable interest in drug discovery targeting the aggregation of α-synuclein (αSyn) since this molecular process is closely associated with Parkinson's disease. However, inhibiting αSyn aggregation remains a major challenge because of its highly dynamic nature which makes it difficult to form a stable binding complex with a drug molecule. Here, by exploiting Random non-standard Peptides Integrated Discovery (RaPID) system, we identified a macrocyclic peptide, BD1, that could interact with immobilized αSyn and inhibit the formation of fibrils. Furthermore, improving the solubility of BD1 suppresses the co-aggregation with αSyn fibrils while it kinetically inhibits more effectively without change in their morphology. We also revealed the molecular mechanism of kinetic inhibition, where peptides bind to fibril ends of αSyn, thereby preventing further growth of fibrils. These results suggest that our approach for generating non-standard macrocyclic peptides is a promising approach for developing potential therapeutics against neurodegeneration.     

Coordination of Platinum to α-Synuclein Inhibits Filamentous Aggregation in Solution

Accumulation of filamentous aggregates of α-synuclein (AS) in Lewy bodies and neurites is characteristic of neurodegenerative diseases such as Parkinson's disease. Inhibition of AS fibrillation is helpful for understanding of AS aggregate structure and for developing chemical therapies. Herein, we report that the Pt^II-containing antitumor drug cisplatin suppresses filamentous aggregation of AS in solution. Pt^II thus contrasts strongly with reported transition-metal ions such as Mn^II, Fe^III, and Cu^II, which accelerate AS aggregation. Interaction between Pt^II and the side chains of methionine and histidine residues was essential for inhibition of AS fibrillation. Binding of Pt^II to AS did not change the protein′s overall random coil structure, as indicated by solution-state two-dimensional NMR and circular dichroism spectroscopy; and a solution of the AS ⋅ Pt^II complex remained free of filamentous aggregates. Our results constitute interesting new information about the biological chemistry of metal ions in Parkinson's disease and might open new lines of research into the suppression of filamentous aggregation.     

Chromenopyrazole−Peptide Conjugates as Small-Molecule Based Inhibitors Disrupting the Protein−RNA Interaction of LIN28-let-7

Targeting the protein−RNA interaction of LIN28 and let-7 is a promising strategy for the development of novel anticancer therapeutics. However, a limited number of small-molecule inhibitors disrupting the LIN28-let-7 interaction with potent efficacy are available. Herein, we developed a novel LIN28-inhibiting strategy by targeting selective hotspot amino acids at the LIN28-let-7 binding interface with small-molecule-based bifunctional conjugates. Starting from reported small-molecule LIN28 inhibitors, we identified a feasible linker-attachment position after performing a structure-activity relationship exploration based on the LIN28-targeting chromenopyrazoles. In parallel, a virtual alanine scan identified hotspot residues at the protein−RNA binding interface, based on which we designed a set of peptides to enhance the interaction with the identified hotspot residues. Conjugation of the tailor-designed peptides with linker-attached chromenopyrazoles yielded a series of bifunctional small-molecule-peptide conjugates, represented by compound 83 (PH-223), as a new LIN28-targeting chemical modality. Our result demonstrated an unexplored rational design approach using bifunctional conjugates to target protein−RNA interactions.     

Electronic Properties of Amyloid β-Based Peptide Filaments with Different Non-Natural Heterocyclic Side Chains

The incorporation of non-canonical amino acids with aromatic side chains is considered to be a promising way to improve and control the electronic properties of self-assembled peptide nanostructures. In this work, we have studied the influence of aromatic ring heteroatom on the electronic properties of amyloid β-derived peptide fiber networks. We show that the incorporation of furan instead of thiophene side chains results in only small changes in the resistivity of the peptide network, with a threefold increase in the sheet resistance and a small decrease in the contact resistance. These changes can be explained by a twofold decrease in the diameter of the self-assembled fibers. These characteristics open the way to the use of furan- instead of thiophene-based analogues as non-natural side-chain modifications of peptides for electronic applications; this makes the fibrils more biodegradable and biorenewable.     

Synthesis of Novel Pyridine-Carboxylates as Small-Molecule Inhibitors of Human Aspartate/Asparagine-β-Hydroxylase

The human 2-oxoglutarate (2OG)-dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) is a potential medicinal chemistry target for anticancer therapy. AspH is present on the cell surface of invasive cancer cells and accepts epidermal growth factor-like domain (EGFD) substrates with a noncanonical (i. e., Cys 1–2, 3–4, 5–6) disulfide pattern. We report a concise synthesis of C-3-substituted derivatives of pyridine-2,4-dicarboxylic acid (2,4-PDCA) as 2OG competitors for use in SAR studies on AspH inhibition. AspH inhibition was assayed by using a mass spectrometry-based assay with a stable thioether analogue of a natural EGFD AspH substrate. Certain C-3-substituted 2,4-PDCA derivatives were potent AspH inhibitors, manifesting selectivity over some, but not all, other tested human 2OG oxygenases. The results raise questions about the use of pyridine-carboxylate-related 2OG analogues as selective functional probes for specific 2OG oxygenases, and should aid in the development of AspH inhibitors suitable for in vivo use.     

The Design of Potent,Selective and Drug-Like RGD αvβ1 Small-Molecule Inhibitors Derived from non-RGD α4β1 Antagonists

Up to 45 % of deaths in developed nations can be attributed to chronic fibroproliferative diseases, highlighting the need for effective therapies. The RGD (Arg-Gly-Asp) integrin αvβ1 was recently investigated for its role in fibrotic disease, and thus warrants therapeutic targeting. Herein we describe the identification of non-RGD hit small-molecule αvβ1 inhibitors. We show that αvβ1 activity is embedded in a range of published α4β1 (VLA-4) ligands; we also demonstrate how a non-RGD integrin inhibitor (of α4β1 in this case) was converted into a potent non-zwitterionic RGD integrin inhibitor (of αvβ1 in this case). We designed urea ligands with excellent selectivity over α4β1 and the other αv integrins (αvβ3, αvβ5, αvβ6, αvβ8). In silico docking models and density functional theory (DFT) calculations aided the discovery of the lead urea series.     

Chemoenzymatic Semi-synthesis Enables Efficient Production of Isotopically Labeled α-Synuclein with Site-Specific Tyrosine Phosphorylation

Post-translational modifications (PTMs) can affect the normal function and pathology of α-synuclein (αS), an amyloid-fibril-forming protein linked to Parkinson's disease. Phosphorylation of αS Tyr39 has recently been found to display a dose-dependent effect on fibril formation kinetics and to alter the morphology of the fibrils. Existing methods to access site-specifically phosphorylated αS for biochemical studies include total or semi-synthesis by native chemical ligation (NCL) as well as chemoenzymatic methods to phosphorylate peptides, followed by NCL. Here, we investigated a streamlined method to produce large quantities of phosphorylated αS by co-expressing a kinase with a protein fragment in Escherichia coli. We also introduced the use of methyl thioglycolate (MTG) to enable one-pot NCL and desulfurization. We compare our optimized methods to previous reports and show that we can achieve the highest yields of site-specifically phosphorylated protein through chemoenzymatic methods using MTG, and that our strategy is uniquely well suited to producing ¹⁵N-labeled, phosphorylated protein for NMR studies.     

Structure-Guided Synthesis and Evaluation of Small-Molecule Inhibitors Targeting Protein–Protein Interactions of BRCA1 tBRCT Domain

The tandem BRCT domains (tBRCT) of BRCA1 engage phosphoserine-containing motifs in target proteins to propagate intracellular signals initiated by DNA damage, thereby controlling cell cycle arrest and DNA repair. Recently, we identified Bractoppin, the first small-molecule inhibitor of the BRCA1 tBRCT domain, which selectively interrupts BRCA1-mediated cellular responses evoked by DNA damage. Here, we combine structure-guided chemical elaboration, protein mutagenesis and cellular assays to define the structural features responsible for Bractoppin's activity. Bractoppin fails to bind mutant forms of BRCA1 tBRCT bearing K1702A, a key residue mediating phosphopeptide recognition, or F1662R or L1701K that adjoin the pSer-recognition site. However, the M1775R mutation, which engages the Phe residue in the consensus phosphopeptide motif pSer-X-X-Phe, does not affect Bractoppin binding, confirming a binding mode distinct from the substrate phosphopeptide binding. We explored these structural features through structure-guided chemical elaboration and characterized structure–activity relationships (SARs) in biochemical assays. Two analogues, CCBT2088 and CCBT2103 were effective in abrogating BRCA1 foci formation and inhibiting G2 arrest induced by irradiation of cells. Collectively, our findings reveal structural features underlying the activity of a novel inhibitor of phosphopeptide recognition by the BRCA1 tBRCT domain, providing fresh insights to guide the development of inhibitors that target protein–protein interactions.     

Virtual Pharmacophore Screening Identifies Small-Molecule Inhibitors of the Rev1-CT/RIR Protein–Protein Interaction

Translesion synthesis (TLS) has emerged as a mechanism through which several forms of cancer develop acquired resistance to first-line genotoxic chemotherapies by allowing replication to continue in the presence of damaged DNA. Small molecules that inhibit TLS hold promise as a novel class of anticancer agents that can serve to enhance the efficacy of these front-line therapies. We previously used a structure-based rational design approach to identify the phenazopyridine scaffold as an inhibitor of TLS that functions by disrupting the protein–protein interaction (PPI) between the C-terminal domain of the TLS DNA polymerase Rev1 (Rev1-CT) and the Rev1 interacting regions (RIR) of other TLS DNA polymerases. To continue the identification of small molecules that disrupt the Rev1-CT/RIR PPI, we generated a pharmacophore model based on the phenazopyridine scaffold and used it in a structure-based virtual screen. In vitro analysis of promising hits identified several new chemotypes with the ability to disrupt this key TLS PPI. In addition, several of these compounds were found to enhance the efficacy of cisplatin in cultured cells, highlighting their anti-TLS potential.     

Synthesis of α-Ketoamide-Based Stereoselective Calpain-1 Inhibitors as Neuroprotective Agents

Calpain inhibitors have been proposed as drug candidates for neurodegenerative disorders, with ABT-957 entering clinical trials for Alzheimer's disease and mild cognitive impairment. The structure of ABT-957 was very recently disclosed, and trials were terminated owing to inadequate CNS concentrations to obtain a pharmacodynamic effect. The multistep synthesis of an α-ketoamide peptidomimetic inhibitor series potentially including ABT-957 was optimized to yield diastereomerically pure compounds that are potent and selective for calpain-1 over papain and cathepsins B and K. As the final oxidation step, with its optimized synthesis protocol, does not alter the configuration of the substrate, the synthesis of the diastereomeric pair (R)-1-benzyl-N-((S)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide ( 1 c ) and (R)-1-benzyl-N-((R)-4-((4-fluorobenzyl)amino)-3,4-dioxo-1-phenylbutan-2-yl)-5-oxopyrrolidine-2-carboxamide ( 1 g ) was feasible. This allowed the exploration of stereoselective inhibition of calpain-1, with 1 c (IC₅₀=78 nM) being significantly more potent than 1 g . Moreover, inhibitor 1 c restored cognitive function in amnestic mice.     

Copolymerization of α-methylstyrene with N-alkylmaleimides

Summary Alternating copolymers of -methylstyrene (-MeSt) with N-alkylmaleimides (RMI; R=Et, n-Pr, iso-Pr, n-Bu, n-Hex) were prepared in Calvet differential microcalorimeter under different monomer-to-monomer ratios in the feed using AIBN as initiator. The equilibrium constants of CT-complex monomers have low values: 0.02–0.05 L.mol^-1 but the mechanism of copolymerization indicates the participation of CT-complex. Equilibrium constants and rate of decomposition under the TGA conditions are not dependent on steric factors, but the rate of copolymerization decreases with the increase of bulkiness of alkyl substituent. In high conversion copolymerization it was observed that in the presence of an excess of homopolymerizable RMI, alternating copolymers are quantitatively formed prior to the formation of poly(RMI).Dedicated to Professor Dragutin Fle on the occasion of his 70th birthday     

Alteration of Dynein Function Affects α-Synuclein Degradation via the Autophagosome-Lysosome Pathway

Growing evidence suggests that dynein dysfunction may be implicated in the pathogenesis of neurodegeneration. It plays a central role in aggresome formation, the delivery of autophagosome to lysosome for fusion and degradation, which is a pro-survival mechanism essential for the bulk degradation of misfolded proteins and damaged organells. Previous studies reported that dynein dysfuntion was associated with aberrant aggregation of α-synuclein, which is a major component of inclusion bodies in Parkinson’s disease (PD). However, it remains unclear what roles dynein plays in α-synuclein degradation. Our study demonstrated a decrease of dynein expression in neurotoxin-induced PD models in vitro and in vivo, accompanied by an increase of α-synuclein protein level. Dynein down-regulation induced by siRNA resulted in a prolonged half-life of α-synuclein and its over-accumulation in A53T overexpressing PC12 cells. Dynein knockdown also prompted the increase of microtubule-associated protein 1 light chain 3 (LC3-II) and sequestosome 1 (SQSTM1, p62) expression, and the accumulation of autophagic vacuoles. Moreover, dynein suppression impaired the autophagosome fusion with lysosome. In summary, our findings indicate that dynein is critical for the clearance of aberrant α-synuclein via autophagosome-lysosome pathway.     

The Amyloid-β Peptide in Amyloid Formation Processes: Interactions with Blood Proteins and Naturally Occurring Metal Ions

This review describes interactions between the amyloid-β peptide (Aβ) involved in Alzheimer's disease (AD) and endogenous metal ions and proteins, with an emphasis on future potential drug therapies and targets. AD is characterised by loss of neurons, memory, and cognitive functions, and by formation of cerebral senile plaque deposits. These plaques consist mainly of aggregated Aβ peptides. AD pathology includes a) on the molecular level imbalanced concentrations of Aβ peptides and metal ions, and formation of amyloid structures, and b) on the physiological level a combination of inflammatory responses and oxidative stress effects causing neuronal death. Interestingly, certain blood proteins and metal ions can affect the Aβ amyloid aggregation process. These interactions are the topics of the present review. A deeper understanding of these interactions could facilitate new therapeutic strategies against AD. Previous therapeutic approaches and trials are also briefly described.     

Real-Time BODIPY-Binding Assay To Screen Inhibitors of the Early Oligomerization Process of Aβ1–42 Peptide

Misfolding and aggregation of amyloid β1–42 peptide (Aβ1–42) play a central role in the pathogenesis of Alzheimer's disease (AD). Targeting the highly cytotoxic oligomeric species formed during the early stages of the aggregation process represents a promising therapeutic strategy to reduce the toxicity associated with Aβ1–42. Currently, the thioflavin T (ThT) assay is the only established spectrofluorometric method to screen aggregation inhibitors. The success of the ThT assay is that it can detect Aβ1–42 aggregates with high β-sheet content, such as protofibrils or fibrils, which appear in the late aggregation steps. Unfortunately, by using the ThT assay, the detection of inhibitors of early soluble oligomers that present a low β-sheet character is challenging. Herein, a new, facile, and robust boron-dipyrromethene (BODIPY) real-time assay suitable for 96-well plate format, which allows screening of compounds as selective inhibitors of the formation of Aβ1–42 oligomers, is reported. These inhibitors decrease the cellular toxicity of Aβ1–42, although they fail in the ThT assay. The findings have been confirmed and validated by structural analysis and cell viability assays under comparable experimental conditions. It is demonstrated that the BODIPY assay is a convenient method to screen and discover new candidate compounds that slow down or stop the pathological early oligomerization process and are active in the cellular assay. Therefore, it is a suitable complementary screening method of the current ThT assay.     

Terminal Alkynes as Raman Probes of α-Synuclein in Solution and in Cells

Conformational changes in α-synuclein (α-syn) are central to its biological function and Parkinson's disease pathology. Here, terminal alkynes (homopropargylglycine) were employed as environmentally sensitive Raman probes at residues 1, 5, 116, and 127 to characterize soluble (disordered), micelle-bound (α-helical), and fibrillar (β-sheet) α-syn. Along with the full-length protein, a disease-related C-terminal truncation (1–115) was also studied. For the first time, β-sheet α-syn amyloid structure was detected by the amide-I band in N27 dopaminergic rat cells, where a reciprocal relationship between levels of fibrils and lipids was seen. Site-specific spectral features of the terminal alkynes also revealed the heterogeneity of the cellular environment. This work shows the versatility of Raman microspectroscopy and the power of unnatural amino acids in providing structural and residue-level insights in solution and in cells.     

Targeted Suppression of Chaperone-Mediated Autophagy by miR-320a Promotes α-Synuclein Aggregation

Chaperone-mediated autophagy (CMA) is involved in wild-type α-synuclein degradation in Parkinson’s disease (PD), and LAMP2A and Hsc 70 have recently been indicated to be deregulated by microRNAs. To recognize the regularory role of miR-320a in CMA and the possible role in α-synuclein degradation, in the present study, we examined the targeting and regulating role of miR-320 in Hsc 70 expression. We first constructed an α-synuclein-overexpressed human neuroblastoma cell line, SH-SY5Y-Syn(+), stably over-expressing wild-type α-synuclein and sensitive to an autophagy inhibitor, which exerted no effect on the expression of LAMP2A and Hsc 70. Then we evaluated the influence on the CMA by miR-320a in the SH-SY5Y-Syn(+) cells. It was shown that miR-320a mimics transfection of specifically targeted Hsc 70 and reduced its expression at both mRNA and protein levels, however, the other key CMA molecule, LAMP2A was not regulated by miR-320a. Further, the reduced Hsc 70 attenuated the α-synuclein degradation in the SH-SY5Y-Syn(+) cells, and induced a significantly high level of α-synuclein accumulation. In conclusion, we demonstrate that miR-320a specifically targeted the 3'' UTR of Hsc 70, decreased Hsc 70 expression at both protein and mRNA levels in α-synuclein-over-expressed SH-SY5Y cells, and resulted in significant α-synuclein intracellular accumulation. These results imply that miR-320a might be implicated in the α-synuclein aggravation in PD.     

Sorption of Acid Dyes onto Silica Modified with Cetyltrimethylammonium Cations

The sorption behavior of acid dyes onto cetyltrimethylammonium bromide (CTAB)- modified silica as a function of pH in the aqueous medium was studied. Single- and multi-solute sorption equilibria of orange Ⅱ(OR), phenol red (PR) and Eriochrome Black T (EBT) were studied at pH 3, unbuffered water pH and pH 11. Sorption behavior of EBT could not be conducted at pH 3 due to its aggregation in acidic medium. All the reaction conditions, experimental protocols and techniques remained the same throughout the sorption process. Sorption isotherms for single-solute system were fitted by the Langmuir model, while Langmuir competitive model (LCM) and the ideal adsorbed solution theory (IAST) coupled with Langmuir model (IAST/Langmuir) were used for the prediction of multisolute competitive sorption. Sorption affinities influenced by the factors like physical interactive forces between the molecules of CTA on silica and sorbate, structural limitations of the dyes based on their geometrical arrangement were investiga     

Rheological behavior of liquefied α-cellulose with phenol

The steady rheological behavior of phenol-liquefied cellulose product (LCP) at varying liquefaction times was investigated at three testing temperatures. The LCP presented interesting rheological behavior. When liquefaction time was longer than 45?min, the LCP showed stronger shear-thinning behavior with increasing temperature, and the viscosity at high testing temperature was higher than that at low testing temperature at low shear rates. An experiment with a heating and cooling cycle indicated that LCP would form reversible association structures when temperature increased. The mechanism of the reversed rheological behavior of LCP may be attributed to the association of alkyl chain parts of liquefied cellulose when heating.