Amyloid Fibril Inhibitors Research Articles

Relaxation of Steric Strains of TTR-Type Amyloid Fibril Inhibitors Radically Changes the Results of Their Virtual Screening

Relaxation of Steric Strains of TTR-Type Amyloid Fibril Inhibitors Radically Changes the Results of Their Virtual Screening

Structural Insights into the Amyloid Fibril Polymorphism Using an Isotope-Edited Vibrational Circular Dichroism Study at the Amino Acid Residue Level.

Polymorphism is common in both in vitro and in vivo amyloid fibrils formed by the same peptide/protein. However, the differences in their self-assembled structures at the amino acid level remain poorly understood. In this study, we utilized isotope-edited vibrational circular dichroism (VCD) on a well-known amyloidogenic peptide fragment (N22FGAIL27) of human islet amyloid polypeptide (IAPf) to investigate the structural polymorphism. Two individual isotope-labeled IAPf peptides were used, with a 13C label on the carbonyl group of phenylalanine (IAPf-F) and glycine (IAPf-G). We compared the amyloid-like nanofibril of IAPf induced by solvent casting (fibril B) with our previous report on the same IAPf peptide fibril but with a different fibril morphology (fibril A) formed in an aqueous buffer solution. Fibril B consisted of entangled, laterally fused amyloid-like nanofibrils with a relatively shorter diameter (15-50 nm) and longer length (several microns), while fibril A displayed nanofibrils with a higher diameter (30-60 nm) and shorter length (500 nm-2 μm). The isotope-edited VCD analysis indicated that fibrils B consisted of anti-parallel β-sheet arrangements with glycine residues in the registry and phenylalanine residues out of the registry, which was significantly different from fibrils A, where a mixture of parallel β-sheet and turn structure with the registry at phenylalanine and glycine residues was observed. The VCD analysis, therefore, suggests that polymorphism in amyloid-like fibrils can be attributed to the difference in the packing/arrangement of the individual β-strands in the β-sheet and the difference in the amino acid registry. Our findings provide insights into the structural aspects of fibril polymorphism related to various amyloid diseases and may aid in designing amyloid fibril inhibitors for therapeutic purposes.

Effects of amino acid-functionalized silver nanoparticles on lysozyme amyloid fibrillogenesis

Amyloid fibrillogenesis of proteins have been considered as the major cause behind several degenerative diseases. Nanoparticles have been found to have great potential as a substrate for designing amyloid fibril inhibitors and/or modulators. This is mainly due to its ideal characteristics, such as small size, high surface area-to volume ratio, and modifiable surface. This study is aimed at examining the influence of amino acid-modified silver nanoparticles on amyloid fibril formation. We first synthesized and characterized the proline- and cysteine-functionalized silver nanoparticles (Pro-AgNP and Cys-AgNP). Next, the effects of functionalized silver nanoparticles on amyloid fibrillogenesis and structural changes of hen lysozyme were examined using various spectroscopic and biophysical tools. Our results showed that Pro-AgNP/Cys-AgNP dose-dependently suppressed lysozyme amyloid fibrillogenesis, whereas negligible fibril-inhibiting effect was observed in the bare AgNP or proline/cysteine. Moreover, reduced solvent exposure and α-to-β transition were detected in lysozyme upon the addition of Pro-AgNP/Cys-AgNP. Finally, the nature of interactions between lysozyme and Pro-AgNP/Cys-AgNP was further examined using fluorescence quenching and molecular docking. By altering the properties of nanoparticles to design suitable nanoprobes, we have shown that amyloid fibrillogenesis can be controlled.

A generic class of amyloid fibril inhibitors.

Amyloid fibrils are large ordered fibrillar aggregates formed from mis-folded proteins. A number of human diseases are linked to the presence of amyloid deposits, including Alzheimer's disease, Parkinson's disease and type II diabetes. One therapeutic strategy for treating amyloid related diseases involves inhibiting fibril formation. Amyloid fibrils are β-sheet rich fibrillar aggregates that associate through hydrophobic interactions between precursor units. In this study, these generic physical properties of amyloid fibrils have been exploited to design a universal class of amphiphilic macromolecular inhibitors. A naturally occurring macromolecule of this structure is arabinogalactan protein (AGP), a component of gum arabic (GA). In addition, two synthetic polymers based on the proposed amphiphilic structure were synthesized and tested. These synthetic mimics, referred to as poly(norbornene glucose ester) (PNGE) and poly(norbornene gluconamide) (PNGA), possess hydrophobic polynorbornene backbones and pendent hydrophilic cyclic and open-chain glucose units, respectively. AGP, PNGE and PNGA all show inhibitory effects on in vitro amyloid fibril formation in bovine insulin (BI), hen egg white lysozyme (HEWL) and amyloid beta 1-40 (Aβ) proteins. Circular dichroism (CD) spectra of the proteins in the presence of the inhibitors suggests that amyloid fibril formation is inhibited by stabilization of the native α-helices of the proteins, as well as binding of the inhibitors to the β-sheet precursors. Based upon these results, glycosylated hydrophobic macromolecules are identified as a promising class of therapeutic agents for amyloid related diseases. Furthermore, we have determined that the intensity of the fluorescent probe thioflavin T (ThT) is dependent on both fibril morphology and the presence of the inhibitors, and is therefore not a quantitative measure of protein conversion to fibrils.

ChemInform Abstract: Synthesis of 4H‐1,4‐Oxazines as Transthyretin Amyloid Fibril Inhibitors.

4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of the interactions between known small molecule inhibitors and TTR by molecular docking. A series of 2,4,6-triaryl-4H-1,4-oxazines was synthesized by the cyclization of N,N-bis(phenacyl)anilines with POCl3 in pyridine. Inhibition of TTR amyloid fibril was evaluated by a fibril formation assay. The results indicate that 4H-1,4-oxazines significantly inhibit TTR amyloid fibril at a concentration of 7.2 μM.

Synthesis of 4H-1,4-oxazines as transthyretin amyloid fibril inhibitors

4H-1,4-oxazines were designed as transthyretin (TTR) amyloid fibril inhibitors based on an analysis of the interactions between known small molecule inhibitors and TTR by molecular docking. A series of 2,4,6-triaryl-4H-1,4-oxazines was synthesized by the cyclization of N,N-bis(phenacyl)anilines with POCl3 in pyridine. Inhibition of TTR amyloid fibril was evaluated by a fibril formation assay. The results indicate that 4H-1,4-oxazines significantly inhibit TTR amyloid fibril at a concentration of 7.2 μM.

Design, Synthesis, and Evaluation of Oxazole Transthyretin Amyloidogenesis Inhibitors.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Design, synthesis, and evaluation of oxazole transthyretin amyloidogenesis inhibitors

Ten oxazoles bearing a C(4) carboxyl group were synthesized and evaluated as transthyretin (TTR) amyloid fibril inhibitors. Substituting aryls at the C(2) position of the oxazole ring reveals that a 3,5-dichlorophenyl substituent significantly reduced amyloidogenesis. The efficacy of these inhibitors was enhanced further by installing an ethyl, a propyl, or a CF 3 group at the C(5) position. The CF 3 substitution at C(5) also improves the TTR binding selectivity over all the other proteins in human blood.

Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action.

Benzoxazoles as transthyretin amyloid fibril inhibitors: synthesis, evaluation, and mechanism of action.

Benzoxazoles as Transthyretin Amyloid Fibril Inhibitors: Synthesis, Evaluation, and Mechanism of Action

Benzoxazoles as Transthyretin Amyloid Fibril Inhibitors: Synthesis, Evaluation, and Mechanism of Action

Screening Transthyretin Amyloid Fibril Inhibitors: Characterization of Novel Multiprotein, Multiligand Complexes by Mass Spectrometry

Tetrameric transthyretin is involved in transport of thyroxine and, through its interactions with retinol binding protein, vitamin A. Dissociation of these structures is widely accepted as the first step in the formation of transthyretin amyloid fibrils. Using a mass spectrometric approach, we have examined a series of 18 ligands proposed as inhibitors of this process. The ligands were evaluated for their ability to bind to and stabilize the tetrameric structure, their cooperativity in binding, and their ability to compete with the natural ligand thyroxine. The observation of a novel ten-component complex containing six protein subunits, two vitamin molecules, and two synthetic ligands allows us to conclude that ligand binding does not inhibit association of transthyretin with holo retinol binding protein.

Evaluating the binding selectivity of transthyretin amyloid fibril inhibitors in blood plasma.

Transthyretin (TTR) tetramer dissociation and misfolding facilitate assembly into amyloid fibrils that putatively cause senile systemic amyloidosis and familial amyloid polyneuropathy. We have previously discovered more than 50 small molecules that bind to and stabilize tetrameric TTR, inhibiting amyloid fibril formation in vitro. A method is presented here to evaluate the binding selectivity of these inhibitors to TTR in human plasma, a complex biological fluid composed of more than 60 proteins and numerous small molecules. Our immunoprecipitation approach isolates TTR and bound small molecules from a biological fluid such as plasma, and quantifies the amount of small molecules bound to the protein by HPLC analysis. This approach demonstrates that only a small subset of the inhibitors that saturate the TTR binding sites in vitro do so in plasma. These selective inhibitors can now be tested in animal models of TTR amyloid disease to probe the validity of the amyloid hypothesis. This method could be easily extended to evaluate small molecule binding selectivity to any protein in a given biological fluid without the necessity of determining or guessing which other protein components may be competitors. This is a central issue to understanding the distribution, metabolism, activity, and toxicity of potential drugs.

Structure-Based Design, Synthesis and Evaluation of Amyloid Fibril Inhibitors

Structure-Based Design, Synthesis and Evaluation of Amyloid Fibril Inhibitors

Structure-Based Design of N-Phenyl Phenoxazine Transthyretin Amyloid Fibril Inhibitors

Starting with the published 2.0 A X-ray crystal structure of the transthyretin·(flufenamic acid)2 complex, a simple structure-based ligand design strategy was employed to conceive of N-phenyl phenoxazine transthyretin (TTR) amyloid fibril inhibitors. Fifteen N-phenyl phenoxazines were chemically synthesized and evaluated using a quantitative amyloid fibril assay in vitro. The structure of one of the two most active phenoxazines, 4, bound to TTR was solved to a resolution of 1.9 A to understand the structural basis of its efficacy. N-phenyl phenoxazine 4 binds similar to the orientation anticipated, although not as deeply into the channel as expected. Like flufenamic acid, 4 mediates binding-induced conformational changes that enable intersubunit H-bonding in tetrameric TTR which may be important for preventing fibril formation. Analytical ultracentrifugation analysis demonstrates that 4 blocks the first step of TTR amyloid fibril formation, that is, tetramer dissociation to the alternatively folded amyloi...

Synthesis and evaluation of inhibitors of transthyretin amyloid formation based on the non-steroidal anti-inflammatory drug, flufenamic acid

A light scattering-based amyloid fibril formation assay was employed to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. Twenty nine aromatic small molecules, some with homology to flufenamic acid (a known non-steroidal anti-inflammatory drug) were tested to identify important structural features for inhibitor efficacy. The results of these experiments and earlier data suggest that likely inhibitors will have aromatic-based structures with at least two aromatic rings. The ring or fused ring system occupying the outermost TTR binding pocket needs to be substituted with an acidic functional group (e.g. a carboxylic acid) to interact with complimentary charges in the TTR binding site. The promising TTR amyloid fibril inhibitors ranked in order of efficacy are: 2>4≈7>3>9>6>21 (see Fig. 5 ). ©

Synthesis and evaluation of anthranilic acid-based transthyretin amyloid fibril inhibitors

Eight small molecules were synthesized to evaluate the structure activity relationships (SAR) of N-substituted anthranilic acids. The molecules were synthesized by benzylation or arylation of methyl anthranilate. A light scattering-based amyloid fibril formation assay was used to evaluate potential inhibitors of transthyretin (TTR) amyloid fibril formation in vitro. The m-carboxyphenylated and o-trifluoromethylphenylated anthranilic acids are potent inhibitors that will be subjected to further SAR and structural analysis.

Discovering transthyretin amyloid fibril inhibitors by limited screening

Insoluble protein fibrils, resulting from the self-assembly of a conformational intermediate are implicated to be the causative agent in several human amyloid diseases including familial amyloid polyneuropathy (FAP) and senile systemic amyloidosis (SSA). These diseases are associated with transthyretin (TTR) amyloid fibrils, which appear to form in the acidic partial denaturing environment of a lysosome or endosome. Here we identify several structural classes of small molecules that are capable of inhibiting the TTR conformational changes facilitating amyloid fibril formation. A small molecule inhibitor that stabilizes the normal conformation of a protein is desirable as a promising approach to treat amyloid diseases and to rigorously test the amyloid hypothesis, the apparent causative role of amyloid fibrils in amyloid disease.