Katarina Siposova

7-Methoxytacrine-Adamantylamine Heterodimers as Cholinesterase Inhibitors in Alzheimer’s Disease Treatment — Synthesis, Biological Evaluation and Molecular Modeling Studies

A structural series of 7-MEOTA-adamantylamine thioureas was designed, synthesized and evaluated as inhibitors of human acetylcholinesterase (hAChE) and human butyrylcholinesterase (hBChE). The compounds were prepared based on the multi-target-directed ligand strategy with different linker lengths (n = 2–8) joining the well-known NMDA antagonist adamantine and the hAChE inhibitor 7-methoxytacrine (7-MEOTA). Based on in silico studies, these inhibitors proved dual binding site character capable of simultaneous interaction with the peripheral anionic site (PAS) of hAChE and the catalytic active site (CAS). Clearly, these structural derivatives exhibited very good inhibitory activity towards hBChE resulting in more selective inhibitors of this enzyme. The most potent cholinesterase inhibitor was found to be thiourea analogue 14 (with an IC50 value of 0.47 µM for hAChE and an IC50 value of 0.11 µM for hBChE, respectively). Molecule 14 is a suitable novel lead compound for further evaluation proving that the strategy of dual binding site inhibitors might be a promising direction for development of novel AD drugs.

Depolymerization of insulin amyloid fibrils by albumin-modified magnetic fluid

Pathogenesis of amyloid-related diseases is associated with the presence of protein amyloid deposits. Insulin amyloids have been reported in a patient with diabetes undergoing treatment by injection of insulin and causes problems in the production and storage of this drug and in pplication of insulin pumps. We have studied the interference of insulin amyloid fibrils with a series of 18 albumin magnetic fluids (MFBSAs) consisting of magnetite nanoparticles modified by different amounts of bovine serum albumin (w/w BSA/Fe₃O₄ from 0.005 up to 15). We have found that MFBSAs are able to destroy amyloid fibrils in vitro. The extent of fibril depolymerization was affected by nanoparticle physical-chemical properties (hydrodynamic diameter, zeta potential and isoelectric point) determined by the BSA amount present in MFBSAs. The most effective were MFBSAs with lower BSA/Fe₃O₄ ratios (from 0.005 to 0.1) characteristic of about 90% depolymerizing activity. For the most active magnetic fluids (ratios 0.01 and 0.02) the DC50 values were determined in the range of low concentrations, indicating their ability to interfere with insulin fibrils at stoichiometric concentrations. We assume that the present findings represent a starting point for the application of the active MFBSAs as therapeutic agents targeting insulin amyloidosis.

Binding of glyco-acridine derivatives to lysozyme leads to inhibition of amyloid fibrillization.

While amyloid-related diseases are at the center of intense research efforts, no feasible cure is currently available for these diseases. The experimental and computational techniques were used to study the ability of glyco-acridines to prevent lysozyme amyloid fibrillization in vitro. Fluorescence spectroscopy and atomic force microscopy have shown that glyco-acridines inhibit amyloid aggregation of lysozyme; the inhibition efficiency characterized by the half-maximal inhibition concentration IC50 was affected by the structure and concentration of the derivative. We next investigated relationship between the binding affinity and the inhibitory activity of the compounds. The semiempirical quantum PM6-DH+ method provided a good correlation pointing to the importance of quantum effects on the binding of glyco-acridine derivatives to lysozyme. The contribution of linkers may be explained by the valence bond theory. Our data provide a basis for the development of new small molecule inhibitors effective in therapy of amyloid-related diseases.

Amyloid aggregation of lysozyme: The synergy study of red wine polyphenols

The amyloidoses are diseases associated with nonnative folding of proteins and characterized by the presence of protein amyloid aggregates. The ability of quercetin, resveratrol, caffeic acid, and their equimolar mixtures to affect amyloid aggregation of hen egg white lysozyme in vitro was detected by Thioflavin T fluorescence assay. The anti‐amyloid activities of tested polyphenols were evaluated by the median depolymerization concentrations DC50 and median inhibition concentrations IC50. Single substances are more efficient (by at least one order) in the depolymerization of amyloid aggregates assay than in the inhibition of the amyloid formation with IC50 in 10−4 to 10−5M range. Analyzed mixture samples showed synergic or antagonistic effects in both assays. DC50 values ranged from 10−5 to 10−8M and IC50 from 10−5 to 10−9M, respectively. We observed that certain mixtures of studied polyphenols can synergistically inhibit production of amyloids aggregates and are also effective in depolymerization of the aggregates. Synergic or antagonistic effects of studied mixtures were correlated with protein–small ligand docking studies and AFM results. Differences in these activities could be explained by binding of each polyphenol to a different amino acid sequence within the protein. Our results indicate that synergic/antagonistic anti‐amyloid effects of studied mixtures depend on the selective binding of polyphenols to the known amyloidogenic sequences in the lysozyme chain. Our findings of the effective reduction of amyloid aggregation of lysozyme by polyphenol mixtures in vitro are of the utter physiological relevance considering the bioavailability and low toxicity of tested phenols. Proteins 2013;

Structure-activity relationship of acridine derivatives to amyloid aggregation of lysozyme.

BACKGROUND Amyloid-related diseases (such as Alzheimers disease or diabetes type II) are associated with self-assembly of protein into amyloid aggregates. METHODS Spectroscopic and atomic force microscopy were used to determine the ability of acridines to affect amyloid aggregation of lysozyme. RESULTS We have studied the effect of acridine derivatives on the amyloid aggregation of lysozyme to investigate the acridine structure-activity relationship. The activity of the effective planar acridines was characterized by the half-maximum depolymerization concentration DC(50) and half-maximal inhibition concentration IC(50). For the most effective acridine derivatives we examined their interaction with DNA and their effect on cell viability in order to investigate their eventual influence on cells. We thus identified planar acridine derivatives with intensive anti-amyloid activity (IC(50) and DC(50) values in micromolar range), low cytotoxicity and weak ability to interfere with the processes in the cell. CONCLUSIONS Our findings indicate that both the planarity and the tautomerism of the 9-aminoacridine core together with the reactive nucleophilic thiosemicarbazide substitution play an important role in the anti-amyloid activities of studied derivatives. GENERAL SIGNIFICANCE The present findings favor the application of the selected active planar acridines in the treatment of amyloid-related diseases.

Design, synthesis and in vitro testing of 7-methoxytacrine-amantadine analogues: a novel cholinesterase inhibitors for the treatment of Alzheimer’s disease

Abstract A series of cholinesterase inhibitors acting as dual binding site heterodimers for the management of Alzheimer’s disease were developed. The series of 7-methoxytacrine (7-MEOTA)-amantadine ureas (11–17) was designed, prepared evaluated in vitro towards human acetyl/butyryl cholinesterase (hAChE, hBChE) and compared with the series of 7-MEOTA-amantadine thioureas (4–10). The heterodimers have different length of linkers combining 7-MEOTA and amantadine moieties. In comparison with 7-MEOTA, the newly synthesized compounds were better inhibitors of both cholinesterases. The urea analogues did not have the anticipated benefit of increased inhibitory activity and have comparable IC50 values with thiourea derivatives.

In Silico and in Vitro Study of Binding Affinity of Tripeptides to Amyloid β Fibrils: Implications for Alzheimer’s Disease

Self-assembly of Aβ peptides into amyloid aggregates has been suggested as the major cause of Alzheimers disease (AD). Nowadays, there is no medication for AD, but experimental data indicate that reversion of the process of amyloid aggregation reduces the symptoms of disease. In this paper, all 8000 tripeptides were studied for their ability to destroy Aβ fibrils. The docking method and the more sophisticated MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) method were employed to calculate the binding affinity and mode of tripeptides to Aβ fibrils. The ability of these peptides to depolymerize Aβ fibrils was also investigated experimentally using atomic force microscopy and fluorescence spectroscopy (Thioflavin T assay). It was shown that tripeptides prefer to bind to hydrophobic regions of 6Aβ9-40 fibrils. Tripeptides WWW, WWP, WPW and PWW were found to be the most potent binders. In vitro experiments showed that tight-binding tripeptides have significant depolymerizing activities and their DC50 values determined from dose-response curves were in micromolar range. The ability of nonbinding (GAM, AAM) and weak-binding (IVL and VLA) tripeptides to destroy Aβ fibrils was negligible. In vitro data of tripeptide depolymerizing activities support the predictions obtained by molecular docking and all-atom simulation methods. Our results suggest that presence of multiple complexes of heterocycles forming by tryptophan and proline residues in tripeptides is crucial for their tight binding to Aβ fibrils as well as for extensive fibril depolymerization. We recommend PWW for further studies as it has the lowest experimental binding constant.

On the determination of the helical structure parameters of amyloid protofilaments by small-angle neutron scattering and atomic force microscopy

The helical structure of amyloid protofilaments of hen egg white lysozyme was analyzed by small-angle neutron scattering (SANS) and atomic force microscopy (AFM). The structure of these formations in bulk solutions was adequately described by SANS in terms of a simplified model of a helix with spherical structural units. The found main helix parameters (pitch and effective diameter) are consistent with the results of AFM analysis for amyloid fibrils adsorbed on a mica surface. Both methods reveal a strong isotope effect on the structure of amyloid fibrils with respect to the substitution of heavy for light water in the solvent. Specific details responsible for the structural differences when comparing SANS and AFM data are discussed from the viewpoint of methodological aspects, the influence of different (native and adsorbed) amyloid states and sample preparation.

Inhibition of insulin amyloid fibrillization by glyco-acridines: an in vitro and in silico study

The formation of insulin amyloid fibrils leads to accumulation of protein aggregates at the sites of insulin injection and interferes with insulin delivery for treatment of diabetes. We investigated the ability of small molecules, aromatic glyco-acridine derivatives, to prevent insulin fibrillization. Fluorescence spectroscopy and atomic force microscopy have shown that glyco-acridines interfere with insulin aggregation and that their inhibitory activity depends on their structure. The binding free energies, estimated by all-atom molecular dynamics simulations, indicate that the non-polar interaction is the key factor controlling the binding affinity of glyco-acridine derivatives to insulin. We introduced, for the first time, geometrical descriptors that allowed us to distinguish the binding affinities of stereo-isomers. The binding free energies correlate with the distance between the planes of the acridine tricyclic core and the side parts in the unbound and bound states. In addition, the aromatic part of glyco-acridines is important for directing the ligand–dimer insulin interaction. Our findings may provide a basis for the development of new small moleculeinhibitors for the treatment of amyloid-related diseases.

Polymorphism of hen egg white lysozyme amyloid fibrils influences the cytotoxicity in LLC-PK1 epithelial kidney cells.

The polymorphism of amyloid fibrils is potentially crucial as it may underlie the natural variability of amyloid diseases and could be important in developing a fuller understanding of the molecular basis of protein deposition disorders. This study examines morphological differences in lysozyme fibrils and the implications of these differences in terms of cytotoxicity. The structural characteristics of amyloid fibrils formed under two different experimental conditions (acidic and neutral) were evaluated using spectroscopic methods, atomic force microscopy and image analysis. Growth curves and apoptotic/necrotic assays were used to determine the cytotoxic effect of fibrils on the LLC-PK1 renal cells. The results reveal that both types of mature lysozyme amyloid fibrils are actively involved in the cytotoxic process, however each exhibit different levels of cytotoxicity. Fibrils formed at acidic pH affect cell growth in a dose-dependent manner, but a threshold-dependent inhibition of cell growth was observed in the case of lysozyme fibrils prepared at neutral pH. Experiments examining the mechanism of the cell death suggest that both types of mature lysozyme fibrils trigger late apoptosis/necrosis at different fibril concentrations. Our findings clearly indicate that the intrinsic differences between amyloid fibrils due to their polymorphism result in different degrees of cytotoxicity.