Rosmarinic Acid Restores Complete Transparency of Sonicated Human Cataract Ex Vivo and Delays Cataract Formation In Vivo.

Cataract, the leading cause of vision impairment worldwide, arises from abnormal aggregation of crystallin lens proteins. Presently, surgical removal is the only therapeutic approach. Recent findings have triggered renewed interest in development of non-surgical treatment alternatives. However, emerging treatments are yet to achieve full and consistent lens clearance. Here, the first ex vivo assay to screen for drug candidates that reduce human lenticular protein aggregation was developed. This assay allowed the identification of two leading compounds as facilitating the restoration of nearly-complete transparency of phacoemulsified cataractous preparation ex vivo. Mechanistic studies demonstrated that both compounds reduce cataract microparticle size and modify their amyloid-like features. In vivo studies confirmed that the lead compound, rosmarinic acid, delays cataract formation and reduces the severity of lens opacification in model rats. Thus, the ex vivo assay may provide an initial platform for broad screening of potential novel therapeutic agents towards pharmacological treatment of cataract.

Inhibition of the Aggregation and Toxicity of the Minimal Amyloidogenic Fragment of Tau by Its Pro-Substituted Analogues.

Inhibiting the toxic aggregation of amyloid-ß and the tau protein, the key pathological agents involved in Alzheimer's, is a leading approach in modulating disease progression. Using an aggregative tau-derived model peptide, Ac-PHF6-NH2 , the substitution of its amino acids with proline, a known efficient ß-breaker, is shown to reduce self-assembly. This effect is attributed to the steric hindrance created by the proline substitution, which results in disruption of the ß-sheet formation process. Moreover, several of the proline-substituted peptides inhibit the aggregation of Ac-PHF6-NH2 amyloidogenic peptide. Two of these modified inhibitors also disassemble pre-formed Ac-PHF6-NH2 fibrils and one inhibits induced cytotoxicity of the fibrils. Taken together, these lead ß-breaker peptides may be developed into novel Alzheimer's disease therapeutics.

Self-Assembled Cyclic d,l-α-Peptides as Generic Conformational Inhibitors of the α-Synuclein Aggregation and Toxicity: In Vitro and Mechanistic Studies.

Many peptides and proteins with large sequences and structural differences self-assemble into disease-causing amyloids that share very similar biochemical and biophysical characteristics, which may contribute to their cross-interaction. Here, we demonstrate how the self-assembled, cyclic d,l-α-peptide CP-2, which has similar structural and functional properties to those of amyloids, acts as a generic inhibitor of the Parkinson's disease associated α-synuclein (α-syn) aggregation to toxic oligomers by an "off-pathway" mechanism. We show that CP-2 interacts with the N-terminal and the non-amyloid-ß component region of α-syn, which are responsible for α-syn's membrane intercalation and self-assembly, thus changing the overall conformation of α-syn. CP-2 also remodels α-syn fibrils to nontoxic amorphous species and permeates cells through endosomes/lysosomes to reduce the accumulation and toxicity of intracellular α-syn in neuronal cells overexpressing α-syn. Our studies suggest that targeting the common structural conformation of amyloids may be a promising approach for developing new therapeutics for amyloidogenic diseases.

Resolution of Two Sub-Populations of Conformers and Their Individual Dynamics by Time Resolved Ensemble Level FRET Measurements.

Most active biopolymers are dynamic structures; thus, ensembles of such molecules should be characterized by distributions of intra- or intermolecular distances and their fast fluctuations. A method of choice to determine intramolecular distances is based on Förster resonance energy transfer (FRET) measurements. Major advances in such measurements were achieved by single molecule FRET measurements. Here, we show that by global analysis of the decay of the emission of both the donor and the acceptor it is also possible to resolve two sub-populations in a mixture of two ensembles of biopolymers by time resolved FRET (trFRET) measurements at the ensemble level. We show that two individual intramolecular distance distributions can be determined and characterized in terms of their individual means, full width at half maximum (FWHM), and two corresponding diffusion coefficients which reflect the rates of fast ns fluctuations within each sub-population. An important advantage of the ensemble level trFRET measurements is the ability to use low molecular weight small-sized probes and to determine nanosecond fluctuations of the distance between the probes. The limits of the possible resolution were first tested by simulation and then by preparation of mixtures of two model peptides. The first labeled polypeptide was a relatively rigid Pro7 and the second polypeptide was a flexible molecule consisting of (Gly-Ser)7 repeats. The end to end distance distributions and the diffusion coefficients of each peptide were determined. Global analysis of trFRET measurements of a series of mixtures of polypeptides recovered two end-to-end distance distributions and associated intramolecular diffusion coefficients, which were very close to those determined from each of the pure samples. This study is a proof of concept study demonstrating the power of ensemble level trFRET based methods in resolution of subpopulations in ensembles of flexible macromolecules.