Potency of a tau fibrillization inhibitor is influenced by its aggregation state

Abstract

Tau fibrillization is a potential therapeutic target for Alzheimer's and other neurodegenerative diseases. Several small-molecule inhibitors of tau aggregation have been developed for this purpose. One of them, 3,3'-bis(beta-hydroxyethyl)-9-ethyl-5,5'-dimethoxythiacarbocyanine iodide (N744), is a cationic thiacarbocyanine dye that inhibits recombinant tau filament formation when present at submicromolar concentrations. To prepare dosing regimens for testing N744 activity in biological models, its full concentration-effect relationship in the range 0.01-60muM was examined in vitro by electron microscopy and laser light scattering methods. Results revealed that N744 concentration dependence was biphasic, with fibrillization inhibitory activity appearing at submicromolar concentration, but with relief of inhibition and increases in fibrillization apparent above 10muM. Therefore, fibrillization was inhibited 50% only over a narrow concentration range, which was further reduced by filament stabilizing modifications such as tau pseudophosphorylation. N744 inhibitory activity also was paralleled by changes in its aggregation state, with dimer predominating at inhibitory concentrations and large dye aggregates appearing at high concentrations. Ligand dimerization was promoted by the presence of tau protein, which lowered the equilibrium dissociation constant for dimerization more than an order of magnitude relative to controls. The results suggest that ligand aggregation may play an important role in both inhibitory and disinhibitory phases of the concentration-effect curve, and may lead to complex dose-response relationships in model systems.

Potent inhibition of tau fibrillization with a multivalent ligand

“…Glutaraldehyde, uranyl acetate, and 300 mesh carbon-coated copper grids were from Electron Microscopy Sciences (Ft. Washington, PA). Cyanine dye N744 [8][9][10] was custom synthesized by deCODE Genetics (Lemont, IL).…”

“…Cyanine structure was assessed using absorbance spectroscopy [10]. Compound 2 was analyzed (Varian Cary 50 Bio) over the wavelength range 400 -650 nm in water, methanol, or assembly buffer containing ODS inducer and either 4 μM htau40 or 0.6 μM mixed histones [6].…”

“…Tau fibrillization antagonists identified by in *Corresponding Author: Jeff Kuret, Ph.D., Center for Molecular Neurobiology, 1060 Carmack Rd., Columbus, OH 43210, Fax: 614-292-5379, Email address: kuret.3@osu.edu (J. Kuret). 1 These authors contributed equally to this work vitro screens involving surfactant inducers include thiacarbocyanine dyes such as N744 [8][9][10]. Cyanines are highly prone to self-association reactions that form dimers and higher order aggregates, leading to shifts in absorbance spectra relative to dye monomer [11].…”

“…Although both classes of aggregate are composed of parallel dye molecules stacked plane-to-plane, they differ in the angle of slippage between successive molecular planes [9]. The degree of dye aggregation appears to modulate tau aggregation antagonist activity [10].…”

“…The reaction is intramolecular, and so population of the closed structure is independent of ligand concentration. Because H-dimers have been implicated in the action of cyanine-mediated inhibition of tau aggregation [10], formation of closed clamshell ligands may have especially potent activity. Together these data predict that multivalent forms of thiacarbocyanines could have potent tau aggregation inhibitor activity, and may represent a novel route to more efficacious inhibitors.…”