Selective interaction of lansoprazole and astemizole with tau polymers: potential new clinical use in diagnosis of Alzheimer's disease

Abstract

We describe the interactions of two benzimidazole derivatives, astemizole (AST) and lansoprazole (LNS), with anomalous aggregates of tau protein (neurofibrillary tangles). Interestingly, these compounds, with important medical applications in the treatment of allergies and gastrointestinal disorders respectively, specifically bind to aggregated variants of tau protein and to paired helical filaments isolated from brains of Alzheimer's disease (AD) patients. These ligands appear to be a powerful tool to tag brain-isolated tau-aggregates and heparin-induced polymers of recombinant tau. The interactions of AST and LNS with tau aggregates were assessed by classical radioligand assays, surface plasmon resonance, and bioinformatic approaches. The affinity of AST and LNS for tau aggregates was comparatively higher than that for amyloid-beta polymers according to our data. This is relevant since senile plaques are also abundant but are not pathognomonic in AD patients. Immunochemical studies on paired helical filaments from brains of AD patients and surface plasmon resonance studies confirm these findings. The capacity of these drugs to penetrate the blood-brain barrier was evaluated: i) in vitro by parallel artificial membrane permeability assay followed by experimental Log P determinations; and ii) in vivo by pharmacokinetic studies comparing distribution profiles in blood and brain of mice using HPLC/UV. Importantly, our studies indicate that the brain/blood concentration ratios for these compounds were suitable for their use as PET radiotracers. Since neurofibrillary tangles are positively correlated with cognitive impairment, we concluded that LNS and AST have a great potential in PET neuroimaing for in vivo early detection of AD and in reducing the formation of neurofibrillary tangles.

Fig. 1

Characterization of AD-PHF and tau filaments by TEM. Filaments obtained from recombinant tau protein induced by 200 μM heparin (A) and AD-PHF from AD patients (B) were characterized by transmission electronic microscopy (TEM). In these macrographs, yellow arrowheads point torsion of the polymers. In the preparations obtained from AD patients, single straight filaments were predominant as compared with the PHF preparation where PHF are predominant (Bars = 80 nm).

Fig. 2

Astemizole displays differential affinities for tau aggregates and Aβ peptide aggregates. Scatchard analyses of [³H]-AST were performed in order to determine Bmax/Kd index. The data shows that atemizole displays differential affinities for amyloid aggregates (A), heparine induced tau filaments (B), and AD-PHF (C). Each point of the curve is the mean of three replicas. Kinetic data is summarized in Table D. Inhibition studies with non labeled LNS and [3H]-AST showed high affinity of LNS for aggregated forms of tau (E).

Fig. 3

Binding data obtained by surface plasmon resonance (SPR). Sensograms of aggregated amyloid peptide and AD-PHFs covalently inmobilized on a C5 biosensor surface. PHF-1 antibody (A) and 6E10 antibody (C) were injected into the chip to verify the presence of AD-PHFs and amyloid aggregates. Both amyloid (B) and PHF (D) surfaces were also reactive to the injection of ThS, which corroborates the presence of polymeric forms of the amyloid and tau protein immobilized on the chip surface. AST displayed higher signals in its interactions with AD-PHFs (E) than with amyloid (F). LNS differential interaction with AD-PHFs (G) and aggregated amyloid (H) was also verified by SPR.

Fig. 4

Structures of the drugs astemizole and lansoprazole and their binding interactions with the C-terminal hexapeptide 386TDHGAE391 of PHF core. (A) The interactions of the drugs with the core PHF-tau fragment drawn as an isosurface. The interaction of AST with PHF fragment shows a high fitting on the tau-PHF surface when it is compared with LNS. The H-bonds are drawn in white dashed lines. (B) Detailed view of the interactions between PHF-tau fragment and the drugs studied by docking experiments. Residues of the PHF fragment are shown in licorice representation and drugs are presented in ball-and-stick representation, with carbon atoms colored in yellow (PHF fragment) and cyan (drugs). The hydrogen bonds are depicted as black dashed lines. The same orientation has been used for the two snapshots (A,C and B,D) for the sake of clarity.

Fig. 5

Immunofluorescence characterization of the interaction of AD-PHFs and lansoprazole. A suspension of isolated PHF or tau polymers was fixed to a glass slide (see scheme below) and processed for double staining with immunofluorescence using PHF-1 antibody and LNS 0.1 %.

Fig. 6

Comparative pharmacokinetic analysis of astemizole and lansoprazole. The concentration of LNS and AST was measured in blood (A, C) and brain (B, D) at different time points (0, 30, 60, 90, 180 min) after intravenous injection. Brain/blood concentration ratio (A, C) were also determined. The results are the mean of three independent experiments.