iododiflunisal and Alzheimer-Disease

Transthyretin (TTR) has a well-established role in neuroprotection in Alzheimer's Disease (AD). We have setup a drug discovery program of small-molecule compounds that act as chaperones enhancing TTR/Amyloid-beta peptide (Aβ) interactions. A combination of computational drug repurposing approaches and in vitro biological assays have resulted in a set of molecules which were then screened with our in-house validated high-throughput screening ternary test. A prioritized list of chaperones was obtained and corroborated with ITC studies. Small-molecule chaperones have been discovered, among them our lead compound Iododiflunisal (IDIF), a molecule in the discovery phase; one investigational drug (luteolin); and 3 marketed drugs (sulindac, olsalazine and flufenamic), which could be directly repurposed or repositioned for clinical use. Not all TTR tetramer stabilizers behave as chaperones in vitro. These chemically diverse chaperones will be used for validating TTR as a target in vivo, and to select one repurposed drug as a candidate to enter clinical trials as AD disease-modifying drug.

Topics: Alzheimer Disease; Calorimetry; Dose-Response Relationship, Drug; Drug Discovery; Humans; Models, Molecular; Molecular Chaperones; Molecular Structure; Prealbumin; Small Molecule Libraries; Software; Structure-Activity Relationship

Transthyretin (TTR) is a tetrameric, amyloid-β (Aβ)-binding protein, which reduces Aβ toxicity. The TTR/Aβ interaction can be enhanced by a series of small molecules that stabilize its tetrameric form. Hence, TTR stabilizers might act as disease-modifying drugs in Alzheimer's disease.. We monitored the therapeutic efficacy of two TTR stabilizers, iododiflunisal (IDIF), which acts as small-molecule chaperone of the TTR/Aβ interaction, and tolcapone, which does not behave as a small-molecule chaperone, in an animal model of Alzheimer's disease using positron emission tomography (PET).. Female mice (AβPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n = 7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100 mg/Kg/day) was started at 5 months of age. Treatment efficacy assessment was based on changes in longitudinal deposition of Aβ in the hippocampus (HIP) and the cortex (CTX) and determined using PET-[18F]florbetaben. Immunohistochemical analysis was performed at age = 14 months.. Standard uptake values relative to the cerebellum (SUVr) of [18F]florbetaben in CTX and HIP of non-treated animals progressively increased from age = 5 to 11 months and stabilized afterwards. In contrast, [18F]florbetaben uptake in HIP of IDIF-treated animals remained constant between ages = 5 and 11 months and significantly increased at 14 months. In the tolcapone-treated group, SUVr progressively increased with time, but at lower rate than in the non-treated group. No significant treatment effect was observed in CTX. Results from immunohistochemistry matched the in vivo data at age = 14 months.. Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions.

Topics: Administration, Oral; Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Diflunisal; Female; Hippocampus; Longitudinal Studies; Mice; Mice, 129 Strain; Mice, Inbred C3H; Mice, Transgenic; Molecular Imaging; Positron Emission Tomography Computed Tomography

The absence of transthyretin (TTR) in AD mice decreases brain Aβ clearance and reduces the low-density lipoprotein receptor-related protein 1 (LRP1). It is possible that neuroprotection by TTR is dependent on its tetramer structural stability, as studies using TTR mutants showed that unstable L55P TTR has low affinity for Aβ, and TTR tetrameric stabilizers such as iododiflunisal ameliorate AD features in vivo.. We firstly investigated TTR folding status in human plasma measuring the resistance to urea denaturation. The importance of TTR stability on Aβ internalization was studied in human cerebral microvascular endothelial (hCMEC/D3) and hepatoma cells (HepG2), by flow cytometry. To investigate the fate of Aβ at the blood-brain barrier, Aβ efflux from hCMEC/D3 cells seeded on transwells was measured using ELISA. Further, to assess Aβ colocalization with lysosomes, Lysotracker was used. Moreover, levels of LRP1 were assessed in the liver and plasma of mice with different TTR backgrounds or treated with iododiflunisal.. We showed that TTR stability is decreased in AD and that WT TTR and drug-stabilized L55P TTR are able to increase uptake of Aβ. Furthermore, measurement of Aβ efflux showed that stable or stabilized TTR increased Aβ efflux from the basolateral to the apical side. Moreover, HepG2 cells incubated with Aβ in the presence of WT TTR, but not L55P TTR, showed an increased number of lysosomes. Further, in the presence of WT TTR, Aβ peptide colocalized with lysosomes, indicating that only stable TTR assists Aβ internalization, leading to its degradation. Finally, we demonstrated that only stable TTR can increase LRP1 levels.. TTR stabilization exerts a positive effect on Aβ clearance and LRP1 levels, suggesting that TTR protective role in AD is dependent on its stability. These results provide relevant information for the design of TTR-based therapeutic strategies for AD.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Blood-Brain Barrier; Cell Line; Diflunisal; Escherichia coli; Humans; Liver; Low Density Lipoprotein Receptor-Related Protein-1; Lysosomes; Mice, Transgenic; Prealbumin; Presenilin-1; Protein Denaturation; Protein Multimerization; Protein Stability; Receptors, LDL; Recombinant Proteins; Tumor Suppressor Proteins; Urea

Several strategies against Alzheimer disease (AD) are directed to target Aβ-peptides. The ability of transthyretin (TTR) to bind Aβ-peptides and the positive effect exerted by some TTR stabilizers for modulating the TTR-Aβ interaction have been previously studied. Herein, key structural features of the interaction between TTR and the Aβ(12-28) peptide (3), the essential recognition element of Aβ, have been unravelled by STD-NMR spectroscopy methods in solution. Molecular aspects related to the role of the TTR stabilizer iododiflunisal (IDIF, 5) on the TTR-Aβ complex have been also examined. The NMR results, assisted by molecular modeling protocols, have provided a structural model for the TTR-Aβ interaction, as well as for the ternary complex formed in the presence of IDIF. This basic structural information could be relevant for providing light on the mechanisms involved in the ameliorating effects of AD symptoms observed in AD/TTR

Topics: Alzheimer Disease; Amyloid beta-Peptides; Crystallography, X-Ray; Diflunisal; Humans; Magnetic Resonance Spectroscopy; Molecular Docking Simulation; Prealbumin; Protein Interaction Maps

Alzheimer's disease (AD) is the most common form of dementia and now represents 50-70% of total dementia cases. Over the last two decades, transthyretin (TTR) has been associated with AD and, very recently, a novel concept of TTR stability has been established in vitro as a key factor in TTR/amyloid-β (Aβ) interaction. Small compounds, TTR stabilizers (usually non-steroid anti-inflammatory drugs), bind to the thyroxine (T4) central binding channel, increasing TTR tetrameric stability and TTR/Aβ interaction. In this work, we evaluated in vivo the effects of one of the TTR stabilizers identified as improving TTR/Aβ interaction, iododiflunisal (IDIF), in Aβ deposition and other AD features, using AβPPswe/PS1A246E transgenic mice, either carrying two or just one copy of the TTR gene (AD/TTR+/+ or AD/TTR+/-, respectively), available and characterized in our laboratory. The results showed that IDIF administered orally bound TTR in plasma and stabilized the protein, as assessed by T4 displacement assays, and was able to enter the brain as revealed by mass spectrometry analysis of cerebrospinal fluid. TTR levels, both in plasma and cerebrospinal fluid, were not altered. In AD/TTR+/- mice, IDIF administration resulted not only in decreased brain Aβ levels and deposition but also in improved cognitive function associated with the AD-like neuropathology in this mouse model, although no improvements were detectable in the AD/TTR+/+ animals. Further, in AD/TTR+/- mice, Aβ levels were reduced in plasma suggesting TTR promoted Aβ clearance from the brain and from the periphery. Taken together, these results strengthen the importance of TTR stability in the design of therapeutic drugs, highlighting the capacity of IDIF to be used in AD treatment to prevent and to slow the progression of the disease.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Animals; Brain; Cognition Disorders; Diflunisal; Humans; Maze Learning; Mice; Mice, Transgenic; Nootropic Agents; Peptide Fragments; Plaque, Amyloid; Prealbumin; Presenilin-1

Transthyretin (TTR) protects against A-Beta toxicity by binding the peptide thus inhibiting its aggregation. Previous work showed different TTR mutations interact differently with A-Beta, with increasing affinities correlating with decreasing amyloidogenecity of the TTR mutant; this did not impact on the levels of inhibition of A-Beta aggregation, as assessed by transmission electron microscopy. Our work aimed at probing differences in binding to A-Beta by WT, T119M and L55P TTR using quantitative assays, and at identifying factors affecting this interaction. We addressed the impact of such factors in TTR ability to degrade A-Beta. Using a dot blot approach with the anti-oligomeric antibody A11, we showed that A-Beta formed oligomers transiently, indicating aggregation and fibril formation, whereas in the presence of WT and T119M TTR the oligomers persisted longer, indicative that these variants avoided further aggregation into fibrils. In contrast, L55PTTR was not able to inhibit oligomerization or to prevent evolution to aggregates and fibrils. Furthermore, apoptosis assessment showed WT and T119M TTR were able to protect against A-Beta toxicity. Because the amyloidogenic potential of TTR is inversely correlated with its stability, the use of drugs able to stabilize TTR tetrameric fold could result in increased TTR/A-Beta binding. Here we showed that iododiflunisal, 3-dinitrophenol, resveratrol, [2-(3,5-dichlorophenyl)amino] (DCPA) and [4-(3,5-difluorophenyl)] (DFPB) were able to increase TTR binding to A-Beta; however only DCPA and DFPB improved TTR proteolytic activity. Thyroxine, a TTR ligand, did not influence TTR/A-Beta interaction and A-Beta degradation by TTR, whereas RBP, another TTR ligand, not only obstructed the interaction but also inhibited TTR proteolytic activity. Our results showed differences between WT and T119M TTR, and L55PTTR mutant regarding their interaction with A-Beta and prompt the stability of TTR as a key factor in this interaction, which may be relevant in AD pathogenesis and for the design of therapeutic TTR-based therapies.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Cell Line, Tumor; Diflunisal; Dinitrophenols; Humans; Mutation; Phthalic Acids; Prealbumin; Protein Binding; Resveratrol; Stilbenes