Oral Treatment with Iododiflunisal Delays Hippocampal Amyloid-β Formation in a Transgenic Mouse Model of Alzheimer's Disease: A Longitudinal in vivo Molecular Imaging Study

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. Objective: 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). Methods: Female mice (AβPPswe/PS1A246E/TTR+/-) were divided into 3 groups (n=7 per group): IDIF-treated, tolcapone-treated, and non-treated. The oral treatment (100mg/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. Results: 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. Conclusion: Our work provides encouraging preliminary results on the ability of small-molecule chaperones to ameliorate Aβ deposition in certain brain regions

Radiochemical examination of transthyretin (TTR) brain penetration assisted by iododiflunisal, a TTR tetramer stabilizer and a new candidate drug for AD

It is well settled that the amyloidogenic properties of the plasma protein transporter transthyretin (TTR) can be modulated by compounds that stabilize its native tetrameric conformation. TTR is also present in cerebrospinal fluid where it can bind to Aβ-peptides and prevent Aβ aggregation. We have previously shown that treatment of Alzheimer’s Disease (AD) model mice with iododiflunisal (IDIF), a TTR tetramer stabilizing compound, prevents AD pathologies. This evidence positioned IDIF as a new lead drug for AD. In dissecting the mechanism of action of IDIF, we disclose here different labeling strategies for the preparation of 131I-labeled IDIF and 131I- and 124I-labeled TTR, which have been further used for the preparation of IDIF-TTR complexes labeled either on the compound or the protein. The biodistribution of all labeled species after intravenous administration has been investigated in mice using ex vivo and in vivo techniques. Our results confirm the capacity of TTR to cross the blood brain barrier (BBB) and suggest that the formation of TTR-IDIF complexes enhances BBB permeability of both IDIF and TTR. The increased TTR and IDIF brain concentrations may result in higher Aβ-peptide sequestration capacity with the subsequent inhibition of AD symptoms as we have previously observed in mice. © 2019, The Author(s).The work was supported by a grant from the Fundació Marató de TV3 (Neurodegenerative Diseases Call, Project Reference 20140330-31-32-33-34, http://www.ccma.cat/tv3/marato/en/ projectes-financats/2013/212/). The group at CIC biomaGUNE also acknowledges MINECO (Spain) for funding through Grant CTQ2017-87637-R. I. Cardoso worked under the Investigator FCT Program which is financed by national funds through the Foundation for Science and Technology (FCT, Portugal) and co-financed by the European Social Fund (ESF) through the Human Potential Operational Programme (HPOP), type 4.2 - Promotion of Scientific Employment.Peer reviewe

The Changing Landscape for Stroke\ua0Prevention in AF: Findings From the GLORIA-AF Registry Phase 2

Background GLORIA-AF (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients with Atrial Fibrillation) is a prospective, global registry program describing antithrombotic treatment patterns in patients with newly diagnosed nonvalvular atrial fibrillation at risk of stroke. Phase 2 began when dabigatran, the first non\u2013vitamin K antagonist oral anticoagulant (NOAC), became available. Objectives This study sought to describe phase 2 baseline data and compare these with the pre-NOAC era collected during phase 1. Methods During phase 2, 15,641 consenting patients were enrolled (November 2011 to December 2014); 15,092 were eligible. This pre-specified cross-sectional analysis describes eligible patients\u2019 baseline characteristics. Atrial fibrillation disease characteristics, medical outcomes, and concomitant diseases and medications were collected. Data were analyzed using descriptive statistics. Results Of the total patients, 45.5% were female; median age was 71 (interquartile range: 64, 78) years. Patients were from Europe (47.1%), North America (22.5%), Asia (20.3%), Latin America (6.0%), and the Middle East/Africa (4.0%). Most had high stroke risk (CHA2DS2-VASc [Congestive heart failure, Hypertension, Age  6575 years, Diabetes mellitus, previous Stroke, Vascular disease, Age 65 to 74 years, Sex category] score  652; 86.1%); 13.9% had moderate risk (CHA2DS2-VASc = 1). Overall, 79.9% received oral anticoagulants, of whom 47.6% received NOAC and 32.3% vitamin K antagonists (VKA); 12.1% received antiplatelet agents; 7.8% received no antithrombotic treatment. For comparison, the proportion of phase 1 patients (of N = 1,063 all eligible) prescribed VKA was 32.8%, acetylsalicylic acid 41.7%, and no therapy 20.2%. In Europe in phase 2, treatment with NOAC was more common than VKA (52.3% and 37.8%, respectively); 6.0% of patients received antiplatelet treatment; and 3.8% received no antithrombotic treatment. In North America, 52.1%, 26.2%, and 14.0% of patients received NOAC, VKA, and antiplatelet drugs, respectively; 7.5% received no antithrombotic treatment. NOAC use was less common in Asia (27.7%), where 27.5% of patients received VKA, 25.0% antiplatelet drugs, and 19.8% no antithrombotic treatment. Conclusions The baseline data from GLORIA-AF phase 2 demonstrate that in newly diagnosed nonvalvular atrial fibrillation patients, NOAC have been highly adopted into practice, becoming more frequently prescribed than VKA in Europe and North America. Worldwide, however, a large proportion of patients remain undertreated, particularly in Asia and North America. (Global Registry on Long-Term Oral Antithrombotic Treatment in Patients With Atrial Fibrillation [GLORIA-AF]; NCT01468701

Preparative Scale Production of Recombinant Human Transthyretin for Biophysical Studies of Protein-Ligand and Protein-Protein Interactions

© 2020 by the authors.Human transthyretin (hTTR), a serum protein with a main role in transporting thyroid hormones and retinol through binding to the retinol-binding protein, is an amyloidogenic protein involved in familial amyloidotic polyneuropathy (FAP), familial amyloidotic cardiomyopathy, and central nervous system selective amyloidosis. hTTR also has a neuroprotective role in Alzheimer disease, being the major Aβ binding protein in human cerebrospinal fluid (CSF) that prevents amyloid-β (Aβ) aggregation with consequent abrogation of toxicity. Here we report an optimized preparative expression and purification protocol of hTTR (wt and amyloidogenic mutants) for in vitro screening assays of TTR ligands acting as amyloidogenesis inhibitors or acting as molecular chaperones to enhance the TTR:Aβ interaction. Preparative yields were up to 660 mg of homogenous protein per L of culture in fed-batch bioreactor. The recombinant wt protein is mainly unmodified at Cys10, the single cysteine in the protein sequence, whereas the highly amyloidogenic Y78F variant renders mainly the S-glutathionated form, which has essentially the same amyloidogenic behavior than the reduced protein with free Cys10. The TTR production protocol has shown inter-batch reproducibility of expression and protein quality for in vitro screening assays.Work supported in part by grant PID2019-104350RB-I00 from MINECO and grant 2017SGR-727 from the Generalitat de Catalunya (to A.P.). G.A. acknowledges a grant from Fundació Marató de TV3, Spain (Project ref. 20140330-31-32-33-34). E.Y.C. acknowledges a contract funded by the project of Fundació Marató de TV3 and a contract from Ford España—Fundación Apadrina la Ciencia.Peer reviewe

An Assay for Screening Potential Drug Candidates for Alzheimer's Disease That Act as Chaperones of the Transthyretin and Amyloid‐β Peptides Interaction

The protein transthyretin (TTR) modulates amyloid‐β (Aβ) peptides deposition and processing and this physiological effect is further enhanced by treatment with iododiflunisal (IDIF), a small‐molecule compound (SMC) with TTR tetramer stabilization properties, which behaves as chaperone of the complex. This knowledge has prompted us to design and optimize a rapid and simple high‐throughput assay that relies on the ability of test compounds to form ternary soluble complexes TTR/Aβ/SMC that prevent Aβ aggregation. The method uses the shorter Aβ(12–28) sequence which is cheaper and simpler to use while retaining the aggregation properties of their parents Aβ(1–40) and Aβ(1–42). The test is carried out in 96‐plate wells that are UV monitored for turbidity during 6 h. Given its reproducibility, we propose that this test can be a powerful tool for efficient screening of SMCs that act as chaperones of the TTR/Aβ interaction that may led to potential AD therapies.I.C. worked under the Investigator FCT Program which is financed by national funds through FCT and co‐financed by ESF through HPOP, type 4.2–Promotion of Scientific Employment and currently works under the Program CEEC Institucional 2017. G.A. from IQAC‐CSIC acknowledges a grant from Fundació Marató de TV3, Spain (Project ref. 20140330‐31–32–33–34) and financial support from the Spanish Ministry of Economy (CTQ2016‐76840‐R). E.Y.C. acknowledges a contract from Ford‐Fundación Apadrina la Ciencia. CIC bioGUNE acknowledges the European Research Council for financial support (ERC‐2017‐AdG, 788143‐RECGLYC‐ANMR), Instituto de Salud Carlos III of Spain, ISCIII (PRB3 IPT17/0019 to A.G.), Agencia Estatal Investigación of Spain, AEI (CTQ2015‐64597‐C2‐1‐P and RTI2018‐094751‐B‐C21) and the Severo Ochoa Excellence Accreditation (SEV‐2016‐0644). J.L. from CIC biomaGUNE acknowledges the Spanish Ministry of Economy and Competitiveness for financial support through grant CTQ2017‐87637. R.G.A. from IQAC‐CSIC acknowledges Dr. Maria José Bleda (CID‐CSIC, Spain) for full support on statistical analysis, and Prof. Antoni Planas (IQS‐URL) for full technical support on the TTR production; and Dr. David Amabilino for language editing and revision of this manuscript.Peer reviewe

Insights on the Interaction between Transthyretin and Aβ in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal

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± animal models after IDIF treatment and eventually for designing new molecules toward AD therapeutic drugs. © 2017 American Chemical Society.The work was supported by a grant from the FundacióMarató de TV3 (Neurodegenerative Diseases Call, Project Reference 20140330-31-32-33-34, http://www.ccma.cat/tv3/marato/en/ projectes-financats/2013/212/). The group at CIC bioGUNE also acknowledges MINECO (Spain) for funding through Grant CTQ2015-64597-C2-1-P and a Juan de la Cierva contract to A.G. The group at IBMC-i3S also acknowledges funding through Grant Norte-01-0145-FEDER-000008-Porto Neurosciences and Neurologic Disease Research Initiative at I3S, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (FEDER). I.C. works under the Investigator FCT Program which is financed by national funds through FCT and cofinanced by ESF through HPOP, Type 4.2, Promotion of Scientific Employment. M.A. is currently a recipient of a Research Fellowship (BIM) funded by the project of FundacióMarató de TV3, Spain, and L.M.S. is currently a recipient of a fellowship from Norte 2020. IQAC-CSIC acknowledges a contract to E.Y.C. funded by the project of FundacióMaratóde TV3, Spain.Peer reviewe

Binding of common organic UV-filters to the thyroid hormone transport protein transthyretin using in vitro and in silico studies: Potential implications in health

Several anthropogenic contaminants have been identified as competing with the thyroid hormone thyroxine (T4) for binding to transport proteins as transthyretin (TTR). This binding can potentially create toxicity mechanisms posing a threat to human health. Many organic UV filters (UVFs) and paraben preservatives (PBs), widely used in personal care products, are chemicals of emerging concern due to their adverse effects as potential thyroid-disrupting compounds. Recently, organic UVFs have been found in paired maternal and fetal samples and PBs have been detected in placenta, which opens the possibility of the involvement of TTR in the transfer of these chemicals across physiological barriers. We aimed to investigate a discrete set of organic UVFs and PBs to identify novel TTR binders. The binding affinities of target UVFs towards TTR were evaluated using in vitro T4 competitive binding assays. The ligand-TTR affinities were determined by isothermal titration calorimetry (ITC) and compared with known TTR ligands. In parallel, computational studies were used to predict the 3-D structures of the binding modes of these chemicals to TTR. Some organic UVFs, compounds 2,2',4,4'-tetrahydroxybenzophenone (BP2, Kd = 0.43 μM); 2,4-dihydroxybenzophenone (BP1, Kd = 0.60 μM); 4,4'-dihydroxybenzophenone (4DHB, Kd = 0.83 μM), and 4-hydroxybenzophenone (4HB, Kd = 0.93 μM), were found to display a high affinity to TTR, being BP2 the strongest TTR binder (ΔH = -14.93 Kcal/mol). Finally, a correlation was found between the experimental ITC data and the TTR-ligand docking scores obtained by computational studies. The approach integrating in vitro assays and in silico methods constituted a useful tool to find TTR binders among common organic UVFs. Further studies on the involvement of the transporter protein TTR in assisting the transplacental transfer of these chemicals across physiological barriers and the long-term consequences of prenatal exposure to them should be pursued.I. Cardoso works under the Investigator FCT Program which is financed by national funds through FCT and co-financed by ESF through HPOP, type 4.2 - Promotion of Scientific Employment. IQAC-CSIC acknowledges a contract to Ellen Y. Cotrina funded by the project of Fundació Marató de TV3 (Project ref. 20140330-31-32-33-34), Spain and a contract from Ford España - Fundación Apadrina la Ciencia. G. Arsequell acknowledges Dr. Rafel Prohens from Unitat de Polimorfisme i Calorimetria, Centres Científics i Tecnològics (University of Barcelona) for his supervision and assistance in ITC studies, and acknowledges Prof. Antoni Planas (IQS-URL) for full support on the TTR production. X. Biarnés acknowledges support from the Generalitat de Catalunya (2017 SGR 727, GQBB, Grup de Química Biològica i Biotecnologia) and M.S. Díaz-Cruz acknowledges support from the Generalitat de Catalunya (2017 SGR 01404, ENFOCHEM, Water, Environmental and Food Chemistry).Peer reviewe

Optimization of kinetic stabilizers of tetrameric transthyretin: A prospective ligand efficiency-guided approach

In the past few years, attempts have been made to use decision criteria beyond Lipinski’s guidelines (Rule of five) to guide drug discovery projects more effectively. Several variables and formulations have been proposed and investigated within the framework of multiparameter optimization methods to guide drug discovery. In this context, the combination of Ligand Efficiency Indices (LEI) has been predominantly used to map and monitor the drug discovery process in a retrospective fashion. Here we provide an example of the use of a novel application of the LEI methodology for prospective lead optimization by using the transthyretin (TTR) fibrillogenesis inhibitor iododiflunisal (IDIF) as example. Using this approach, a number of compounds with theoretical efficiencies higher than the reference compound IDIF were identified. From this group, ten compounds were selected, synthesized and biologically tested. Half of the compounds (5, 6, 7, 8 and 10) showed potencies in terms of IC50 inhibition of TTR aggregation equal or higher than the lead compound. These optimized compounds mapped within the region of more efficient candidates in the corresponding experimental nBEI-NSEI plot, matching their position in the theoretical optimization plane that was used for the prediction. Due to their upstream (North-Eastern) position in the progression lines of NPOL = 3 or 4 of the nBEI-NSEI plot, three of them (5, 6 and 8) are more interesting candidates than iododiflunisal because they have been optimized in the three crucial LEI variables of potency, size and polarity at the same time. This is the first example of the effectiveness of using the combined LEIs within the decision process to validate the application of the LEI formulation for the prospective optimization of lead compounds.We thank Dr. Lluís Bosch for help on the synthesis work. Funding Sources. This work was supported by a Grant 080530/31/32 from the Fundació Marató de TV3, Barcelona, Spain (to G.A, A.P., and J.Q.) and a Grant from Plan Nacional, Ministerio de Economía y Competitividad (Ref: CTQ2010-20517-C02-02) to G.A.Peer reviewe

Calorimetric Studies of Binary and Ternary Molecular Interactions between Transthyretin, Aβ Peptides, and Small-Molecule Chaperones toward an Alternative Strategy for Alzheimer's Disease Drug Discovery

Transthyretin (TTR) modulates the deposition, processing, and toxicity of Abeta (Aβ) peptides. We have shown that this effect is enhanced in mice by treatment with small molecules such as iododiflunisal (IDIF, 4), a good TTR stabilizer. Here, we describe the thermodynamics of the formation of binary and ternary complexes among TTR, Aβ(1-42) peptide, and TTR stabilizers using isothermal titration calorimetry (ITC). A TTR/Aβ(1-42) (1:1) complex with a dissociation constant of K = 0.94 μM is formed; with IDIF (4), this constant improves up to K = 0.32 μM, indicating the presence of a ternary complex TTR/IDIF/Aβ(1-42). However, with the drugs diflunisal (1) or Tafamidis (2), an analogous chaperoning effect could not be observed. Similar phenomena could be recorded with the shorter peptide Aβ(12-28) (7). We propose the design of a simple assay system for the search of other chaperones that behave like IDIF and may become potential candidate drugs for Alzheimer's disease (AD)

Insights on the Interaction between Transthyretin and Aβ in Solution. A Saturation Transfer Difference (STD) NMR Analysis of the Role of Iododiflunisal

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.The group at CIC bioGUNE acknowledges MINECO (Spain) for funding through Grant CTQ2015-64597-C2-1-P and a Juan de la Cierva contract to A.G