Query String: TAFAMIDIS
US10377729, Compound Tafamidis Tafamidis CHEBI:78538 BDBM50197883 US20240150297, Compound Tafamadis Vyndaqel FX-1006
- Binding of Examples to Transthyretin - EC50 Determination TTR SPA binding assays were performed in a final volume of 60 mI containing 100 ng human TTR (biotinylated recombinant protein) coupled to 25 pg SPA beads (streptavidin coated, Perkin Elmer, RPNQ0007) and 50 nM [3H] tafamidis (Moravek, MT-1003033), plus varying concentrations of test compound or vehicle. Briefly, assays were prepared at room temperature in 384-well plates (Corning, 3767) containing 200 nl_ of test compound in DMSO (or DMSO as vehicle). The plates also contained wells with a saturating concentration of unlabeled ligand (200 nl_ of 3 mM tafamidis or 3 mM thyroxine in DMSO) for measuring non-specific binding. Assays were initiated by addition of 20 mI of 5 mg/mL TTR protein in assay buffer (10 mM Tris pH 7.5, 150 mM NaCI, 0.25% Triton X-100) and 20 mI_ of 150 nM [3H] tafamidis in assay buffer. The plates were incubated 1 hour prior to addition of 20 mI_ of 1.25 mg/ml_ SPA beads diluted in assay buffer. The assays were incubated an additional 10 hours to allow binding to reach equilibrium and the amount of receptor-ligand complex was determined by liquid scintillation counting using a 1450 Microbeta Trilux (Wallac). The % effect values for test wells were calculated based on the total binding (vehicle, 0% effect) and non-specific binding (unlabeled ligand, 100% effect) wells on each assay plate. ECso values were then determined using a standard 4 parameter logistic dose response equation.
- KD Determination by SPR Affinity and Reversibility -The binding affinity and kinetics of binding were measured using Surface Plasmon Resonance based binding assay. These experiments were carried out on Bruker SPR MASS-1 and MASS-2 instruments. There was no significant difference in results obtained on both these instruments. Bap-tagged TTR protein was captured on a Streptavidin coated sensor chip to achieve about 2000 to 3000 RUs of surface density. All the samples were prepared in buffer consisting of 10 mM Sodium Phosphate, pH 7.6, 100 mM KCI, 0.005% Tween-20 and 2% DMSO. The same buffer was used as the running buffer during the experiments. Compound samples were injected at a flow rate of 30 pL/min for 90 seconds of association time followed by at least 240 seconds of dissociation period. The compounds were tested in a concentration series consisting of at least 6 samples (usually 10) made with 5-fold, 3-fold, or 2-fold dilution. The highest concentration was 10 mM or selected based on compound binding affinity observed in a previous experiment. Multiple blank injections were run before and after each compound series to allow double reference subtraction during data processing and analysis. Tafamidis or another compound with >10 replicates was tested in every experiment as a positive control to assess activity of the captured protein on the surface. A DMSO curve was run during each experiment to properly correct for excluded volume. The data were processed and analyzed using Bruker Analyzer and Scrubber to calculate binding affinities by fitting the data to 1:1 binding model. The binding parameters obtained for tafamidis binding to TTR (n=24) are listed below. Tafamidis binds to TTR in a reversible manner with calculated residence time of around 40 seconds.
- In vitro evaluation of inhibitory activity against WT-TTR In vitro evaluation of inhibitory activity against WT-TTR amyloid fibril formation of AT09 and reference compounds. Reference compounds (thyroxine, tafamidis and 2OH PCB80) are represented by dash bars and AT09 compounds by solid-gray bars. All compounds were analyzed at 2×, 1× and 0.5× the molar concentration of wild-type TTR (3.6 μM). Upon assay completion at 72 hours incubation at 37° C., the percentage of amyloid fibril formation was normalized against the positive control (black bar) corresponding to 100% of amyloid formation in the absence of test compounds.