| Assay Method Information | |
| | In Vitro Apo(a) Binding Assay |
| Description: | The in vitro binding affinity of compounds to the intended target human Apo(a) protein is tested in a competitive binding assay. Human Apo(a) protein containing 17 Kringle repeats is affinity purified from conditioned media of transiently transfected HEK-293F cells. All reagents are prepared in assay buffer containing 50 mM Tris-HCl pH 7.4, 0.1% BSA. The binding assay is conducted by adding to each well of a clear-bottom plate 50 each of (1) test compound in dilution series (final concentration 0.3210000 nM), (2) Apo(a) protein (6 ng/well), (3) re-suspended Wheat Germ Agglutinin Polyvinyltoluene SPA beads (20 mg/ml), and (4) the radioligand, tritium-labeled (2S)-3-[3-[2-oxo-3-[3-(tritritiomethoxy)phenyl]imidazolidin-1-yl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride (final concentration 0.52 nM). Plates are incubated for 60 minutes at RT and counted on TRILUX LSC. Non-specific binding, defined as binding in the presence of 10 M cold (i.e. non-radiolabeled) ligand: (2S)-3-[3-[2-oxo-3-[3-(methoxy)phenyl]imidazolidin-1-yl]phenyl]-2-[(3R)-pyrrolidin-3-yl]propanoic acid; hydrochloride is subtracted to determine specific binding. Data are analyzed by fitting to a standard single site binding model and the IC50 for the Example test compound is determined. These results are summarized in Table 1, and indicate that the Example test compounds bind to human Apo(a) protein. Inhibition of the assembly of the LDL particle with apo(a) through binding to the Apo(a) protein supports a reduction in Lp(a) levels. |
| Affinity data for this assay | |
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