Construction Of Membrane Transport Proteins As A Target For Drug Screening Model And Applied Research

Atherosclerosis is the principal pathogenesis for many critical cardiovascular diseases (e.g., coronary heart disease, angina pectoris, myocardial infarction and stroke).The characteristic component of the atherosclerotic plaque is the macrophage derived foam cell. Intracellular free cholesterol can be toxic to the cell and therefore an efficient cholesterol efflux mechanism in macrophage is mandatory to prevent cholesterol accumulation. The ATP-binding cassette transporter A1 (ABCA1), which belongs to a superfamily of ATP-binding cassette transporters (ABCs), mediates the rate-controlling step in the HDL particle formation and in the assembly of free cholesterol and phospholipids with lipid-poor apolipoprotein A-I (apoA-I). Several laboratories have demonstrated that lipid-poor apolipoprotein A-I (apoA-I) can interacts with ABCA1 to mediate cellular cholesterol and nascent HDL assembly, which suggests a possible receptor-ligand type interaction between ABCA1 and apoA-I. In the current study, a high-throughput assay cell-ELISA-like method for ABCA1 regulators targeted at the binding apoA-I was developed and used for screening a synthetic and natural compound library. The cell-ELISA based high throughput screen is conducted in a 96-well format using CHO cells stably transfected with ABCA1 pIRE2-EGFP expression vector and calibrated with reference ABCA1 inhibitor Glibenclamide. Among 2600 compounds,1 xanthone compound 2026791 was picked up as hits by the highthroughput screening assay, and the compound was further identified as potential accelerator of cholesterol efflux via ABCA1 by flow cytometry assay and western blot analysis. Na,K-ATPase is an integral membrane protein found in most mammalian cells. It was initially discovered as an energy transducing ion pump that transports 3 Na+out and 2 K+ into the cell by hydrolysis of one molecule of ATP. The ligands of Digitalis-like glycosides have been reported to be the specific inhibitors of Na,K-ATPase which are most accessible to the E2-P form on the extracellular side, resulting in the specific inhibition of ATP hydrolysis and cation transport. Recently we and others have found that Na,K-ATPase can interact with Src to form a functional signaling compex which can be as the receptor of endogenous cardiotonic glycosides and mediate extracellular signal transduction into the cell so as to regulate cell growth and expression of various genes. In order to develop new promising chemicals targeted at Na,K-ATPase which benefit the cardiovascular diseases and cancer, A high throughput screen assay in a multiwell format was established. A library of 2600 compounds was screened using the developed assay, and a class of hydroxyxanthones with different inhibition of the Na,K-ATPase was identified. Further study of one compound MB5 indicate that the compound favors El like conformations and can compete for ouabain binding to the Na/K-ATPase. Taken together, we have identified a new class of Na/K-ATPase ligand that may function as a CTS antagonist.