High-throughput analysis identifying drugs that reduce oxidative and ER stress in human coronary artery endothelial cells

Abstract Endoplasmic reticulum (ER) stress as well as oxidative stress have been shown to play important roles in metabolic and cardiovascular disease, and drugs that counteract the effects of ER and oxidative stresses maybe clinically useful. To identify novel compounds that ameliorate ER and oxidative stresses, we screened two drug libraries purchased from Evotec, San Francisco, CA; the NIH clinical collection 1 (446 compounds) and the NIH clinical collection 2 (281 compounds). Human coronary artery endothelial cells (HCAEC) were tested for ER and oxidative stress. ER stress was measured with an ER stress-sensitive secreted alkaline phosphatase (SAP) assay. The cells were transfected with the plasmid pSAP2. Control, expressing a heat-resistant form of SAP, and treated with the ER stress inducer tunicamycin in the presence or absence of each of the various compounds for 24-h, at which time SAP activity was measured. Compounds exhibiting significant increases in SAP activity (41 compounds out of a total of 727 tested; 5.6%) were then assayed for their ability to suppress superoxide (SO) anion generation in cells treated with 27.5 mM dextrose. SO generation was measured using the superoxide-reactive probe 2-methyl-6-(4-methoxyphenyl)-3,7-dihydroimidazo[1,2-A]pyrazin-3-one hydrochloride chemiluminescence. Of the 41 compounds identified as ER stress reducers, only 33 (80.5%) suppressed dextrose-induced SO anion generation. Interestingly, 51% of the compounds found to be dual-stress modifiers consisted of cardioprotective drugs, including statins, angiotensin receptor blockers, angiotensin-converting enzyme inhibitors as well as β-blockers. Future studies to validate the clinical effectiveness of these agents remain to be performed in pre-clinical and clinical trials.