TY - JOUR
T1 - Development of a dimethylarginine dimethylaminohydrolase (DDAH) assay for high-throughput chemical screening
AU - Ghebremariam, Yohannes T.
AU - Erlanson, Daniel A.
AU - Yamada, Keisuke
AU - Cooke, John P.
N1 - Funding Information:
The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported in part by grants to JPC from the NIH (RC2HL103400, 1U01HL100397, and K12HL087746), AHA (11IRG5180026), Stanford SPARK Program, and the Tobacco-Related Disease Research Program of the University of California (18XT-0098). YTG was a recipient of the Stanford School of Medicine Dean’s fellowship (1049528-149- KAVFB) and is currently supported by the Tobacco-Related Disease Research Program of the University of California (20FT-0090). Keisuke Yamada was a visiting scholar.
PY - 2012/6
Y1 - 2012/6
N2 - Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.
AB - Nitric oxide (NO) is a potent signaling molecule that needs to be tightly regulated to maintain metabolic and cardiovascular homeostasis. The nitric oxide synthase (NOS)/dimethylarginine dimethylaminohydrolase (DDAH)/asymmetric dimethylarginine (ADMA) pathway is central to this regulation. Specifically, the small-molecule ADMA competitively inhibits NOS, thus lowering NO levels. The majority of ADMA is physiologically metabolized by DDAH, thus maintaining NO levels at a physiological concentration. However, under pathophysiological conditions, DDAH activity is impaired, in part as a result of its sensitivity to oxidative stress. Therefore, the application of high-throughput chemical screening for the discovery of small molecules that could restore or enhance DDAH activity might have significant potential in treating metabolic and vascular diseases characterized by reduced NO levels, including atherosclerosis, hypertension, and insulin resistance. By contrast, excessive generation of NO (primarily driven by inducible NOS) could play a role in idiopathic pulmonary fibrosis, sepsis, migraine headaches, and some types of cancer. In these conditions, small molecules that inhibit DDAH activity might be therapeutically useful. Here, we describe optimization and validation of a highly reproducible and robust assay successfully used in a high-throughput screen for DDAH modulators.
KW - asymmetric dimethylarginine
KW - diabetes
KW - hypertension
KW - idiopathic pulmonary fibrosis
KW - nitric oxide
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U2 - 10.1177/1087057112441521
DO - 10.1177/1087057112441521
M3 - Article
C2 - 22460174
AN - SCOPUS:84861825417
SN - 1087-0571
VL - 17
SP - 651
EP - 661
JO - Journal of Biomolecular Screening
JF - Journal of Biomolecular Screening
IS - 5
ER -