| The innate antiviral response in higher vertebrates is mediated in part by 2-5A dependent endoribonuclease, RNase L. In humans, RNase L maps to the Hereditary Prostate Cancer 1 (HPC1) gene suggesting that it may have a tumor suppressor role. RNase L activation occurs upon binding unique 2' 5' linked oligoadenylates (2-5A). 2-5A is produced by 2-5A synthetases in response to viral infections. Pharmacologic properties of 2-5A render it unsuitable as a therapeutic agent. It is unstable in serum, cells, and the digestive tract due to the action of phosphodiesterases and phosphatases, and it requires a cationic lipid to enter the cells. Our hypothesis is that the identification of small molecule activators of RNase L, will have improved pharmacological properties compared to 2-5A. To identify small molecule activators of RNase L, we have designed and implemented a Convenient and Sensitive Fluorescence Resonance Energy Transfer (FRET) assay to measure RNase L activity in vitro. We identified seven small molecule activators of RNase L in High Throughput Screening (HTS) of thirty-two thousand chemicals (ChemBridge DiverSet(TM)). We confirmed the activity of hits in an alternative radiolabeled RNA cleavage assays. Our Structure Activity Relationship (SAR) studies of lead compounds resulted in four related compounds. We studied the mechanism of RNase L activation using two lead compounds and compared it to that of 2-5A. We determined the binding constants of two lead compounds in the range of 10 to 30muM in Surface Plasmon Resonance (SPR) studies. SPR analysis suggest that two hits compete with 2-5A probably for the same binding pocket in RNase L. We showed that RNase L dimerizes during activation by small molecules, which is a prerequisite for endonuclease activity. Computational docking of the two lead compounds validates our experimental results, as they seem to bind in the 2-5A binding pocket of RNase L. Compound 1 or 2 are benign to the cells in culture, and in addition both compounds significantly suppressed Human ParaInfluenza Virus type 3 (HPIV3), Vesicular Stomatitis Virus (VSV) and Encephalomyocarditis Virus (EMCV) replication in culture. Our findings provide a promising avenue for further development of small molecule mimics of 2-5A that may eventually lead to novel drug discovery for prostate cancer and viral infections. |
