Characterization of SARS Main Protease and Facile Assay for Inhibitors
by Using a Fluorogenic Substrate
Chih-Jung Kuo §, Ping-Fang Huang §, John T.A. Hsu ‡, Wen-Bin Yang †, Ming-Feng Hsu §, Tsung-Yi Wu †, Chun-Hung Lin §, Chun-Cheng Lin # , Jim-Min Fang *, Chi-Huey Wong †,
Andrew H.-J. Wang §, and Po-Huang Liang §
Department of Chemistry,
SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. The enzyme expressed in E. coli was characterized using a HPLC assay to monitor the formation of products from 11 peptide substrates covering the cleavage sites found in the SARS viral genome. In this study, a peptide with fluorescent quench pair (Dabcyl and Edans) at both ends of the peptide substrate was prepared and utilized this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorescent peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Sufficient reducing power is found critical in preparing the fully active enzyme. Under our assay condition, the enzyme stays as an active dimmer without dissociating into monomer with a small Kd value. This enzyme and fluorogenic peptide substrate together provide us a suitable tool for identifying potent inhibitors.
Multiphoton Polarization and Generalized Polarization (GP) Microscopy Reveals Oleic Acid Induced Structural Changes in Intercellular
Lipid Layers of the Skin
C. Y. Hsiao2,
In this work, we will present the results elucidating the actions of chemical enhancer oleic acid on the mechanism of transdermal delivery using multiphoton fluorescence polarization and generalized polarization (GP) microscopy. In our approach, model drugs such as sulforhodamine B (hydrophilic) and RBHE (hydrophobic) is combined with membrane probes such as laurdan to image the change in the stratum corneum in the presence and absence of oleic acid. By analyzing Laurdan fluorescence, both the polarization and GP (at 440 nm and 490 nm emission) images show that the treatment of oleic acid results in a skin surface with a more random packing of the lipid molecules which allows easier water penetration. Most likely, this results in easing the delivery of chemicals across the skin. Our results are significant in understanding the biophysical transport pathways across the skin and can lead to better drug and gene delivery methodologies.