Master of Science / Department of Diagnostic Medicine/Pathobiology / Kyeong-Ok Chang / Positive sense RNA viruses include diverse groups of viruses that cause a wide variety of diseases in humans and animals. Most of these viruses encode proteases that cleave the viral polyprotein into intermediate or mature functional proteins during virus replication. As these proteases play a critical role in virus replication, they represent an attractive target for the development of antiviral drugs. In this study, the main goal was to establish assay systems and characterize the enzymatic activity of related proteases from Norwalk virus (NV), poliovirus, and transmissible gastroenteritis virus (TGEV). These proteases share several common characteristics including a typical chymotrypsin-like fold, a Cys residue as a nucleophile in the catalytic triad (or dyad) composed of Cys, His and Glu (or Asp) residues, and a preference for a Glu or Gln residue at the P1 position on the substrate. We cloned and expressed proteases from these viruses and characterized their enzymatic activities using a fluorescence resonance energy transfer (FRET) assay using a specific FRET substrate corresponding to each viral protease. First, assay conditions of the FRET assay was optimized for each virus protease. Second, inhibition profiles of each virus protein were investigated using five commercially available standard protease inhibitors (chymostatin, leupeptin, antipain, TPCK, and TLCK). The inhibition studies showed that TPCK inhibited NV, poliovirus, and TGEV proteases with varying strength, and chymostatin inhibited only NV protease. All other inhibitors had little effects on the virus proteases. The established FRET assays should facilitate screening potential antivirals.

Sortase A is a housekeeping enzyme of Gram-positive bacteria that catalyses the anchoring of surface proteins to the bacterial peptidoglycan. The enzyme works to establish an interaction between bacteria and host cells and is essential for pathogenesis. This makes Sortase A a potential suitable target for inhibition, in order to treat bacterial infections. In this degree project Sortase A from Staphylococcus aureus was explored and potential inhibitors were investigated by performing enzyme activity and bacterial binding assays. A robust FRET assay was developed and optimized for a recombinant version of the enzyme and serves as a good starting point for studying inhibition.