病毒3C及3C-Like蛋白酶的鑑定、抑制和改造

Abstract

Severe acute respiratory syndrome (SARS) is an emerging infectious disease caused by a novel human coronavirus (CoV), which infected more than 8000 people during the 2003 outbreak. The viral maturation requires a main protease (3CLpro) to cleave the virus-encoded polyproteins. Accordingly, in human picornavirus (PV) family which consists of over 200 medically relevant viruses, a chymotrypsin-like protease (3Cpro) is required for viral replication through the processing the polyproteins. As a result, the 3CLpro and 3Cpro are regarded as anti-viral drug targets. However, known inhibitors (AG7088) against PV 3Cpro failed to inhibit SARS-CoV 3CLpro, indicating differences in their active-site structures. In this study, we have prepared the recombinant SARS 3CLpro without redundant residues at both N- and C-termini and characterized its kinetic property using a fluorogenic substrate. Combined with our crystallography data, we proposed a model to illustrate the maturation process of SARS 3CLpro. In addition, we evaluated several types of inhibitors and investigated their inhibitory mechanisms. Additionally, the recombinant 3Cpro from PV (including entervirus, coxsackievirus, and rhinovirus, abbreviated as EV, CV, and RV, respectively) were purified and characterized as well. We have identified several inhibitors which show their potencies against viral replication. Furthermore, we tested 6800 small molecules by high-throughput screening for anti-SARS agents and found one hit and its analogues could serve as the common inhibitors against CoV 3CLpro and PV 3Cpro. By computer modeling, the structural features of these compounds were elucidated to enhance our knowledge for developing anti-viral agents against PV and CoV. In order to determine the amino acid residues essential for the substrate specificity and engineer 3CLpro as a tool for tag removal of the recombinant fusion proteins, we developed a mutant 3CLpro (T25G) which has altered substrate specificity to cleave Gln↓Met. We have also constructed E. coli and yeast vectors to express recombinant fusion proteins with the T25G 3CLpro recognition site (Ala-Val-Leu-Gln↓Met) between the tags and the target proteins for tag removal.