Quaternary Structure, Substrate Selectivity and Inhibitor Design for SARS 3C-Like Proteinase

Abstract

The SARS coronavirus 3C-like proteinase is recognized as a potential drug design target for the treatment of severe acute respiratory syndrome. In the past few years, much work has been done to understand the catalytic mechanism of this target protein and to design its selective inhibitors. The protein exists as a dimer/monomer mixture in solution and the dimer was confirmed to be the active species for the enzyme reaction. Quantitative dissociation constants have been reported for the dimer by using analytic ultracentrifuge, gel filtration and enzyme assays. Though the enzyme is a cysteine protease with a chymotrypsin fold, SARS 3C-like proteinase follows the general base catalytic mechanism similar to chymotrypsin. As the enzyme can cut eleven different sites on the viral polyprotein, the substrate specificity has been studied by synthesized peptides corresponding or similar to the cleavage sites on the polyprotein. Predictive model was built for substrate structure and activity relationships and can be applied in inhibitor design. Due to the lack of potential drugs for the treatment of SARS, the discovery of inhibitors against SARS 3C-like proteinase, which can potentially be optimized as drugs appears to be highly desirable. Various groups have been working on inhibitor discovery by virtual screening, compound library screening, modification of existing compounds or natural products. High-throughput in vitro assays, auto-cleavage assays and viral replication assays have been developed for inhibition activity tests. Inhibitors with IC50 values as low as 60 nM have been reported.

Maturation Mechanism of Severe Acute Respiratory Syndrome (SARS) Coronavirus 3C-like Proteinase

“…The dissociation constant of pure SARS-3CL pro was measured to be 14 M using sedimentation equilibrium. However, for most in vitro assays for SARS-3CL pro , the enzyme concentration was around 1 M. As our previous study (4) and various other studies (19) have shown, only dimer is active for this enzyme. This brought a dilemma as the dimer concentration must be very low under the assay condition assuming a K d of 14 M. However, when substrate was present, the dimerization ability of SARS-3CL pro was significantly increased with a K d around 1 M under the experimental conditions (24).…”

“…For SARS 3CL pro , much has been learned about its catalytic mechanism (17), substrate specificity (18), as well as inhibitor design (19,20). However, few studies have been reported on its maturation mechanism.…”

The spike protein of SARS-CoV — a target for vaccine and therapeutic development

“…The trypsin cleavage site occurs at R667–S668 (REF. 36), whereas cathepsin L cleavage is mapped to T678-M679 in the S protein35. Cathepsin L cleaves the S protein of SARS-CoV upstream of, rather than adjacent to, the fusion peptide, and the cleavage is required for activation of the membrane fusion domain of the S protein following entry into target cells35.…”

Structure-based virtual screening against SARS-3CLpro to identify novel non-peptidic hits

“…Current drug design efforts 30,31 against this enzyme can be broadly classified into two categories: peptidic and small molecule based inhibitors. Figure 1 shows some of the latest peptidic 29,32-34 and small molecule 24,35,36 based inhibitors devised using the structural information and the substrate specificity profile of the SARS-3CL pro enzyme.…”