The N-terminal octapeptide acts as a dimerization inhibitor of SARS coronavirus 3C-like proteinase.
The 3C-like proteinase of severe acute respiratory syndrome (SARS) coronavirus has been proposed to be a key target for structural-based drug design against SARS. Accurate determination of the dimer dissociation constant and the role of the N-finger (residues 1-7) will provide more insights into the enzyme catalytic mechanism of SARS 3CL proteinase. The dimer dissociation constant of the wild-type protein was determined to be 14.0microM by analytical ultracentrifugation method. The N-finger fragment of the enzyme plays an important role in enzyme dimerization as shown in the crystal structure. Key residues in the N-finger have been studied by site-directed mutagenesis, enzyme assay, and analytical ultracentrifugation. A single mutation of M6A was found to be critical to maintain the dimer structure of the enzyme. The N-terminal octapeptide N8 and its mutants were also synthesized and tested for their potency as dimerization inhibitors. Peptide cleavage assay confirms that peptide N8 is a dimerization inhibitor with a K(i) of 2.20mM. The comparison of the inhibitory activities of N8 and its mutants indicates that the hydrophobic interaction of Met-6 and the electrostatic interaction of Arg-4 contribute most for inhibitor binding. This study describes the first example of inhibitors targeting the dimeric interface of SARS 3CL proteinase, providing a novel strategy for drug design against SARS and other coronaviruses.
Wei
Ping
P
State Key Laboratory for Structural Chemistry of Stable and Unstable Species, College of Chemistry, Peking University, Beijing 100871, China.
Fan
Keqiang
K
Chen
Hao
H
Ma
Liang
L
Huang
Changkang
C
Tan
Lei
L
Xi
Dong
D
Li
Chunmei
C
Liu
Ying
Y
Cao
Aoneng
A
Lai
Luhua
L
D001665:Binding Sites; D003198:Computer Simulation; D000086782:Coronavirus 3C Proteases; D003546:Cysteine Endopeptidases; D019281:Dimerization; D015195:Drug Design; D010450:Endopeptidases; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000037:antagonists & inhibitors*
D004791:Enzyme Inhibitors; D010455:Peptides; D014764:Viral Proteins; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D000086782:Coronavirus 3C Proteases