Characterization of SARS main protease and inhibitor assay using a fluorogenic substrate

Abstract

SARS main protease is essential for life cycle of SARS coronavirus and may be a key target for developing anti-SARS drugs. Recently, the enzyme expressed in Escherichia 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. This protease easily dissociated into inactive monomer and the deduced Kd of the dimer was 100 microM. In order to detect enzyme activity, the assay needed to be performed at micromolar enzyme concentration. This makes finding the tight inhibitor (nanomolar range IC50) impossible. In this study, we prepared a peptide with fluorescence quenching pair (Dabcyl and Edans) at both ends of a peptide substrate and used this fluorogenic peptide substrate to characterize SARS main protease and screen inhibitors. The fluorogenic peptide gave extremely sensitive signal upon cleavage catalyzed by the protease. Using this substrate, the protease exhibits a significantly higher activity (kcat = 1.9 s(-1) and Km = 17 microM) compared to the previously reported parameters. Under our assay condition, the enzyme stays as an active dimer without dissociating into monomer and reveals a small Kd value (15 nM). This enzyme in conjunction with fluorogenic peptide substrate provides us a suitable tool for identifying potent inhibitors of SARS protease.

Fig. 1

SDS–PAGE analysis of the SARS main protease at different stages of purification procedure. Lane 1 represents the molecular mass markers which are 220, 97, 66, 46, 30, 21.5, and 14.3 kDa. Lanes 2 and 3 show the cell lysate without and with IPTG induction to overexpress SARS main protease with tag, respectively. Lane 4 is the tagged protease after Ni–NTA column chromatography. Lane 5 represents the protease treated with FXa to remove the tag. Two extra bands, intact protease and the tag at lower molecular mass, appear on SDS–PAGE. Lane 6 shows the purified untagged protease after using the second Ni–NTA column.

Fig. 2

Measurements of kinetic parameters of SARS main protease. The reaction initial rates of the protease under a variety of different substrate concentrations were plots against substrate concentrations to obtain the V_max and K_m values of the enzyme. KaledaGraph computer program was used to fit the kinetic data using Michaelis–Menten equation.

Fig. 3

Dependence of SARS protease reaction rate on enzyme concentration. The 5–150 nM SARS main protease as shown in (A) and 50–3000 nM as shown in (B) with 60 μM fluorogenic substrate were used to determine the K_d value of dimer–monomer equilibrium. From (A), the K_d was determined to be 15 ± 4 nM by fitting the data to Eq. (3) (see Materials and methods). At the protein concentrations from 50 to 3000 nM (significantly larger than the K_d value), the fitting curve is almost linear.

Fig. 4

Gel filtration study of the SARS protease. At 0.2 (dot-and-dash line) and 4 mg/mL (solid line), the SARS protease shows a major peak corresponding to the dimer on the elution profile of gel filtration column chromatography. The arrows indicate the positions for dimer and monomer of the protease.