Characterization of SARS-CoV main protease and identification of biologically active small molecule inhibitors using a continuous fluorescence-based assay

Abstract

Severe acute respiratory syndrome associated coronavirus main protease (SARS-CoV Mpro) has been proposed as a prime target for anti-SARS drug development. We have cloned and overexpressed the SARS-CoV Mpro in Escherichia coli, and purified the recombinant Mpro to homogeneity. The kinetic parameters of the recombinant SARS-CoV Mpro were characterized by high performance liquid chromatography-based assay and continuous fluorescence-based assay. Two novel small molecule inhibitors of the SARS-CoV Mpro were identified by high-throughput screening using an internally quenched fluorogenic substrate. The identified inhibitors have Ki values at low microM range with comparable anti-SARS-CoV activity in cell-based assays.

Figure 1

(A) Specific cleavage of synthetic peptide SP1 by purified SARS‐CoV M^pro. P1 and P2, cleavage products; S, uncleaved synthetic substrate SP1. (B) Lineweaver–Burk plot for the determination of SARS‐CoV M^pro kinetic parameters in HPLC‐based assays. The K _m and k _cat of the SARS‐CoV M^pro for substrate SP1 were determined by incubation of SP1 at different concentrations varying from 0.6 to 2.4 mM with 200 nM of SARS‐CoV M^pro in 20 mM Tris–HCl, pH 7.3, and 150 mM NaCl under conditions described in Section 2. The initial rates of cleavage were determined under the condition that 5–10% of the total substrate was cleaved. Experiments were carried out in triplicates and data points are expressed as means ± S.D.

Figure 2

(A) Initial fluorescence intensity of fluorogenic substrate SP2. Results are expressed as fluorescence intensity units (FIU). (B) Initial cleavage rate of SP2 by SARS‐CoV M^pro. 0–800 nM of purified SARS‐CoV M^pro were used in the experiment.

Figure 3

HTS of small molecule inhibitors of SARS‐CoV M^pro using fluorogenic substrate SP2. The initial cleavage rates of SARS‐CoV M^pro on fluorogenic substrate SP2 in the presence of the test compounds (104 compounds) were normalized to values in the absence of compounds and expressed as arbitrary units (AU). Dotted line indicates 0.5 AU.

Figure 4

Chemical structures of SARS‐CoV M^pro inhibitors and their chemical analogues. (A) Chemical structure of MP576 (3‐quinolinecarboxylic acid, 1,4,5,6,7,8‐hexahydro‐2‐methyl‐4‐(2‐nitrophenyl)‐5‐oxo‐, 2‐phenylethyl ester). (B) Chemical structure of MP521 (2‐(2‐nitrophenyl)‐1,3‐diphenyl‐imidazolidine). (C) Chemical structure of CB5751. (D) Chemical structure of CB5173. CB5751 is an analogue of MP576 and CB5173 is an analogue of MP521.

Figure 5

Anti‐SARS‐CoV activities of MP576 and MP521. (A) Vero cells infected with 100 TCID₅₀ SARS‐CoV. (B) Vero cells infected with 100 TCID₅₀ SARS‐CoV in the presence of 20 μg/ml of MP576. (C) Vero cells infected with 100 TCID₅₀ SARS‐CoV in the presence of 20 μg/ml of MP521. CPE were recorded 96 h post infection. Experiments were carried out in duplicate and repeated twice.

Figure 6

Inhibitory activity of analogues of MP576 and MP521 on SARS‐CoV M^pro. The cleavage of 10 μM of the fluorogenic substrate SP2 by 200 nM of purified SARS‐CoV M^pro in 20 mM Tris‐HCl, pH 7.3, and 150 mM NaCl at 25 °C was monitored continuously by a fluorescence spectrophotometer in the presence or absence of 50 μM of different compounds. Background fluorescence was subtracted for clarity of comparison. Experiments were carried out in duplicate and mean value of each data point was used for plotting.