key: cord-1006658-jgtbp5r6
authors: Kao, Richard Y.; Tsui, Wayne H.W.; Lee, Terri S.W.; Tanner, Julian A.; Watt, Rory M.; Huang, Jian-Dong; Hu, Lihong; Chen, Guanhua; Chen, Zhiwei; Zhang, Linqi; He, Tian; Chan, Kwok-Hung; Tse, Herman; To, Amanda P.C.; Ng, Louisa W.Y.; Wong, Bonnie C.W.; Tsoi, Hoi-Wah; Yang, Dan; Ho, David D.; Yuen, Kwok-Yung
title: Identification of Novel Small-Molecule Inhibitors of Severe Acute Respiratory Syndrome-Associated Coronavirus by Chemical Genetics
date: 2004-09-17
journal: Chem Biol
DOI: 10.1016/j.chembiol.2004.07.013
sha: 993a4ec640e7411cea67abb9b520ceab732fb883
doc_id: 1006658
cord_uid: jgtbp5r6

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) infected more than 8,000 people across 29 countries and caused more than 900 fatalities. Based on the concept of chemical genetics, we screened 50,240 structurally diverse small molecules from which we identified 104 compounds with anti-SARS-CoV activity. Of these 104 compounds, 2 target the SARS-CoV main protease (M(pro)), 7 target helicase (Hel), and 18 target spike (S) protein-angiotensin-converting enzyme 2 (ACE2)-mediated viral entry. The EC(50) of the majority of the 104 compounds determined by SARS-CoV plaque reduction assay were found to be at low micromolar range. Three selected compounds, MP576, HE602, and VE607, validated to be inhibitors of SARS-CoV M(pro), Hel, and viral entry, respectively, exhibited potent antiviral activity (EC(50) < 10 μM) and comparable inhibitory activities in target-specific in vitro assays.

Introduction screen for small-molecule compounds that perturb the infectivity of the virus. The employment of high-throughput The severe acute respiratory syndrome-associated coscreening (HTS) technologies to generate a collection ronavirus (SARS-CoV) recently emerged as the causof structurally diverse small-molecule compounds perative agent of an endemic atypical pneumonia. Within turbing the pathogenesis of the SARS-CoV will lay down a year, SARS-CoV infected more than 8,000 people the foundation to dissecting the molecular basis of viral across 29 countries and cost more than 900 human lives infections using chemical genetics.

[1]. Lack of knowledge of the novel coronavirus SARS-CoV and the absence of efficacious therapeutic agents were the main reasons for the failure to manage the Results and Discussion outbreak of SARS effectively. After the causative agent of the devastating disease was identified by us and

In a primary screening (at 20 g/ml of each compound), rapidly by several groups [5] [6] [7] . Subsequently, reverse we identified 1003 "hits" (a hit rate of 2%) that protected genetics with SARS-CoV cDNA was accomplished [8] , Vero cells from SARS-CoV-induced CPE. When the hits and ACE2 was identified as a functional receptor for were rearrayed and the concentration of selected compounds was lowered to 10 g/ml for secondary screening, 104 compounds retained consistent protective ef-*Correspondence: rytkao@hkucc.hku.hk

A schematic illustration of major processes involved in the phenotype-based screen is shown. Enlarged images of Vero cells from a typical 384-well tissue culture plate used in screening are also included to indicate the criteria for hit selection. Only those compounds that fully protected the Vero cells from SARS-CoV-induced CPE were selected as hits.

fects against SARS-CoV-induced CPE in Vero cells of the viral M pro . The purified SARS-CoV M pro cleaved efficiently and specifically a synthetic peptide with the ( Figure 1 ). Further evaluation by quantitative plaque reduction assays demonstrated that the EC 50 (median ef-sequence H 2 N-TSAVLQ↓SGFRKW-COOH mimicking the putative autolytic cleavage site (the cleavage site is indi-fective concentration) of the selected compounds were below 10 g/ml, with 78 compounds having an EC 50 cated with a ↓) of the N-terminal part of M pro [22] . Screening of the 104 compounds at a concentration of 20 g/ml below 2 g/ml. For subsequent studies, the concentrations of selected compounds were converted to molar identified two candidates as potential inhibitors of the SARS-CoV M pro . One of the compounds (designated units for more precise comparison of their biological activities. The TC 50 (median toxic concentration) of se-MP576) displayed potent inhibitory activity with an IC 50 (median inhibitory concentration) of 2.5 M (Figure 2A ) lected compounds was determined to be Ͼ50 M by MTT (3-[4,5-dimethylth-iazol-2-yl]-2,5-diphenyltetrazol-and an EC 50 of 7 M in the Vero cell-based SARS-CoV plaque reduction assay ( Figure 2B ). Furthermore, MP576 ium bromide) assay. To test our hypothesis that the 104 selected compounds represent diverse molecular can be docked favorably into the active site of SARS-CoV M pro ( Figure 2C ), consistent with the in vitro cleavage blockers of various biological pathways crucial for SARS-CoV infectivity, we screened for molecules tar-data indicating that MP576 is a novel nonpeptide inhibitor of SARS-CoV M pro . Since SARS-CoV M pro shares a geting viral entry, transcription, and proteolytic processing, the three major processes essential for suc-similar structural fold with serine proteases [21, 23] , we tested the inhibitory activity of MP576 on human neutro-cessful viral replication in the host. phil elastase (HNE). Using azocasein as the substrate, we demonstrated that 100 M of MP576 failed to inhibit Compounds Targeting SARS-CoV M pro SARS-CoV M pro is believed to play a major role in proteo-the proteolytic activity of HNE ( Figure 2D ), suggesting that MP576 is not a general protease inhibitor. lytic processing of the viral polyproteins and is regarded as a prime target for anti-SARS-CoV drug development [20, 21] . The recently available crystal structure of the Compounds Targeting SARS-CoV Hel To identify compounds that interfere with viral compo-SARS-CoV M pro makes it a suitable target for structurebased rational drug design [22] . We cloned the SARS-nents essential for transcription and replication, we selected the SARS-CoV Hel, which we had previously CoV M pro in an E. coli expression system and obtained purified enzyme to examine if any of the 104 selected cloned and characterized [24], for our assay system. It was demonstrated that the SARS-CoV NTPase/helicase compounds targets this vital component of the virus. High-performance liquid chromatography (HPLC)-based had an unstimulated ATPase activity that could be stimulated by the addition of polynucleotides, in particular assays were used to monitor the in vitro cleavage activity nated HE602) exhibited substantial inhibitory activity at 2 g/ml. HE602 was found to strongly inhibit the polynucleotide-stimulated ATPase activity of SARS-CoV Hel with an IC 50 of 6.9 M ( Figure 3A ), but barely inhibited the dT 24 . As the functional conformation of the helicase is likely to be the nucleic acid-bound state, we initially unstimulated ATPase activity even at a concentration of 200 M ( Figure 3B ). In the helicase assay, a duplex screened the 104 active compounds against the polynucleotide-stimulated ATPase activity of SARS-CoV Hel DNA was prepared incorporating a 5Ј overhang that is required for SARS-CoV helicase activity. The shorter strand of the duplex DNA incorporated a 32 P label so that differential migration of the duplex or single-stranded oligonucleotide could be easily observed following polyacrylamide gel electrophoresis. It can be clearly seen that both 200 and 20 M concentrations of HE602 inhibited the helicase activity of the SARS-CoV Hel, while 2 and 0.2 M concentrations did not inhibit the helicase activity ( Figure 3C) . It was only possible to perform a qualitative assay with this system, but these results are fully consistent with the IC 50 value that we measured using our quantitative ATPase assay. Furthermore, HE602 inhibited the viral plaque formation in Vero cell with an EC 50 of 6 M ( Figure 3D ). The effective inhibition of only the stimulated ATPase activity together with its ability to inhibit helicase activity indicates that HE602 has distinct parallels with the pharmacological profiles of inhibitors targeted against the herpes simplex virus (HSV) helicase-primase protein [23, 25] . pounds are indisputable, the precise mode of inhibition Based on the concept of chemical genetics, we have established a HTS platform to identify small-molecule of these novel inhibitors is currently under investigation. In addition, one has to be cautioned that the ultimate compounds that will perturb the pathogenic pathways of SARS-CoV in Vero cells. After screening 50,240 validation of their targets will be the selection of chemical-resistant viruses and subsequent identification of structurally diverse small-molecule compounds against SARS-CoV infection in a cellular model, we have identi-genetic mutations conferring the resistant phenotype.

fection. 

(Novagen). ATPase assays were performed using a phosphomolybdate-malachite green assay to measure phosphate released as de-immediately. Plates were further incubated for 48 hr under identical conditions. Cells were fixed by adding 1 ml of 10% formaldehyde. scribed previously [24] . dT 24 at a concentration of 200 nM was used in the polynucleotide stimulated ATPase assay. For the helicase The agarose plugs were removed subsequently and cells stained with 0.5% crystal violet in 70% methanol and the viral plaques assay, 32 P-labeled "released" oligo (5Ј-GGTGCAGCCGCAGCGGTG CTCG-3Ј) was thermally annealed to oligo "5T20" (5Ј-TTTTTTTTT counted. The poliovirus (type 1) used in the study was a clinical isolate from a patient in 

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