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Discovering Severe Acute Respiratory Syndrome Coronavirus 3CL Protease Inhibitors Virtual Screening Surface Plasmon Resonance and Fluorescence Resonance Energy Transfer Assays LILI CHEN 1 SHUAI CHEN 1 CHUNSHAN GUI 1 JIANHUA SHEN 1 XU SHEN 1 2 and HUALIANG JIANG 1 2 An integrated system has been developed for discovering potent inhibitors of severe acute respiratory syndrome coronavirus 3C like protease SARS CoV 3CL pro by virtual screening correlating with surface plasmon resonance SPR and fluores cence resonance energy transfer FRET technologies based assays The authors screened 81 287 small molecular com pounds against SPECS database by virtual screening 256 compounds were subsequently selected for biological evaluation Through SPR technology based assay 52 from these 256 compounds were discovered to show binding to SARS CoV 3CL pro The enzymatic inhibition activities of these 52 SARS CoV 3CL pro binders were further applied to FRET based assay and IC 50 values were determined Based on this integrated assay platform 8 new SARS CoV 3CL pro inhibitors were discovered The fact that the obtained IC 50 values for the inhibitors are in good accordance with the discovered dissociation equilibrium constants K D s assayed by SPR implied the reliability of this platform Our current work is hoped to supply a powerful approach in the discovery of potent SARS CoV 3CL pro inhibitors and the determined inhibitors could be used as possible lead compounds for further research Journal of Biomolecular Screening 2006 915 921 Key words SARS SARS CoV 3CL pro inhibitor virtual screening SPR FRET 2006 Society for Biomolecular Sciences www sbsonline org 915 T HE LAST OUTBREAK OF THE SEVERE ACUTE RESPIRATORY SYNDROME SARS epidemic in 2003 has led to thousands of lethally infected patients and hundreds of deaths 1 and the seri ous aftereffect has caused major medical and economic con cerns It has been reported that SARS coronavirus SARS CoV is responsible for SARS infection 2 3 and SARS CoV 3C like proteinase SARS CoV 3CL pro is an attractive target for the dis covery of anti SARS agents for its functional importance in the viral life cycle To date varied kinds of SARS CoV 3CL pro inhibitors were discovered 4 6 although no effective SARS CoV 3CL pro inhibitor has yet been reported to treat SARS Therefore it is still a great challenge to explore new chemical classes of SARS CoV 3CL pro inhibitors that can be possibly used in anti SARS research As has been used in the current drug discovery process by using the high performance computation technique to search the large chemical compound databases for the identification of pos sible drug candidates the virtual screening approach is a tech nology that is based on the 3D structure of the target protein It involves the rapid fitting of the chemical library members into the active sites of 3D protein structures and is critical to distin guish active from inactive substances at the primary screening stage To date there have been successful cases of virtual screen ing used in the discovery of lead compound candidates For example we recently reported that by using virtual screening technology with other methods several new small molecular specific cyclophilin A inhibitors were discovered 7 through vir tual screening with surface plasmon resonance SPR technology against a database containing structural information of more than 8000 available drugs Cinanserin SQ 10 643 a well characterized serotonin antagonist that had undergone preliminary clinical test ing in humans in the 1960s was discovered to be the SARS CoV 3CL pro inhibitor 5 Recently SPR technology has been recognized as a powerful tool in monitoring receptor ligand interactions with advantages of no labeling real time noninvasive measurements and low sample 1 Drug Discovery and Design Center State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai China 2 School of Pharmacy East China University of Science and Technology Shanghai China Received Mar 28 2006 and in revised form Jul 7 2006 Accepted for publica tion Jul 18 2006 Journal of Biomolecular Screening 11 8 2006 DOI 10 1177 1087057106293295 at TEXAS A 2006 at TEXAS A 2006 www sbsonline org 917 FIG 1 Structures of the 8 compounds screened in this study at TEXAS A 2006 Table 1 Summary of the Properties of the 8 SARS CoV 3CL pro Inhibitors No No No Rule of Molecular Hydrogen Hydrogen 5 Pass Compound Weight Bond Donors Bond Donors CLogP or Fail DC060015 558 4 2 7 6 5484 Fail DC060087 487 5 1 9 2 9486 Pass DC060159 519 4 4 7 5 6938 Fail DC060170 541 3 2 9 4 551 Pass DC060180 522 1 2 7 4 507 Pass DC060245 536 6 1 7 7 056 Fail DC060251 470 5 2 7 5 7126 Pass DC060256 420 3 1 5 3 2525 Pass FIG 2 The binding model of the 8 inhibitors against SARS CoV 3CL pro in the active site DC060251 is rendered in sticks whereas others are shown in lines FIG 3 Schematic representation of SARS CoV 3CL pro interaction with DC060251 DC060251 and protein residues are rendered in ball and stick and sticks respectively at TEXAS A 2006 www sbsonline org 919 FIG 4 Structures of 3 compounds A B C from MDL Drug Data Report MDDR Comprehensive Medicinal Chemistry CMC and China Natural Products Database CNPD databases DC060251 shows 90 structural similarity to them Table 2 Summary of Concentration of Inhibitor Producing 50 Inhibition IC 50 Values and Equilibrium Dissociation Constants K D s for SARS CoV 3CL pro Inhibitors Molecular Compound Weight Da IC 50 M K D M DC060015 558 4 49 96 4 62 18 60 1 18 DC060087 487 5 80 46 2 07 42 50 2 10 DC060159 519 4 42 19 3 02 19 14 2 03 DC060170 541 3 6 86 0 91 4 23 0 81 DC060180 522 1 9 19 2 34 6 50 0 23 DC060245 536 6 21 49 2 97 23 91 5 63 DC060251 470 5 9 56 1 75 4 78 0 29 DC060256 420 3 56 24 3 48 49 91 3 98 at TEXAS A 113 701 702 2 Drosten C Preiser W Gunther S Schmitz H Doerr HW Severe acute res piratory syndrome identification of the etiological agent Trends Mol Med 2003 9 325 327 3 Fouchier 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inhibition and kinetic assay curves for the 3 compounds from the screening procedure IC 50 values were extracted from the curves using nonlinear regression analysis and dissociation equilibrium constant K D values were analyzed using a global data analysis program of Biacore 3000 A B C represent the dose response inhibition curves Inset the responding binding curve of the compounds DC060170 DC060180 DC060251 respectively at TEXAS A 11 1445 1453 7 Li J Chen J Gui C Zhang L Qin Y Xu Q et al Discovering novel chem ical inhibitors of human cyclophilin A virtual screening synthesis and bioassay Bioorg Med Chem 2006 14 2209 2224 8 Cooper MA Optical biosensors in drug discovery Nat Rev Drug Discov 2002 1 515 528 9 Markgren PO Hamalainen M Danielson UH Screening of compounds interacting with HIV 1 proteinase using optical biosensor technology Anal Biochem 1998 265 340 350 10 Du L Zhang Z Luo X Chen K Shen X Jiang H Binding investigation of human 5 lipoxygenase with its inhibitors by SPR 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State Key Laboratory of Drug Research Shanghai Institute of Materia Medica Chinese Academy of Sciences Shanghai 201203 E mail xshen or hljiang SARS Coronavirus 3CL Protease Inhibitors Journal of Biomolecular Screening 11 8 2006 www sbsonline org 921 at TEXAS A M INTL UNIV on March 16 Downloaded from