【病毒外文文献】2005 Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acut

Abstract lighted on screening screening anti TGEV f w screening K 1 caused al outbreak pounds and consensus are replication ral al 0166 0934 doi 10 1016 j jviromet 2005 05 010 Journal of Virological Methods 129 2005 56 63 Development of a homogeneous screening assay for automated detection of antiviral agents active against severe acute respiratory syndrome associated coronavirus Tania Ivens Christel Van den Eynde Koen Van Acker Erik Nijs G ery Dams Eva Bettens Asa Ohagen Rudi Pauwels Kurt Hertogs Tibotec BVBA Lead Discovery Operations Gen De Wittelaan L 11B 3 Mechelen 2800 Belgium Received 20 January 2005 received in revised form 19 April 2005 accepted 9 May 2005 Available online 14 June 2005 The severity and global spread of the 2003 outbreak of the severe acute respiratory syndrome associated coronavirus SARS CoV high the risks to human health posed by emerging viral diseases and emphasized the need for specific therapeutic agents instead of relying existing broadly active antiviral compounds The development of rapid screening assays is essential for antiviral drug discovery Thus a system for anti SARS CoV agents was developed which evaluated compound potency specificity and cytotoxicity at the initial phase Cell lines were engineered to constitutively express an enhanced green fluorescent protein EGFP and used to detect 1 viral potency in SARS CoV infection tests 2 antiviral specificity in tests using the porcine coronavirus transmissible gastroenteritis virus and 3 cytotoxicity in the same assays without virus challenge The assay system involves minimal manipulation after assay set up acilitates automated read out and minimizes risks associated with hazardous viruses The suitability of this assay system in drug discovery as demonstrated by screening of 3388 small molecule compounds The results show that these assays can be applied to high throughput for identification of inhibitors selectively active against SARS CoV 2005 Elsevier B V All rights reserved eywords SARS CoV High throughput screening Antiviral activity EGFP expression Introduction SARS is an infectious disease with a high mortality rate by the human coronavirus SARS CoV Ksiazek et 2003 The severity and global spread of the 2003 SARS has led to the search for specific antiviral com against SARS CoV Results from both in vitro studies case reports from the clinic have been published but no treatment has yet been established Major efforts being made to understand the mechanism of SARS CoV in order to identify drug targets of specific antivi compounds Chu et al 2004 Liu et al 2004 Thiel et 2003 Vastag 2003 Wu et al 2004 Coronaviruses are Corresponding author Tel 32 15 293 445 fax 32 15 401 257 E mail address khertog1 K Hertogs en tion con via F tious gene replicase transcriptase responsible the those brane 2002 viral such tion see front matter 2005 Elsevier B V All rights reserved veloped positive strand RNA viruses that initiate infec of susceptible cells by binding to human angiotensin verting enzyme 2 ACE 2 on the host cell membrane the viral S protein Li et al 2003 Xu et al 2004 ollowing receptor binding and membrane fusion the infec genomic RNA is delivered into the cytoplasm and viral expression is initiated resulting in the translation of two polyproteins These two enzymes are for the replication of the viral genome as well as generation of a multiple subgenomic mRNAs including encoding the structural proteins S spike M mem E envelope and N nucleocapsid De Haan et al A complex set of regulatory mechanisms controls the gene expression and additional virus encoded enzymes as helicase and proteases are crucial for the produc of infectious virus particles Thiel et al 2003 Progeny ological virions apparatus viral steps for the against also dard infection c throughput ef describes to can virus replication c EGFP specificity CoV ing using in chemical disco 2 2 1 monk K Institute ti in BioScience with 1 tamycin PT67 Alto plemented viral under kind SARS CoV ters and Beerse A don 2 2 e cells In The and duced clones and ing to by monitoring EGFP and 2 3 a inoculated SARS CoV bator cells by until determined T Ivens et al Journal of Vir are assembled in the endoplasmatic reticulum golgi and released from infected cells by exocytosis The entry and assembly processes as well as the enzymatic involved in viral replication constitute potential targets antiviral therapy Understanding the mechanism of viral replication is not only key step towards identification of effective drugs a virus Development of rapid screening assays is essential for antiviral drug discovery The present stan infectivity assay for SARS CoV is based on viral of VeroE6 cells followed by visual monitoring of ytopathic effects CPE Ksiazek et al 2003 The low nature of this assay format limits its use for ficient screening of large chemical libraries This study the development of a stable cell line susceptible SARS CoV that constitutively expresses EGFP which be used for the automated screening of inhibitors of activity A high degree of correlation between virus and reduced EGFP signal is expected for highly ytopathic viruses such as SARS CoV where the background signal is rapidly reduced by extensive cell death The and selectivity of compounds active in this SARS screening assay were determined by parallel screen for cytotoxicity and inhibition of a porcine coronavirus similar assay formats This set of assays was used the screening of a test panel of 3388 randomly chosen compounds to demonstrate its application to drug very Materials and methods Cells viruses and reagents The porcine kidney cell line LLC PK1 and the African ey kidney cell line VeroE6 were kindly provided by Dr Andries J Beerse Belgium and Dr G Van Ham for Tropical Medicine Antwerp Belgium respec vely The LLC PK1 and VeroE6 cell lines were maintained Dulbecco s modified Eagle s medium DMEM Cambrex Walkersville Walkersville MD supplemented 10 fetal calf serum FCS Highclone Logan UT l glutamine Invitrogen Carlsbad CA and 0 02 gen Invitrogen The packaging cell line RetroPack was purchased from BD Biosciences Clontech Palo CA and grown in RPMI 1640 medium Invitrogen sup with 10 FCS and 0 02 gentamycin The retro vector pLNCE containing the EGFP coding sequence the cytomegalovirus immediate early promotor was a gift of Dr E Kandel University of Illinois Chicago IL strain 200300592 was obtained from the Cen for Disease Control and Prevention CDC Atlanta GA TGEV strain purdue112 was obtained from J NY The plates were sealed with gas permeable and incubated in a humidified incubator at 37 C 5 CO 2 After 5 days the wells were examined for EGFP xpression using an argon laser scanning microscope The settings were excitation at 488 nm and emission 510 nm and the fluorescence images of the wells were con erted into signal values The results were expressed as EC 50 alues defined as the concentration of compound achieving inhibition of the virus reduced EGFP signals as com to the untreated virus infected control cells Cytotoxicity screening assays To test for cytotoxicity cells were incubated with serial dilutions as described above but in the absence virus challenge The 50 cytotoxic concentration CC 50 as determined by comparing the EGFP signal from treated 58 ological wells calculated 2 6 toxicity anti where w CoV SARS CoV EC 2 7 virus stock on performed lo signal added nals 25 000 CCID and with The ing repeated ation the day lating on cient virus infected reproducibility the dilutions b parallel assays 2 8 acti anti agents lar al ti ing ha CoV 2004 of T Reference Compound Riba AZT lopina A DS5000 No Glycirrhizin S P38 TGF A T Ivens et al Journal of Vir to that of control wells containing untreated cells and similarly to EC 50 Selectivity indices Two selectivity indices SI were calculated The cyto viral activity SI provides a measure of the range a compound is effective without being cytotoxic and as calculated as the ratio CC 50 EC 50 The TGEV SARS SI represents the specificity of a compound in inhibiting over TGEV and was calculated as the ratio TGEV 50 SARS CoV EC 50 Validation and characterization of screening assays The validity of reduced EGFP expression as a marker for activity was determined by titration of a SARS CoV on VeroE6 EGFP cells and by calculating titers based both EGFP signal and CPE Similar experiments were using TGEV and LLC PK1 EGFP cells To determine the sensitivity of the assays defined as the west virus titer resulting in a detectable decrease in EGFP serially diluted SARS CoV or TGEV stocks were to their respective indicator cells and the EGFP sig were recorded as described for the screening assay To determine intra and inter experiment reproducibility VeroE6 EGFP cells ml were added together with 300 50 of SARS CoV into 192 wells of a 384 well plate an additional 192 wells of the same plate were seeded 25 000 uninfected VeroE6 EGFP cells ml as controls EGFP signal of each well was recorded 5 days post plat as described for the screening assay The experiment was on three separate days and intra experimental vari was determined by calculating mean EGFP signals for tricarboxylic No were SARS CoV 2003 ral able 1 compounds tested in the screening assay Source Reported activity virin Sigma Nucleoside analog acti range of virus Sequoia Research Products HIV and HTLV 1 RT vir ritonavir a Sequoia Research Products and Toronto Research Chemicals HIV PR inhibitor TA Sigma Broadly acting with multiple viral targets Specs and biospecs Non specific virus entry varon Milliken chemical Broadly acting microbiocide SARS CoV activity TCI Europe SARS CoV replication Nitroso N acetylpenicillamine Sigma SARS CoV replication MAP kinase inhibitors 1 6 J Sigma St Louis MO and analyzed in in the SARS CoV TGEV and cytotoxicity screening as described above Selection and preparation of compounds To identify potential classes of pharmacological agents ve against SARS CoV a range of previously described viral compounds was tested Table 1 The antiviral included compounds targeting both viral and cellu factors Ribavirin So et al 2003 and AZT Furman et 1986 Zhang et al 2001 were included as representa ves of the widely used class of nucleoside analogs inhibit viral polymerases Selected antiviral protease inhibitors ve been implicated as being active against the SARS Yamamoto et al 2004 Wu et al 2004 Chu et al meriting the inclusion of lopinavir ritonavir A range non specific antiviral reagents was included such as aurin acid ATA dextran sulfate 5000 DS5000 and varon Glycirrhizin and S nitroso N acetylpenicillamine included due to previously reported activities against by unclear mechanisms of action Cinatl et al Keyaerts et al 2004 Additionally a variety of antivi compounds acting on cellular targets were included such Reference ve against a diverse So et al 2003 inhibitor Furman et al 1986 Zhang et al 2001 Mangum and Graham 2001 reported binding to Zhang et al 1999 Cushman and Sherman 1992 Schols et al 1990 inhibitor Zhang et al 1999 Schols et al 1990 with Toagosei Press Release 2003 Yoshida et al 1999 inhibitor Cinatl et al 2003 inhibitor Keyaerts et al 2004 of HIV and HCV Balasubramanian et al 2003 of HCV symptoms Ray et al 2003 ACE Internal screening data Research Products was mixed at a ratio of 4 1 ological Methods 129 2005 56 63 59 as A compounds All ide to 3 3 1 LLC PK1 throughput e cell rying cell were select cence e virus SARS CoV virus of ilar sho cell an density o density in period of sho sity 3 2 e pathic were reporter determined based e EGFP ods e viruses titer T Ivens et al Journal of Vir p38 MAP kinase inhibitors TGF H9252 inhibitors and 21 CE inhibitors Table 1 Separately 3388 randomly chosen from a proprietary chemical library were tested compounds were dissolved at 20 mM dimethylsulphox DMSO Sigma and then diluted in cell culture medium a final DMSO concentration below 0 5 RESULTS Development of EGFP expressing VeroE6 and cells To provide convenient indicator cells for the high screening assays stable cell lines constitutively xpressing EGFP were generated VeroE6 and LLC PK1 lines were transduced with a packaging retrovirus car the EGFP gene and 100 clones were isolated for each line for subsequent characterization studies The cells subjected to FACS analysis of EGFP expression to those clones exhibiting a profile of high mean fluores intensity and homogeneous signal Fig 1 The EGFP xpressing clones were further evaluated for susceptibility to infection showing all clones were susceptible to either or TGEV as measured by CPE induction after challenge data not shown The growth characteristics the VeroE6 EGFP and LLC PK1 EGFP clones were sim to the parental VeroE6 and LLC PK1 cell lines data not wn indicating that the EGFP expression did not affect proliferation As expected the transduced cells showed exponential growth that was dependent on the initial cell a representative example is shown in Fig 2 More ver the growth experiments showed that a final seeding of 25 000 cells ml was suitable for keeping the cells the exponential growth phase throughout a 5 day assay Taking all selection criteria into account clone 21 VeroE6 EGFP and clone 45 of LLC PK1 EGFP data not wn were selected as indicator cells to be seeded at a den of 25 000 cells ml in the screening assays Validation of screening assays To ensure a reliable screening platform the reduced EGFP xpression was validated as a marker for virus induced cyto activity and the robustness and specificity of the assays determined The validity of using quantification of a gene such as EGFP to measure viral activity was by virus titration experiments calculating titers on both CPE and EGFP measurements The titration xperiments showed a virus concentration dependent loss of expression and that virus titers scored by both meth were in good agreement Table 2 confirming that EGFP xpression can serve as a marker for activity of these two The assay sensitivity was determined by the lowest virus resulting in an at least two fold decrease in EGFP signal Fig 1 EGFP expression profiles of VeroE6 EGFP clones FACS analysis with arbitrarily set gates M1 and M2 showed that cell clone 21 A exhib ited a preferred EGFP expression pattern as compared to clone 61 B as demonstrated by the higher mean fluorescence intensity 925 vs 672 and higher fraction of bright cells 97 vs 89 within the M2 gate Percentages represent the fractions of events recorded within each gate Fig 2 Growth characteristics of VeroE6 EGFP clone 21 The cells showed an exponential growth that was dependent on the initial seeding density Data points and error bars represent mean and S D obtained in one single experiment using 80 replica wells per cell concentration and time point 60 ological T Comparati TGEV T 99 034 The signal 1 2 of of assessed and control 19 8 ral selecti an in potent tions virus when sho viral sho cific 3 3 compounds test included inhibitors One ti city anti PK1 EGFP c cell lular assay acti not throughput were Sixty four and acti The T Intra SARS CoV Uninfected T Ivens et al Journal of Vir able 2 ve virus titration using CPE and EGFP as read out CCID 50 well iter a scored using CPE Titer a scored using EGFP 8129 116 536 32 715 a Results represent mean S D of two independent titration experiments indicator cells showed a significant reduction of EGFP at virus titers as low as 2 0 CCID 50 SARS CoV and CCID 50 TGEV Fig 3 confirming that the measurement EGFP expression in these cells allows sensitive detection low levels of virus Assay robustness or intra and inter assay variation was by multiple EGFP measurements on the same day over several days The signal ratio between uninfected cells and virus infected cells varied between 4 1 and suggesting a sufficient dynamic range to measure antivi activity of test compounds Table 3 The specificity of the assay defined as the ability to detect ve SARS CoV inhibitors was determined by testing anti ACE 2 antibody specific for the SARS CoV receptor the screening assays The antibody showed a selective and inhibition of SARS CoV at non cytotoxic concentra Fig 4 and Table 4 The antiviral EC 50 value was 1 43H9262M when measuring inhibition based on EGFP signal Table 4 and 1 56H9262M using CPE as read out data not shown This further ws the close correlation between EGFP expression and cytopathic activity Together the validation experiments wed that the screening system was reproducible and spe for the detection of agents active against SARS CoV Screening of antiviral agents and chemical library The SARS CoV screening system was initially used to a selection of pharmacological agents The compounds agents targeting different viral factors as well as of cellular p38 kinase TGF H9252 and ACE Table 1 of the 21 ACE inhibitors Table 4 showed a selec ve antiviral activity against SARS CoV using a cytotoxi viral activity SI criterion of 3 However the LLC the 2 of pounds the the 4 eases to get w set up rently were tification This screening able 3 and inter experimental reproducibility Experiment 1 infected cells Mean a 146328 S D a 6329 CV 4 31 cells Mean a 2594210 S D a 89300 CV 3 48 Ratio b 19 81 a Mean and S D values are given as EGFP signal arbitrary units of fluorescence well b EGFP signal ratio for uninfected control cells versus virus infected cells Methods 129 2005 56 63 SARS CoV CCID 50 well Titer a scored using CPE Titer a scored using EGFP 329 964 38 427 329 964 38 427 cell line appeared to be more sensitive to the ytotoxic effects of the compound than the VeroE6 EGFP line indicating that the ACE inhibitor may exhibit cel effects not observed in the SARS CoV VeroE6 EGFP system The other tested agents showed no antiviral vity below the cytotoxic concentration Table 4 and data shown To test the possible use of the assay system in a high setting a total of 3388 small molecule compounds tested for selective antiviral activity against SARS CoV compounds were active in the SARS CoV assay 18 of these compounds exhibited a cytotoxicity antiviral vity SI of at least 3 listed as compounds A R in Table 4 EC 50 values ranged between 0 83 and 9 57H9262M Out of 18 anti SARS CoV active and non cytotoxic compounds showed a TGEV SARS CoV SI of at least 3 in the absence cytotoxic effects on any of the two indicator cell lines com N and P in Table 4 Together these results show that combination of assays used in this screening system has potential to provide an efficient basis for drug discovery Discussion An important aspect of drug screening for new viral dis is the choice of assay system A drug candidate has fulfill a number of requirements regarding potency tar specificity and cytotoxicity Thus a screening system as designed with three parallel types of assays with similar and read out to evaluate all these parameters concur at the initial screening phase Susceptible cell lines equipped with EGFP reporter genes and used for quan of cytotoxicity SARS CoV and TGEV activity assay system can be readily applied to high throughput and has many advantages over assays based on Experiment 2 Experiment 3 Inter experiment 176300 405419 242682 31787 55651 126894 8 13 752 3 2515356 1580705 2230090 222138 112550 505836 87 122 7 4 74 114 7 T Ivens et al Journal of Virological Methods 129 2005 56 63 61 laborious and subjective scoring of CPE or plaque formation The assay system described in this study requires minimal manipulation after assay set up and no staining washing fixation or manual inspection of the tissue culture plates which is especially advantageous when working with haz ardous viruses such as SARS CoV The strategy to use parallel counter screening of chemical compounds on a porcine specific member of the coronavirus family was employed to select drug candidates with high specificity compounds viral acti tified allel the than stream potent pounds against other coronaviruses may be selected from the data obtained in this screening system and analyzed separately Analyzing a panel of reported antiviral com