【病毒外文文献】2015 Testing of Middle East Respiratory Syndrome Coronavirus Replication Inhibitors for the Ability To Block Viral Entry

LETTER TO THE EDITOR 1 2 Testing of MERS CoV replication inhibitors for their ability to block 3 viral entry 4 5 6 Qi Liu a c Shuai Xia a Zhiwu Sun a Qian Wang a Lanying Du b Lu Lu a Shibo Jiang a b 7 8 Key Lab of Medical Molecular Virology of MOE MOH Shanghai Medical College 9 Fudan University 130 Dong An Rd Xuhui District Shanghai 200032 China a Lindsley 10 F Kimball Research Institute New York Blood Center New York NY 10065 USA b 11 Department of Medical Microbiology and Immunology School of Basic Medicine Dali 12 University Dali 671000 China c 13 14 Address correspondence to Shibo Jiang shibojiang or Lu Lu 15 lul 16 17 18 AAC Accepts published online ahead of print on 20 October 2014 Antimicrob Agents Chemother doi 10 1128 AAC 03977 14 Copyright 2014 American Society for Microbiology All Rights Reserved As of 23 July 2014 837 laboratory confirmed cases of MERS CoV infection 19 including 291 deaths had been reported to WHO 20 http www who int csr disease coronavirus infections en raising concerns about its 21 pandemic potential and calling for the development of vaccines and therapeutics against 22 MERS CoV infection 23 We previously identified potent peptidic HIV 1 and SARS CoV fusion inhibitors 24 5 7 leading to the development of MERS CoV spike S protein mediated cell cell 25 fusion and six helix bundle 6 HB formation assays Using these assays we identified a 26 peptide from the of MERS CoV S protein HR2 region termed HR2P which potently 27 inhibited 6 HB formation cell cell fusion and MERS CoV replication 8 To identify 28 small molecule MERS CoV fusion inhibitors we used the cell cell fusion assay to screen 29 1 280 compounds from an FDA approved drug library obtained from MicroSource 30 Discovery Systems Inc Gaylordsville CT but none of the compounds at 10 M could 31 significantly inhibit MERS CoV S mediated membrane fusion 32 Most recently de Wilde et al 2 and Dyall et al 3 used the cytopathogenic effect 33 CPE assay to screen several hundreds of compounds from FDA approved drug libraries 34 and identified a series of compounds inhibiting both MERS CoV and SARS CoV 35 replication in the low micromolar range Although their mechanisms of action have not 36 been defined both groups suggested that some of them such as chlorpromazine a 37 clathrin mediated endocytosis inhibitor might block virus entry 2 3 38 Coronavirus enters into the target cell via endocytosis or plasma membrane fusion 39 while the latter is the main pathway for MERS CoV entry 8 To determine whether 40 these reported MERS CoV replication inhibitors also block virus entry via plasma 41 membrane fusion we tested 16 compounds with MERS CoV replication inhibiting 42 activity available in the FDA approved drug library from MicroSource and ribavirin and 43 mycophenolic acid Sigma Aldrich that were reported to inhibit MERS CoV replication 44 1 for their inhibitory activity on MERS CoV S mediated cell cell fusion using HR2P as 45 a control Cell cell fusion inhibition assay was performed as we described before 8 46 Briefly Huh 7 cells were used as the target cells and 293T cells that instantaneously 47 express MERS CoV S protein and EGFP 293T MERS EGFP used as the effector cells 48 The 293T MERS EGFP cells were cocultured with HR2P or compounds at graded 49 concentrations initial concentration at 40 M for 30min then added into Huh 7 cells at 50 37 C for 2 to 4 hours 8 As expected HR2P inhibited cell cell fusion with IC 50 half 51 maximal inhibitory concentration value of 1 M and effectively blocked 6 HB 52 formation In contrast most of these compounds at 40 M exhibited no significant 53 inhibitory activity except the three neurotransmitter inhibitors chlorpromazine 54 promethazine and fluphenazine showing moderate inhibitory activity with IC 50 values of 55 about 20 20 and 29 M respectively on cell cell fusion Table 1 56 Subsequently we determined the inhibitory ability of these compounds 40 M on 57 6 HB formed between HR1P and HR2P FITC using a fluorescence native 58 polyacrylamide gel electrophoresis FN PAGE adapted from the FN PAGE assay for 59 testing HIV fusion inhibitors 6 As expected HR1P showed no band because it carries 60 net positive charges thus migrating up and off the gel under native electrophoresis 61 condition which is consistent with the results of HR1 peptides from HIV 1 6 and 62 SARS CoV 7 while HR2P FITC showed a band at a lower position The mixture of 63 HR1P and HR2P FITC showed a band at a higher position suggesting the formation of 64 an HR1P HR2P FITC complex possibly the 6 HB band Fig 1 In the presence of 65 HR2P the upper band disappeared while lower HR2P FITC band displayed suggesting 66 that HR2P binds to HR1P and block the 6 HB formation between HR2P FITC and HR1P 67 However none of the MERS CoV replication inhibitors at 40 M could block the 6 HB 68 formation by HR2P FITC and HR1P Fig 1 and Table 1 69 The cytotoxicity of these MERS CoV replication inhibitors to the Huh 7 cells which 70 were used as the target cells in cell cell fusion assay was determined using Cell Counting 71 Kit 8 CCK 8 Dojindo Kumamoto Japan as previously described 8 Except Emetine 72 dihydrochloride Triflupromazine hydrochloride and Clomipramine hydrochloride with 73 CC 50 the concentration of a compound causing 50 cytotoxicity at 28 63 33 58 and 40 40 40 Loperamide Opioid receptor agonist 5 90 2 40 40 40 Chloroquine diphosphate Antiparasitic agent 4 10 2 6 28 3 40 40 40 Hydroxychloroquine sulfate Antiparasitic agent 8 28 3 40 40 40 Amodiaquine dihydrochloride Antiparasitic agent 6 21 3 40 40 40 Chlorpromazine hydrochloride Neurotransmitter inhibitor 8 80 2 9 51 3 23 33 2 89 7 24 2 55 40 Promethazine hydrochloride Neurotransmitter inhibitor 11 80 3 16 67 7 22 7 48 4 53 40 Fluphenazine hydrochloride Neurotransmitter inhibitor 5 86 3 15 00 4 33 3 23 2 79 40 Thiothixene Neurotransmitter inhibitor 9 30 3 40 5 74 2 51 40 Astemizole Neurotransmitter inhibitor 4 88 3 40 3 48 1 34 28 63 1 94 Triflupromazine hydrochloride Neurotransmitter inhibitor 5 76 3 40 3 32 1 51 33 58 2 37 Clomipramine hydrochloride Neurotransmitter inhibitor 9 33 3 40 8 79 2 35 40 Emetine dihydrochloride Antibacterial agent 0 01 3 40 5 40 7 46 2 74 40 Cycloheximide Protein processing inhibitor 0 19 3 40 40 40 Dasatinib Kinase signaling inhibitor 5 47 3 40 40 40 Ribavirin Nucleoside analogue 9 99 1 40 40 40 Mycophenolic acid MPA Immunosuppressant agent 0 17 1 40 40 40 HR2P Fusion inhibitor 0 60 8 1 64 0 75 14 28 5 57 40 The samples were tested in triplicate and the experiment was repeated twice The data are presented as 175 mean SD 176 If a compound at 40 M had no more than 50 inhibition its IC 50 was recorded as 40 M If a 177 compound at 40 M had no more than 50 cytotoxicity its CC 50 was recorded as 40 M If a compound 178 at 40 M could or could not inhibit 6 HB formation its activity was recorded as or respectively 179 No inhibitory activity at its CC 50 value 5 M 180 181 e e HR1P HR2P FITC s phate n e sulfate d r o chloride y dr ochlorid e r ochloride d r o chloride y dr ochlorid h loride MP A C v ir m ide m ide q uine d ipho s y chlor o qui n a quine dihy d r omazine h y h azine hyd r x ene r amine hy d r omazine h y e dihydr o c h z ole f en c itrate e ximide i b i n h enolic a cid H R2P FIT C F ITC Lopina v Lopera m Lopera m Chlor o q Hydr ox y Amodi a Chlorp r Pr omet h Thiothi x Clomip r T r iflup r Emetin e Astemi z T a moxi f Cycloh e Dasatin i Ribavir i Mycop h HR2P HR1P H HR2P F HR1P 6 HB I h ibiti f 6 HB f t i b HR2P d HR2P FITC I n hibiti on o f 6 HB f orma ti on b y HR2P or compoun d s