key: cord-269285-2r40mico
authors: Resnick, Samuel J.; Iketani, Sho; Hong, Seo Jung; Zask, Arie; Liu, Hengrui; Kim, Sungsoo; Melore, Schuyler; Nair, Manoj S.; Huang, Yaoxing; Tay, Nicholas E.S.; Rovis, Tomislav; Yang, Hee Won; Stockwell, Brent R.; Ho, David D.; Chavez, Alejandro
title: A simplified cell-based assay to identify coronavirus 3CL protease inhibitors
date: 2020-08-29
journal: bioRxiv
DOI: 10.1101/2020.08.29.272864
sha: 
doc_id: 269285
cord_uid: 2r40mico

We describe a mammalian cell-based assay capable of identifying coronavirus 3CL protease (3CLpro) inhibitors without requiring the use of live virus. By enabling the facile testing of compounds across a range of coronavirus 3CLpro enzymes, including the one from SARS-CoV-2, we are able to quickly identify compounds with broad or narrow spectra of activity. We further demonstrate the utility of our approach by performing a curated compound screen along with structure-activity profiling of a series of small molecules to identify compounds with antiviral activity. Throughout these studies, we observed concordance between data emerging from this assay and from live virus assays. By democratizing the testing of 3CL inhibitors to enable screening in the majority of laboratories rather than the few with extensive biosafety infrastructure, we hope to expedite the search for coronavirus 3CL protease inhibitors, to address the current epidemic and future ones that will inevitably arise.

We next conducted dose-response profiling for two additional SARS-CoV-2 3CLpro inhibitors, 112 compound 4 and compound 11a, and observed reversal of the toxic effect of the protease in a dose-113 dependent manner (Fig. 2b-c) 25, 26 . In agreement with the results obtained with GC376, the EC 50 114 value for compound 4 was comparable to those obtained with live virus, 0.98 µM and 3.023 µM, 115 respectively (Table 1) 24 . Unexpectedly, we calculated an EC 50 of 6.89 µM for 11a, which is 116 approximately 10-fold higher than the literature reported value of 0.53 µM, based on viral plaque 117 assay 26 . We have noticed that literature reported EC 50 values from live virus testing could range over 118 an order of magnitude depending on the exact method employed, as is the case for GC376 (Table   119 1). To resolve this discrepancy between the transfection-based approach and the live virus assay, 120 we conducted live virus testing of 11a using the commonly employed readout of cytopathic effect in 121 Vero E6 cells and observed closer concordance with our transfection-based results (Supplementary 122 Figure 3 and Table 1 ) 20,24,27 . To measure the toxicity of each compound, we exposed EYFP-123 transfected cells to each molecule and determined CC 50 values (Fig. 2) . We also calculated the 124 selectivity index (SI) for each compound tested in this study (Supplementary Table 1 ).

We hypothesized that the assay would be able to distinguish between compounds that are only 127 active on the purified SARS-CoV-2 3CLpro and those that are able to inhibit the live virus through 128 protease inhibition. In general, we observe concordance between compounds showing activity within 129 this transfection-based 3CL assay and live virus studies ( Supplementary Fig. 4a -e) 13 . However, We hypothesized that this assay may be used to study other coronavirus 3CLpros to enable users to 139 identify broad-acting inhibitors, as constructs containing other 3CLpro enzymes could be readily variable amino acid identity compared with SARS-CoV-2 3CLpro ( Supplementary Fig. 5a ). For each 143 of these proteases, we confirmed that expression in mammalian cells resulted in toxicity that is 144 dependent upon the enzyme's catalytic activity ( Supplementary Fig. 5b ). Next, we tested GC376, 145 compound 4, and 11a across this panel of proteases. GC376, a drug originally identified for use 146 against the Feline Infectious Peritonitis virus, showed EC 50 <10 µM for the most, but not all of 147 proteases tested 30 . Unexpectedly, compound 4, which was originally designed as a SARS-CoV 148 3CLpro inhibitor showed particular potency against IBV 3CLpro (EC 50 = 0.058 µM) along with broad 149 activity (EC 50 <10 µM) for all other 3CL proteases tested. In contrast to GC376 and compound 4, 11a 150 had a relatively narrow activity spectrum with EC 50 <10 µM against only SARS-CoV and SARS-CoV-151 2 3CLpro enzymes (Fig. 3) . Of note, in all cases where previous live virus data was available, the 152 EC 50 values obtained from this transfection-based assay were similar (Table 1) .

Having further determined the assay's ability to examine the effects of active individual compounds, 156 we sought to determine its suitability for small molecule screening. Before performing the screen, we

We noted that GC373 is structurally similar to its prodrug GC376, except for the change of the 165 bisulfide salt adduct to an aldehyde warhead 22,33 . Additional testing of GC373 revealed it to have a 166 similar EC 50 as GC376 in both the transfection assay and when tested against live SARS-CoV-2 167 virus, suggesting that the differences in structure has a minimal effect on their potency 168 (Supplementary Fig. 7 and Table 1 ), although solubility may be affected 33 . The other hit from the 169 screen, GRL-0496, shares structural similarity to several other compounds within the library, one of 170 which is a previously reported 3CLpro inhibitor (MAC-5576) that failed to show activity against 5.05 µM against SARS-CoV-2 3CLpro within our transfection-based assay (Fig. 4c ). To verify GRLexamined and observed a narrow range of activity, with EC 50 <10 µM only observed against SARS- Table 1 ) 27 . We also tested GC373 against the full panel of 3CLpro enzymes and observed 178 concordance with GC376, with SARS-CoV 3CLpro, MERS-CoV 3CL pro, and IBV 3CL pro demonstrating an EC 50 <10 µM ( Supplementary Fig. 8 ).

Given the potential for protease inhibitors in the treatment of viral illnesses, small molecule inhibitors 183 of coronavirus 3CL proteases represent a promising avenue for treating infections caused by this 184 large family of viruses. Here, we present a simplified assay to identify candidate inhibitors under 185 physiologic cellular conditions. This approach presents significant advantages over other methods to 186 detect 3CL protease inhibitory activity with its ease of use and ability to be performed with equipment 187 and reagents commonly available to many biomedical research laboratories. While conventional 188 methods for identifying 3CL protease inhibitors make use of in vitro purified protease, the isolation of 189 sufficiently pure enzyme in its native state can be costly and labor intensive. Furthermore, assays 190 using purified protease fail to consider cell permeability and the influence of the extracellular and 191 . CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint intracellular milieu on compound activity. In comparison to live virus-based assays, the outlined approach does not require extensive biosafety containment. These data also suggest that the 193 approach described here is applicable to a number of coronaviruses for which live virus assays may 194 not be available or would be deemed ethically challenging to be performed even with extensive biosafety infrastructure 35, 36 . Finally, because the phenotype assayed within this approach is driven 196 solely by protease activity, it may enable the distinction between compounds with multiple biological 197 targets and subsequent potential for off-target toxicity from those that function primarily as 3CLpro (Table 1 ). These differences appear to be driven by variation in experimental setup such 212 as cell line used, assay readout, incubation period, and initial concentration of virus added. While we 213 have observed agreement between the EC 50 values obtained from the described transfection-based 214 method and those reported in the literature, given the differences in EC 50 across assays, we suggest 215 caution when comparing results across studies. By developing this transfection-based 3CLpro 216 testing platform, we hope to facilitate the discovery of new coronavirus inhibitors while also 217 facilitating the comparison of existing inhibitors within a single simplified assay system. Furthermore,

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint we propose that this cellular protease assay system could be industrialized to screen and optimize a 219 large number of compounds to discover potential treatments for future viral pandemics.

Cell Lines and Cell Culture

HEK293T and HEK293 cells used in this study were obtained from ATCC. Cells were maintained at 224 37°C in a humidified atmosphere with 5% CO 2 . HEK293T and HEK293 cells were grown in 

During the drug screen, within each of the four plates screened, two positive controls wells were 306 included to ensure assay reliability, along with several wells with the negative control 0.1% DMSO For hit selection, we employed a robust z-score method. We first normalized data using a robust z- The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint of saturated NaHCO 3 was added. The cloudy mixture was extracted with CH 2 Cl 2 , dried over Na 2 SO 4 , 437 and concentrated in vacuo to give GC373 as a colorless oil (4.0 mg).

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint

All reagents generated in this study are without restriction. Plasmids generated in this study will be some of the molecules described in this work. B.R.S. is an inventor on additional patents and patent 466 applications related to small molecule therapeutics, and co-founded and serves as a consultant to 467 Inzen Therapeutics and Nevrox Limited.

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a 

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a 

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint Absorbance . CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint . CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint 

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint

Compound 4 11a

Bat-CoV-HKU9 3CLpro

HCoV-NL63 3CLpro IBV 3CLpro -6 -4 -2 0 2 4 Fig. 4 . Small-scale drug screen and structure-activity profiling at 10 µM identify two 

. CC-BY-NC-ND 4.0 International license (which was not certified by peer review) is the author/funder. It is made available under a The copyright holder for this preprint this version posted August 29, 2020. . https://doi.org/10.1101/2020.08.29.272864 doi: bioRxiv preprint

A new coronavirus associated with human respiratory disease in China