Inhibitors of Coronavirus 3CL Proteases Protect Cells from Protease-Mediated Cytotoxicity 2 3 Samuel J

1 Inhibitors of coronavirus 3CL proteases protect cells from protease-mediated cytotoxicity 2 3 Samuel J. Resnick1,2, Sho Iketani3,4, Seo Jung Hong1, Arie Zask5, Hengrui Liu6, Sungsoo Kim1, Schuyler 4 Melore1, Fang-Yu Lin7, Manoj S. Nair3, Yaoxing Huang3, Sumin Lee6, Nicholas E.S. Tay6, Tomislav Rovis6, 5 Hee Won Yang1, Li Xing7, Brent R. Stockwell5,6*, David D. Ho3*, Alejandro Chavez1* 6 7 1 Department of Pathology and Cell Biology, Columbia University Irving Medical Center, New York, NY, 8 10032, USA

9 2 Medical Scientist Training Program, Columbia University Irving Medical Center, New York, NY, 10032, 10 USA

11 3 Aaron Diamond AIDS Research Center, Columbia University Irving Medical Center, New York, NY, 10032, 12 USA

13 4 Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 14 10032, USA

15 5 Department of Biological Sciences, Columbia University, New York, NY, 10027, USA

16 6 Department of Chemistry, Columbia University, New York, NY, 10027, USA

17 7 WuXi AppTec, Cambridge, MA 02142, USA 18

19 *Correspondence: [email protected]; [email protected]; 20 [email protected] 21

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 22 Abstract 23 We describe a mammalian cell-based assay to identify coronavirus 3CL protease (3CLpro) inhibitors. This 24 assay is based on rescuing protease-mediated cytotoxicity and does not require live virus. By enabling the 25 facile testing of compounds across a range of 15 distantly related coronavirus 3CLpro enzymes, we identify 26 compounds with broad 3CLpro inhibitory activity. We also adapt the assay for use in compound screening 27 and in doing so uncover additional SARS-CoV-2 3CLpro inhibitors. We observe strong concordance 28 between data emerging from this assay and those obtained from live virus testing. The reported approach 29 democratizes the testing of 3CLpro inhibitors by developing a simplified method for identifying coronavirus 30 3CLpro inhibitors that can be used by the majority of laboratories, rather than the few with extensive 31 biosafety infrastructure. We identify two lead compounds, GC376 and compound 4, with broad activity 32 against all 3CL proteases tested including 3CLpro enzymes from understudied zoonotic coronaviruses. 33 34 Importance 35 Multiple coronavirus pandemics have occurred over the last two decades. This has highlighted a need to be 36 proactive in the development of therapeutics that can be readily deployed in the case of future coronavirus 37 pandemics. We develop and validate a simplified cell-based assay for the identification of chemical inhibitors 38 of 3CL proteases encoded by a wide range of coronaviruses. This assay is reporter-free, does not require 39 specialized biocontainment, and is optimized for performance in high-throughput screening. By testing 40 reported 3CL protease inhibitors against a large collection of 3CL proteases with variable sequence 41 similarity, we identify compounds with broad activity against 3CL proteases and uncover structural insights 42 that contribute to their broad activity. Furthermore, we demonstrate this assay is suitable for identifying 43 chemical inhibitors of proteases from families other than 3CL proteases.

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 44 Introduction 45 The outbreak of a novel coronavirus (SARS-CoV-2) infection has paralyzed countries around the world [1,2]. 46 This crisis is further exacerbated by the dearth of approved therapeutics, leaving physicians with few proven 47 treatment options. In the past two decades, the world has suffered from two other major coronavirus 48 outbreaks, Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS), 49 suggesting that coronaviruses represent a real and ever-present threat to global health that must be 50 addressed [3]. Yet, even if therapeutics against the existing epidemic strains are identified, there are several 51 hundred other coronaviruses in active circulation within animal populations, many of which are understudied, 52 but have the theoretical potential to infect humans. To help identify therapeutics for the current epidemic 53 along with preparing for the next, there is a need for readily-deployable small molecule screening assays 54 that enable the identification of therapeutics that are broad-acting across a large collection of coronavirus 55 strains. 56 57 During coronavirus infection, the RNA genome is delivered into cells and translated into a pair of 58 polyproteins [4]. These polyproteins are then processed by a set of virally encoded proteases, of which the 59 three-chymotrypsin-like protease (3CLpro) performs the majority of cleavage events [4]. As a result of its 60 essential role in viral replication and high degree of conservation across all coronaviruses, 3CLpro enzymes 61 represent important targets for therapeutic drug development [5,6]. Previous work expressing a variety of 62 viral proteases within yeast and mammalian cells have shown that protease expression can lead to cellular 63 toxicity, which can be rescued by the addition of protease inhibitors [7-13]. We hypothesized that the 64 expression of coronavirus 3CLpro enzymes within mammalian cells may lead to a similar toxic phenotype as 65 a result of its proteolytic activity. If 3CL protease inhibitors rescue the toxic phenotype, this could form the 66 basis of a cell-based assay to detect 3CL protease inhibitors. While multiple assays exist for evaluating 67 protease inhibitors, an assay of the nature has clear advantages, as it requires minimal upfront cost or effort, 68 is accessible to many biomedical research labs, does not involve the use of live virus, and requires no 69 specialized reporter to read out protease activity. In contrast, in vitro protease assays using purified protein 70 have formed the backbone of inhibitor screening, but require upfront efforts to isolate the pure protease and 71 are not conducted under physiologic cellular conditions [14,15]. In addition, if one desires to identify broad- 72 acting coronavirus inhibitors, one must purify and identify experimental conditions suitable for testing each 73 protease in vitro. An alternative approach for identifying protease inhibitors is the use of live virus which is 74 performed under more biologically relevant conditions, assuming relevant host cell systems can be 75 identified, but requires intensive safety training and specialized biosafety protocols [16]. In addition, for 76 many coronaviruses, no live virus assay exists, limiting the ability to test compounds within mammalian cell 77 systems to a small subset of all coronaviruses [17]. Furthermore, compounds with activity against live virus 78 may function through a number of mechanisms other than 3CLpro inhibition which cannot be readily

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 79 determined when performing the assay, and may lead to undesired off-target activities which are not 80 realized until much later in the drug development process [18,19]. 81 82 Here, we report a mammalian cell-based assay for identifying coronavirus 3CLpro inhibitors that does not 83 require the use of live virus. We demonstrate the utility of the assay by characterizing a variety of SARS-

84 CoV-2 3CLpro inhibitors and obtaining EC50 values that are highly concordant with the results from live virus 85 testing. We then establish the generality of our approach across a diverse set of 15 3CLpros from a wide 86 range of coronaviruses, and in doing so identify a set of key structural features shared among broadly active 87 3CLpro inhibitors. We next perform a small molecule screen, along with structure-activity profiling of a set of 88 compounds to find those with enhanced antiviral activity. Finally, we provide data that suggest our approach 89 is applicable to other protease families and thus represents a general platform for viral protease inhibitor 90 studies. 91 92 Results 93 Expression of the SARS-CoV-2 3CLpro in HEK293T cells results in protease-mediated cytotoxicity 94 that can be rescued by protease inhibitors 95 Motivated by prior work demonstrating protease-mediated cytotoxicity, we sought to determine the effect of 96 transfecting an expression plasmid encoding the SARS-CoV-2 3CLpro into HEK293T cells. Utilizing a cost- 97 effective crystal-violet-based approach to quantify cell abundance, we observed that SARS-CoV-2 3CLpro 98 expression results in significant growth inhibition as compared to a control construct containing enhanced 99 yellow fluorescent protein (EYFP) (Fig. 1a-b) [20]. This suppression of growth was dependent upon the 100 catalytic function of the enzyme, as mutating cysteine 145, which is essential for the enzyme’s proteolytic 101 activity, abolished the growth defect (Fig. 1a-b). We confirmed expression of active and inactive SARS-CoV- 102 2 3CLpro enzymes in HEK293T cells with western blotting (Fig 1c). We next determined if the observed 103 growth defect could be rescued by incubating cells with GC376, a previously reported SARS-CoV-2 3CLpro 104 inhibitor [21]. In comparison to untreated control cells, the addition of GC376 led to a robust increase in cell 105 growth (Fig. 1d-e). To ensure reproducibility between transfections and to select for cells expressing 3CL 106 protease, expression constructs contained a puromycin resistance marker and puromycin resistance was 107 selected for 24 h after transfection. 108 109 Compound rescue of transfected 3CLpro cytotoxicity mimics the results obtained with live virus

110 We next tested if this transfection-based assay could be used to determine compound EC50 values and 111 whether the values showed any correlation with those obtained with live virus assays. After incubating

112 SARS-CoV-2 3CLpro transfected cells with a range of GC376 concentrations, we calculated an EC50 of 3.30

113 μM, which is similar to published values using live virus on Vero E6 cells (EC50 4.48 μM, 3.37 μM, 2.2 μM, 114 0.9 μM, 0.18 μM) (Fig. 2a and Table 1) [21-26]. We then investigated the assay’s tolerance to deviation by

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 115 varying the amount of plasmid transfected or the number of cells seeded into wells containing compound

116 (Supplementary Fig. 1). In all cases, the assay was robust to variation, delivering a similar EC50 for GC376 117 across all conditions. We also tested an orthogonal method of quantifying cell abundance based on 118 fluorescence microscopy and observed agreement with the results obtained with crystal violet staining 119 (Supplementary Fig. 2). In the fluorescence microscopy approach, EYFP labeled cells are transfected with 120 the SARS-CoV-2 3CLpro expression construct. Rather than reading out the cellular abundance using crystal 121 violet staining, the area occupied by the cells under various treatment conditions is measured with a 122 fluorescent microscope. As a whole these data suggest that our approach provides consistent results across 123 methods of measurement, although with the crystal violet assay we observed lower measurement variability 124 and higher changes in relative growth and thus, the crystal violet approach was chosen for further validation. 125 126 We next conducted dose-response profiling for two additional SARS-CoV-2 3CLpro inhibitors, compound 4 127 and 11a, and observed reversal of the toxic effect of the protease in a dose-dependent manner (Fig. 2b-c)

128 [27,28]. In agreement with the results obtained with GC376, the EC50 value for compound 4 was comparable 129 to those obtained with live virus, 0.98 µM and 3.023 µM, respectively (Table 1) [24]. Unexpectedly, we

130 calculated an EC50 of 6.89 μM for 11a, which is approximately 10-fold higher than the literature reported

131 value of 0.53 μM, based on viral plaque assay [27]. We have noticed that literature reported EC50 values

132 from live virus testing can show over an order of magnitude difference in the reported EC50 values 133 depending on the exact method employed, as is the case for GC376 (Table 1). To resolve the discrepancy 134 between the transfection-based approach and the live virus assay for 11a, we conducted live virus testing of 135 11a using the commonly employed readout of protection from cytopathic effect (CPE) in Vero E6 cells and

136 observed closer concordance with our transfection-based results, with a reported EC50 of 3.83 μM 137 (Supplementary Fig. 3 and Table 1) [21,24,29]. During the course of our studies, we also measured the

138 toxicity of each compound by exposing EYFP-transfected cells to each molecule and determining CC50 139 values (Fig. 2). Using these data, we calculated the selectivity index (SI) for each compound, observing that 140 both GC376 and compound 4 show moderate selectivity (SI > 10) for SARS-CoV-2 (Supplementary Table 141 1). 142 143 As our assay requires that inhibitors successfully engage their protease target within the intracellular milieu, 144 we hypothesized that it would be able to distinguish between compounds that are only active on the in vitro 145 purified SARS-CoV-2 3CLpro and those that inhibit viral replication by blocking 3CLpro activity in situ. In 146 general, we observe concordance between compounds showing activity within this transfection-based 147 3CLpro assay and live virus studies (Supplementary Fig. 4a-e) [14,30]. However, within our assay we did 148 not observe activity for ebselen, a small molecule with demonstrated in vitro activity against purified SARS- 149 CoV-2 3CLpro and data showing inhibition of SARS-CoV-2 live virus (Supplementary Fig. 4f). We suggest 150 that this may be due to ebselen targeting more than 3CLpro within the live virus assay, which is in line with

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 151 reports showing that ebselen is highly reactive and readily forms selenosulfide bonds with numerous 152 proteins including the SARS-CoV-2 papain-like protease (PLP) [19,31-33]. 153 154 Assay is applicable to a range of coronavirus 3CLpro enzymes 155 We next sought to determine if our simplified testing paradigm could be used to study other coronavirus 156 3CLpros, to enable users to identify broad-acting compounds. To test the assay’s generality, we created 157 expression constructs for 3CL proteases from five other coronaviruses (SARS-CoV, MERS-CoV, Bat-CoV- 158 HKU9, HCoV-NL63 and IBV) with variable amino acid identity compared with SARS-CoV-2 3CLpro 159 (Supplementary Fig. 5a). For each of these proteases, we confirmed that expression in mammalian cells 160 resulted in toxicity that is dependent upon the enzyme’s catalytic activity (Supplementary Fig. 5b). We also 161 observed expression of each construct by western blot (Supplementary Fig. 5c). Next, we tested GC376, 162 compound 4, and 11a across this panel of proteases. GC376, a drug originally identified for use against the

163 Feline Infectious Peritonitis virus, showed EC50 <10 μM for most, but not all of proteases tested [34]. 164 Unexpectedly, compound 4, which was originally designed as a SARS-CoV 3CLpro inhibitor showed

165 particular potency against IBV 3CLpro (EC50 = 0.058 μM) along with broad activity (EC50 <10 μM) for all 166 other 3CL proteases tested. In contrast to GC376 and compound 4, 11a had a relatively narrow activity

167 spectrum with EC50 <10 μM against only SARS-CoV and SARS-CoV-2 3CLpro enzymes (Fig. 3). Of note, in

168 all cases where previous live virus data was available, the EC50 values obtained from this transfection-based 169 assay were similar (Table 1). 170 171 Differences in the 3CLpro S2 pocket explain variable susceptibility to 11a 172 Given similarities between 11a and our other tested inhibitors, we were intrigued by its narrow spectrum of 173 activity and sought to uncover the mechanism underlying this observation. In examining published crystal 174 structures, we observed striking variability in the S2 pocket between 3CLpros, with some having large, 175 flexible, and nonpolar S2 pockets (e.g. SARS and SARS-CoV-2) and others showing narrower, less flexible, 176 and more polar S2 pockets (e.g. MERS-CoV, IBV, and HCoV-NL63) (Fig 4a-b and Supplementary Fig. 6). 177 We hypothesized these differences in the S2 pocket explain why 11a, with its larger cyclohexylmethyl P2 178 group which interacts with the S2 pocket, is unable to potently inhibit the majority of tested 3CLpros. In 179 contrast, GC376 and compound 4 both have a smaller isobutyl P2 group and are less restricted by 180 differences in the S2 pocket, and are able to interact with a larger number of 3CLpros. To test this 181 hypothesis, we took the broadly activity inhibitor, GC376, and synthesized an analog, SL-4-241, which 182 substitutes the isobutyl P2 group for the bulkier cyclohexylmethyl moiety from 11a (Fig. 4c). When tested 183 against SARS and MERS 3CLpros, SL-4-241 only showed activity against SARS-CoV-2 with its more 184 accommodating S2 pocket (Fig. 4d-e). These data suggest that broad acting inhibitors should avoid the use 185 of bulky P2 groups, as these will limit their activity to only a small subset of all 3CLpros. 186

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 187 Rapid testing of protease inhibitors elucidates structure-function relationships 188 Having demonstrated the assay’s performance when testing individual compounds, we sought to determine 189 its suitability for small molecule screening. Before performing the screen, we optimized the testing 190 parameters to ensure suitable performance characteristics (Supplementary Fig. 7 and Methods) [35]. We 191 compiled a collection of 162 diverse protease inhibitors, along with compounds with reported in vitro activity 192 against 3CLpro enzymes or structural similarity to known 3CLpro inhibitors (Supplementary Table 2 and 193 Supplementary Table 3). Of the nearly 200 compounds tested against the SARS-CoV-2 3CLpro, two potent 194 hits were identified, GC373 and GRL-0496 (Fig. 5a, Supplementary Table 2) [36]. Also included in the 195 compounds screened were several apoptosis inhibitors. Notably, we did not call hits for these compounds 196 which suggests that apoptosis inhibitors do not give false positive results in our assay (Supplementary Table 197 2). 198 199 Our first hit, GC373, is structurally similar to its prodrug GC376, except for the change of the bisulfite salt

200 adduct to an aldehyde warhead [26,37]. Additional testing of GC373 revealed it to have a similar EC50 as 201 GC376 across a range of 3CLpros within our transfection-based assay and when tested against live SARS- 202 CoV-2 virus, suggesting that the difference in structure has a minimal effect on potency (Supplementary Fig. 203 8 & 9 and Table 1), although solubility may be affected [37]. The other hit from the screen, GRL-0496 was

204 further tested and was found to have an EC50 of 5.05 μM against SARS-CoV-2 3CLpro in our transfection- 205 based assay (Fig. 5c). To verify GRL-0496’s activity, we tested it against live SARS-CoV-2 virus, and

206 confirmed its potency (EC50 = 9.12 μM) (Fig. 5d). We next tested GRL-0496 against the full panel of 3CLpro

207 enzymes we previously examined and observed a narrow range of activity, with EC50 <10 μM only observed 208 against SARS-CoV 3CLpro and SARS-CoV-2 3CLpro, in agreement with previous live virus testing 209 (Supplementary Fig. 9 and Table 1) [29]. 210 211 Further analysis of the screened compounds revealed a large number that were structurally similar to GRL- 212 0496, with one being a previously reported 3CLpro inhibitor (MAC-5576) that failed to show activity within 213 our transfection-based assay, in agreement with recent live virus studies (Fig. 5b) [24,38]. We hypothesize 214 that the difference in activity between these compounds is due to the indole group in GRL-0496 forming a 215 more stable inhibitory thioester bond with the 3CLpro catalytic cysteine as compared to the more 216 unstable thioester bonds formed by MAC-5576, BTB07408, and BTB07417 (see Supplementary Note 1 for 217 further discussion). 218 219 Further testing against 3CLpros confirms a pair of broadly active lead compounds 220 Two compounds from our studies, GC376 and compound 4, demonstrated activity across the six 3CLpros 221 tested and also harbor small P2 substituents that may be less likely to restrict target engagement. To further 222 characterize the extent to which these two compounds may serve as valuable lead candidates, we tested

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 223 them against 3CLpros from nine other coronavirus species. For these additional studies, we focused mostly 224 on testing additional members from the betacoronavirus and alphacoronavirus lineages, as these are the 225 genera which are known to infect humans. Within these genera, many coronaviruses have no established 226 live virus assay to enable small molecule testing. We validated that expression of these 3CLpros within 227 HEK293T cells results in protease-mediated cytotoxicity dependent upon the enzyme’s catalytic function 228 (Supplementary Fig. 10). Next, we conducted dose-response curves with GC376 and compound 4 for each 229 of these additional 3CLpros. Both compounds demonstrated activity against all additional 3CLpros tested

230 (Fig. 6). Compound 4 had an EC50 of <10 μM for 13/15 3CLpros while GC376 had an EC50 of <10 μM for 231 12/15 3CLpros, suggesting that these compounds represent promising leads capable of inhibiting 3CLpro 232 within a wide range of coronaviruses including zoonotic coronaviruses with the potential to transmit to 233 humans. 234 235 Expansion of assay to other protease families 236 Having established the power of our assay to rapidly characterize 3CLpros inhibitors, we sought to explore 237 whether our approach was generalizable for other protease families. We expressed the 3C protease from 238 Human Rhinovirus B14 (HRV-B14 3C) and the SARS-CoV papain-like protease (SARS-CoV PLP) within 239 HEK293T cells as both of these proteases are well characterized with documented small molecule inhibitors 240 tested in live virus assays. In cells transfected with the protease containing constructs, we observed marked 241 cytotoxicity and a dose-dependent reversal of toxicity when cells were incubated with either a known 3C

242 protease or PLP inhibitor, respectively (Supplementary Figure 11). As with our 3CLpro studies, the EC50 243 values obtained from our transfection-based approach showed excellent concordance with live virus studies 244 for both of these proteases, suggesting that our approach to compound testing may be generalizable to 245 many other protease families (Table 1) [39-41]. 246 247 Discussion 248 Given the essentiality of the coronavirus 3CL protease for viral replication and the success of protease 249 inhibitors in the treatment of viral illness, the chemical inhibition of coronavirus 3CL proteases represent a 250 promising avenue for treating infections caused by this large family of viruses. Here, we present a simplified 251 assay to identify and characterize candidate inhibitors under physiologic cellular conditions. While 252 conventional methods for identifying 3CL protease inhibitors make use of in vitro purified protease, the 253 isolation of sufficiently pure enzyme in its native state can be costly and labor intensive. Furthermore, 254 assays using purified protease fail to consider cell permeability and the influence of the extracellular and 255 intracellular milieu on compound activity. In contrast, live virus-based assay are performed under 256 physiologically relevant conditions but require extensive biosafety containment while many coronaviruses, 257 particularly those of zoonotic origin, do not have existing live virus assays [42,43]. In comparison, our 258 approach presents significant advantages over these traditional approaches, given its physiologic relevance

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 259 and ability to be performed with equipment, reagents, and safety infrastructure commonly available to the 260 majority of biomedical research laboratories. The phenotype assayed within our approach is driven solely by 261 protease activity, and thus it is not subject to the same confounders as live virus assays. In live virus assays, 262 a tested compound may function against multiple cellular targets to inhibit viral replication, which has been 263 demonstrated for 3CLpro inhibitors that are also active against Cathepsin L, an endopeptidase with a role in 264 SARS-CoV-2 replication [19,21,44]. Generally toxic compounds may also result in observed activity during 265 live virus assays as a result of cellular perturbations that prevent viral replication [45]. Additionally, cell line 266 specific effects of live virus assays have been observed, most notably for hydroxychloroquine, wherein 267 compounds may inhibit viral replication in certain cell lines but are not active in other cell lines [46]. These 268 properties of live virus assays may complicate drug screening results and lead to uncertainty about 269 compound mechanism. In contrast, our approach allows users to identify compounds whose function is 270 squarely dependent on 3CLpro inhibition and may be less likely to demonstrate off-target activity during 271 further development or when tested in the live virus setting. Other 3CL protease cell-based assays have also 272 been developed, such as the FlipGFP and pGlo assays, which are based on reporters that become 273 fluorescent or can activate luminescence when cut by the 3CL protease, respectively [22,47,48]. Our

274 approach performs similarly to these assays wherein the FlipGFP assay reports an EC50 of 5.5 μM for the

275 interaction between GC376 and SARS-CoV-2 3CL in comparison to our reported EC50 of 3.30 μM. The pGlo

276 assay has reported an EC50s of 2.68 μM and 3.41 μM for the interaction between GRL-0496 and SARS-CoV 277 3CLpro and SARS-CoV-2 3CLpro, respectively, which are in line with our reported values of 7.84 μM and 278 5.05 μM. With similar performance, our assay provides advantages over these cell-based assays in that it 279 has been extensively validated across 3CL proteases and a wide range of compounds, has been optimized 280 for high-throughput screening, and does not require reporters to be modified when testing different 281 proteases. 282

283 Within the literature, EC50 values obtained for a 3CLpro inhibitor against live virus can show a broad range of

284 reported potencies, with EC50 values at times ranging across multiple orders of magnitude (Table 1). These 285 differences appear to be driven by variation in experimental setup such as cell line used, assay readout, 286 incubation period, and initial concentration of virus added. While we have observed agreement between the

287 EC50 values obtained from the described transfection-based method and those reported in the literature,

288 given the differences in EC50 across assays, we suggest caution when comparing results across studies. By 289 developing this transfection-based 3CLpro testing platform, we hope to facilitate the discovery of new 290 coronavirus inhibitors while also facilitating the comparison of existing inhibitors within a single simplified 291 assay system. Furthermore, we propose that this cellular protease assay system could be industrialized to 292 screen and optimize a large number of compounds to discover potential treatments for future viral 293 pandemics. 294

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 295 During our studies, we observed protease-mediated cytotoxicity and small molecule rescue for 15 3CLpros, 296 suggesting that our assay is widely applicable to this family of proteases. The plasticity of the assay across 297 various 3CLpros is of particular significance given the myriad of coronaviruses that have no live virus assay 298 and therefore have few options available for testing compound efficacy within mammalian cells. The 299 approach is also compatible with small molecule screening and allows for comparisons across 3CLpros to 300 obtain structural insights into compound activity such as our studies of 11a, GC376, and SL-4-241 that 301 demonstrated the role of the P2 substituent in dictating compound specificity. 302 303 Our findings have important implications for the manner in which small molecule protease inhibitors are 304 studied. We propose that given our assay’s breadth and ease of use it is well suited to form the backbone of 305 a forward-thinking pandemic preparedness strategy. The goal of such a strategy would be the proactive 306 identification of inhibitors capable of addressing not only the current human coronaviral strains, but also 307 small molecule leads against zoonotic strains with the highest potential to transmit into humans. Such a 308 strategy if properly implemented would provide the biomedical community with a series of high value 309 chemical leads upon which to perform additional focused chemical optimization or if already passed through 310 preclinical testing a set of compounds ready for rapid translation into humans. 311 312 Materials and Methods 313 Cell Lines and Cell Culture 314 HEK293T and HEK293 cells used in this study were obtained from ATCC. Cells were maintained at 37°C in

315 a humidified atmosphere with 5% CO2. HEK293T and HEK293 cells were grown in Dulbecco’s Modified 316 Eagle Medium (DMEM, Invitrogen) which was supplemented with 10% fetal bovine serum (Gibco) and 317 penicillin-streptomycin (Invitrogen). HEK293T and HEK293 cells were confirmed to be free of mycoplasma 318 contamination with the Agilent MycoSensor PCR Assay Kit. 319 320 To obtain HEK293 cells stably expressing EYFP, for fluorescent imaging-based studies, cells were co- 321 transfected with EYFP plasmids cloned within a piggyBac transposon (pPB bsr2-EYFP) and pCMV-mPBase 322 (mammalian codon-optimized PBase) encoding a piggyBac transposase using Lipofectamine 2000 323 (Invitrogen) according to the manufacturer’s instructions. One day after transfection, the transfected cells 324 were selected with 10 μg/mL of blasticidin (Invitrogen). 325 326 Transfections and Drug Selections 327 24 h prior to transfection, 293T cells were seeded at 40-60% confluency into 24-well plates coated for 30 328 min with a 0.1 mg/mL solution of poly-D-lysine (MP Biomedicals Inc.) and washed with PBS (Gibco) once 329 prior to media addition. The next day, 500 ng of 3CLpro expression plasmid, unless otherwise stated, was 330 incubated with Opti-MEM and Lipofectamine 2000 for 30 min at room temperature prior to dribbling on cells,

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 331 as per manufacturer’s protocol. For plating into drug conditions, 20 h after transfection, cells were washed 332 once with PBS and 200 μL Trypsin-EDTA 0.25% (Gibco) was added to cells to release them from the plate. 333 Trypsinized cell slurry was pipetted up and down repeatedly to ensure a single cell suspension. 96-well 334 plates were coated with poly-D-lysine, either coated manually with 1 μg/mL poly-D-lysine in PBS solution for 335 30 min or purchased pre-coated with poly-D-lysine (Corning). Wells were filled with 100 μL of media ± drug 336 and 1 μg/mL puromycin to select for protease expressing cells and were seeded with 9 μL of trypsinized cell 337 slurry. For data analysis the relative growth in the drug treated condition was compared to the DMSO or 338 lowest drug treated condition to further control for any batch to batch variation in transfection efficiency or 339 other sources of variation between experiments. For higher throughput experiments, multiple individually 340 transfected wells of a 24-well plate were combined after trypsinization and prior to seeding in drug. After 341 seeding into wells containing drug and puromycin, cells were incubated for 48 h unless otherwise specified. 342 343 Plasmids 344 Protease constructs used for compound testing were cloned into the pLEX307 backbone containing a 345 puromycin resistance marker (Addgene #41392) using Gateway LR II Clonase Enzyme mix (Invitrogen). 346 3CL proteases used in this study were generated using gene fragments ordered from Twist Biosciences. 347 Start codons were added upstream of the P1’ (5’) serine residue and stop codons were added downstream 348 of the P1 (3’) glutamine residue. Sequences were not codon optimized for expression in mammalian cells, 349 but were codon optimized when necessary for synthesis. Inactive 3CL proteases were generated by site 350 directed mutagenesis of the essential catalytic cysteine. DNA was transformed into NEB 10-beta high 351 efficiency competent cells. Sanger sequencing to verify proper inserts were done for all plasmids used in this 352 study (Genewiz). 353 Plasmid DNA was isolated using standard miniprep buffers (Omega Biotek) and silica membrane columns 354 (Biobasic). To reduce batch-to-batch variability between plasmid DNA isolations and its subsequent impact 355 on transfection efficiency, multiple plasmid DNA extractions were conducted in parallel, diluted to 50 ng/μL 356 and pooled together. 357 358 For western blotting, proteases were cloned into the pGCS-N3 backbone which expresses proteases with an 359 N-terminal 3xHA tag (Addgene #85720) using LR II Clonase. 360 361 Crystal Violet Staining and Quantification 362 The crystal violet staining protocol was adapted from Feoktistova et al. [20]. Briefly, after compound 363 incubation with 3CLpro expressing cells in 96-well plates, the medium was discarded and cells were washed 364 once with PBS. Cells were incubated with 50 μL of crystal violet staining solution (0.5% crystal violet in 80% 365 water and 20% methanol) and rocked gently for 30 min. The staining solution was removed and cells were 366 washed four times with water using a multichannel pipette. Stained cells were left to dry for ≥4 h on the

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 367 laboratory bench or within the chemical hood (to speed up drying rate). The crystal violet staining solution 368 was eluted by the addition of 200 μL of methanol followed by 30 min of gentle rocking. Plates were sealed 369 with parafilm to mitigate methanol evaporation. 100 μL of eluted stain from each well was transferred to a 370 new 96-well plate for reading in a Tecan Infinite F50 plate reader. Absorbance was measured at 595 nm 371 twice and values were averaged between replicate measurements. Blank wells were included in each batch 372 of experiments, and absorbance values were normalized by background levels of staining from blank wells. 373 374 Fluorescence measurements of cell density 375 Transfected protease expressing cells were plated at ~50% confluency on poly-D-lysine coated 96-well 376 plates (Greiner Bio-OneTM) 48 hours prior to imaging and were washed once immediately before imaging. 377 EYFP fluorescence imaging was performed using an Axio Observer 7 microscope (Zeiss) equipped with a 378 Plan-Apochromat 10X objective (0.45 N.A.) with 1-by-1 pixel binning. Optical Illumination bias was 379 empirically derived by sampling background areas and subsequently used to flatten images. For each well, 9 380 1.32 mm x 1.32 mm images were taken covering approximately 60% of the well area. After a global 381 background subtraction, cell density was calculated based on area of EYFP intensity. All images were 382 analyzed using custom Matlab scripts. 383 384 Western Blotting 385 For detection of protease expression from HA-tagged protease constructs, 72 h after transfection, HEK293T 386 cells were harvested in RIPA buffer (Alfa Aesar) supplemented with Halt Protease Inhibitor Cocktail on ice 387 (Thermo Scientific). Cells were sonicated for 10 s at 20% amplitude. Sonicated cells were spun at 4°C for 20 388 min at 12,000 g. Supernatant was collected and protein concentration was normalized to 300 ng/μL 389 supplemented with LDS loading buffer and boiled (Invitrogen). 3 μg of total protein was loaded into a 390 polyacrylamide gel (Invitrogen). 391 392 For detection of HA tagged proteases, an HA Tag monoclonal (clone 2-2.2.14; Thermo Scientific catalogue 393 number 26183) antibody was used at a 1:5000 dilution. 394 395 Statistical Analysis of Dose Response Curves 396 For analysis of crystal violet staining experiments, relative growth was calculated from background 397 normalized absorbance values. Test wells containing drug were divided by average background normalized 398 values from wells where cells were expressing protease and exposed to vehicle, when available. Otherwise, 399 values were normalized by values from protease-expressing cells exposed to the lowest concentration of 400 drug included in the dose-response curve. When there were significant deviations from protease-expressing 401 cells exposed to no drug and protease-expressing cells exposed to lowest concentrations of drug included in 402 the dose-response curve, experiments were repeated with normalization by protease-expressing cells

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 403 exposed to no drug. CC50 values were calculated in Prism using the nonlinear regression functionality and 404 derived from dose-response curves with EYFP transfected cells. A nonlinear curve fitting function 405 accounting for variable curve slopes was calculated by plotting the normalized response as a function of

406 log(compound). Similarly, EC50 values were calculated in GraphPad Prism also using the nonlinear 407 regression functionality. A nonlinear curve fitting function measuring the stimulatory response of a

408 compound as a function of an unnormalized response was used to calculate the EC50. All reported values

409 were confirmed to not have ambiguous curve fitting. The 95% confidence interval of EC50 calculations was 410 also calculated and included. 411

412 For analysis of live virus experiments, EC50 values were determined by fitting a nonlinear curve to the data 413 with the assumption of a normalized response (GraphPad Prism). Cells were confirmed as mycoplasma 414 negative prior to use. 415 416 Compound Screening 417 For screening condition optimization, we measured the Z-Factor for replicates of positive controls GC376, 418 tested at 50 μM, and compound 4, tested at 20 µM. Replicate measurements were recorded for DMSO 419 negative controls and positive control compounds after 48, 72, and 96 h of incubation with drug. Background 420 normalized crystal violet absorbance values at each timepoint were collected. 421 During the drug screen, within each of the four plates screened, two positive controls wells were included to 422 ensure assay reliability, along with several wells with the negative control 0.1% DMSO condition. All 423 compounds were screened at 10 µM resuspended in DMSO (Fisher Scientific). 424 For hit selection, we employed a robust z-score method. We first normalized data using a robust z-score that 425 uses median and median absolute deviation (MAD) instead of mean and standard deviation. We then used 426 a threshold of 3.5 MAD to determine which drugs rescued the cytotoxicity imposed by expression of the viral 427 protease [49]. 428 429 Live Virus Assay 430 The SARS-CoV-2 strain 2019-nCoV/USA_WA1/2020 was grown and titered in Vero-E6 cells. One day 431 before the experiment, Vero-E6 cells were seeded at 30,000 cells/well in 96 well-plates. Serial dilutions of 432 the test compound were prepared in media (EMEM + 10% FCS + penicillin/streptomycin), pipetted onto 433 cells, and virus was subsequently added to each well at an MOI of 0.2. Cells were incubated in a humidified

434 environment at 37 °C with 5% CO2 for 72 h after addition of virus. Cytopathic effect was scored by visual 435 inspection of the wells performed by researchers that were blinded to the treatment condition. The reported 436 cytopathic effect value represents the average from two independent reviewers. Percent Inhibition was 437 calculated by comparison to control wells with no inhibitor added. All live virus experiments were conducted 438 in a biosafety level 3 lab.

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 439 440 Compounds and Chemical Synthesis 441 GC376 was purchased from Aobious. Myrecetin, rupintrivir, grazoprevir, saquinavir, fosamprenavir, 442 indinavir, apigenin, quercetin, famotidine, MDL28170, bicailein, betrixaban, and amentoflavone were 443 purchased from Fisher Scientific. Tipranavir was purchased from Cayman Chemical. MAC5576, MAC22272, 444 MAC8120, MAC30731, BTB07404, BTB07408, MWP00332, BTB07417, MWP00508, MWP00333, 445 BTB07407, SPB08384, SPB06613, SPB06636, SPB06591, SPB06593, MWP00709, CC42746, BTB07789, 446 BTB07420, MWP00710, BTB07421, SCR00533, and SEW03089 were purchased from Maybridge. GRL- 447 0496 and GRL-0617 were purchased from Focus Biomolecules. AZVIII-40A (1,2-Benzisothiazol-3(2H)-one) 448 was purchased from Alfa Aesar. Other protease inhibitors listed were purchased from TargetMol: 449 Omarigliptin, Apoptosis Activator 2, Picolamine, Muscone, 2-Aminoethanethiol, Dexibuprofen, Glucosamine, 450 Gabexate mesylate, Zalcitabine, Amiloride hydrochloride, Saxagliptin hydrate, Linagliptin, Sitagliptin, 451 Hexylresorcinol, Arbutin, Diminazene Aceturate, 3-Pyridylacetic acid hydrochloride, Racecadotril, Mizoribine, 452 Sodium etidronate, Monobenzone, Limonin, Betulinic acid, PMSF, Fenofibric acid, Ramelteon, Ritonavir, 453 Alogliptin Benzoate, Bortezomib, Acetohydroxamic acid, Nevirapine, Lopinavir, Penciclovir, AOB2796, 454 Maribavir, Trelagliptin succinate, MLN9708, SC514, Ixazomib, Raltegravir potassium, PSI6206, Cilastatin, 455 Taxifolin, Nafamostat mesylate, Daclatasvir dihydrochloride, Darunavir Ethanolate, Ilomastat, Elvitegravir, 456 Dolutegravir sodium, Astragaloside IV, Arctigenin, Stigmasterol, Nobiletin, Celastrol, Glucosamine sulfate, 457 Picroside I, Alvelestat, N-Ethylmaleimide, DAPT, Trelagliptin, Z-VAD(OMe)-FMK, Abietic Acid, Atazanavir 458 sulfate, Abacavir, Balicatib, Carfilzomib, Atazanavir, Vildagliptin, Dapivirine, SB-3CT, PD 151746, PAC1, 459 Camostat mesilate, Efavirenz, Des(benzylpyridyl) Atazanavi, LY2811376, FLI06, SRPIN340, NSC 405020, 460 Leupeptin Hemisulfate, Stearic acid, Epoxomicin, MG101, lavendustin C, BMS707035, Asunaprevir, 461 Loxistatin Acid, GK921, L-685,458, Tenofovir Disoproxil Fumarate, GSK690693, Ledipasvir, ONX0914, 462 PI1840, (+)-Isocorydine hydrochloride, UAMC 00039 dihydrochloride, PE859, RO4929097, Emricasan, CGS 463 27023A, Talabostat mesylate, Ledipasvir acetone, Batimastat, TOFA, HZ1157, Abacavir sulfate, Sivelestat, 464 Dasabuvir, Calycosin, 4-Methoxysalicylaldehyde, Sebacic acid, Deoxyarbutin, 2-5-dihydroxyacetophenone, 465 Oxyresveratrol, Aloxistatin, Fostemsavir, Tasisulam, Semagacestat, Triciribine, IMR-1A, IMR1, Z-IETD-FMK, 466 VR23, Amprenavir, AA26-9, Dolutegravir, Lomibuvir, Ginsenoside Rh2, UK371804, CA-074 methyl ester, 467 ML281, CP 640186, Hydroumbellic acid, Ethyl gallate, Senegenin, lithospermic acid, Dibenzazepine, 468 LY411575, Paritaprevir, Sofosbuvir, Crenigacestat, Doravirine, Delanzomib, Morroniside, Calycosin-7-O- 469 beta-D-glucoside, Glabridin, Licochalcone A, Velpatasvir, Telaprevir, Odanacatib, Darunavir, Danoprevir, 470 Nelfinavir Mesylate, Oprozomib, AEBSF hydrochloride, Belnacasan, Z-DEVD-FMK, Z-FA-FMK, Trovirdine, 471 MG132, Cabotegravir, and Avagacestat. 472 See Supplementary Materials and Methods for further information with regard to compounds synthesized for 473 this study. 474

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 621 Acknowledgements

622 Compound 11a was synthesized and generously provided by Shi-Xian Deng, Columbia University. This 623 work was supported by a grant from the Jack Ma Foundation to D.D.H. and A.C. and by grants from 624 Columbia Technology Ventures and the Columbia Translational Therapeutics (TRx) program to B.R.S. 625 Further support for these studies comes from a pilot grant award from the Herbert Irving Comprehensive 626 Cancer Center in partnership with the Irving Institute for Clinical and Translational Research at Columbia 627 University to H.Y. and A.C. A.C. is also supported by a Career Awards for Medical Scientists from the 628 Burroughs Wellcome Fund. S.J.R is supported by NIH grant F31NS111851. S.I. is supported by NIH grant 629 T32AI106711.

630 Author contributions 631 S.J.R., S.I, and A.C conceived the project. S.J.R., S.I., B.R.S., D.D.H., and A.C. planned and designed the 632 experiments. S.J.R. performed crystal violet-based assays. S.J.R. and S.K. performed the HEK293-EYFP 633 based assays. S.J.R., S.I., and S.J.H. cloned plasmids. A.Z. and H.L. synthesized compounds and provided 634 compound structure information for synthesized compounds. N.E.S.T. and T.R. synthesized compounds NT- 635 1-21, NT-1-24, and NT-1-32. S.L. and T.R. synthesized compound SL-4-241. M.S.N. and Y.H. conducted 636 the live virus assays. F.L. and L.X. conducted structural modeling and chemical composition analysis. S.K. 637 and H.Y. performed imaging and created the HEK293-EYFP cell line. S.J.R. and S.M. conducted data 638 analysis. S.J.R, S.I., S.J.H, and A.C. wrote the manuscript with input from all authors. 639 640 Competing interests 641 S.I., H.L., A.Z., B.R.S., A.C., and D.D.H. are inventors on a patent application submitted based on some of 642 the molecules described in this work. B.R.S. is an inventor on additional patents and patent applications 643 related to small molecule therapeutics, and co-founded and serves as a consultant to Inzen Therapeutics 644 and Nevrox Limited. 645 646 Data and materials availability 647 All reagents generated in this study are without restriction. Plasmids generated in this study will be 648 deposited to Addgene. Source data for all figures are provided with this manuscript online. All statistics were 649 performed using Prism v.8.4.2. 650

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 651 Figures and Tables

652 Table 1. Comparison of literature reported live virus based EC50 values compared to values 653 generated during this study. CPE = Cytopathic effect. Literature Calculated Protease Drug Reported Method Cell Line Citation EC50 (μM) Value (μM) Ma et al. 3.4 CPE Vero E6 [21] Plaque Vuong et 0.9 Vero E6 Assay al. [26] SARS-CoV-2 Luan et al. GC376 3.3 0.18 qPCR Vero E6 3CLpro [25] Froggatt et 2.2 qPCR Vero E6 al. [22] Iketani et 4.5 CPE Vero E6 al. [24] 3.8 CPE Vero E6 This study SARS-CoV-2 11a 6.89 Plaque Dai et al. 3CLpro 0.5 Vero E6 Assay [27]

SARS-CoV-2 Iketani et compound 4 0.98 3.0 CPE Vero E6 3CLpro al. [24] SARS-CoV-2 GRL-0496 5.05 9.1 CPE Vero E6 This study 3CLpro SARS-CoV Ghosh et GRL-0496 7.84 6.9 CPE Vero E6 3CLpro al. [29] Plaque Vuong et SARS-CoV-2 1.5 Vero E6 GC373 2.8 Assay al. 3CLpro 4.8 CPE Vero E6 This Study SARS-CoV Ratia et al. GRL-0617 5.65 14.5 CPE Vero E6 PLP [41] Binford et 0.021 CPE HRV-B14 H1-HeLa al. [39] Rupintrivir 0.0086 3Cpro Patick et al. 0.013 CPE H1-HeLa [40] 654

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Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 655 Fig. 1. Expression of SARS-CoV-2 3CLpro in HEK293T cells results in toxicity that can be rescued by 656 the protease inhibitor GC376. a. SARS-CoV-2 3CL toxicity is dependent on protease activity and can be 657 visualized with crystal violet staining. C145A represents the catalytically null variant of the protease. b. 658 Quantification of crystal violet staining in a. c. Detection of protease expression via western blotting. d. 659 Treatment of SARS-CoV-2 3CLpro expressing cells with protease inhibitor GC376 results in rescue of 660 cytotoxicity. e. Quantification of d. Data are shown as mean ± s.d. for four technical replicates. a EYFP b

0.6

e c

n 0.4

o s

SARS-CoV-2 3CLpro b 0.2

4 1

0.0 C

P o A o r F r 5 r

p 4 p

Y p

E L 1 L C L 3 C c

o C

-2 r C 3

V p 3

SARS-CoV-2 3CLpro C145A o L 2

C C 2 - - 3 -

S 2 V - V

R V o A o o

S C C

- C -

S - S R S

A R

S R

A A

d e Y

H H SARS-CoV-2 3CLpro + Vehicle 0.6 E

e c

n 0.4

a Anti-HA

r o

SARS-CoV-2 3CLpro + 50 µM GC376 s

b 0.2 A

0.0 Vehicle 50µM GC376 661

pg. 21

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 662 Fig. 2. Dose response curves for SARS-CoV-2 3CLpro can be conducted with transfection-based 663 assays. a-c. SARS-CoV-2 3CLpro can be inhibited by known 3CLpro inhibitors GC376, compound 4, and 664 11a. The toxicity of each compound was determined by treating EYFP-transfected cells with indicated 665 concentrations of drug and is reported as Cell Viability. d. Chemical structures for each of the compounds

666 tested. EC50 values are displayed as best-fit value alongside 95% confidence interval. CC50 values are 667 displayed as best-fit value. Data are shown as mean ± s.d. for four technical replicates. a b EC50 = 3.30 mM EC50 = 0.98 mM 4 95% CI: [1.81 - 6.04] 150 4 95% CI: [0.48 - 2.01] 150 CC = >100 mM CC = 81 mM 50 R 50 C

h h

e t

t el e 3 l 3

w w

100 V

o 100

o a ll

i r

G G

ti V

2 i

e 2 e

v v

t i

y v i i

t t

(

a a

( l

50 %

l 50

e % a

e e

1 )

)

R 1 R G b r ili 0 0 0o 0 t -6 -4 -2 0 2 4 w-6 -4 -2 0 2 4 y log[GC376], µM t log[Compound 4], µM h

c EC50 = 6.89 mM d 4 95% CI: [3.80 - 12.59] 150 CC50 = 48 mM

C h

e t 3

l w

100 V o

i r

a G

GC376 Compound 4

2 i e

i v

t i

y t

(

l 50

% e

1 ) R

0 0 -6 -4 -2 0 2 4 log[11a], µM 11a 668

pg. 22

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 669 Fig. 3. The activity of GC376, compound 4, and 11a show variable effectiveness and potency against 670 the coronavirus 3CL proteases from SARS-CoV, MERS-CoV, Bat-CoV-HKU9, HCoV-NL63, and IBV.

671 EC50 values are displayed as best-fit value alongside 95% confidence interval. CC50 values are displayed as 672 best-fit value. Data are shown as mean ± s.d. for three or four technical replicate

GC376 Compound 4 11a

o EC = 5.38 mM r 4 EC50 = 5.83 mM 150 5 EC50 = 3.17 mM 150 3 50 150

p 95% CI: [3.58 - 9.38] 95% CI: [2.03 - 4.99] 95% CI: [1.63 - 32.5]

L h

4 e

h h

t t

3 l

l C

l w

w w

V 2 100

100 100 o

3 o

i r

a 3 G

b G

i V

i e

2 e e

i v

v v

t i

i y t

y t

t 2

(

a l

(

( 1 50

% l

l 50

C 50

e -

)

) R

1 ) R

R 1

S R 0 0 0 0 0 0

A -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 S log[GC376], µM log[Compound 4], µM log[11a], µM

o 3 EC = 7.44 mM 150 4 EC = 1.40 mM 150

r 50 50 3 150

p 95% CI: [4.01 - 14.04] 95% CI: [0.78 - 2.48]

C C

h h

t t 3 e

l t

l l

w w

C w

2 100 V 100 o

o 2 100 o

i r

r 3

a i r

a G

b G

i 2 e

l e

l V

i e

i v

t v

i v

y i

i t

y o

( a

( l 50

a l 1 50 (

l 1 50

C e 1 )

)

R -

)

R S

R 0 0 0 0 0 0

E -6 -4 -2 0 2 4 -4 -2 0 2 4 -6 -4 -2 0 2 4

M log[GC376], µM log[Compound 4], µM log[11a], µM

o r 3 EC = 11.07 mM 150 EC = 4.11 mM 3 150 p 50 3 50 150

L 95% CI: [6.07 - 20.9] 95% CI: [2.18 - 7.86]

C h

h h

e t

t t

l l

w 3

w w

2 100

2 100 2 100 o

o o

i r

a 9

a G

b G

i e

U e

l e

i v

t y i

i t

K t

(

l 1 50 a

( l 1 50

l 1 50

H e

)

- )

V o 0 0 0 0 0 0 C -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 - -4 -2 0 2 4 t log[Compound 4], µM log[11a], µM

a log[GC376], µM B

o 4 150 4 EC = 4.92 mM 150 3 150 r EC = >50 mM 50 50 EC50 = >20 mM

p 95% CI: [3.53 - 6.85]

C C

L h

e e h

t t

3 t

l l 3

w w

100 V 2 100 o

o 100 o

r r

r 3

G G b

b G

2 e e

2 l

e 3

v v

t v

i i

t t

a a

( ( a

(

l l

50 l 1 50

% 50

e e

)

1 1 )

- V 0 0 0 0 0 0

o -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 C

log[GC376], µM log[Compound 4], µM log[11a], µM H

3 EC50 = 0.58 mM 150 4 EC50 = 0.058 mM 150 3 150 EC50 = >20 mM 95% CI: [0.27 - 1.21] 95% CI: [0.028 - 0.1]

C h

o h

h t

e t

t 3 l r l

l w

w V

V 2 100 o

V 100 o

p 2 100 o

i r

a G

L G

i e

2 l e

i l

i e

l i

v C

t v

i i

i v

i t

3 a

( a

(

l 1 50 l %

50 a

(

l 1 50 e

% e

) e

1 )

V R

)

B I 0 0 0 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -4 -2 0 2 4 log[Compound 4], µM log[11a], µM log[GC376], µM

673

pg. 23

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 674 Fig. 4. Structural differences between SARS-CoV-2 3CLpro and MERS-CoV 3CLpro determine 675 sensitivity to compounds containing large P2 substituents. a. Structure of SARS-CoV-2 3CLpro (PDB: 676 6LZE). b. Structure of MERS-CoV 3CLpro (PDB: 5WKJ). c. Structure of GC376 analog SL-4-241 containing 677 a P2 cyclohexylmethyl substituent. d. Dose-response profiling and cytotoxicity determination of SL-4-241 678 against the SARS-CoV-2 3CLpro. e. Dose-response profiling and cytotoxicity determination of SL-4-241 679 against the MERS-CoV 3CLpro. Data are shown as mean ± s.d. for four technical replicates. 680 a SARS-CoV-2 3CLpro b MERS-CoV 3CLpro

S2 S2

c d e SARS-CoV-2 3CLpro MERS-CoV 3CLpro 150 150 EC50 = 7.15 mM

95% CI: [4.29 - 11.91] C C

h h

e e t t 2 2

l l

w w

100 V 100 V

o o

i i

r r

a a

G G

b b

i i

e e

l l

i i

v v

t t

i i

y y

t t

a a

( ( l l 50 50

% %

e e

1 ) 1 )

R R

0 0 SL-4-241 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[SL-4-241], µM log[SL-4-241], µM 681 682

pg. 24

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 683 Fig. 5. Small-scale drug screen and structure-activity profiling at 10 μM identify two compounds, 684 GC373 and GRL-0496, with activity against the SARS-CoV-2 3CLpro. a. Identification of hits from the 685 drug screen and structure-activity profiling. Positive control compounds were included in each plate and are 686 highlighted. b. Compounds with structural similarity to known inhibitors. Compounds in bold are molecules 687 that show activity against the SARS-CoV-2 3CLpro at 10 μM. c. Dose-response profiling and cytotoxicity 688 determination of GRL-0496 against the SARS-CoV-2 3CLpro. d. Live virus testing of GRL-0496 against

689 SARS-CoV-2. EC50 values are displayed as best-fit value alongside 95% confidence interval. The live virus

690 assay was conducted with two biological replicates, each with three technical replicates and the EC50 value

691 was derived from all replicates. CC50 values are displayed as best-fit value. Data are shown as mean ± s.d. 692 for three or four technical replicates.

pg. 25

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. a 60 GC373 GRL-0496 Controls GC376 - 50 mM Compound 4 - 20 mM 40

e Compound 4 - 10 mM

t 20

b o R 0

-20 Plate 1 Plate 2 Plate 3 Plate 4 b

GC376 AZVIII-41A GC373

MAC-5576 BTB07408 GRL-0496 BTB07417

c d

EC = 5.05 M n

2 50 m

o i

95% CI: [2.99 - 8.56] t 100

i b

CC = 81 uM 150 i h

50 h

t EC = 9.12 mM

l 50 n

I w

V o

2 95% CI: [7.00 - 11.88]

r -

a G

100 V

o e

i v

C i

- t

a S

(

l 1

% R e 50

)

R A

S 0

0 % -6 -4 -2 0 2 4 -3 -2 -1 0 1 2 log[GRL-0496], µM log[GRL-0496], µM 693 694

pg. 26

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. 695 Fig. 6. Compound 4 and GC376 are broadly active 3CL protease inhibitors. EC50 values are displayed 696 as best-fit value alongside 95% confidence interval. Data are shown as mean ± s.d. for four technical 697 replicates. The genus from which each coronavirus is derived is listed below each protease’s name.

FIPV 3CLpro PEDV 3CLpro HCoV-229E 3CLpro alpha alpha alpha 6 4 2.5 EC50 = 4.19 mM EC = 1.61 mM EC > 10 mM

4 50 50

h 95% CI: [2.08 - 8.40]

h t

95% CI: [0.78 - 3.15] h

t d

w 3 2.0

w w

o 4

e 2 1.5

i v

t p

2 t

l e

e 1

e 1.0

R o 0 C 0 0.5 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM 4 2.5

EC50 = 5.27 mM EC50 = 8.88 mM EC50 = 7.51 mM

h h

95% CI: [2.85 - 9.56] 95% CI: [5.60 - 13.94] h 95% CI: [2.71 - 19.16]

t t 4 t

3 2.0

o o

7 2 1.5

i i

2 i

l l

e 1 e

e 1.0

R G

0 0 0.5 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[GC376], µM log[GC376], µM log[GC376], µM

Bat-CoV-HKU4 3CLpro Bat-SL-CoVZC45 3CLpro Civet-CoV010 3CLpro beta beta beta 4 4 5

4 EC50 = 3.24 mM EC50 = 2.53 mM EC50 = 3.69 mM

h h

95% CI: [2.21 - 4.76] h 95% CI: [1.20 - 5.30] 95% CI: [1.89 - 7.23]

t t

d 4

3 3

r r

r 3

G G

2 2

e e

v v

i i

i 2

t t

a a

l l

l e

e 1 m e 1

R o

C 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM 4 4 EC = 2.08 mM 5

EC50 = 3.33 mM 50 EC50 = 6.27 mM

h h

95% CI: [2.11 - 5.26] 95% CI: [1.38 - 3.16] h 95% CI: [3.13 - 12.38]

t t t 4

3 3

r r

7 3

2 2

i i

i 2

l G

e 1

R R

0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[GC376], µM log[GC376], µM log[GC376], µM

HCoV-HKU1 3CLpro HCoV-OC43 3CLpro Beluga-CoV 3CLpro beta beta gamma 3 5 4 4 EC = 2.64 mM EC > 10 mM EC50 = 2.51 mM

50 50

h h

95% CI: [1.67 - 4.22] h 95% CI: [1.24 - 5.09]

t t t 4

o o

2 o

r r

u 3

o 2

p i

i 2

t t

1 a

l m

e 1 e

e 1

R o

C 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM

3 5 4

EC50 = 6.92 mM EC50 = 7.13 mM EC50 = 4.72 mM

h h

95% CI: [4.77- 9.93] h 95% CI: [3.84 - 12.59] 95% CI: [2.17 - 10.01]

t t t 4

6 3

w w w

o o o

7 2

r r

r 3

G G G

e e e

v v v

i i

i 2

t t

1 t

G a a a

l l l

e e e 1

R R R

0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 698 log[GC376], µM log[GC376], µM log[GC376], µM 699

pg. 27

Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. a EYFP b 0.6

0.4

SARS-CoV-2 3CLpro 0.2 Absorbance

0.0 P o A F 5 Y pr 4 E L C C1 3 c 2 SARS-CoV-2 3CLpro C145A Lpro oV- C -C 3 S 2 R - A S oV -C S R A S -SARS-CoV-2 3CLpro -SARS-CoV-2 d e C145A 3CLpro -SARS-CoV-2 EYFP EYFP HA SARS-CoV-2 3CLpro + Vehicle 0.6 HA 35

0.4 Anti-HA

SARS-CoV-2 3CLpro + 50 µM GC376 0.2 Absorbance

0.0 Vehicle 50µM GC376 Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. a m b EC50 = 3.30 M 4 95% CI: [1.81 - 6.04] 150 m CC50 = >100 M Cell Viability (%) 3 100

50 1 Relative Growth Relative

0 0 -6 -4 -2 0 2 4 log[GC376], µM

EC = 6.89 mM c 50 d 4 95% CI: [3.80 - 12.59] 150 m CC50 = 48 M Cell Viability (%) 3 100

2 GC376 Compound 4

50 1 Relative Growth Relative

0 0 -6 -4 -2 0 2 4 log[11a], µM 11a Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. a SARS-CoV-2 3CLpro b MERS-CoV 3CLpro

S2 S2

c d e SARS-CoV-2 3CLpro MERS-CoV 3CLpro

m 150 150 EC50 = 7.15 M

95% CI: [4.29 - 11.91] Cell Viability (%) Cell Viability (%) 2 2 100 100

50 50 1 1 Relative Growth Relative Growth Relative

0 0 SL-4-241 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[SL-4-241], µM log[SL-4-241], µM Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. GC376 Compound 4 11a

m m EC = 5.38 mM 4 EC50 = 5.83 M 150 5 EC50 = 3.17 M 150 3 50 150 95% CI: [3.58 - 9.38] 95% CI: [2.03 - 4.99] 95% CI: [1.63 - 32.5] Cell Viability (%) Cell Viability (%) Cell Viability (%) 4 3 100 100 2 100 3 2 2 50 50 1 50 -CoV -CoV 3CLpro

1 Growth Relative Relative Growth Relative Relative Growth Relative 1

0 0 0 0 0 0 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 SARS log[GC376], µM log[Compound 4], µM log[11a], µM

m m 3 EC50 = 7.44 M 150 4 EC50 = 1.40 M 150 3 150 95% CI: [4.01 - 14.04] 95% CI: [0.78 - 2.48] Cell Viability (%) Cell Viability (%) Cell Viability (%) 3 2 100 100 2 100 2

1 50 50 1 50 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative

0 0 0 0 0 0 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 MERS-CoV MERS-CoV 3CLpro log[GC376], µM log[Compound 4], µM log[11a], µM

m m 3 EC50 = 11.07 M 150 3 EC50 = 4.11 M 150 3 150 95% CI: [6.07 - 20.9] 95% CI: [2.18 - 7.86] Cell Viability (%) Cell Viability (%) Cell Viability (%)

2 100 2 100 2 100

1 50 1 50 1 50 Relative Growth Relative Relative Growth Relative Relative Growth Relative

0 0 0 0 0 0 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[1 log[GC376], µM log[Compound 4], µM 1a], µM Bat-CoV-HKU9 3CLpro 3CLpro Bat-CoV-HKU9

m 4 m 150 4 EC50 = 4.92 M 150 3 150 EC50 = >50 M EC = >20 mM 95% CI: [3.53 - 6.85] 50 Cell Viability (%) Cell Viability (%) Cell Viability (%) 3 3 100 100 2 100

2 2

50 50 1 50 1 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative

0 0 0 0 0 0 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[1 HCoV-NL63 3CLpro 3CLpro HCoV-NL63 log[GC376], µM log[Compound 4], µM 1a], µM

m EC = 0.058 mM 3 150 3 EC50 = 0.58 M 150 4 50 150 m EC50 = >20 M 95% CI: [0.27 - 1.21] 95% CI: [0.028 - 0.1] Cell Viability (%) Cell Viability (%) Cell Viability (%) 3 2 100 100 2 100 2

1 50 50 1 50 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative IBV 3CLpro 0 0 0 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -4 -2 0 2 4 log[Compound 4], µM log[11a], µM log[GC376], µM Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. a 60 GRL-0496 GC373 Controls GC376 - 50 mM Compound 4 - 20 mM 40 Compound 4 - 10 mM

Robust Z Score Z Robust 0

-20 Plate 1 Plate 2 Plate 3 Plate 4 b

GC376 AZVIII-41A GC373

MAC-5576 BTB07408 GRL-0496 BTB07417 c d EC = 5.05 mM 2 50 95% CI: [2.99 - 8.56] 100

150 Cell Viability (%) CC50 = 81 uM m EC50 = 9.12 M 95% CI: [7.00 - 11.88] 100 50

1 50 Relative Growth Relative 0

0 Inhibition SARS-CoV-2 % -6 -4 -2 0 2 4 -3 -2 -1 0 1 2 log[GRL-0496], µM log[GRL-0496], µM Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36. FIPV 3CLpro PEDV 3CLpro HCoV-229E 3CLpro alpha alpha alpha 6 EC = 4.19 mM 4 m 2.5 m 50 EC50 = 1.61 M EC50 > 10 M 95% CI: [2.08 - 8.40] 95% CI: [0.78 - 3.15] 3 2.0 4

2 1.5 2 1 1.0 Relative Growth Relative Relative Growth Relative Relative Growth Relative 0 Compound Compound 4 0 0.5 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM m 4 m 2.5 m EC50 = 5.27 M EC50 = 8.88 M EC50 = 7.51 M 4 95% CI: [2.85 - 9.56] 95% CI: [5.60 - 13.94] 95% CI: [2.71 - 19.16] 3 2.0

2 1.5 2 1 1.0 Relative Growth Relative GC376 GC376 Relative Growth Relative Relative Growth Relative

0 0 0.5 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[GC376], µM log[GC376], µM log[GC376], µM

Bat-CoV-HKU4 3CLpro Bat-SL-CoVZC45 3CLpro Civet-CoV010 3CLpro beta beta beta 4 m 4 m 5 m EC50 = 3.24 M EC50 = 2.53 M EC50 = 3.69 M 95% CI: [2.21 - 4.76] 95% CI: [1.20 - 5.30] 4 95% CI: [1.89 - 7.23] 3 3 3 2 2 2

1 1 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative

Compound Compound 4 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM 4 4 m EC = 2.08 mM 5 m EC50 = 3.33 M 50 EC50 = 6.27 M 95% CI: [2.11 - 5.26] 95% CI: [1.38 - 3.16] 4 95% CI: [3.13 - 12.38] 3 3

3 2 2 2

GC376 GC376 1 1 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative

0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[GC376], µM log[GC376], µM log[GC376], µM HCoV-HKU1 3CLpro HCoV-OC43 3CLpro Beluga-CoV 3CLpro beta beta gamma 4 3 m 5 m EC = 2.51 mM EC50 = 2.64 M EC50 > 10 M 50 95% CI: [1.24 - 5.09] 95% CI: [1.67 - 4.22] 4 3 2 3 2 2 1 1 1 Relative Growth Relative Relative Growth Relative Relative Growth Relative

Compound Compound 4 0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[Compound 4], µM log[Compound 4], µM log[Compound 4], µM

3 m 5 m 4 m EC50 = 6.92 M EC50 = 7.13 M EC50 = 4.72 M 95% CI: [4.77- 9.93] 4 95% CI: [3.84 - 12.59] 95% CI: [2.17 - 10.01] 3 2 3 2 2 1 GC376 GC376 1 1 Relative Growth Relative Growth Relative Growth Relative

0 0 0 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 -6 -4 -2 0 2 4 log[GC376], µM log[GC376], µM log[GC376], µM Downloaded from https://journals.asm.org/journal/jvi on 22 June 2021 by 108.29.98.36.