key: cord-0428837-u8trmlhf
authors: Mediouni, Sonia; Mou, Huihui; Otsuka, Yuka; Jablonski, Joseph Anthony; Adcock, Robert Scott; Batra, Lalit; Chung, Dong-Hoon; Rood, Christopher; de Vera, Ian Mitchelle S.; Rahaim, Ronald; Ullah, Sultan; Yu, Xuerong; Nguyen, Tu-Trinh; Hull, Mitchell; Chen, Emily; Bannister, Thomas D.; Baillargeon, Pierre; Scampavia, Louis; Farzan, Michael; Valente, Susana T.; Spicer, Timothy P.
title: Identification of Potent Small Molecule Inhibitors of SARS-CoV-2 Entry
date: 2021-08-05
journal: bioRxiv
DOI: 10.1101/2021.08.05.455262
sha: 4309728082a51378b6d690e66aa9657b4f08a238
doc_id: 428837
cord_uid: u8trmlhf

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, Calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals Calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

The severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) is the pathogen 48 responsible for the COVID-19 disease which reached pandemic designation in early 49 2020. Even with the release of FDA-approved vaccines in early 2021, the pandemic 50 remains the number one worldwide public health threat with massive negative social 51 and economic impacts. Much of the world still faces widespread spikes in confirmed 52 cases, hospitalizations, demands on critical care, and mortality 1 . The highly infectious 53 delta variant has become the predominant strain in the United States. Those that 54 remain vaccine resistant are taking the brunt of infections but, breakthrough cases are 55 occurring more frequently. Infection rates vary by location, but in almost all areas, 56 episodes of disease reemergence have been observed. People with underlying medical 57 conditions, especially elderly that are immunosuppressed, have been heavily impacted. 58

In fact, adults over 65 years of age represent 80% of hospitalizations with a 23-fold 59 higher risk of death 2 . 60

Common symptoms include high fever, cough, fatigue, breathing difficulties, and loss of 61 taste and smell. Severe cases also exhibit clinical manifestations such as pneumonia, 62 acute respiratory distress syndrome and clotting disorders 3,4 .The disease spread is 63 primarily via micro droplet dispersion, typically by inhalation, and thus measures that 64 reduce or eliminate exposures to infected people are highly recommended. These 65 include regular wearing of masks, social distancing, avoiding groups of people other 66 than the close family unit, self-isolation, and avoidance of indoor spaces with 67 substandard ventilation 5-7 . Such preventive measures, in some cases, have been aided 68 by electronic apps to identify exposures 5 , which seem effective yet compliance over 69 The plate was centrifuged at 4°C, 3000g for 30 mins (spinoculation) and incubated for 2 186 hours at 37°C and 5% CO 2 20 . Virus containing media was then aspirated and 100 µL of 187 fresh media with 1 µg/mL puromycin was added to the wells. The plate was further 188 incubated for 48 hours at 37°C and 5% CO 2 . Then, 100 µL/well of OneGlo (Promega) 189 was added and luminescent signal was read. The average and 3 standard deviations of 190 the signal level for media only wells were calculated and used as a cutoff. TCID 50 was 191 calculated using Reed & Muench Calculator 21 . 192

Compounds were pre-spotted into 1536 well plates (Greiner BioOne part 789173-F) at 194 either 5 nL for 10 mM stocks of compounds at CALIBR for the ReFRAME library or 20 195 nL for either 1 mM or 2.5 mM stocks of compounds for the Cathepsin L, Pathogen Box 196 or TargetMol library, at Scripps Molecular Screening Center 16, 22 . The HEK293T-ACE2 197 cells were seeded at 2,000 cells/well, using an Aurora FRD (Aurora Discovery), at 2.5 198 µL/well. Plates were incubated for 1 hour at 37°C and 5% CO 2 . Pseudotyped virus was 199 then dispensed at 2.5 µL/well, at a multiplicity of infection (MOI) of 0.1 for primary and 200 confirmation assays and 0.5 for titration assays. After the 48-hour incubation period at 201 37°C and 5% CO 2 , plates were removed from the incubator and allowed to equilibrate at 202 room temperature for 15 mins. OneGlo (Promega) Luciferase reagent was then added 203 at 5 µL/well and incubated for 10 mins at room temperature. The luminescence was 204 subsequently measured using a ViewLux (PerkinElmer) for 5 secs. The process is 205 summarized in Fig.1B . High control wells contained HEK293T-ACE2 cells + Media + 206 vehicle (DMSO), and the low control and data wells had HEK293T-ACE2 cells + 207 SARS2-S + test compound or vehicle. 208

The first counterscreen assay followed the same protocol as the SARS2-S entry assay 210 but instead uses VSV-G pseudotyped virus, to identify non-specific entry inhibitors. In 211 addition, and in parallel, the cytotoxicity of selected compounds was also tested during 212 the campaign using CellTiter-Glo (Promega). The controls for the VSV-G assay followed 213 the exact logic of the primary assay while the toxicity assay incorporated no cells (high 214 control) vs cells treated with vehicle only (low control) as controls. 215 We then surveyed our Scripps Drug Discovery Library of greater than 665K small 233 molecules to identify ~ 450 compounds that overlapped with a Tanimoto score >80% 234 identity with these inhibitors. These compounds were cherry-picked and registered into 235 source plate for screening. 236

Data files were uploaded into the Scripps institutional HTS database (Symyx 238

Technologies, Santa Clara, CA) for plate QC and hit identification. Activity for each well 239 was normalized on a per-plate basis using the following equation: 240

where "High Control" represents wells containing HEK293T-ACE2 cells + assay Media 243 + vehicle (DMSO) wells, while "Low Control" represents wells containing HEK293T-244 ACE2 cells + SARS2-S + vehicle and finally the "Data Wells" contain HEK293T-ACE2 245 cells + SARS2-S + test compounds. The Z' and S:B were calculated using the High 246

Control and Low Control wells. A Z' value greater than 0.5 was required for a plate to be 247 considered acceptable 23 . Z-score was also calculated to show the distribution of the 248 well-to-well results by using following equation: 249

A positive Z-score indicates the raw score is higher than average 24 . 251

HEK293T-ACE2 cells, HEK293T-ACE2-TMPRSS2 cells or Vero CCL81 cells were 253 seeded at 1-1.5x10 4 cells/well in a 96-well plate. The next day, compounds (or DMSO 254 control) were added to cells at multiple concentrations, in presence of pseudotyped 255

viruses. This was done separately using SARS2-S, different mutants of SARS2-S, VSV-256 G and finally with pseudotyped virus containing the S protein from SARS-CoV-1 257 "SARS1-S". The MOI of each virus was chosen based on an equivalent level of 258 luciferase production. After a 48-hour incubation at 37°C and 5% CO 2 , plates were 259 removed from the incubator and allowed to equilibrate to room temperature for 15 mins. Bright-Glo (Promega) Luciferase reagent was then added at 100 µL/well. After a 2-261 minute incubation period at room temperature, luminescence was measured using a 262

PerkinElmer plate reader. Cytotoxicity was performed using the same cells but without 263 virus and monitored using CellTiter-Glo according to the manufacturer's instructions. 264

Time-of-drug addition assay was performed in Vero CCL81 cells. Cells were plated at 266 1×10 4 cells/well in a 96-well plate and infected with SARS2-S. Compounds SR-914 (10 267 µM) and E64D (20 µM) were added to the wells at different time points post-infection. 268

The infection proceeded for 48 hours. Bright-Glo (Promega) Luciferase reagent was 269 used to quantify viral replication. 270

Total RNA was extracted using the RNA extraction kit (Qiagen) following the 272 manufacturers instruction. Contaminating DNA was removed using Turbo-DNAse kit 273 (Ambion). cDNA was synthesized using Sensifast (Bioline) following the manufacturer's 274 instructions. Real time qPCR was performed with an aliquot of cDNA as template, using 275

Sensifast SYBR green (Bioline) in a 20 μ L reaction according to the manufacturer's 276 instructions. The same validated primer sequences of GAPDH, ACE2 and TMPRSS2 277 were implemented as previously described 25 . 278

The cytopathic effect (CPE) assay was determined using Vero E6 cells expressing 280 different levels of ACE2 cells and seeded at 12,000 cells/well in 96-well plates. The next 281 day, compounds diluted in cell culture media were added. After 2 hours of incubation, 282 Using these criteria, the hit-cutoff was determined to be 55.7% activation, which yielded 345 990 compounds that exceeded that value ("hit" , Fig.1C) . 346

To select compounds specifically targeting SARS2-S entry, we compared the activity of 347 the 990 hits against other COVID-19 directed screens completed at Scripps. These 348 screens were done using the same libraries and were tested against SARS-CoV-2 3CL 349 protease (3CLpro) and SARS-CoV-2 papain-like protease (PLpro) 26 incorporating CellTiter-Glo detection reagent. This assay yielded an average Z' of 0.96 361 ± 0.01 and a S:B of 141.9 ± 1.7 (Fig.1G ). 227 hits were selected (54.2 %) using a cutoff 362 of 12.0 % inhibition; and upon merging entry and cytotoxicity data, 108 compounds 363 were found to inhibit SARS2-S entry without being overtly cytotoxic (Fig.1C ). Next, 364 these selected compounds were titrated using 10-point dose-response titrations (3-fold 365 dilutions), in triplicate. The SARS2-S entry titration assay performance was consistent 366 with an average Z' of 0.60 ± 0.035 and a S:B of 157.0 ± 9.5. (Fig.1G ). Of the 108 367 compounds tested, 74 compounds demonstrated nominal potency (IC 50 < 10 µM) in the 368 SARS2-S entry assay and were considered active. The cytotoxicity counterscreen 369 identified 6 compounds with a CC 50 < 10 µM, and the VSV-G counterscreen identified 370 42 compounds with an IC 50 < 10 µM (Fig.1C) . At the conclusion of the HTS phase, we 371 found 6 small molecules of interest from the ReFRAME collection with a therapeutic 372 index (TI=CC 50 /IC 50 ) higher than 15 (Table.S1 ). However, upon further inspection, we 373 learned that these 6 ReFRAME compounds including their analogs had recently been 374

identified by other groups when tested in whole virus CPE assays 16,28,29 , which 375 confirmed the robustness of our assay but diminished our interest in pursuing these 376 compounds further. As such, we focused our attention on the hits identified from the 377 other collections. Cathepsin L library (Fig.1E) . From a total of 111 hits, all but two compounds were 387 available for titration assays. As with the ReFRAME library, all 109 compounds from 388 these libraries were tested in 10-point dose-response titrations (3-fold dilutions) in 389 triplicate. All compounds had IC 50 values < 10 µM in this assay. The cytotoxicity 390 counterscreen identified 61 active compounds, while the VSV-G counterscreen titration 391 assay identified 90 compounds with an IC 50 < 10 µM (Fig.1E) . 392

Out of the 91 hits, 11 showed a TI higher than 20 and a least ~10-fold higher 393 sensitivity to SARS2-S as compared to VSV-G (Table 1) . Interestingly, SR-914, SR-372 394 and SR728 had a TI and specificity toward SARS2-S higher than achieved by any 395 ReFRAME hit. As compared to the best ReFRAME hit, SR-806 (VBY-825), SR-914 and 396 SR-372 showed 7.1 and 3.5-fold higher potency, and a specificity higher than 7.1 and 3-397 fold, respectively ( Table 1 and S1). Inhibitors of calcium dependent protein kinase were also among the hits we identified. 411

Coronaviruses are reported to use calcium ions during entry into host cells 37,38 , and 412 experimental depletion of calcium seems to reduce viral entry 38 . It has been reported 413 that SARS-CoV-1 S protein stimulates cyclooxygenase-2 (COX-2) expression via both 414 calcium-dependent and calcium-independent protein kinase C pathways 39 . This inhibition was observed when SR-914 was present during the "full-time" condition. 471

Interestingly, SR-914 blocked equally well during both entry and post-entry stages 472 3C ). In the case of the "entry" condition, compound was only present during the 473 first hour prior to removal of excess virus by washing. In the "post-entry" condition, 474 compound was only present after the washing step, where the virus had already 475 permeated the cell membrane. Interestingly, VBY-825, which is a cathepsin inhibitor 476 from the ReFRAME library, previously shown to be an entry inhibitor, presented a 477 similar pattern as SR-914 49 . However, E64d showed a better potency at the entry than 478 post-entry (Fig.3C) . This result suggests a dual mechanism of action for SR-914 at the 479 entry and post-entry steps. 480

Cathepsin L and K). To verify its specificity toward the entry of SARS2, we tested its 482 activity against the SARS-CoV-2 non-structural proteases 3CLpro and PLpro in a 483 luciferase complementation reporter assay, which incorporates the unique peptide 484 cleavage site specific for each of the proteases (Fig.3D ). In this sense, inhibitors of 485 these assays would diminish the luminescence response generated upon 486 implementation of the firefly luciferase substrate. SR-914 showed no activity against 487 3CLpro and PLpro (Fig.3E ), further supporting a specific role for SR-914 in the inhibition 488 of the early events involved in SARS-CoV-2 entry. 489

CoV-1 and highly virulent emerging strains of SARS-CoV-2. The breath of SR-914 491 activity was first investigated in entry assays in HEK293T-ACE2 cells of MLV reporter 492 viruses pseudotyped with the S proteins of SARS-1 (Fig.4A ). SR-914 showed potent 493 activity against SARS1-S protein with an IC 50 of 77.18 ± 4.69 nM. However, it was found 494 to be ~8 fold less potent than it is against SARS2-S ( Fig.4A and Table 1 ). This lower 495 activity may be derived from the fact that only 68% similarity is shared between the two 496 S proteins 50,51 , suggesting specificity of SR-914 to the SARS-CoV-2 entry step. 497

New SARS-CoV-2 variants have been emerging worldwide ( Fig.4B and C) . The United 498

Kingdom (UK) variant, known as B.1.1.7, has large number of variations but noteworthy 499 is the N501Y-D614G mutation located in the S protein, which has been associated with 500 increased risk of transmissibility 10,52 . The South Africa (SA) variant, B.1.351 also has 501 these 2 substitutions and additional mutations at residues K417N and E484K that 502 weaken the neutralization by current vaccines 52,11 . Residues 417, 484 and 501 are 503 located in the crucial 438-506 region of the RBD involved in the binding of the S protein 504 with the ACE2 receptor 53 . Although the mutation 614 is far from this region, it was 505 shown to affect their interaction 54 . All these variants have now been detected in 506 numerous countries, including the United States. The D614G mutation variants overtook 507 the wildtype SARS-CoV-2 over the year of 2020 but importantly in 2021 an increased 508 number of populations are affected by N501Y-D614G variants (Fig.4C) . Delta, or 509 B.1.617.2, is now circulating in 98% of the countries world wide and is considerably 510 more transmissible (~2 fold) than the first Wuhan strain. It has ~10 mutations in the S 511 glycoprotein, with the four of them associated to higher virulence (L452R, T478K, 512 D614G and P681R 55 ). These may support the increase in vaccine breakthrough 513 recently observed with this variant. In fact, the WHO currently regards the Delta variant 514 as the fittest and fastest variant so far. Notably, at the time that most of the work 515 associated with this manuscript was done, the delta variant didn't exist, again alluding to 516 the expediency with which incredibly infectious variants arise. 517

We thus investigated the activity of SR-914 in entry assays in HEK293T-ACE2 cells 518 infected with pseudotyped viruses expressing the S protein mutated in residues D614G, 519 N501Y-D614G or presenting the mutations from UK and SA variants ( Fig.4D and E) . 520

Pseudotyped viruses were used at the same TCID 50 . 521

In the absence of Calpeptin, all construct presenting inclusive of the mutation D614G 522 showed a significant increase of luciferase expression, when used at equivalent MOI, 523 compared to Wild Type (WT, Fig.4D ). This increased activity related to the mutation 524 D614G was previously shown by Choe's group 54 . Interestingly, the combination of this 525 mutation with N501Y further amplified the luciferase signal and confirms the importance 526 of this mutation in the increased spread of the virus (Fig.4D ). To our knowledge this has 527 never been demonstrated in vitro. Only a recent computational analysis of mutational 528 strains 56 described N501Y as a more prone to disease compared to D614G. The 529 mutation of D614G for secondary structure prediction shows no changes in secondary 530 structure, while remaining in the coil region, whereas the mutation N501Y changes from 531 coil structure to extended strand. This results in a higher affinity to human ACE2 protein 532 compared to D614G based on this docking study. Remarkably, N501Y-D614G 533 mutations present in UK and SA strains did not induce the same increase of activity 534 As we mentioned earlier, we hypothesized that Calpeptin may have a dual mechanism 542 of action that inhibit virus entry and post entry steps. To visualize how calpeptin inhibits 543 entry steps, we performed molecular docking of the compound SR-914 to wildtype RBD 544 in the context of the ACE2 receptor (Fig.S3) . Accordingly, in silico ligand docking 545 studies revealed that Calpeptin binds to the WT RBD (PDB ID: 6M0J), with the highest 546 affinity (-4.38 kcal/mol), by forming two hydrogen bonds with S494, a single hydrogen 547 bond with Y453, a π-π stacking interaction with Y505, and a p-cation interaction with 548 R403 (Fig.S3D) . These residues are all critical to forming the intermolecular interaction 549 with the ACE2 receptor 53 , and therefore viral entry. As expected, an inactive analog of 550

Calpeptin showed poor binding to WT RBD (-3.46 kcal/mol, docking GlideScore is similar to WT. Consequently, the double mutant is the only outlier 569 in the linear correlation plot (Fig.S5B ). Both mutants form hydrogen bonds with S494 570 and G496, though the introduction of the N501Y mutation, which allows the formation of 571 a hydrogen bond and an additional π-π stacking interaction bond between Calpeptin and 572 the Y501 residue. Additionally, the introduction of the N501Y mutation created an 573 additional π-π stacking bond between Calpeptin and Y505, likely as a result of a 574 conformational perturbation originating from the Y501 mutation. 575

Mutant RBD models derived from PDB ID: 7KDK contained some missing residues 576 within the RBD that were assigned from the protein peptide sequence using Prime in 577 Schrodinger Maestro. Notably, the missing residues in the conformations are not 578 derived from a crystal structure, and hence the models may stray from the actual 579 structure of the mutant spike proteins. However, plotting the GlideScores obtained 580 through in silico docking against the logarithmic IC 50 of the ligand obtained in cell-based 581 assays resulted in a strong linear correlation with a R 2 of 0.878, validating the models 582 created. 583

In summary, in this study, calpeptin showed inhibitory activity specifically to SARS2 584 entry as well as post-entry step. This strongly suggests calpeptin may a dual 585 mechanism. For SARS entry inhibition, we hypothesized a direct binding between the 586 RBD and Calpeptin that affects the S-ACE2 interaction. In support of this hypothesize, Taken together, our study demonstrates the success of a large-scale drug repurposing 630 effort, confirming the outcomes of others with respect to hit-to-lead efficacy. We 631 identified Calpeptin as a novel lead that appears to be a potent inhibitor of the early 632 events in SARS-CoV-2 entry and confirmed its broad activity against whole virus and 633 pseudotyped virus containing novel alarming coronavirus variants. Our future efforts will 634 be focused on determining the precise mechanism of action, testing its activity in human 635 tissues, and performing structure-activity relationship along with using it as a component 636 f  u  n  c  t  i  o  n  a  l  l  y  c  l  e  a  v  e  s  t  h  e  s  e  v  e  r  e  a  c  u  t  e   724   r  e  s  p  i  r  a  t  o  r  y  s  y  n  d  r  o  m  e  c  o  r  o  n  a  v  i  r  u  s  c  l  a  s  s  I  f  u  s  i  o  n  p  r  o  t  e  i  n  u  p  s  t  r  e  a  m  o  f  r  a  t  h  e  r  t  h  a  n  a  d  j  a  c  e  n  t  t  o  t  h  e   725   f  u  s  i  o  n  p  e  p  t  i  d  e  .   J  V  i  r  o  l   8  2  ,  8  8  8  7  -8  8  9  0  ,  d  o  i  :  1  0  .  1  1  2  8  /  J  V  I  .  0  0  4  1  5  -0  8  (  2  0  0  8  )  .   726   3  5  D  r  e  w  s  ,  K  .   e  t  a  l  .   G  l  u  c  o  s  y  l  c  e  r  a  m  i  d  e  s  y  n  t  h  a  s  e  m  a  i  n  t  a  i  n  s  i  n  f  l  u  e  n  z  a  v  i  r  u  s  e  n  t  r  y  a  n  d  i  n  f  e  c  t  i  o I  n  h  i  b  i  t  i  o  n  o  f  C  a  l  p  a  i  n  s  P  r  o  t  e  c  t  s  M  n  -I  n  d  u  c  e  d  N  e  u  r  o  t  r  a  n  s  m  i  t  t  e  r  r  e  l  e  a  s  e  d  i  s  o  r  d  e  r  s  i  n   817   S  y  n  a  p  t  o  s  o  m  e  s  f  r  o  m  M  i  c  e  :  I  n  v  o  l  v  e  m  e  n  t  o  f  S  N  A  R  E  C  o  m  p  l  e  x  a  n  d  S  y  n  a  p  t  i  c  V  e  s  i  c  l  e  F  u  s  i  o  n  .   S  c  i  R  e  p  818   7  ,  3  7  0  1  ,  d  o  i  :  1  0  .  1  0  3  8  /  s  4  1  5  9  8  -0  1  7  -0  4  0  1  7  -9  (  2  0  1  7  )  .   819   7  2  d  e  l  a  F  u  e  n  t  e  ,  S  .  ,  S  a  n  s  a  ,  A  .  ,  P  e  r  i  y  a  k  a  r  u  p  p  i  a  h  ,  A  .  ,  G  a  r  c  e 

Its activity against SARS2-S Entry, 3CLpro and PLpro. C-: negative control. C+; positive 968 control. Shown is the mean ± SD of 3 independent experiments. One-way ANOVA

Tukey's post-test were used for statistical comparisons

HEK293T-ACE2 cells were incubated with 1022 different concentrations of Calpeptin, then infected with SARS1-S. Luciferase was 1023 measured 48 hours later, using Bright-Glo. Shown is the mean ± SEM of n=2 1024 independent experiments. B. Schematic of the substituted residues in the S protein 1025 of the highest threat of SARS-CoV-2 strains. C. Evolution of the S protein 1026 residues at the position 417, 484, 501 and 614 from

Activity of the new emergent variants. HEK293T-1029 ACE2 cells were infected with different mutants of SARS2-S. The day after, a medium 1030 change was performed. Luciferase was measured 48 hours later

Shown is the mean ± SEM of n=3 independent experiments. WT: wild type

Similar experiment 1034 than D but Calpeptin was added during infection and after medium change. Shown is 1035 the mean ± SEM of n=2-5 independent experiments. Two-way ANOVA

Dunnett's post-test were used for statistical comparisons

Anti-antiviral activity of the compounds with therapeutic index higher than 100

Vero E6 cell treated with test compounds for two hours was infected with SARS-CoV-2 1101 virus at an MOI of 0.05, then incubated for three days in the presence of compound

Cell viability (protection from virus-induced CPE) was measured with CellTiter-Glo

Cytotoxicity was tested in the same conditions with cell culture media instead of the 1104 virus. IC 50 and CC 50 were calculated with the 4-parameter Logistic model (XLFit fit 1105 model 205) and the standard errors

Activity of SR-914, SR-372 and E64d in Vero CCL81 infected with SARS2-S 1135

Cells were incubated with different concentrations of drugs, then infected with SARS2-S 1136 or VSV-G. Luciferase was measured 48 hours later, using Bright-Glo. Toxicity was 1157 1158 1159 1160 1161 1162 1163 1164 Fig

ACE2 + 1173 cells were gated on the basis of Goat IgG Isotype Control (R&D Systems, Catalog 1174 # IC108P) (1), and sorted on FACSAria III. Approximately 23% of the cell population 1175 were determined as ACE-positive (2). The cell fraction sorted by the gating was further 1176 validated for purity before expansion (3). B. Total RNA was extracted, and first-strand 1177 cDNA was quantified by qPCR using primers directed to ACE2 or TMPRSS2. Results 1178 were normalized as copies of viral mRNA per copy of GAPDH mRNA. The arbitrary 1179 value of 100 was assigned to the amount of viral mRNA generated in HEK293T-ACE2-1180 TMPRSS2 cells

Calpeptin docked to the RBD. B. 3D view of Calpeptin docked to 1188 RBD. Molecular surface colored according to residue electrostatic potential. Calpeptin 1189 shown in yellow. C. Calpeptin's interactions with critical residues within the RBD. D 1190 Types of interaction between Calpeptin and RBD. Hydrogen bonds are shown in 1191 orange, pi-pi stacking bons are shown in purple and pi-cation bonds are shown in green 1192 E. Calpeptin docked within the RBD-ACE2 connective interface

Docking of Calpeptin to the mutants RBD, using a modified PDB 6M0J and 1211 7KDK structures. Calpeptin docked to the D614G, N501Y-D614G, UK and SA variants 1212 of SARS-CoV-2 RBD. Residues and types of interactions are shown

Docking of Calpeptin to RBDs. A. An inactive analog of Calpeptin binds 1222 poorly to wild type RBD. Z LVG CHN2 was tested in SARS-2 entry assay in HTS. 1223 Activity and toxicity were measured. Shown is the mean experiments. B. Correlation plot between Glidescore and IC50 of Calpeptin 1225 against the different mutants of RBD

throughput assay. Compounds were pre-spotted in 1536-well plates. Next, 2,000 860HEK293T-ACE2 cells were added to each well and pre-incubated with each compound 861 for 1 h, followed by infection with MLV reporter luciferase virus pseudotyped with the 862 SARS-CoV-2 Spike protein (SARS2-S) or VSV-G protein (VSV-G