key: cord-0256008-ufpt1juo authors: Ma, Chunlong; Tan, Haozhou; Choza, Juliana; Wang, Yuying; Wang, Jun title: Validation and invalidation of SARS-CoV-2 main protease inhibitors using the Flip-GFP and Protease-Glo luciferase assays date: 2021-08-30 journal: bioRxiv DOI: 10.1101/2021.08.28.458041 sha: 0a90bc5886bcffbbbd793c383f3c737f84645ee6 doc_id: 256008 cord_uid: ufpt1juo SARS-CoV-2 main protease (Mpro) is one of the most extensive exploited drug targets for COVID-19. Structurally disparate compounds have been reported as Mpro inhibitors, raising the question of their target specificity. To elucidate the target specificity and the cellular target engagement of the claimed Mpro inhibitors, we systematically characterize their mechanism of action using the cell-free FRET assay, the thermal shift-binding assay, the cell lysate Protease-Glo luciferase assay, and the cell-based Flip-GFP assay. Collectively, our results have shown that majority of the Mpro inhibitors identified from drug repurposing including ebselen, carmofur, disulfiram, and shikonin are promiscuous cysteine inhibitors that are not specific to Mpro, while chloroquine, oxytetracycline, montelukast, candesartan, and dipyridamole do not inhibit Mpro in any of the assays tested. Overall, our study highlights the need of stringent hit validation at the early stage of drug discovery. Graphical abstract Flip-GFP and Protease-Glo luciferase assays, coupled with the FRET and thermal shift binding assays, were applied to validate the reported SARS-CoV-2 Mpro inhibitors. The HCV protease inhibitors have been proven a rich source of SARS-CoV-2 M pro 171 inhibitors 2, 22, 43 . From screening a focused protease library using the FRET assay, we 172 discovered simeprevir, boceprevir, and narlaprevir as SARS-CoV-2 M pro inhibitors with IC50 173 values of 13.74, 4.13, and 5.73 µM, respectively, while telaprevir was less active (31% inhibition 174 at 20 µM) 2 . The binding of boceprevir to M pro was characterized by thermal shift assay and 175 native mass spectrometry. Boceprevir inhibited SARS-CoV-2 viral replication in Vero E6 cells 176 with EC50 values of 1.31 and 1.95 µM in the primary CPE and secondary viral yield reduction 177 assays, respectively (Table 2) In the current study, we found that boceprevir showed moderate inhibition in the cellular 182 Flip-GFP M pro assay with an IC50 of 18.33 µM ( Fig. 2A and B) , a more than 4-fold increase 183 compared to the IC50 in the FRET assay (4.13 µM). The IC50 value of boceprevir in the cell 184 lysate Protease-Glo luciferase assay was 4.49 µM (Fig. 2E ). In comparison, telaprevir and 185 narlaprevir showed weaker inhibition than boceprevir in both the Flip-GFP and Protease-Glo 186 luciferase assays ( Fig. 2A , C, D, F, and G), which is consistent with their weaker potency in the 187 FRET assay (Table 2) . Overall, boceprevir, telaprevir, and narlaprevir have been validated as 188 SARS-CoV-2 M pro inhibitors in both the cellular Flip-GFP assay and the cell lysate Protease-Glo 189 luciferase assay. Therefore, the antiviral activity of these three compounds against SARS-CoV- cobicistat inhibited M pro with an IC50 of 6.7 µM in the FRET assay 33 . Cobicistat was also reported 235 to have antiviral activity against SARS-CoV-2 with an EC50 of 2.74 ± 0.20 µM using the SARS-236 CoV-2-Nluc reporter virus 17 . However, our FRET assay showed that ritonavir, nelfinavir, and 237 cobicistat did not inhibit M pro in the FRET assay (IC50 > 20 µM), which was further confirmed by 238 the lack of binding to M pro in the thermal shift assay ( Table 2) was provided. When we repeated the FRET assay, the IC50 was 64.2 µM (Table 2) . Manidipine 266 also did not show binding to M pro in the thermal shift assay. Furthermore, manidipine showed no 267 activity in either the Flip-GFP assay or the Protease-Glo luciferase assay (Fig. 4A, B, and F) . (Fig. 4G, H) . However, calpain inhibitor II had no effect on the cellular 289 M pro activity as shown by the lack of inhibition in the Flip-GFP assay (IC50 > 60 µM) (Fig. 4A, C) , 290 while calpain inhibitor XII showed weak activity (IC50 = 38.71 µM) (Fig. 4A, D) . A recent study by 291 Liu et al using a M pro trigged cytotoxicity assay similarly found the lack of cellular M pro inhibition 292 by calpain inhibitors II and XII 51 . These results contradict to the potent antiviral activity of both 293 compounds in Vero E6 cells 2 . It is noted that calpain inhibitors II and XII are also potent 294 inhibitors of cathepsin L with IC50 values of 0.41 and 1.62 nM, respectively 3 . One possible 295 explanation is that the antiviral activity of calpain inhibitors II and XII against SARS-CoV-2 might 296 be cell type dependent, and the observed inhibition in Vero E6 cells might be due to cathepsin L 297 inhibition instead of M pro inhibition. Vero E6 cells are TMPRSS2 negative, and SARS-CoV-2 298 enters cell mainly through endocytosis and is susceptible to cathepsin L inhibitors 52 . To further 299 evaluate the antiviral activity of calpain inhibitors II and XII against SARS-CoV-2, we tested 300 them in Calu-3 cells using the immunofluorescence assay (Fig. 4G , K, L). Calu-3 is TMPRSS2 301 positive and it is a close mimetic of the human primary epithelial cell 53 . As expected, calpain 302 inhibitors II and XII displayed much weaker antiviral activity against SARS-CoV-2 in Calu-3 cells 303 than in Vero E6 cells with EC50 values of 30.34 and 14.78 µM, respectively (Fig. 4K, L) . These 304 results suggest that the Flip-GFP assay can be used to faithfully predict the antiviral activity of 305 M pro inhibitors. The lower activity of calpain inhibitors II and XII in the Flip-GFP assay and the 306 Calu-3 antiviral assay might due to the competition with host proteases, resulting in the lack of 307 cellular target engagement with M pro . 308 In conclusion, calpain inhibitors II and XII are validated as M pro inhibitors but their antiviral 309 activity against SARS-CoV-2 is cell type dependent. Accordingly, TMPRSS2 positive cell lines 310 such as Calu-3 should be used to test the antiviral activity of calpain inhibitors II and XII 311 Ebselen is among one of the most frequently reported promiscuous M pro inhibitors. It was 313 first reported by Yang et al that ebselen inhibits SARS-CoV-2 M pro with an IC50 of 0.67 µM and 314 the SARS-CoV-2 replication with an EC50 of 4.67 µM 8 . However, it was noted that no reducing 315 reagent was added in the FRET assay, and we reasoned that the observed inhibition might be 316 due to non-specific modification of the catalytic cysteine 145 by ebselen. To test this hypothesis, 317 we repeated the FRET assay with and without reducing reagent DTT or GSH, and found that 318 was found that selenium coordinates directly to Cys145 and forms a S-Se bond 36 . Accordingly, 344 a mechanism involving hydrolysis of the organoselenium compounds was proposed. Similar to 345 their previous study, the M pro enzymatic reaction buffer (50 mM Tris pH 7.3, 1 mM EDTA) did 346 not include the reducing reagent DTT. Therefore, the M pro inhibition by these ebselen analogs 347 might be non-specific and the antiviral activity might arise from other mechanisms. 36 348 Overall, it can be concluded that ebselen is not a specific M pro inhibitor, and its antiviral 349 activity against SARS-CoV-2 might involve other drug targets such as nsp13 or nsp14. either the Flip-GFP assay or the Protease-Glo luciferase assay (Fig. 5A, B, N) . CoV-2 Papain-like Protease Inhibitors through a Combination of High Screening and a FlipGFP-Based Reporter Assay Development of a Fluorescence-Based CoV-2 3CL(pro) Reporter Assay SARS-CoV-2 by inactivating viral particles in cellular models Fluorogenic Protease Reporter by Flipping a Beta Strand of GFP for Imaging Apoptosis 579 in Animals A novel 581 bioluminescent protease assay using engineered firefly luciferase A 584 nanoluciferase SARS-CoV-2 for rapid neutralization testing and screening of anti-585 infective drugs for COVID-19 efficaciously inhibit SARS-CoV-2 by targeting its main protease SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption 591 ionization mass spectrometry Feline 593 coronavirus drug inhibits the main protease of SARS-CoV-2 and blocks virus replication SARS-CoV-2 and human rhinovirus with protease inhibitors in clinical development Hepatitis C virus 599 drugs that inhibit SARS-CoV-2 papain-like protease synergize with remdesivir to 600 suppress viral replication in cell culture Malleability of 602 the SARS-CoV-2 3CL M(pro) Active-Site Cavity Facilitates Binding of Clinical Antivirals Identification of existing 605 pharmaceuticals and herbal medicines as inhibitors of SARS-CoV-2 infection SARS-CoV-2 M(pro) inhibitors 608 with antiviral activity in a transgenic mouse model Direct Observation 610 of Protonation State Modulation in SARS-CoV-2 Main Protease upon Inhibitor Binding 611 with Neutron Crystallography SARS-CoV-2 main protease by the anti-HCV drug narlaprevir Completely Blocks SARS-CoV-2 Infection lopinavir, emetine, and homoharringtonine inhibit SARS-CoV-2 replication in vitro chloroquine, montelukast sodium, candesartan oxytetracycline, and atazanavir are not SARS-CoV-2 main protease inhibitors Identify potent SARS-CoV-2 main 625 protease inhibitors via accelerated free energy perturbation-based virtual screening of 626 existing drugs Alone or in Combination with Ritonavir CoV-2 Replication and Proinflammatory Cytokine Production. Antimicrob Agents 630 Chemother Screening and Biochemical Validation to Discover a Potential Inhibitor of the SARS-633 CoV-2 Main Protease Identification of 14 Known Drugs as Inhibitors of the Main Protease of 636 SARS-CoV-2 PX-12, Tideglusib, and Shikonin Are Nonspecific Promiscuous 639 SARS-CoV-2 Main Protease Inhibitors Inhibition 641 mechanism of SARS-CoV-2 main protease by ebselen and its derivatives Inhibition 644 mechanism of SARS-CoV-2 main protease by ebselen and its derivatives SARS-CoV-2 main protease by antineoplastic drug carmofur Scutellaria baicalensis extract and 650 baicalein inhibit replication of SARS-CoV-2 and its 3C-like protease in vitro Anti-SARS-CoV-2 activities in 653 vitro of Shuanghuanglian preparations and bioactive ingredients Remdesivir and chloroquine 656 effectively inhibit the recently emerged novel coronavirus (2019-nCoV) in vitro Hydroxychloroquine, a less toxic 659 derivative of chloroquine, is effective in inhibiting SARS-CoV-2 infection in vitro Malleability of 662 the SARS-CoV-2 3CL Mpro Active-Site Cavity Facilitates Binding of Clinical Antivirals Oral SARS-CoV-2 Mpro Inhibitor Clinical Candidate for the Treatment of 666 lopinavir/ritonavir in the treatment of SARS: initial virological and clinical findings A Trial of Lopinavir-Ritonavir in 671 Adults Hospitalized with Severe Covid-19 Lopinavir-ritonavir in patients admitted to hospital with COVID-19 (RECOVERY): a 674 randomised, controlled, open-label, platform trial Approved Drugs against SARS-CoV-2 Infection in Ferrets Pharmacological and therapeutic properties of ritonavir-678 boosted protease inhibitor therapy in HIV-infected patients Small molecules 681 targeting severe acute respiratory syndrome human coronavirus SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics Have Broad-Spectrum Antiviral Activity against Coronaviruses SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically 691 SARS-CoV-2 3C-like protease inhibitors using self-assembled monolayer desorption 694 ionization mass spectrometry Synergistic 696 Inhibition of SARS-CoV-2 Replication Using Disulfiram/Ebselen and Remdesivir Multi-targeting of 699 functional cysteines in multiple conserved SARS-CoV-2 domains by clinically safe Zn-700 ejectors Identification of ebselen and its analogues as potent covalent inhibitors of papain-like 703 protease from SARS-CoV-2 Ebsulfur and Ebselen as highly 705 potent scaffolds for the development of potential SARS-CoV-2 antivirals Structural basis for 708 inactivation of Giardia lamblia carbamate kinase by disulfiram Disulfiram is a 711 direct and potent inhibitor of human O 6 -methylguanine-DNA methyltransferase 712 (MGMT) in brain tumor cells and mouse brain and markedly increases the alkylating 713 DNA damage Disulfiram can 715 inhibit MERS and SARS coronavirus papain-like proteases via different modes Inhibit SARS-CoV-2 RNA-Dependent-RNA Polymerase protease inhibitors via accelerated free energy perturbation-based virtual screening of 721 existing drugs Protease Inhibitors by a Quantitative High-Throughput Screening Expedited Approach 726 toward the Rational Design of Noncovalent SARS-CoV-2 Main Protease Inhibitors The M pro is perhaps the most extensive exploited drug target for SARS-CoV-2. A variety of 412 drug discovery techniques have been applied to search for M pro inhibitors. Researchers around 413 the world are racing to share their findings with the scientific community to expedite the drug 414 discovery process. However, the quality of science should not be compromised by the speed. 415The mechanism of action of drug candidates should be thoroughly characterized in biochemical, 416 binding, and cellular assays. Pharmacological characterization should address both target 417 specificity and cellular target engagement. For target specificity, the drug candidates can be 418 counter screened against unrelated cysteine proteases such as the viral EV-A71 2A pro , EV-D68 419 2A pro , the host cathepsins B, L, and K, caspase, calpains I, II, and III, and etc. Compounds 420 inhibit multiple cysteine proteases non-discriminately are most likely promiscuous compounds 421 that act through redox cycling, inducing protein aggregation, or alkylating catalytic cysteine 422 residue C145. For cellular target engagement, the Flip-GFP and Protease-Glo luciferase assays 423 can be applied. Both assays are performed in the presence of competing host proteins at the 424 cellular environment. Collectively, our study reaches the following conclusions: 1) for validated 425 M pro inhibitors, the IC50 values with and without reducing reagent should be about the same in 426 24 the FRET assay; 2) validated M pro inhibitors should show consistent results in the FRET assay, 427 thermal shift binding assay, and the Protease-Glo luciferase assay. For compounds that are not 428 cytotoxic, they should also be active in the Flip-GFP assay; 3) compounds that have antiviral 429 activity but lack consistent results from the FRET, thermal shift, Flip-GFP, and Protease-Glo 430 luciferase assays should not be classified as M pro inhibitors; 4) compounds that non-specifically 431 inhibit multiple unrelated viral or host cysteine proteases are most likely promiscuous inhibitors 432 that should be triaged. 5) X-ray crystal structures cannot be used to justify the target specificity 433 or cellular target engagement. Promiscuous compounds have been frequently co-crystallized 434 with M pro including ebselen, carmofur, and shikonin (Table 2) . 435Overall, we hope our studies will promote the awareness of the promiscuous SARS-CoV-2 436 M pro inhibitors and call for more stringent hit validation. 437 438 Protein Expression and Purification. The tag-free SARS CoV-2 M pro protein with native N-440 and C-termini was expressed in pSUMO construct as described previously 3 .