key: cord-0983042-y4lqwp3j
authors: Cao, Wenyue; Cho, Chia-Chuan Dean; Geng, Zhi Zachary; Ma, Xinyu R.; Allen, Robert; Shaabani, Namir; Vatansever, Erol C.; Alugubelli, Yugendar R.; Ma, Yuying; Ellenburg, William H.; Yang, Kai S.; Qiao, Yuchen; Ji, Henry; Xu, Shiqing; Liu, Wenshe Ray
title: Cellular Activities of SARS-CoV-2 Main Protease Inhibitors Reveal Their Unique Characteristics
date: 2021-06-09
journal: bioRxiv
DOI: 10.1101/2021.06.08.447613
sha: ef3e3dbd435098d3d3e245fe22e13e8b4646ccf9
doc_id: 983042
cord_uid: y4lqwp3j

As an essential enzyme of SARS-CoV-2, the pathogen of COVID-19, main protease (MPro) triggers acute toxicity to its human cell host, an effect that can be alleviated by an MPro inhibitor with cellular potency. By coupling this toxicity alleviation with the expression of an MPro-eGFP fusion protein in a human cell host for straightforward characterization with fluorescent flow cytometry, we developed an effective method that allows bulk analysis of cellular potency of MPro inhibitors. In comparison to an antiviral assay in which MPro inhibitors may target host proteases or other processes in the SARS-CoV-2 life cycle to convene strong antiviral effects, this novel assay is more advantageous in providing precise cellular MPro inhibition information for assessment and optimization of MPro inhibitors. We used this assay to analyze 30 literature reported MPro inhibitors including MPI1-9 that were newly developed aldehyde-based reversible covalent inhibitors of MPro, GC376 and 11a that are two investigational drugs undergoing clinical trials for the treatment of COVID-19 patients in United States, boceprevir, calpain inhibitor II, calpain inhibitor XII, ebselen, bepridil that is an antianginal drug with potent anti-SARS-CoV-2 activity, and chloroquine and hydroxychloroquine that were previously shown to inhibit MPro. Our results showed that most inhibitors displayed cellular potency much weaker than their potency in direct inhibition of the enzyme. Many inhibitors exhibited weak or undetectable cellular potency up to 10 μM. On contrary to their strong antiviral effects, 11a, calpain inhibitor II, calpain XII, ebselen, and bepridil showed relatively weak to undetectable cellular MPro inhibition potency implicating their roles in interfering with key steps other than just the MPro catalysis in the SARS-CoV-2 life cycle to convene potent antiviral effects. characterization of these molecules on their antiviral mechanisms will likely reveal novel drug targets for COVID-19. Chloroquine and hydroxychloroquine showed close to undetectable cellular potency to inhibit MPro. Kinetic recharacterization of these two compounds rules out their possibility as MPro inhibitors. Our results also revealed that MPI5, 6, 7, and 8 have high cellular and antiviral potency with both IC50 and EC50 values respectively below 1 μM. As the one with the highest cellular and antiviral potency among all tested compounds, MPI8 has a remarkable cellular MPro inhibition IC50 value of 31 nM that matches closely to its strong antiviral effect with an EC50 value of 30 nM. Given its strong cellular and antiviral potency, we cautiously suggest that MPI8 is ready for preclinical and clinical investigations for the treatment of COVID-19.

COVID-19 is an ongoing pandemic that has paralyzed much of the world. As of May 26 th , 2021, the total confirmed infections have reached above 167 million and the total death toll has exceeded 3.4 million worldwide. 1 The disease is ongoingly devastating countries including Brazil and India. With vaccines available for COVID-19, many countries have been conducting immunization campaigns hoping that herd immunity will be achieved when the majority of the population is vaccinated. 2 Current COVID-19 vaccines are targeting the membrane Spike protein of SARS-CoV-2, the pathogen of COVID-19. 3 Spike is a weakly conserved protein in a highly mutable RNA virus. Although SARS-CoV-2 shares overall 82% genome sequence identity with SARS-CoV, Spike has only 76% protein sequence identity shared between two origins. 4 The highly mutable nature of Spike has also been corroborated by the continuous identification of new SARS-CoV-2 strains that have Spike mutations. 5 The most notable are UK, South African, and currently Indian strains. Accumulated evidences have shown attenuated activity of developed vaccines against some newly emerged SARS-CoV-2 strains. 6 Booster vaccines might be developed for new virus strains. However, the situation will likely turn into an incessant race between the emergence of new virus strains and the development of new vaccines. The focus on vaccine development and immunization that are preventative toward COVID-19 has largely obscured the development of targeted therapeutics that are direly needed for the treatment of patients with severe symptoms. By targeting a conserved gene in SARS-CoV-2, a small molecule medication can potentially turn more successful than a vaccine in containing the COVID-19 pandemic in both prevention and treatment since it is generally easier to manufacture, store, deliver, and administer a small molecule than a vaccine and the high conservativeness of the targeted gene will also make it hard for the virus to evade the small molecule.

One demonstrated drug target in SARS-CoV-2 is its main protease (M Pro ). 7, 8 Unlike Spike that is highly mutable, M Pro is highly conserved. Its 96% protein sequence identity shared between SARS-CoV and SARS-CoV-2 is much higher than the overall 82% genome sequence identify shared between the two viruses. 3 Much work has also been done in the development of M Pro inhibitors. [9] [10] [11] A general strategy that most researchers have been following in the development of M Pro inhibitors is to synthesize an active site inhibitor, test its enzymatic inhibition, and then carry out its crystallographic and antiviral analysis to obtain information for next round optimization. For most medicinal chemists, the bottleneck in this drug discovery process is the antiviral assay that requires the use of a BSL3 facility and is often not accessible.

The antiviral assay itself may also lead to misleading results about the real mechanism of an M Pro inhibitor. The life cycle of SARS-CoV-2 ( Figure 1A ) requires a number of proteases that are from either the host or the virus itself. It has been shown that transmembrane protease serine 2 (TMPRSS2) serves a critical function to prime Spike for interactions with the human cell host receptor ACE2 during the virus entry process. 12 After SARS-CoV-2 is internalized into an endosome, cathepsin L (CtsL) potentiates its membrane fusion with the endosome for the release of the virus RNA genome into the host cytosol. 13 Other cathepsin proteins such as cathepsin B

(CtsB) have also been suggested serving a role in the SARS-CoV-2 entry. 14 After the SARS-CoV-2 genomic RNA is released into the host cytosol, it is translated by the host ribosome to form two large polypeptides, ORF1a and ORF1ab. The processing of OFR1a and ORF1ab to 15 mature nonstructural proteins (nsps) requires proteolytic functions of two internally coded protease fragments, nsp3 and nsp5 that are also called papain-like protease (PL Pro ) and main protease (M Pro ) respectively. Some nsps package into an RNA replicase complex that replicates both genomic and subgenomic RNAs. Translation of subgenomic RNAs leads to essential structural proteins for packaging new virions. Furin is a host protease that can hydrolyze Spike to prime it for new virion packaging and release. 15 Based on our current understanding of SARS-CoV-2 pathogenesis and replication, there are at least three host and two viral proteases serving critical roles in the SARS-CoV-2 life cycle. Inhibition of any of these enzymes will potentiate a strong antiviral effect. Catalytic similarity between these enzymes also makes it likely that a developed small molecule is unselective toward these enzymes. M Pro , PL Pro , CtsB, and CtsL are cysteine proteases with a similar catalytic mechanism. TMPRSS2 and furin are serine proteases.

Although serine proteases are mechanistically different from cysteine proteases, many currently developed M Pro inhibitors have covalent warheads such as aldehyde and ketone making them prone to form covalent adducts with TMPRSS2 and furin as well to exert potent inhibition. 16, 17 All these proteases are also localized in different parts of the host cell. Their inhibition requires different characteristics in their inhibitors such as cellular permeability and pH sensitivity. A simple antiviral assay of a developed M Pro inhibitor will likely lead to a positive result that reflects inhibition not necessarily of M Pro and therefore causes misunderstanding that can be detrimental to further rounds of lead optimization. Therefore, an assay system that directly reflects M Pro inhibition in the host cell is critical for both assessment and optimization of M Pro inhibitors. In the current work, we will describe such a system and its application in revealing unique characteristics of a number of developed and repurposed M Pro inhibitors.

A typical antiviral assay for SARS-CoV-2 is its triggering of strong cytopathogenic effect (CPE) in host cells leading to death that can be quantified by counting formed viral plaques ( Figure 1B ). An M Pro inhibitor with high cellular potency will suppress this strong CPE leading to host cell survival. A good cellular M Pro inhibition assay will need to mimic this CPE suppression process to a large extent. Our original design for a cellular M Pro inhibition assay was to express M Pro in host cells that is fused with a N-terminal cyan fluorescent protein (CFP) and a C-terminal yellow fluorescent protein (YFP) and test the inhibition of autocleavage of this fusion protein in the presence of an inhibitor. M Pro natively cuts off its fused protein at the C-terminus.

We put an M Pro digestion site between CFP and M Pro for its cleavage as well. CFP and YFP form a Förster resonance energy transfer (FRET) pair. 18 Without an inhibitor, both CFP and YFP will be cleaved from the fusion protein in host cells leading to no FRET signal. In the presence of a potent inhibitor, the fusion protein will be intact in host cells leading to strong FRET signals.

However, transfection of 293T cells with pECFP-M Pro -EYFP (SI Appendix, Fig. S1 ), a plasmid containing a gene coding the CFP-M Pro -YFP fusion protein led to death of most transfected cells.

Repeating this transfection process all led to the exact same result. It is evident that M Pro can exert acute toxicity to its human cell host. The same observation has been made by others as well. 19 MPI8 is an M Pro inhibitor that our lab developed previously. 16 Therefore, we decided to adopt this new way for the analysis of cellular potency of M Pro inhibitors. Since a FRET system is not necessary for cellular potency analysis of M Pro inhibitors, we modified our plasmid to express an M Pro -eGFP fusion protein ( Figure 1C ) in host cells that can be easily analyzed using fluorescent flow cytometry. The expression of M Pro -eGFP in host cells will trigger cell death and weak fluorescence. This process will be reversed by adding a potent inhibitor with cellular activity. In order to use eGFP fluorescence to accurately represent expressed M Pro , we introduced a Q306G mutation in M Pro to abolish its cleavage of the Cterminal eGFP. M Pro requires a free N-terminal serine for strong activity. To achieve this, we built two constructs as shown in Figure Figure 3D . The data showed obvious MPI8-induced saturation of M Pro -eGFP expression and fit nicely to a three-parameter dose dependent inhibition mechanism in Prism 9 for IC 50 determination. The determined cellular M Pro inhibition IC 50 value of MPI8 is 31 nM. As presented later, an antiviral assay in Vero E6 cells showed an EC 50 value of 30 nM for MPI8 in inhibiting SARS-CoV-2. This high similarity between cellular M pro inhibition IC 50 and antiviral EC 50 values of MPI8 validates that cellular M Pro inhibition potency of an inhibitor represents closely its antiviral potency through M Pro inhibition.

Since MPI8 is highly effective in inhibiting M Pro in cells, we used it in combination with pLVX-M Pro -eGFP-2 to make stable 293T cells that continuously expressed M Pro -eGFP. Using this stable cell line, we characterized M Pro -induced apoptosis that was detected by anti-annexin.

After we withdrew MPI8 from the growth media that we used to culture stable cells, strong apoptotic effect started to show after 24 h and continued to increase (SI Appendix, Fig. S4 ).

Since MPI8 is a reversible covalent inhibitor, the relatively long incubation time for the observation of apoptosis is likely due to its slow release from the M Pro active site. Due to concerns about residual MPI8 and its potential slow release from M Pro in stable cells, we chose to perform cellular potency characterization of all M Pro inhibitors by doing transient transfection of 293T cells and then growth in the presence of different inhibitor concentrations.

MPI8 was one of 9 β -(S-2-oxopyrrolidin-3-yl)-alaninal (Opal)-based, reversible covalent M Pro inhibitors MPI1-9 we previously developed ( Figure 3A ). 16 GC376 is a prodrug that dissociates quickly in water to release its Opal component. 20 11a is another Opal-based, reversible covalent M Pro inhibitor that was developed in 2020. 9 All 11 compounds showed high potency in inhibiting M Pro in an enzymatic assay. 16 Besides MPI8, we went on to test cellular potency of all other 10 Opal inhibitors in their cellular inhibition of M Pro as well by following the exact same procedure that we did for MPI8. As shown in Figure Drug repurposing research has led to the identification of a number of both FDA-approved and investigational medications as M Pro inhibitors. These include boceprevir, telaprevir, and calpain inhibitor XII that have an α -ketoamide moiety for the formation of a reversible covalent adduct and calpeptin, MG-132, and calpain inhibitor II that has an aldehyde for a reversible covalent interaction with the M Pro active site cysteine. 17, 21, 22 Some of these compounds display potency in inhibiting SARS-CoV-2 replication in host cells as well. We went on to characterize cellular potency of these inhibitors using our developed cellular assay. K777 is a known CtsL inhibitor with high potency in inhibiting SARS-CoV-2 replication in human cell host. 23 It has a vinylsulfonate moiety. Due to its propensity to form a permanent covalent adduct with the M Pro active site cysteine, we tested its cellular potency in inhibiting M Pro . As shown in Figure 4B 

Drug repurposing research has also shown that carmofur, tideglusib, ebselen, disulfiram, and PX-12 can potently inhibit M Pro . 7 Carmofur is an antineoplastic agent that generates a permanent thiocarbamate covalent adduct with the M Pro active site cysteine. 24 All other four compounds are redox active for covalent conjugation with the M Pro active site cysteine. We applied our cellular potency assay to these drugs as well. As shown in Figure 4C 

Using computational docking analysis in combination with experimental examination to guide drug repurposing for COVID-19, we previously showed that bepridil, an antianginal drug inhibited M Pro and had high potency in inhibiting SARS-CoV-2 replication in host cells. 25 To provide a full picture for understanding the mechanism of bepridil in inhibiting SARS-CoV-2, we used our cellular M Pro inhibition assay to study bepridil as well. As shown in Figure 4D 

The SARS-CoV-2 life cycle requires the involvement of proteases from both the virus and the human cell host. Given high similarity in catalytic mechanisms of these proteases, an inhibitor that is developed for M Pro may also inhibit other proteases in the SARS-CoV-2 pathogenesis and replication pathway to exert an antiviral effect. Therefore, a direct antiviral assay is not optimal to reveal the real antiviral mechanism of an inhibitor and for its structureactivity relationship study for optimization. The strict requirement of a BSL3 facility to handle SARS-CoV-2 also prevents many research groups from conducting an antiviral assay in their labs and therefore causes delays in drug development. The antiviral assay itself is also complicated, lengthy, and difficult to turn high throughput. To resolve these issues, we developed a cellular M Pro inhibition assay that can be easily characterized using fluorescent cell cytometry for bulk analysis of M Pro inhibitors. We applied this assay to analyze 30 claimed M Pro inhibitors and revealed unique features for a number of them.

MPI1-9 were previously developed as potent M Pro inhibitors. All showed enzymatic IC 50 values around or below 100 nM (Table 1) . Among them MPI3 has the most enzymatic inhibition potency with an IC 50 value of 8.5 nM. However, a CPE-based antiviral assay in Vero E6 cells showed that MPI3 weakly inhibited SARS-CoV-2. 16 This observation correlates well with its low antiviral potency. 24 The high chemical reactivity of carmofur likely contributes to its low cellular and antiviral potency. Tideglusib, ebselen, well. 33 We detected close to undetectable cellular M Pro inhibition potency for bepridil up to 10 μ M. This correlates with its relatively high enzymatic IC 50 value. Therefore, it is evident that bepridil must use a mechanism different from the inhibition of M Pro in convening its high antiviral potency. This needs to be investigated. Chloroquine and hydroxychloroquine are two respectively. 34 Although TMPRESS2 was shown as the target of chloroquine and hydroxychloroquine, 35 a previous report showed that chloroquine and hydroxychloroquine potently inhibited M Pro in an enzyme inhibition assay. 26 We tested both drugs using the new cellular assay but revealed close to undetectable cellular M Pro inhibition up to 10 μ M for both drugs. We recharacterized enzymatic inhibition of M Pro by both drugs. However, we were not able to detectable any M Pro inhibition by hydroxychloroquine up to 16 μΜ and chloroquine exhibited weak inhibition of M Pro at 16 μM. Based on our cellular data, enzymatic inhibition data, and data from a separate study, 36 we are confident that both chloroquine and hydroxychloroquine don't potently inhibit M Pro inhibitors. Their antiviral activities are from different mechanism(s).

10-1, 10-2, and 10-3 are three diaryl esters in which 10-1 and 10-2 displayed high potency in inhibiting M Pro enzymatically. All three compounds displayed significant cellular M Pro inhibition potency at 10 μ M but their potency is much lower than MPI5-8. Although 10-3 has much weaker enzymatic inhibition potency than 10-1 and 10-2, its cellular M Pro inhibition potency is slightly higher than that from 10-1 and 10-2. A likely explanation is that 10-3 is more stable than 10-1 and 10-2 leading to a longer cellular time to convene its cellular M Pro inhibition potency. Therefore, we recommend balancing cellular stability and enzymatic inhibition potency for future development of diaryl esters as M Pro inhibitors to achieve optimal antiviral effects. 

We have developed a cellular assay for the determination of cellular potency of SARS-CoV-2 M Pro inhibitors. Unlike an antiviral assay in which the interference of any key step in the SARS-CoV-2 life cycle may lead to a strong antiviral effect, this new cellular assay reveals only cellular M Pro inhibition potency of a compound. It provides more precise information that reflects real M Pro inhibition in cells than an antiviral assay. Using this assay, we characterized 30 M Pro inhibitors. Our data indicated that 11a, boceprevir, ebselen, calpain inhibitor II, calpain inhibitor XII, K777, and bepridil likely interfere with key processes other than the M Pro catalysis in the SARS-CoV-2 pathogenesis and replication pathways to convene their strong antiviral effects.

Our results also revealed that MPI8 has the highest cellular potency among all compounds that were tested. It has a cellular M Pro inhibition IC 50 value of 31 nM. As the compound with the highest antiviral potency with an EC 50 value of 30 nM, we cautiously believe and recommend that MPI8 is ready for preclinical and clinical investigations for COVID-19 treatment. were shown in a previous publication. 16 Plasmid construction. We amplified M Pro with an N-terminal KTSAVLQ sequence using two primers FRET-M pro -for and FRET-M pro -rev primers (Table S1 ) and cloned it into the pECFP-18aa-EYFP plasmid (Addgene, #109330) between XhoI and HindIII restriction sites to afford pECFP-M Pro -EYFP. To construct pLVX-M Pro -eGFP-1, we amplified M Pro with an N-terminal methionine using primers XbaI-Mpro-f and Mpro-HindIII-r (Table S1 ) and eGFP using primers

HindIII-eGFP-f and eGFP-NotI-r. We digested the M Pro fragment using XbaI and HindIII-HF restriction enzymes and the eGFP fragment using HindIII-HF and NotI restriction enzymes. We ligated the two digested fragments together with the pLVX-EF1α-IRES-Puro vector (Takara Bio 631988) that was digested at XbaI and NotI restriction sites. To facilitate the ligation of three fragments, we used a ratio of M Pro , eGFP and pLVX-EF1α-IRES-Puro digested products as 3:3:1.

We constructed pLVX-M Pro -eGFP-2 in the same way as pLVX-M Pro -eGFP-1 except that we amplied the M Pro fragment using primers XbaI-Cut-Mpro-f and Mpro-HindIII-r (Table S1) .

XbaI-Cut-Mpro-f encodes an MKTSAVLQ sequence for its integration to the M Pro N-terminus.

confluency and then transfected them with pECFP-M Pro -EYFP using Lipofectamine 3000. We added 10 μM MPI8 at the same time of transfection. After 72 h incubation, cells were collected and analyzed by flow cytometer as well as fluorescence microscopy. In order to obtain highdefinition image, glass bottom plates were used for microimaging. was determined and used to plot against the MPI8 concentration. Data were fit to the threeparameter dose dependent inhibition mechanism to determine the cellular IC 50 value. 

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Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

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Furin Inhibitors Block SARS-CoV-2 Spike Protein Cleavage to Suppress Virus Production and Cytopathic Effects

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*

GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease

A Genetically Encoded FRET Sensor for Intracellular Heme

A simplified cell-based assay to identify coronavirus 3CL protease inhibitors. bioRxiv

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Both Boceprevir and GC376 efficaciously inhibit SARS-CoV-2 by targeting its main protease

A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV2 main protease

A Clinical-Stage Cysteine Protease Inhibitor blocks SARS-CoV-2 Infection of Human and Monkey Cells

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Dipyridamole, chloroquine, montelukast sodium, candesartan, oxytetracycline, and atazanavir are not SARS-CoV-2 main protease inhibitors

The authors declare no competing financial interests.