Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 2024 Aug 16;33(9):e5139. doi: 10.1002/pro.5139

Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad‐spectrum antiviral compounds

Wioletta Rut 1,✉, Katarzyna Groborz 1,4, Xinyuanyuan Sun 2, Rolf Hilgenfeld 2,3, Marcin Drag 1,✉
PMCID: PMC11328108  PMID: 39150063

Abstract

The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach—HyCoSuL—to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4‐P2 positions. However, chemical tools such as substrates, inhibitors, and activity‐based probes developed for SARS‐CoV‐2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad‐spectrum antiviral compounds.

Keywords: coronaviruses, enteroviruses, substrate specificity, viral proteases

1. INTRODUCTION

The outbreak of the COVID‐19 (coronavirus disease 2019) pandemic and the continuing increase in the number of new coronavirus variants demonstrate the importance of finding new, effective antiviral drugs (Carabelli et al., 2023; Li et al., 2023). Among the effective targets for antiviral therapy are proteases encoded by the virus. These proteins play a pivotal role in the replication and maturation of viruses. They are responsible for cleaving viral polyproteins into individual functional proteins required for viral assembly and replication (Anand et al., 2003; Jin et al., 2020; Steuber & Hilgenfeld, 2010; Zephyr et al., 2021; Zhang et al., 2020a). Additionally, viral proteases are often involved in evading host immune responses by cleaving host proteins involved in antiviral defense mechanisms (Clementz et al., 2010; Devaraj et al., 2007; Frieman et al., 2009; Lei & Hilgenfeld, 2017; Mukherjee et al., 2011). The main protease (Mpro) is highly conserved across different coronaviruses and displays a substrate specificity distinct from that of human host‐cell proteases (Dai et al., 2020; Hegyi & Ziebuhr, 2002; Zhang et al., 2020b). Furthermore, Mpro found in coronaviruses and the 3C protease (3Cpro) present in enteroviruses have similar active‐site architectures, both requiring glutamine at the P1 position of the substrate as a unique characteristic (Anand et al., 2002). Consequently, inhibitors designed to target Mpro and 3Cpro should offer highly specific, broad‐spectrum, and effective treatments against human coronaviruses and enteroviruses (Zhang et al., 2020b).

So far, seven human coronaviruses have been identified, three of which—severe acute respiratory syndrome coronavirus (SARS‐CoV), Middle East respiratory syndrome coronavirus (MERS‐CoV), and severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2)—are highly pathogenic and have caused epidemics in the past 25 years (Hilgenfeld, 2014; Kesheh et al., 2022). The most common human betacoronavirus after SARS‐CoV‐2 is HCoV‐HKU1 (Human coronavirus HKU1) (Woo et al., 2005). Infections caused by these betacoronaviruses typically manifest with fever and respiratory issues (Fung & Liu, 2021). Within the genus Alphacoronavirus, there are human coronaviruses HCoV‐NL63 (Human coronavirus NL63) and 229E (Human coronavirus 229E), which typically result in mild respiratory symptoms in generally healthy individuals (Hamre & Procknow, 1966; van der Hoek et al., 2004). However, they are considerably more prevalent than SARS‐CoV or MERS‐CoV. Another widely spread group of viruses that affect millions of people worldwide every year is enteroviruses. Enteroviruses typically cause mild infections, but they lead to clinical illnesses more frequently than coronaviruses do (except for SARS‐CoV‐2) (Brouwer et al., 2021; Epidemiological update: echovirus 11 infections in neonates, 2023; Park et al., 2021; Pons‐Salort et al., 2018). This suggests that developing an antiviral drug effective against both virus families could be commercially viable.

Throughout the COVID‐19 pandemic, numerous compounds have been discovered to combat SARS‐CoV‐2 through the application of diverse drug discovery methodologies (Boras et al., 2021; Drayman et al., 2021; Kitamura et al., 2022; Ma et al., 2020; Owen et al., 2021; Zhang et al., 2020a). Among the array of compounds, two SARS‐CoV‐2 Mpro inhibitors have been approved to treat COVID‐19 patients: nirmatrelvir (approved by the Food and Drug Administration) and ensitrelvir (approved in Japan) (Li et al., 2022; McCarthy, 2022; Owen et al., 2021; Unoh et al., 2022; Zhao et al., 2022). Ensitrelvir exhibited similar effectiveness to nirmatrelvir and demonstrated encouraging antiviral effects against other human coronaviruses as well as SARS‐CoV‐2 variants of concern (Kawashima et al., 2023; Unoh et al., 2022). To develop compounds with a broad spectrum of antiviral activity, inhibitors of SARS‐CoV‐2 Mpro were tested to assess their effectiveness against other viral main proteases. In these studies, a structure‐based design approach was typically employed (Göhl et al., 2022; Lin et al., 2023; Zhang et al., 2020b). Given that coronaviral Mpro and enteroviral 3Cpro share a number of common features, these proteases might possess a similar substrate specificity profile, which is reflected by the structure of their natural substrates (Zephyr et al., 2021). However, proteases with the same structural fold can exhibit differences in their binding pockets, which can be found through in‐depth biochemical analysis. Using natural amino acids offers only restricted insights into the architecture of the substrate‐binding pocket. To gain much broader insight, a combinatorial strategy involving a wide array of diverse chemical structures is required.

In the present study, we applied the HyCoSuL (Hybrid Combinatorial Substrate Library) approach to determine the full substrate specificity profile of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. Next, we compared these data with a previously obtained specificity matrix for SARS‐CoV and SARS‐CoV‐2 Mpros (through HyCoSuL screening) and assessed the inhibitory potency of SARS‐CoV‐2 Mpro compounds toward selected coronaviral and enteroviral major proteases. Results from our studies clearly indicate that coronaviral Mpro and enteroviral 3Cpro proteases exhibit similar substrate specificities, providing a structural framework for the development of broad‐spectrum, more potent inhibitors targeting viral main and 3C proteases.

2. RESULTS AND DISCUSSION

2.1. Substrate specificity of viral main proteases

To determine the substrate preferences of MERS‐CoV Mpro, HCoV‐NL63 Mpro, and CVB3 3Cpro (Coxsackievirus B3), we employed a hybrid combinatorial substrate library (HyCoSuL) approach that was previously used for SARS‐CoV and SARS‐CoV‐2 Mpro profiling (Rut et al., 2021). This library comprises three sublibraries, each containing a fluorescent tag (ACC—7‐amino‐4‐carbamoylmethylcoumarin), two fixed positions, and two variable positions with an equimolar mixture of 19 amino acids (Mix) (P2 sublibrary: Ac‐Mix‐Mix‐X‐Gln‐ACC, P3 sublibrary: Ac‐Mix‐X‐Mix‐Gln‐ACC, P4 sublibrary: Ac‐X‐Mix‐Mix‐Gln‐ACC, X = 19 natural and over 100 unnatural amino acids). Glutamine was incorporated at the P1 position since this amino acid is preferred by the S1 pocket of all tested enzymes (Fan et al., 2004; Hilgenfeld, 2014; Wang et al., 2016; Zhang et al., 2020a). The library screening results indicated that Mpros from betacoronaviruses and alphacoronaviruses display similar substrate specificity (Figure 1). Leucine is the most favored amino acid at the P2 position for these proteases. HCoV‐NL63 Mpro, like SARS‐CoV Mpro, exhibits lower activity toward other tested amino acids at this position (<30%). Whereas, the S2 pocket of MERS‐CoV and SARS‐CoV‐2 Mpros can accommodate other hydrophobic residues with higher activity, such as 2‐Abz, Phe(4‐NO2), 3‐Abz, β‐Ala, Dht, hLeu, Met, and Ile. Phenylalanine and its derivatives are the most preferred amino acids by CVB3 3Cpro at the P2 position (Phe(NH2), Phe(4‐Me), Phg, Phe(4‐F), Phe, Phe(3,4‐F2)). CVB3 3Cpro also recognized leucine and norleucine at this position but with lower activity (40% and 46%, respectively). These findings can be explained by the available crystal structures of coronaviral Mpro and enteroviral 3Cpro proteases in complex with inhibitors (Anand et al., 2002, 2003; Göhl et al., 2022; Rut et al., 2021; Zhang et al., 2020a; Zhang et al., 2020b). The hydrophobic S2 subsite of SARS‐CoV‐2 Mpro is more flexible compared to alphacoronavirus Mpros, which explains less stringent specificity. The S2 pocket can form hydrophobic interactions with P2 residues that are not only limited to leucine. Furthermore, the size of the S2 pocket varies among different proteases, with the following characteristics: SARS‐CoV (and SARS‐CoV‐2) Mpro, large and covered by a lid; CVB3 3Cpro, large and open; and HCoV‐NL63 Mpro, small and covered (Zhang et al., 2020b). Therefore, phenylalanine and its derivatives are preferred by the enterovirus 3Cpro. All tested proteases exhibit broad substrate specificity at the P3 position. Coronaviral Mpros prefer hydrophobic D and L amino acids and also positively charged residues; the best are: Tle, D‐Phe, D‐Tyr, Orn, hArg, Dab, Dht, Lys, D‐Phg, D‐Trp, Arg, and Met(O)2. Acidic and hydrophobic L amino acids are the most preferred by enteroviral 3Cpro (3‐Pal, Aad, hSer(Bzl), Gla, His(3‐Bom), Tyr(Bzl)). HCoV‐NL63 Mpro and CVB3 3Cpro display narrow substrate specificity profiles at the P4 position (most preferred are threonine and alanine, respectively). Betacoronaviral Mpros exhibit a broad substrate preference at this position. They particularly favor small aliphatic residues like Abu, Val, Ala, and Tle, although they can also accommodate other hydrophobic amino acids.

FIGURE 1.

FIGURE 1

Open in a new tab

Substrate specificity profiles of selected viral Mpros and 3Cpro presented as heat maps. The average relative activity is presented as a percentage of the best‐recognized amino acid (n = 2, where n represents the number of independent experiments).

Since coronaviral Mpro s display similar substrate specificity and CVB3 3Cpro recognizes some amino acids preferred by all tested proteases, we decided to analyze whether substrates designed for SARS‐CoV and SARS‐CoV‐2 Mpros are recognized by selected proteases. Kinetic analysis revealed that almost all tested substrates are hydrolyzed by coronaviral Mpros and CVB3 3Cpro (Figure 2). Only a substrate containing DTyr at the P3 position was not recognized by 3Cpro, and the substrate containing hLeu at the P2 position was not recognized by HCoV‐NL63 Mpro, which is consistent with the substrate specificity profile results for these enzymes. Furthermore, the results from the kinetic analysis enabled us to extract sequences that are efficiently hydrolyzed by all tested proteases (Ac‐Abu‐Tle‐Leu‐Gln‐ACC, Ac‐Val‐Lys‐Leu‐Gln‐ACC, and Ac‐Abu‐Orn‐Leu‐Gln‐ACC). This holds particular significance in protease inhibitor screening, where fluorogenic peptide substrates emerge as the predominant tools. Their usage facilitates a direct and swift assessment of protease inhibitor potency. This leads to a reduction in research costs, as one substrate can be utilized to assess the activity of several viral proteases in the retargeting of already known inhibitors and drugs as potential agents in antiviral therapy. Thus, we applied the Ac‐Abu‐Tle‐Leu‐Gln‐ACC substrate to evaluate the inhibitory potency of selected compounds in subsequent studies.

FIGURE 2.

FIGURE 2

Open in a new tab

The rate of tetrapeptide substrate hydrolysis by Mpros and 3Cpro. ([S] = 5 μM; SARS‐CoV Mpro concentration, 0.2 μM; SARS‐CoV‐2 Mpro concentration, 0.1 μM; MERS‐CoV Mpro concentration, 0.3 μM; HCoV‐NL63 Mpro concentration, 1 μM; CVB3 3Cpro concentration, 5 μM; data presented as mean values ± SD; n = 3, where n represents the number of independent experiments).

2.2. Inhibitors and activity‐based probes of coronaviral Mpros and enteroviral 3Cpro

In the next step of the research, we wanted to verify whether inhibitors, drugs designed for the Mpro of the SARS‐CoV‐2, would also be effective against the main protease of other coronaviruses and enteroviruses. Thus, we determined the potency of the inhibitors and activity‐based probe toward viral proteases by measuring the half‐maximal inhibitory concentration value (IC50,Table 1) (Rut et al., 2021). The probe included the Bodipy FL fluorophore, polyethylene glycol (PEG(4)) as a linker, the selected peptide sequence, and vinyl sulfone (VS) as a reactive group. Kinetic analysis revealed that the designed probe can be utilized to detect major proteases from enterovirus and coronaviruses.

TABLE 1.

IC50 values for inhibitors and activity‐based probe of viral enzymes (Data presented as mean values ± SD; n = 3, where n represents the number of independent experiments).

Compound IC50, μM
SARS‐CoV‐2 Mpro MERS‐CoV Mpro HCoV‐NL63 Mpro CVB3 3Cpro
Ac‐Abu‐Tle‐Leu‐Gln‐VS, Ac‐QS1‐VS 1.45 ± 0.12 1.59 ± 0.11 4.21 ± 0.04 47.75 ± 2.37
Ac‐Abu‐DTyr‐Leu‐Gln‐VS, Ac‐QS5‐VS 1.49 ± 0.02 1.26 ± 0.05 7.53 ± 0.14 >200
Bodipy‐PEG(4)‐Abu‐DTyr‐Leu‐Gln‐VS, Bodipy‐QS5‐VS 1.38 ± 0.12 2.27 ± 0.15 4.12 ± 0.27 16.50 ± 0.28
Open in a new tab

To further validate our probe and test its sensitivity, we performed SDS‐PAGE analysis followed by protein transfer onto membranes and ABP visualization (Figure 3a,b). We observed MERS‐CoV and HCoV‐NL63 Mpros (50 nM) labeling at a concentration of 1 μM. Bodipy‐QS5‐VS was less active toward the 3Cpro from CVB3, and the band from 3Cpro complex with the probe was visible on the membrane at a concentration of 5 μM. Then we performed time‐point analysis of probes (2.5 μM) binding to the viral proteases using different enzyme concentrations (10 and 50 nM, Figure 3c). HCoV‐NL63 Mpro (10 nM) labeling by the activity‐based probe was noticeable after 15 minutes of incubation. MERS‐CoV Mpro (50 nM) was detected by the probe after 5 minutes of incubation. For the 3Cpro, a higher enzyme concentration was required (100 nM). The enzyme‐probe complex was visible after 5 minutes of incubation. Since the Ac‐Abu‐Tle‐Leu‐Gln‐VS was more potent toward viral proteases than Ac‐Abu‐DTyr‐Leu‐Gln‐VS, we decided to test the probe containing Abu‐Tle‐Leu‐Gln sequence. The probe included the cyanine‐5 fluorophore (Cy5), PEG(4), the Abu‐Tle‐Leu‐Gln sequence, and vinyl sulfone (Cy5‐QS1‐VS). To compare both probes, we performed the same time‐point analysis using the Cy5‐QS1‐VS (Figure 3d). All viral proteases were detected by Cy5‐QS1‐VS probe. The most significant difference in detection sensitivity was observed during the labeling of the 3Cpro. The Bodipy‐QS5‐VS probe (2.5 μM) labeled the enzyme at 100 nM after 5 minutes of incubation, whereas the Cy5‐QS1‐VS probe (2.5 μM) detected 10 nM of enzyme after 15 min of incubation. These results indicated that Ac‐Abu‐Tle‐Leu‐Gln‐VS can serve as a scaffold for further optimization to develop broad‐spectrum antiviral compounds. The outcomes from viral protease labeling by the synthesized activity‐based probes reflected those obtained from substrate specificity profiles.

FIGURE 3.

FIGURE 3

Open in a new tab

Coronaviral Mpros and enteroviral 3Cpro detection by activity‐based probes. (a) Structure of activity‐based probes. (b) Viral protease labeling by the probe Bodipy‐QS5‐VS. Time‐point analysis of Bodipy‐QS5‐VS (c) and Cy5‐QS1‐VS (d) binding to the enzymes.

3. CONCLUSION

Viral proteases play essential roles in the replication and maturation of viruses by cleaving viral polyproteins into functional proteins necessary for viral assembly and infection. Additionally, viral proteases have been implicated in modulating host immune responses by cleaving host proteins involved in antiviral defense mechanisms (Zephyr et al., 2021). Understanding the structure, function, and substrate specificity of viral proteases is crucial for developing effective antiviral strategies. By targeting these proteases, viral replication can be disrupted to prevent the spread of viral infections. Therefore, ongoing research on viral proteases is crucial for advancing our understanding of viral pathogenesis and developing novel therapeutic agents against viruses. In our research, we aimed to investigate whether the chemical tools developed for SARS‐CoV‐2 Mpro could be applied to study the main proteases of other coronaviruses and enteroviruses. In the first step, we determined the substrate preferences of selected viral proteases at the P4‐P2 positions using the combinatorial library approach. The obtained substrate specificity profiles and kinetic analysis of individual substrates toward Mpro from coronaviruses and 3Cpro from enterovirus clearly demonstrated that these enzymes display similar substrate specificity; however, some differences between CVB3 3Cpro and coronaviral Mpros were found. In the next step, we utilized the optimal fluorogenic peptide substrate (Ac‐Abu‐Tle‐Leu‐Gln‐ACC) to evaluate the inhibitor potency of selected compounds. The results from the kinetic assays revealed that the inhibitors and activity‐based probes designed for SARS‐CoV‐2 Mpro are also potent toward the main protease from other coronaviruses and enterovirus 3Cpro. Furthermore, protease labeling assays from the synthesized activity‐based probes toward the viral proteases confirmed probe utility to detect viral proteases. The data presented in this work clearly demonstrate that coronaviral main proteases exhibit similar substrate preferences, implying that compounds designed for one protease can be applied to study others. Moreover, drugs designed for SARS‐CoV‐2 Mpro may prove effective in treating infections caused by other coronaviruses or even enteroviruses. The results of our work provide a structural framework for developing broad‐spectrum antiviral compounds.

4. MATERIALS AND METHODS

4.1. Enzyme preparation

The recombinant production of viral proteases is described elsewhere (Göhl et al., 2022; Zhang et al., 2020a; Zhang et al., 2020b).

4.2. Combinatorial library and individual fluorogenic substrate synthesis

Detailed protocols of the hybrid combinatorial substrate library and of tetrapeptide fluorogenic substrate synthesis were described elsewhere (Rut et al., 2021). Each tetrapeptide fluorogenic substrate was purified by HPLC and analyzed using LCMS. The purity of each compound was ≥95%. The individual substrates were dissolved at 10 mM in DMSO and stored at −80°C until use.

4.3. Library and substrate screening

HyCoSuL and substrate screening were performed using a spectrofluorometer (Molecular Devices Spectramax Gemini XPS) in 96‐well plates. Enzymes were diluted in assay buffer (20 mM Tris, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3) and preincubated at 37°C for 10 min prior to adding into the wells on the plate. Assay conditions were 1 μL of substrate in DMSO and 99 μL of enzyme. The final concentrations of enzymes and substates were as follows: library screening—[S] = 100 μM; MERS‐CoV Mpro concentration, 0.3 μM; HCoV‐NL63 Mpro concentration, 3 μM; CVB3 3Cpro concentration, 5 μM; individual substrate screening—[S] = 5 μM; MERS‐CoV Mpro concentration, 0.3 μM; HCoV‐NL63 Mpro concentration, 1 μM; CVB3 3Cpro concentration, 5 μM; SARS‐CoV Mpro concentration, 0.2 μM; SARS‐CoV‐2 Mpro concentrations, 0.1 μM. Substrate hydrolysis was measured for 40 minutes at the appropriate wavelength (λ ex = 355 nm, λ em = 460 nm). The library screening was carried out two times. Individual substrate hydrolysis assay was performed three times, and the results were reported as averages with standard deviation.

4.4. Activity‐based probe and inhibitor synthesis

Inhibitors and activity‐based probes were synthesized using solution and solid‐phase synthesis methods as previously described (Rut et al., 2021). Compounds were purified by HPLC and analyzed using analytical HPLC and HRMS. After lyophilization, each compound was dissolved at 20 mM in DMSO and stored at −80°C until use.

4.5. IC50 determination for inhibitors and activity‐based probe

Each enzyme was preincubated in assay buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3) for 10 min at 37°C. Subsequently, the enzyme was added to wells containing different concentrations of inhibitor or activity‐based probe (ranging from 500 to 0.5 μM). Then, the enzyme was incubated with an inhibitor or activity‐based probe for 30 min at 37°C. The measurement was started immediately after adding the substrate (QS1, 50 μM) and conducted for 30 min. Each assay was repeated at least three times, and the IC50 value was calculated using the GraphPad Prism software.

4.6. Protease labeling assay

The main proteases from coronaviruses and enteroviruses (50 nM) were incubated with probes at different concentrations (50–5000 nM) in assay buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 7.3) for 30 minutes, followed by hydrolysis in 3x SDS loading buffer for 5 minutes at 95°C. The samples were loaded onto a 4%–12% Bis‐Tris Plus gel (Life Technologies), and SDS‐PAGE separation was run for 35 minutes at 200 V. Next, the samples were transferred into a 0.2 μm nitrocellulose membrane for 60 minutes at 10 V, and the membrane was blocked with 2.5% BSA in Tris‐buffered saline with 0.1% (v/v) Tween 20 (TBS‐T) for 1 hour at room temperature. Then, the enzyme was visualized at 488 nm using an Azure Biosystems SapphireTM Biomolecular Imager and Azure Spot Analysis Software. To perform a time‐point analysis, enzymes (10 and 50 nM) were incubated with Bodipy‐QS5‐VS or Cy5‐QS1‐VS (2.5 μM) for different durations of time in the range from 5 to 60 minutes. Then the samples were boiled and run on a gel. Electrophoresis, protein transfer to a nitrocellulose membrane, and probe visualization were conducted in the same manner as described above.

AUTHOR CONTRIBUTIONS

Wioletta Rut: Conceptualization; investigation; writing – original draft; methodology; validation; visualization. Katarzyna Groborz: Methodology; writing – review and editing; formal analysis; validation; investigation. Xinyuanyuan Sun: Writing – review and editing; resources. Rolf Hilgenfeld: Writing – review and editing; resources. Marcin Drag: Conceptualization; funding acquisition; validation; writing – review and editing; project administration; supervision; resources.

CONFLICT OF INTEREST STATEMENT

Wroclaw University of Science and Technology has filed a patent application covering compounds: Ac‐Abu‐Tle‐Leu‐Gln‐VS, Ac‐Abu‐DTyr‐Leu‐Gln‐VS, Biotin‐PEG(4)‐Abu‐Tle‐Leu‐Gln‐VS, and Cy5‐PEG(4)‐Abu‐Tle‐Leu‐Gln‐VS, as well as related compounds with W.R. and M.D. as inventors.

ACKNOWLEDGMENTS

This work was supported by the “TEAM/2017‐4/32” project, which is carried out within the TEAM program of the Foundation for Polish Science, cofinanced by the European Union under the European Regional Development Fund.

Rut W, Groborz K, Sun X, Hilgenfeld R, Drag M. Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad‐spectrum antiviral compounds. Protein Science. 2024;33(9):e5139. 10.1002/pro.5139

Review Editor: Aitziber L. Cortajarena

Contributor Information

Wioletta Rut, Email: wioletta.rut@pwr.edu.pl.

Marcin Drag, Email: marcin.drag@pwr.edu.pl.

REFERENCES

  1. Anand K, Palm GJ, Mesters JR, Siddell SG, Ziebuhr J, Hilgenfeld R. Structure of coronavirus main proteinase reveals combination of a chymotrypsin fold with an extra α‐helical domain. EMBO Journal. 2002;21(13):3213–3224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Anand K, Ziebuhr J, Wadhwani P, Mesters JR, Hilgenfeld R. Coronavirus main proteinase (3CLpro) structure: basis for design of anti‐SARS drugs. Science. 2003;300(5626):1763–1767. [DOI] [PubMed] [Google Scholar]
  3. Boras B, Jones RM, Anson BJ, Arenson D, Aschenbrenner L, Bakowski MA, et al. Preclinical characterization of an intravenous coronavirus 3CL protease inhibitor for the potential treatment of COVID19. Nat Commun. 2021;12(1):6055. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Brouwer L, Moreni G, Wolthers KC, Pajkrt D. World‐wide prevalence and genotype distribution of enteroviruses. Viruses. 2021;13(3):434. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Carabelli AM, Peacock TP, Thorne LG, et al. SARS‐CoV‐2 variant biology: immune escape, transmission and fitness. Nature Reviews Microbiology. 2023;21(3):162–177. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clementz MA, Chen Z, Banach BS, Wang Y, Sun L, Ratia K, et al. Deubiquitinating and interferon antagonism activities of coronavirus papain‐like proteases. Journal of Virology. 2010;84(9):4619–4629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dai W, Zhang B, Jiang XM, et al. Structure‐based design of antiviral drug candidates targeting the SARS‐CoV‐2 main protease. Science. 2020;368(6497):1331–1335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Devaraj SG, Wang N, Chen Z, Chen Z, Tseng M, Barretto N, et al. Regulation of IRF‐3‐dependent innate immunity by the papain‐like protease domain of the severe acute respiratory syndrome coronavirus. Journal of Biological Chemistry. 2007;282(44):32208–32221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Drayman N, DeMarco JK, Jones KA, Azizi SA, Froggatt HM, Tan K, et al. Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS‐CoV‐2. Science. 2021;373(6557):931–936. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. European Centre for Disease Prevention and Control. Epidemiological update: echovirus 11 infections in neonates. https://www.ecdc.europa.eu/en/news-events/epidemiological-update-echovirus-11-infections-neonates. Accessed 19 Jul 2023. [Google Scholar]
  11. Fan K, Wei P, Feng Q, Chen S, Huang C, Ma L, et al. Biosynthesis, purification, and substrate specificity of severe acute respiratory syndrome coronavirus 3C‐like proteinase. Journal of Biological Chemistry. 2004;279(3):1637–1642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Frieman M, Ratia K, Johnston RE, Mesecar AD, Baric RS. Severe acute respiratory syndrome coronavirus papain‐like protease ubiquitin‐like domain and catalytic domain regulate antagonism of IRF3 and NF‐kappaB signaling. Journal of Virology. 2009;83(13):6689–6705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fung TS, Liu DX. Similarities and dissimilarities of COVID‐19 and other coronavirus diseases. Annual Review of Microbiology. 2021;75:19–47. [DOI] [PubMed] [Google Scholar]
  14. Göhl M, Zhang L, el Kilani H, Sun X, Zhang K, Brönstrup M, et al. From repurposing to redesign: optimization of boceprevir to highly potent inhibitors of the SARS‐CoV‐2 Main protease. Molecules. 2022;27(13):4292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hamre D, Procknow JJ. A new virus isolated from the human respiratory tract. Proceedings of the Society for Experimental Biology and Medicine. 1966;121(1):190–193. [DOI] [PubMed] [Google Scholar]
  16. Hegyi A, Ziebuhr J. Conservation of substrate specificities among coronavirus main proteases. Journal of General Virology. 2002;83:595–599. [DOI] [PubMed] [Google Scholar]
  17. Hilgenfeld R. From SARS to MERS: crystallographic studies on coronaviral proteases enable antiviral drug design. FEBS Journal. 2014;281(18):4085–4096. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jin Z, du X, Xu Y, Deng Y, Liu M, Zhao Y, et al. Structure of M(pro) from SARS‐CoV‐2 and discovery of its inhibitors. Nature. 2020;582(7811):289–293. [DOI] [PubMed] [Google Scholar]
  19. Kawashima S, Matsui Y, Adachi T, Morikawa Y, Inoue K, Takebayashi S, et al. Ensitrelvir is effective against SARS‐CoV‐2 3CL protease mutants circulating globally. Biochemical and Biophysical Research Communications. 2023;645:132–136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kesheh MM, Hosseini P, Soltani S, Zandi M. An overview on the seven pathogenic human coronaviruses. Review in Medical Virology. 2022;32(2):e2282. [DOI] [PubMed] [Google Scholar]
  21. Kitamura N, Sacco MD, Ma C, Hu Y, Townsend JA, Meng X, et al. Expedited approach toward the rational Design of Noncovalent SARS‐CoV‐2 Main protease inhibitors. Journal of Medicinal Chemistry. 2022;65(4):2848–2865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Lei J, Hilgenfeld R. RNA‐virus proteases counteracting host innate immunity. FEBS Letters. 2017;591(20):3190–3210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li G, Hilgenfeld R, Whitley R, de Clercq E. Therapeutic strategies for COVID‐19: progress and lessons learned. Nature Reviews Drug Discovery. 2023;22(6):449–475. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li J, Lin C, Zhou X, Zhong F, Zeng P, Yang Y, et al. Structural basis of the Main proteases of coronavirus bound to drug candidate PF‐07321332. J Virol. 2022;96(8):e0201321. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lin C, Jiang H, Li W, Zeng P, Zhou X, Zhang J, et al. Structural basis for the inhibition of coronaviral main proteases by ensitrelvir. Structure. 2023;31(9):1016–1024 e3. [DOI] [PubMed] [Google Scholar]
  26. Ma C, Sacco MD, Hurst B, Townsend JA, Hu Y, Szeto T, et al. Boceprevir, GC‐376, and calpain inhibitors II, XII inhibit SARS‐CoV‐2 viral replication by targeting the viral main protease. Cell Research. 2020;30(8):678–692. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. McCarthy MW. Ensitrelvir as a potential treatment for COVID‐19. Expert Opinion on Pharmacotherapy. 2022;23(18):1995–1998. [DOI] [PubMed] [Google Scholar]
  28. Mukherjee A, Morosky SA, Delorme‐Axford E, Dybdahl‐Sissoko N, Oberste MS, Wang T, et al. The coxsackievirus B 3C protease cleaves MAVS and TRIF to attenuate host type I interferon and apoptotic signaling. PLOS Pathogens. 2011;7(3):e1001311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Owen DR, Allerton CMN, Anderson AS, Aschenbrenner L, Avery M, Berritt S, et al. An oral SARS‐CoV‐2 M(pro) inhibitor clinical candidate for the treatment of COVID‐19. Science. 2021;374(6575):1586–1593. [DOI] [PubMed] [Google Scholar]
  30. Park SW, Pons‐Salort M, Messacar K, Cook C, Meyers L, Farrar J, et al. Epidemiological dynamics of enterovirus D68 in the United States and implications for acute flaccid myelitis. Science Translational Medicine. 2021;13(584). [DOI] [PubMed] [Google Scholar]
  31. Pons‐Salort M, Oberste MS, Pallansch MA, Abedi GR, Takahashi S, Grenfell BT, et al. The seasonality of nonpolio enteroviruses in the United States: patterns and drivers. Proceedings of the National Academy of Sciences of the U SA. 2018;115(12):3078–3083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rut W, Groborz K, Zhang L, Sun X, Zmudzinski M, Pawlik B, et al. SARS‐CoV‐2 M(pro) inhibitors and activity‐based probes for patient‐sample imaging. Nature Chemical Biology. 2021;17(2):222–228. [DOI] [PubMed] [Google Scholar]
  33. Steuber H, Hilgenfeld R. Recent advances in targeting viral proteases for the discovery of novel antivirals. Current Topics in Medicinal Chemistry. 2010;10(3):323–345. [DOI] [PubMed] [Google Scholar]
  34. Unoh Y, Uehara S, Nakahara K, Nobori H, Yamatsu Y, Yamamoto S, et al. Discovery of S‐217622, a noncovalent Oral SARS‐CoV‐2 3CL protease inhibitor clinical candidate for treating COVID‐19. Journal of Medicinal Chemistry. 2022;65(9):6499–6512. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. van der Hoek L, Pyrc K, Jebbink MF, Vermeulen‐Oost W, Berkhout RJM, Wolthers KC, et al. Identification of a new human coronavirus. Nature Medicine. 2004;10(4):368–373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Wang F, Chen C, Tan W, Yang K, Yang H. Structure of Main protease from human coronavirus NL63: insights for wide Spectrum anti‐coronavirus drug design. Scientific Reports. 2016;6:22677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Woo PC, Lau SK, Chu CM, et al. Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia. Journal of Virology. 2005;79(2):884–895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zephyr J, Kurt Yilmaz N, Schiffer CA. Viral proteases: structure, mechanism and inhibition. Enzyme. 2021;50:301–333. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Zhang L, Lin D, Sun X, et al. Crystal structure of SARS‐CoV‐2 main protease provides a basis for design of improved α‐ketoamide inhibitors. Science. 2020a;368(6489):409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhang L, Lin D, Kusov Y, et al. α‐Ketoamides as broad‐Spectrum inhibitors of coronavirus and enterovirus replication: structure‐based design, synthesis, and activity assessment. Journal of Medicinal Chemistry. 2020b;63(9):4562–4578. [DOI] [PubMed] [Google Scholar]
  41. Zhao Y, Fang C, Zhang Q, Zhang R, Zhao X, Duan Y, et al. Crystal structure of SARS‐CoV‐2 main protease in complex with protease inhibitor PF‐07321332. Protein Cell. 2022;13(9):689–693. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES