The Structure of the Porcine Deltacoronavirus Main Protease Reveals a Conserved Target for the Design of Antivirals

The existing zoonotic coronaviruses (CoVs) and viral genetic variants are important microbiological pathogens that cause severe disease in humans and animals. Currently, no effective broad-spectrum antiviral drugs against existing and emerging CoVs are available. The CoV main protease (Mpro) plays an essential role in viral replication, making it an ideal target for drug development. However, the structure of the Deltacoronavirus Mpro is still unavailable. Porcine deltacoronavirus (PDCoV) is a novel CoV that belongs to the genus Deltacoronavirus and causes atrophic enteritis, severe diarrhea, vomiting and dehydration in pigs. Here, we determined the structure of PDCoV Mpro complexed with a Michael acceptor inhibitor. Structural comparison showed that the backbone of PDCoV Mpro is similar to those of alpha-, beta- and gamma-CoV Mpros. The substrate-binding pocket of Mpro is well conserved in the subfamily Coronavirinae. In addition, we also observed that Mpros from the same genus adopted a similar conformation. Furthermore, the structure of PDCoV Mpro in complex with a Michael acceptor inhibitor revealed the mechanism of its inhibition of PDCoV Mpro. Our results provide a basis for the development of broad-spectrum antivirals against PDCoV and other CoVs.


Introduction
Coronaviruses (CoVs) are round or oval enveloped viruses with a positive-sense RNA genome [1]. CoVs are among the most dangerous microbiological pathogens that infect mammals, such as humans, mice, cats, and pigs, as well as birds, such as sparrows, and they are responsible for a large number of gastric, enteric and respiratory syndromes [2][3][4][5][6]. In 2003, an outbreak of severe acute respiratory syndrome (SARS) led to an international epidemic, and severe acute respiratory syndrome coronavirus (SARS-CoV) was demonstrated to be the etiological agent [7][8][9][10]. In 2012, a novel CoV, Middle East respiratory syndrome coronavirus (MERS-CoV), was reported in Saudi Arabia [11]. MERS-CoV infection can cause patients to develop acute renal failure. In late December 2019, a novel coronavirus (SARS-CoV-2) was identified in Wuhan, Hubei Province. This infectious pneumonia has spread worldwide, and as of January 2022, more than 318 million people have been infected, and 5.5 million have died from the disease [12]. The ceaseless emergence of new pathogenic CoVs indicates that CoVs remain an enormous threat to public health security. However, at peptidomimetic inhibitors carrying a Michael acceptor warhead are effective against the M pro s of all CoVs.

Gene Expression and Protein Purification
The PDCoV M pro coding sequence was cloned into the BamHI and XhoI restriction sites of the pET-28b_SUMO vector and then transformed into Escherichia coli strain BL21 (DE3). The fusion protein SUMO-PDCoV M pro was purified by Ni-affinity chromatography (GE Healthcare, Uppsala, Sweden) and then cleaved with ULP protease. M pro was further purified using anion exchange chromatography (HiTrap Q, GE Healthcare, Uppsala, Sweden) with a linear gradient from 2.5 to 500 mM NaCl (20 mM Tris-HCl pH 8.0) and size exclusion chromatography (Superdex 75 10/300 GL, GE Healthcare, Uppsala, Sweden) in 10 mM HEPES pH 7.5 and 150 mM NaCl.

Crystallization, Data Collection, Structure Determination and Refinement
Crystals of the complex were obtained by cocrystallization following the incubation of 1 mg mL −1 PDCoV M pro and 10 mM N3 in the buffer of 10 mM HEPES pH 7.5 and 150 mM NaCl at 4 • C at a molar ratio of 1:5 for 12 h. The complex was concentrated to 9 mg mL −1 and then crystallized by the microbatch-under-oil method at 291 K. The successful crystal growth conditions were 0.1 M sodium citrate (pH 5.1) and 4% (w/v) polyethylene glycol 6000. Crystals were cryoprotected with 20% glycerol, 0.1 M sodium citrate (pH 5.1) and 4% (w/v) polyethylene glycol 6000 and flash-frozen in liquid nitrogen. Data were collected at the Shanghai Synchrotron Radiation Facility (SSRF) beamline BL19U1 at 100 K using an ADSC Q315r detector with a wavelength of 0.97923 Å. The crystal belonged to space group P6 1 with unit cell dimensions a = b = 122.3 Å and c = 289.8 Å. Diffraction data were processed with HKL3000 (version 721.3, HKL Research, Inc., Charlottesville, VA, USA) (44). The complex structure was solved by molecular replacement using the structure of PEDV M pro (PDB ID 5GWZ) [27] as a search model through the PHASER [39] program from the CCP4 package [40]. Model building and refinement were performed using PHENIX (version 1.14) [41] and COOT (version 0.8.9) [42]. The R work and R free of the final model were 19.21% and 24.14%, respectively.

Enzyme Activity and Inhibition Assays
Enzymatic assays were carried out as previously reported [15,28,29]. A fluorogenic substrate of PDCoV M pro , MCA-AVLQ↓SGFR-Lys(Dnp)-Lys-NH 2 (>95% purity, GL Biochem Shanghai Ltd., Shanghai, China), was used to assess enzyme activity by measuring fluorescence intensity with excitation and emission wavelengths of 320 nm and 405 nm, respectively. The assay was performed at 30 • C, and the buffer used consisted of 50 mM Tris-HCl (pH 7.3) and 1 mM EDTA. The K m and k cat of PDCoV M pro and K i and k 3 of N3 were determined according to the methods used in our previous work [15,29]. The values of K i and k 3 were obtained following the addition of PDCoV M pro . The enzyme and substrate concentration were set at 2 µM and 50 µM, respectively. The inhibitor concentration varied among seven different concentrations (6-24 µM). Data were analyzed with the program GraphPad Prism (version 5.0, GraphPad, San Diego, CA, USA). The enzymatic assay used to test M14 and M25 was similar to that used to test N3.

Overall Structure
We cocrystallized PDCoV M pro with a Michael acceptor inhibitor, named N3, and determined the structure of the complex at 2.60 Å resolution ( Table 1). The crystal structure contained six M pro molecules per asymmetric unit. In the crystals, two neighboring molecules, protomer A and protomer B, formed a typical homodimer. Each protomer contains three domains: domain I (residues 1-97), domain II (residues 98-186) and domain III (residues 200-304). Domain I and II each have a chymotrypsin-like fold, and domain III is composed of five α-helixes and contributes to the formation of a homodimer ( Figure 1A). The substrate-binding pocket, which contains a catalytic dyad (His-41 and Cys-144), is located in the cleft between domains I and II ( Figure 1A). The superimposition of M pro s [15,[21][22][23][24][25][26][27][28][29] from four different CoV genera shows that the PDCoV M pro shares a similar overall structure and backbone with other CoV M pro s ( Figure 1B). Domain I (residues 1-97) and domain II (residues 98-186) of M pro from PDCoV are well conserved, with Cα root-mean-square deviations (RMSDs) of 1.4-1.7 Å, 1.3-1.4 Å and 1.1 Å in comparison with those of alpha-, beta-, and gamma-CoV, respectively. Structural overlay of the M pro s from four CoV genera shows that domain III (residues 200-304) of PDCoV has a similar orientation to that of the other CoV M pro s. The Cα RMSDs between different CoVs and PDCoV are summarized in Table 2.     To provide more insight into the properties of PDCoV M pro , we analyzed the substratebinding pocket between domain I and domain II.
The S1 binding pocket of PDCoV M pro is composed of residues Phe-139, His-162, Glu-165, and His-171 and the backbones of the other amino acids, such as Leu-140, Asn-141, His-163 and Ile-164. Sequence analysis showed that the M pro cleavage sites at the P1 position in the identified PDCoV were all glutamines, indicating that the S1 substratebinding pocket of PDCoV M pro has an extremely strong preference for glutamine residues. In our previously reported structure of the complex between a SARS-CoV M pro H41A mutant and an 11-peptidyl substrate, the Nε2 atom of His-163 and the main chain carbonyl oxygen of Phe-140 in the S1 binding pocket form three hydrogen bonds with glutamine at the P1 position [28]. Structural superposition of the S1 binding pocket in M pro s from the CoVs of four genera showed that in PDCoV M pro , the carbonyl oxygen atom of Phe-139, the imidazole ring NH of His-162 and the carbonyl oxygen atoms of residues at position 163 in PDCoV M pro , PEDV M pro , SARS-CoV-2 M pro , SARS-CoV M pro and IBV M pro are extremely conserved. In addition, we found that the key residues that form the S1 binding pocket, His-171 and Glu-165, also share a similar structure ( Figure 2A). For the above reasons, we concluded that the Deltacoronavirus PDCoV M pro shares a conserved S1 binding pocket with M pro s from the other three genera. The evolutionary conservation of amino acids plays a crucial role in drug design.
In  [28]. The backbone atoms of the residues that form the S1 pocket of PDCoV M pro are similar to the corresponding sequences in the other three genera. Sequence alignment showed that the residues at position 25 of M pro from PDCoV, PEDV, SARS-CoV-2, SARS-CoV and IBV are different from the consensus of the CoV M pro s from the four genera; these residues are Thr, Met, Asn and Ser, respectively ( Figure 2B,D). interact with the P1′ residue of the substrate via van der Waals interactions [28]. The backbone atoms of the residues that form the S1′ pocket of PDCoV M pro are similar to the corresponding sequences in the other three genera. Sequence alignment showed that the residues at position 25 of M pro from PDCoV, PEDV, SARS-CoV-2, SARS-CoV and IBV are different from the consensus of the CoV M pro s from the four genera; these residues are Thr, Met, Asn and Ser, respectively ( Figure 2B,D).

The Peptidomimetic Inhibitor N3 Efficiently Inhibits PDCoV M pro
We determined the K m and k cat of PDCoV M pro to be 56.6 ± 1.9 µM and 0.030 ± 0.009 s −1 , respectively (Table 3). This K m value is close to that of HCoV-NL63 M pro (50.8 ± 3.4 µM) and TGEV M pro (61 ± 5 µM), lower than that of mouse hepatitis virus A59 (MHV-A59) M pro (77 ± 5 µM), HCoV-HKU1 M pro (83.2 ± 13.3 µM), SARS-CoV M pro (129 ± 7 µM) and IBV M pro (139 ± 15 µM), and higher than that of HCoV-229E M pro (29.8 ± 0.9 µM) and FIPV M pro (13.5 ± 1.8 µM) [15,26,29] (Table 3). Structural analysis showed that the substrate-binding pocket of PDCoV M pro shares several features in common with M pro s from CoVs in the other three genera, especially the S1, S2 and S4 subsites. The key residues at these sites are almost completely conserved ( Figure 2). The N3 is a peptidomimetic inhibitor designed against various M pro s, such as those from SARS-CoV, HCoV-229E and FIPV [15]. Therefore, we deduced that the Michael acceptor and peptidomimetic inhibitor N3 may inhibit PDCoV M pro . An enzymatic assay showed that N3 inactivated PDCoV M pro . The calculated K i and k 3 were 11.98 ± 0.13 µM and 72.91 ± 7.05 (10 −3 s −1 ), respectively. The k 3 is approximately 23-fold larger than that of SARS-CoV M pro , which indicates that N3 inactivates PDCoV M pro faster than it does SARS-CoV.

Binding of N3 to PDCoV M pro
In the crystal structures, N3 is bound to each protomer of the M pro dimer. We thus only discuss the binding mode in one of the protomers. The inhibitor is located in the substrate-binding pocket, which adopts the canonical conformation seen in other M pro -N3 complex structures. As an irreversible inhibitor, the Cβ atom of the vinyl group on N3 is bound to the Sγ atom of Cys-144 through a 1.8 Å covalent bond ( Figure 3A,B). The lactam ring of the glutamine analog N3 at the P1 site inserts into the S1 pocket and forms 3.2 Å, 2.5 Å and 2.9 Å hydrogen bonds with the carbonyl oxygen of Phe-139, the imidazole ring NH of His-162 and the Oε1 atom of Glu-165, respectively. The side chain of Leu at the P2 position extends into the S2 pocket and participates in hydrophobic interactions with the hydrophobic amino acids Trp, Ile and Phe. The valine side chain of N3 at the P3 position is exposed to solvent. The alanine residue in the P4 position inserts into a pocket composed of the residues Pro-183 and Tyr-184, leading to a hydrophobic interaction among these residues. The isoxazole at the P5 position, Gln-167 and Ile-190 form a "sandwich structure" by van der Waals interaction. The P2 and P4 sites insert into the S2 subsite and S4 subsite well. Moreover, the backbone NH of Cys-144, the carbonyl oxygen atoms of His-163 and Glu-165, the Oε1 atom of Glu-188 and the NH group of Glu-165 form hydrogen bonds with the inhibitor N3, which ensures tight binding between the M pro and the inhibitor, as shown in Figure 3. We concluded that peptidomimetic inhibitors carrying the Michael acceptor warhead N3 are effective against the M pro of PDCoV. "sandwich structure" by van der Waals interaction. The P2 and P4 sites insert into the S2 subsite and S4 subsite well. Moreover, the backbone NH of Cys-144, the carbonyl oxygen atoms of His-163 and Glu-165, the Oε1 atom of Glu-188 and the NH group of Glu-165 form hydrogen bonds with the inhibitor N3, which ensures tight binding between the M pro and the inhibitor, as shown in Figure 3. We concluded that peptidomimetic inhibitors carrying the Michael acceptor warhead N3 are effective against the M pro of PDCoV.

The P1′ Position May Play an Important Role in the Interaction between PDCoV M pro and Inhibitors
Previously, we have designed 16 N3 derivatives that target PEDV M pro (the detailed structures and their chemical synthesis were described in our previous paper) [27]. Next, we evaluated the inhibitory activity of these compounds against PDCoV M pro . Among these compounds, M14 and M25 exhibited stronger inhibition than N3 ( Table 4). The k3/Ki values of M14 and M25 were 13.8 and 9.9, respectively, indicating that they have much better inhibitory activity than N3, which had a k3/Ki of 6.1. The detailed inhibition parameters of N3, M14 and M25 are listed in Table 5. Interestingly, we found that the three compounds shared the same side groups at all positions except the P1′ position ( Table 5). The benzyl group at the P1′ position of N3 interacts with Ser-25 and Leu-27 through van der Waals forces. Therefore, we suggest that rational design of the P1′ position could dramatically enhance the interaction between the substrate-binding pocket and the inhibitor. Future modification of peptidomimetic inhibitors at the P1′ position has the potential to control acute gastroenteritis in pigs infected with PDCoV. Table 4. Evaluation of the inhibitory activity of compounds targeting PDCoV M pro . The inhibition ratio (Ir) is defined as the percent inactivation of the initial enzymatic activity of PDCoV M pro .

The P1 Position May Play an Important Role in the Interaction between PDCoV M pro and Inhibitors
Previously, we have designed 16 N3 derivatives that target PEDV M pro (the detailed structures and their chemical synthesis were described in our previous paper) [27]. Next, we evaluated the inhibitory activity of these compounds against PDCoV M pro . Among these compounds, M14 and M25 exhibited stronger inhibition than N3 ( Table 4). The k 3 /K i values of M14 and M25 were 13.8 and 9.9, respectively, indicating that they have much better inhibitory activity than N3, which had a k 3 /K i of 6.1. The detailed inhibition parameters of N3, M14 and M25 are listed in Table 5. Interestingly, we found that the three compounds shared the same side groups at all positions except the P1 position ( Table 5). The benzyl group at the P1 position of N3 interacts with Ser-25 and Leu-27 through van der Waals forces. Therefore, we suggest that rational design of the P1 position could dramatically enhance the interaction between the substrate-binding pocket and the inhibitor. Future modification of peptidomimetic inhibitors at the P1 position has the potential to control acute gastroenteritis in pigs infected with PDCoV.

Discussion
The M pro is an ideal target for drug design against CoVs. Since IBV, the first CoV to be described was discovered in 1937, four genera of CoVs have been identified. Currently, we have a thorough understanding of the M pro structures of alpha-, beta-and gamma-CoVs; however, we know little about the Deltacoronavirus M pro . In this paper, we present the first structure of the M pro of a newly emerged Deltacoronavirus (PDCoV) in complex with a Michael acceptor inhibitor.
As observed in the previously reported M pro structures, PDCoV M pro presented a functional homodimer and conserved His-Cys dyads. Furthermore, a detailed comparison of the M pro structures showed that PDCoV M pro shares a similar overall structure and a relatively conserved substrate-binding pocket with the M pro s of the other three CoV genera, especially the key residues located at the S1, S4, and S2 subsites ( Figure  4). Meanwhile, the irreversible inhibitor N3 in our structure, designed based on the structure of SARS M pro in complex with its substrate, could inactivate PDCoV and multiple CoV M pro s. These results also proved the conservation of the overall structures and substrate binding pockets of M pro s. As demonstrated, emerging zoonotic viruses such 11

Discussion
The M pro is an ideal target for drug design against CoVs. Since IBV, the first CoV to be described was discovered in 1937, four genera of CoVs have been identified. Currently, we have a thorough understanding of the M pro structures of alpha-, beta-and gamma-CoVs; however, we know little about the Deltacoronavirus M pro . In this paper, we present the first structure of the M pro of a newly emerged Deltacoronavirus (PDCoV) in complex with a Michael acceptor inhibitor.
As observed in the previously reported M pro structures, PDCoV M pro presented a functional homodimer and conserved His-Cys dyads. Furthermore, a detailed comparison of the M pro structures showed that PDCoV M pro shares a similar overall structure and a relatively conserved substrate-binding pocket with the M pro s of the other three CoV genera, especially the key residues located at the S1, S4, and S2 subsites ( Figure  4). Meanwhile, the irreversible inhibitor N3 in our structure, designed based on the structure of SARS M pro in complex with its substrate, could inactivate PDCoV and multiple CoV M pro s. These results also proved the conservation of the overall structures and substrate binding pockets of M pro s. As demonstrated, emerging zoonotic viruses such 74% + + + + a Percentage inhibitory activity: + + + + +, >80%; + + + +, 70%−80%; + + +, 50%−70%; + +, 30%−50%; +, <30%. b No inhibition was observed. c The detailed structures and chemical synthesis of the compounds were described in reference [27].

Discussion
The M pro is an ideal target for drug design against CoVs. Since IBV, the first CoV to be described was discovered in 1937, four genera of CoVs have been identified. Currently, we have a thorough understanding of the M pro structures of alpha-, beta-and gamma-CoVs; however, we know little about the Deltacoronavirus M pro . In this paper, we present the first structure of the M pro of a newly emerged Deltacoronavirus (PDCoV) in complex with a Michael acceptor inhibitor.
As observed in the previously reported M pro structures, PDCoV M pro presented a functional homodimer and conserved His-Cys dyads. Furthermore, a detailed comparison of the M pro structures showed that PDCoV M pro shares a similar overall structure and a relatively conserved substrate-binding pocket with the M pro s of the other three CoV genera, especially the key residues located at the S1, S4, and S2 subsites ( Figure  4). Meanwhile, the irreversible inhibitor N3 in our structure, designed based on the structure of SARS M pro in complex with its substrate, could inactivate PDCoV and multiple CoV M pro s. These results also proved the conservation of the overall structures and substrate binding pockets of M pro s. As demonstrated, emerging zoonotic viruses such 8.80 ± 0.15 86.8 ± 5.1 9.9

Discussion
The M pro is an ideal target for drug design against CoVs. Since IBV, the first CoV to be described was discovered in 1937, four genera of CoVs have been identified. Currently, we have a thorough understanding of the M pro structures of alpha-, beta-and gamma-CoVs; however, we know little about the Deltacoronavirus M pro . In this paper, we present the first structure of the M pro of a newly emerged Deltacoronavirus (PDCoV) in complex with a Michael acceptor inhibitor.
As observed in the previously reported M pro structures, PDCoV M pro presented a functional homodimer and conserved His-Cys dyads. Furthermore, a detailed comparison of the M pro structures showed that PDCoV M pro shares a similar overall structure and a relatively conserved substrate-binding pocket with the M pro s of the other three CoV genera, especially the key residues located at the S1, S4, and S2 subsites ( Figure 4). Meanwhile, the irreversible inhibitor N3 in our structure, designed based on the structure of SARS M pro in complex with its substrate, could inactivate PDCoV and multiple CoV M pro s. These results also proved the conservation of the overall structures and substrate binding pockets of M pro s. As demonstrated, emerging zoonotic viruses such as SARS-CoV-2, SARS-CoV and MERS-CoV are a potential threat to public health because of the existing viral variants. As an important pathogen of piglets, the nonhuman animal virus PDCoV poses the risk of cross-species transmission to humans as well [30,31]. Therefore, the conserved CoV M pro we identified could be considered a drug target in the event of genetic changes during human-to-human or animal-to-human transmission of CoVs.
as SARS-CoV-2, SARS-CoV and MERS-CoV are a potential threat to public health because of the existing viral variants. As an important pathogen of piglets, the nonhuman animal virus PDCoV poses the risk of cross-species transmission to humans as well [30,31]. Therefore, the conserved CoV M pro we identified could be considered a drug target in the event of genetic changes during human-to-human or animal-to-human transmission of CoVs.  Figure 2B. The background is PDCoV M pro . Red: identical residues among all ten CoV M pro s; orange: substituted in two CoV M pro s. The S1, S2, S4, and S1′ pockets and the residues that form the substrate-binding pocket are labeled. N3 is shown in green.
Interestingly, the structure and conformation of M pro s presented a stable characteristic evolution and obvious species correlation. We superposed the determined structures of M pro s from CoVs in four different genera and found some loops, especially for the region from 41-51, that exhibited corresponding features ( Figure 5). For example, this loop in Alphacoronavirus and Betacoronavirus forms a 310 helix, while in Gammacoronavirus IBV, it forms a short loop. Surprisingly, the loop from residues 41-51 of PDCoV M pro adopts a conformation similar to that of IBV M pro , which supports that the Deltacoronavirus may be closely related to Gammacoronavirus [6]. Since the loop from 41-51 is associated with the outer wall of the S2 pocket, our structural information will support reasonable broad-spectrum peptidomimetic drug design based on the evolutionary conservation of M pro s from CoVs.  Figure 2B. The background is PDCoV M pro . Red: identical residues among all ten CoV M pro s; orange: substituted in two CoV M pro s. The S1, S2, S4, and S1 pockets and the residues that form the substrate-binding pocket are labeled. N3 is shown in green.
Interestingly, the structure and conformation of M pro s presented a stable characteristic evolution and obvious species correlation. We superposed the determined structures of M pro s from CoVs in four different genera and found some loops, especially for the region from 41-51, that exhibited corresponding features ( Figure 5). For example, this loop in Alphacoronavirus and Betacoronavirus forms a 3 10 helix, while in Gammacoronavirus IBV, it forms a short loop. Surprisingly, the loop from residues 41-51 of PDCoV M pro adopts a conformation similar to that of IBV M pro , which supports that the Deltacoronavirus may be closely related to Gammacoronavirus [6]. Since the loop from 41-51 is associated with the outer wall of the S2 pocket, our structural information will support reasonable broadspectrum peptidomimetic drug design based on the evolutionary conservation of M pro s from CoVs.
The peptidomimetic inhibitor N3, which carries a Michael acceptor warhead, was also effective against M pro of PDCoV, the newly emerging Deltacoronavirus. Peptidomimetic compounds are attractive inhibitors for the development of novel antiviral therapies. These compounds target proteases that are essential for viral replication. For example, boceprevir, telaprevir and simeprevir are peptidomimetic drugs that act as viral NS3/4A serine protease inhibitors of hepatitis C virus (HCV) [43][44][45], while saquinavir, indinavir, nelfinavir, ritonavir, and amprenavir are clinically approved human immunodeficiency virus (HIV) protease inhibitors, which have a similar molecular structure to the protease substrate [46,47]. Furthermore, these peptidomimetic drugs were derived from lead compounds identified based on viral protease structures. For instance, boceprevir, which is a tripeptide derivative that forms a covalent bond with Ser-139 to inactivate the NS3/4A protease [45], was designed based on an undecapeptide alpha-ketoamide inhibitor identified from compound libraries. Hence, after multiple rounds of modification, the inhibitor N3 is a currently available compound for broad-spectrum drug design. It is worth noting that P1 may be a key position of compound modification for broad-spectrum drug design because of the side chains variability in the amino acid at position 25, which directly participates in the interaction with the inhibitor. In our study, both N3 derivatives (M14 and M25) with improved inhibitory activity against PDCoV M pro presented a unique group at P1 . Therefore, it is necessary to balance the relatively conserved substrate binding pockets during rational drug design. Furthermore, we found that M25 exhibited potent inhibition of both PDCoV and PEDV M pro proteins [27]. The two main emerging swine CoVs, PDCoV and PEDV, account for the majority of lethal watery diarrhea in neonatal pigs in the past decade. More recently, the epidemiological evidence shows that the rate of PDCoV coinfection with PEDV has increased up to 51% in China [32,48]. Therefore, M25 could be further developed to combat both PDCoV and PEDV infection in the swine industry. The peptidomimetic inhibitor N3, which carries a Michael acceptor warhead, was also effective against M pro of PDCoV, the newly emerging Deltacoronavirus. Peptidomimetic compounds are attractive inhibitors for the development of novel antiviral therapies. These compounds target proteases that are essential for viral replication. For example, boceprevir, telaprevir and simeprevir are peptidomimetic drugs that act as viral NS3/4A serine protease inhibitors of hepatitis C virus (HCV) [43][44][45], while saquinavir, indinavir, nelfinavir, ritonavir, and amprenavir are clinically approved human immunodeficiency virus (HIV) protease inhibitors, which have a similar molecular structure to the protease substrate [46,47]. Furthermore, these peptidomimetic drugs were derived from lead compounds identified based on viral protease structures. For instance, boceprevir, which is a tripeptide derivative that forms a covalent bond with Ser-139 to inactivate the NS3/4A protease [45], was designed based on an undecapeptide alpha-ketoamide inhibitor identified from compound libraries. Hence, after multiple rounds of modification, the inhibitor N3 is a currently available compound for broad-spectrum drug design. It is worth noting that P1′ may be a key position of compound modification for broad-spectrum drug design because of the side chains variability in the amino acid at position 25, which directly participates in the interaction with the inhibitor. In our study, both N3 derivatives (M14 and M25) with improved inhibitory activity against PDCoV M pro presented a unique group at P1′. Therefore, it is necessary to balance the relatively conserved substrate binding pockets during rational drug design. Furthermore, we found that M25 exhibited potent inhibition of both PDCoV and PEDV M pro proteins [27]. The two main emerging swine CoVs, PDCoV and PEDV, account for the majority of lethal watery diarrhea in neonatal pigs in In summary, the structure of PDCoV M pro in complex with the Michael acceptor inhibitor N3 provides a basis for the inactivation of Deltacoronavirus viral proteases. The structural comparison of different viral enzymes identified a conserved substrate-binding pocket in all CoV M pro s; this pocket is a target for the development of broad-spectrum antivirals against all existing and emerging CoVs.  Data Availability Statement: Atomic coordinates for the crystal structure of PDCoV M pro in complex with N3 can be accessed using PDB code 7WKU in the RCSB Protein Data Bank (https://doi.org/10 .2210/pdb7WKU/pdb. accessed on 25 January 2022). Authors will release the atomic coordinates and experimental data upon article publication.