Structural basis of main proteases of HCoV-229E bound to inhibitor PF-07304814 and PF-07321332

Abstract

PF-07321332 and PF-07304814, inhibitors against SARS-CoV-2 developed by Pfizer, exhibit broad-spectrum inhibitory activity against the main protease (M^pro) from various coronaviruses. Structures of PF-07321332 or PF-07304814 in complex with M^pros of various coronaviruses reveal their inhibitory mechanisms against different M^pros. However, the structural information on the lower pathogenic coronavirus M^pro with PF-07321332 or PF-07304814 is currently scarce, which hinders our comprehensive understanding of the inhibitory mechanisms of these two inhibitors. Meanwhile, given that some immunocompromised individuals are still affected by low pathogenic coronaviruses, we determined the structures of lower pathogenic coronavirus HCoV-229E M^pro with PF-07321332 and PF-07304814, respectively, and analyzed and defined in detail the structural basis for the inhibition of HCoV-229E M^pro by both inhibitors. Further, we compared the crystal structures of multiple coronavirus M^pro complexes with PF-07321332 or PF-07304814 to illustrate the differences in the interaction of M^pros, and found that the inhibition mechanism of lower pathogenic coronavirus M^pro was more similar to that of moderately pathogenic coronaviruses. Our structural studies provide new insights into drug development for low pathogenic coronavirus M^pro, and provide theoretical basis for further optimization of both inhibitors to contain potential future coronaviruses.

1. Introduction

Although the pandemics caused by viruses of coronaviruses are in the process of receding, given the history of coronaviruses, in the future we are probably in danger of new diseases caused by coronaviruses [1,2]. Consequently, knowledge of the mechanisms involved in treating coronaviruses would contribute to our preparedness for possible future homologous diseases. Current studies have shown that the coronaviridae family capable of infecting humans is divided into two main genera, including alpha coronavirus (alphaCoV) and beta coronavirus (betaCoV) [3]. HCoV-229E and HCoV-NL63 were classified as alphaCoV, HCoV-OC43, while eHCoV-HKU1, SARS-CoV, SARS-CoV-2 and MERS-CoV were classified as betaCoV [3]. In terms of pathogenicity, HCoV-229E and HCoV-NL63 belong to the lower pathogenicity strains, HCoV-OC43 and HCoV-HKU1 to the moderate pathogenicity strains (A lineage), and SARS-CoV, SARS-CoV-2 (B lineage) and MERS-CoV (C lineage) to the high pathogenicity strains [[4], [5], [6], [7], [8]]. Highly pathogenic strains are associated with acute respiratory distress syndrome and pose a tremendous threat to human health [9]. Despite the low pathogenicity of the lower and moderately pathogenic strains, they continue to be prevalent in human populations and maintain a greater incidence of lower respiratory tract infections in immunocompromised individuals [8,10].

The main protease of coronaviruses (M^pro or 3CL^pro) is highly conserved in different genera, integral to the virus life cycle, and is considered an ideal target for the development of broad-spectrum targeted drugs [11,12]. In addition, no homologues of coronavirus M^pro have been found in humans, thus targeting M^pro as an inhibitor would have fewer side effects in humans, which also makes M^pro an attractive target for inhibitor development [13]. Inhibitor development for coronaviruses tends to be achieved through high-throughput screening, computer-based assisted drug design, structure-based drug design and repurposing of drugs [[14], [15], [16], [17], [18], [19]]. A variety of inhibitors have been designed or developed to act against coronaviruses, such as YH-53, GC376, CMK, boceprevir, Spirocyclic Thiopyrimidinones [14,17,[20], [21], [22]]. Despite the large number of inhibitors designed and developed, few have actually been used for clinical treatment of coronaviruses due to unmet potency, toxicity or metabolic properties. Pfizer has developed PF-07321332 and PF-07304814, which are effective inhibitors of SARS-CoV-2. PF-07321332 and ritonavir (together named PAXLOVIDTM) are currently approved for the early treatment of patients with mild to moderate COVID-19 [23]. PF-07304814 is also highly promising for drug development as an inhibitor of anti-SARS-CoV-2, and its safety and efficacy are still being studied in clinical trials.

PF-07321332 is an orally-administered inhibitor of anti-SARS-CoV-2, and co-administration with ritonavir could slow down the metabolic degradation of PF-07321332 in vivo and exhibits favorable antiviral activity, safety, and pharmacokinetic properties [24]. In accordance with the Berger and Schechter nomenclature [25], PF-07321332 comprises five primary components, namely P1’ and P1 to P4 (Fig. 1 A). A nitrile warhead is located at position P1’, a γ-lactam ring at position P1, a dimethyl cyclopropyl proline (DMCP) moiety at position P2, a tert-leucine residue at position P3, and a trifluoromethyl group at position P4 (Fig. 1A). Currently, the structures of M^pro of SARS-CoV-2, SARS-CoV and MSARS-CoV in complex with PF-07321332 have been reported, and the structural basis for the inhibition of highly pathogenic strains of coronavirus by PF-07321332 has been elucidated [26,27].

Fig. 1.

2D structure of PF-07321332 and PF-07304814. (A) 2D structure of PF-07321332, Structural compositions (P1′ and P1 to P4) are labeled. (B) 2D structure of PF-07304814, Structural compositions (P1′ and P1 to P3) are labeled.

Intravenous infusion is the preferred route of administration for PF-07304814, which is a covalent inhibitor based on hydroxymethyl ketone (HMK) [19]. PF-07304814 comprises four primary components, namely P1 to P3 and P1’ (Fig. 1B) [25]. A phosphate group is located at the P1’ position, a lactam ring at the P1 position, a leucine moiety at the P2 position, and an indole group at the P3 position (Fig. 1B). Previously, the structures of M^pro of SARS-CoV-2, SARS-CoV, MSARS-CoV, HCoV-NL63 in their complex with PF-07304814 have all been investigated, respectively, and the structural basis for the inhibition of highly pathogenic and moderately pathogenic strains of coronavirus by PF-07304814 has been elaborated [23,28].

The structure of the complex of low pathogenic coronavirus strains M^pro with PF-07321332 or PF-07304814 has not been reported and a proportion of immunocompromised individuals have difficulty in defending against low pathogenic coronavirus strains, which affects the understanding of the broad-spectrum suppression mechanisms of PF-07321332 and PF-07304814. In this study, we report the structure of Mpro of HCoV-229E with PF-07321332 and PF-07304814, respectively, to elucidate the structural basis of M^pro of HCoV-229E bound to drug candidates PF-07321332 and PF-07304814, providing a structural basis for the mechanism of coronavirus inhibition.

2. Materials and methods

2.1. Expression and purification of HCoV-229E M^pro

The gene of the HCoV-229E M^pro was commercially synthesized by codon optimization and cloned into the pET-28a vector with a 6∗His tag fused to its N-terminal end. The plasmid containing gene encoding M^pro was introduced into competent cell E. coli Rosetta DE3 for protein expression, and the expression and purification were performed according to standard methods previously described in our laboratory [12]. The N-terminal His tag was removed from the M^pro using TEV protease and the M^pro was further purified by size exclusion chromatography.

2.2. Crystallization of HCoV-229E M^pro in complex with PF-07321332 and PF-07304814

HCoV-229E M^pro was concentrated to 10 mg/mL, and incubated on ice with PF-07321332 and PF-07304814 at a molar ratio of 1:3 for 30 min, respectively. Crystallization was performed at 20 °C using the PF-07321332 or PF-07304814 in complex with HCoV-229E M^pro. The final growth condition for crystals of HCoV-229E M^pro in complex with PF-07304814 were 0.15 M Tris pH 8.0, 28% w/v PEG 4000, and the final growth condition for crystals of HCoV-229E M^pro in complex with PF-07304814 were 10% v/v 2-Propanol, 0.1 M BICINE pH 8.5, 30% w/v PEG 1500.

2.3. Data collection, structure determination, and refinement

Crystals were stabilized by the addition of a reservoir solution containing 20% glycerol, cryoprotected, and rapidly cooled in liquid nitrogen. All X-ray diffraction data were collected at 100 K at BL02U1 of the Shanghai Synchrotron Radiation Facility. Diffraction data were automatically processed using aquarium pipeline24 and data processing statistics are presented in Table 1 [29]. Structures of HCoV-229E M^pro in complex with PF-07321332 and PF-07304814 were determined by molecular replacement methods using the program Phaser, respectively [30]. Atomic positions and temperature factors were refined by the maximum likelihood method using Phenix [31]. Atomic models were fitted using the program Coot [32]. The stereochemical quality of the final model was assessed with MolProbity [33]. The structural refinement statistics of the HCoV-229E M^pro in complex with PF-07321332 or PF-07304814 are shown in Table 1. Figures were generated in PyMOL.

Table 1.

Data collection and refinement statistics.

PDB code	HCoV-229E Mpro-PF-07321332	HCoV-229E Mpro-PF-07304814
	7YRZ	8IM6
Data collection
Synchrotron	SSRF	SSRF
Beamline	BL02U1	BL02U1
Space group	P1211	P1211
a, b, c(Å)	53.40, 76.22, 77.27	53.52, 77.01, 74.63
α, β, γ(°)	90.00, 90.79, 90.00	90.00, 90.79, 90.00
Wavelength(Å)	0.97918	0.97918
Resolution(Å)(a)	1.79 (1.89–1.79)	2.01 (2.12–2.01)
Total reflections	231615	152609
Unique reflections	57910	39222
Rmerge(%)(a)	3.6(53.1)	5.3(77.9)
Mean I/σ(I) (a)	4.0(2.8)	9.0(2.5)
Completeness(%)(a)	99.1(99.7)	99.8(99.8)
Multiplicity(a)	4.0(2.8)	3.9(3.5)
Refinement
Resolution(Å)	31.09–1.79	38.90–2.01
Rwork/Rfreeb	19.15/21.96	20.42/24.03
Atoms	4703	4644
Mean temperature factor (Å2)	27.2	33.3
Bond lengths(Å)	0.008	0.007
Bond angles(°)	0.93	0.858
Ramachandran plot
Favored (%)	97.97	97.97
Allowed (%)	2.03	2.03
Outliers (%)	0	0

Rmeas = ℎkln/(n−1)i = 1nIiℎkl−Iℎkl/ℎkliIiℎkl, where Iℎkl is the mean intensity of a set of equivalent reflections.

Rwork = ℎklFobs−｜Fcalc‖/ℎkl｜Fobs｜where F_obs and F_calc are observed and calculated structure factors, respectively.

^aThe values in parentheses are for the outermost shell.

^bR_free is the R_work based on 5% of the data excluded from the refinement.

2.4. Data availability

Structure factors and coordinates have been deposited in the Protein Data Bank under accession codes 7YRZ for HCoV-229E M^pro-PF-07321332, 8IM6 for HCoV-229E M^pro-PF-07304814, respectively.

3. Results

3.1. Crystal structure of HCoV-229E M^pro in complex with PF-07321332

To elucidate the inhibitory mechanisms of PF-07321332 against HCoV-229E M^pro, we determined the structure of HCoV-229E M^pro complexed with PF-07321332. The resolution of the complex structure is 1.79 Å. The remaining structural parameters are shown in Table 1. The structure of HCoV-229E M^pro complexed with PF-07321332 shows that M^pro presents a dimeric form, which is the same as previously reported [21]. HCoV-229E M^pro monomer includes three domains, including domain I (residues 2–99), domain II (residues 100–174), and domain III (residues 200–299), with domain II and III linked by a long loop (residues 175–199). The narrow cavity between domain I and domain II contains PF-07321332, with one of each monomer (Fig. 2 A). Amplifying the narrow cavity, PF-07321332 occupies the S1, S2 and S4 subsites of HCoV-229E M^pro in an extended conformation (Fig. 2B). The substrate binding pocket featuring an atypical dyad of cysteine 144 and histidine 41 is located in the cleft between domains I and II (Fig. 2C). The explicit electron density map shows that PF-07321332 is connected to C144 through a 1.8 Å C–S covalent bond (Fig. 2C), which is the same length as the C–S bond formed between SARS-CoV-2-M^pro and PF-07321332 [26,27]. To further reveal the mechanism of PF-07321332 inhibition of HCoV-229E M^pro, we analyzed the interaction between PF-07321332 and HCoV-229E M^pro (Fig. 2D). At the P1’ position, the nitrile warhead was attacked by thiol group of C144 to form a thioimidate adduct. The imine nitrogen of the thioimidate moiety occupies the oxyanion hole and forms hydrogen bonds with two water molecules (W1 and W2), while W1 forms a hydrogen bond with Gly142 and W2 forms a hydrogen bond with His41. At the P1 position, the γ-lactam ring is inserted into the S1 subsite, and the nitrogen atom of the γ-lactam ring forms hydrogen bonds with the carboxyl groups of F139 and E165, and the oxygen atom of the γ-lactam ring forms a hydrogen bond with Nᶓ2 of H162. In addition, a hydrogen bond is formed between the amide nitrogen at position P1 and the main-chain carbonyl oxygen of Q163. At the P2 position, the DMCP moiety is inserted into the S2 subsite, forming mainly hydrophobic interactions, such as those of I164, Q187 and P188 with the DMCP moiety. The tertiary leucine residue M^pro at position P3 is not inserted into the S3 subsite, but the main-chain carbonyl oxygen forms a hydrogen bond with main-chain NH of E165. Trifluoromethyl group at position P4 is inserted into the S4 subsite, and the amide nitrogen at position P4 forms a hydrogen bond with the main chain carbonyl oxygen of E165. In addition, trifluoromethyl can also form hydrophobic interactions with the S4 site. In general, PF-07321332 occupies the active site of HCoV-229E-M^pro and stabilizes the binding of PF-07321332 by covalently binding to C144, forming hydrogen bonding interactions with F139, H162, Q163, E165 and water, and hydrophobic interactions with A143, I164, Q187, P188, N189 and Q191 (Fig. 2E).

Fig. 2.

Crystal structure of HCoV-229E-M^pro-PF-07321332. (A) Three domains of the M^pro are labeled with black line, and the black dotted box represents substrate-binding pocket. PF-07321332 is shown as sticks, with hot pink carbon atoms, red oxygen atoms, blue nitrogen atoms, and pale cyan fluorine atoms. (B) An enlarged view of the inhibitor-binding pocket. The inhibitor-binding subsites (S1, S2, and S4) with PF-07321332 are marked. (C) A C–S covalent bond forms between the sulfur atom of C144 and the nitrile carbon of PF-07321332, as shown by a blue mesh 2Fo-Fc density map contoured at 1.0 σ. The 2Fo-Fc density map of catalytic C144 and H41 are also shown at 1.0 σ. (D) The detailed interaction between PF-07321332 and M^pro is presented in sticks, with residues involved in inhibitor binding (within 3.5 Å) highlighted. Two water molecules are also visible, labeled with W, and hydrogen bond interactions are depicted as yellow dashed lines (E) A schematic interaction between PF-07321332 and M^pro is provided, with hydrogen bond interactions shown as blue dashed lines and blue boxed residues, with hydrophobic interaction shown as magenta boxed residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.2. Crystal structure of HCoV-229E M^pro in complex with PF-07304814

To investigate the inhibitory mechanisms of PF-07304814 against HCoV-229E M^pro, we also determined the structure of HCoV-229E M^pro in complex with PF-07304814, and the detailed structural information is shown in Table 1. As shown in Fig. 3 A, HCoV-229E M^pro is also present as a dimer in the structure of the complex with PF-07304814. Each monomer contains three structural domains and one PF-07304814. PF-07304814 similarly binds to the active site between domain I and II in an extended conformation, occupying the substrate binding pocket. By enlarging the cleft at the substrate binding pocket, it can be seen that PF-07304814 occupies the S1, S2 and S3 subsites of HCoV-229E M^pro, but the typical S4 site of this protease is not occupied (Fig. 3B), which is identical to SARS-CoV-2 M^pro [23]. Similarly, the atypical dyad electron density map of C144 and H41 is clearly visible in the substrate binding pocket (Fig. 3C). In addition, the electron density map indicated that PF-07304814 (carbonyl carbon of hydroxymethyl ketone) forms a 2.1 Å C–S covalent bond with the sulfur atom of the M^pro active site cysteine (Cys144). To further analyze the inhibitory mechanisms of PF-07304814 against HCoV-229E M^pro, we listed the hydrogen bonding and hydrophobic interactions of PF-07304814 with HCoV-229E M^pro (Fig. 3D). At the P1’ position, a hydroxyl group of the phosphate group forms a hydrogen bonding interaction with the amide NH of Gly142, while the double-bonded oxygen atom of phosphate group forms a hydrogen bond with amide NH of Asn141 and the single-bonded oxygen atom forms a hydrogen bond with His41. In addition, both Gly142 and Cys144 can form hydrogen bonds with the hydroxyl of the main chain. At the P1 position, the lactam ring is deeply embedded in the S1 position of HCoV-229E-M^pro, and the lactam NH forms hydrogen bonding interactions with the side chain oxygen of Glu165 and the backbone oxygen of Phe139, while the lactam carbonyl group forms hydrogen bonding interactions with His162, which is conserved in SARS-CoV-2 [23]. Furthermore, a hydrogen-bonding interaction exists between the backbone NH and the main-chain carbonyl oxygen of Gln163. At the P2 position, leucine is inserted into the S2 pocket, which maintains the stability of P2 mainly through hydrophobic interactions. At the P3 position, the indole group is inserted into the S3 subsite. The main chain carbonyl oxygen of Glu165 forms a hydrogen bond interaction with the NH of the indole group. The main chain carbonyl oxygen of PF-07304814 forms a hydrogen bond interaction with NH of Glu165. In addition, Pro187 and Asn188 also stabilize the indole group through hydrophobic interactions. Overall, PF-07304814 occupied the active site of HCoV-229E-M^pro by covalently binding to Cys144 and forming hydrogen bonding interactions with His41, Phe139, Asn141, Gly142, Cys144, His162, Gln163, Glu165, and hydrophobic interactions with Ala163, His171, Ile164, Pro188 and Gln189 (Fig. 3E).

Fig. 3.

Crystal structure of HCoV-229E-M^pro-PF-07304814. (A) Three domains of the M^pro are labeled with black line, and the black dotted box represents substrate-binding pocket. PF-07304814 is shown as sticks, with hot yellow carbon atoms, red oxygen atoms, blue nitrogen atoms, and orange phosphorus atoms. (B) An enlarged view of the inhibitor-binding pocket. The inhibitor-binding subsites (S1, S2 and S3) with PF-07304814 are marked. (C) A C–S covalent bond forms between the sulfur atom of C144 and the nitrile carbon of PF-07304814, as shown by a blue mesh 2Fo-Fc density map contoured at 1.0 σ. The 2Fo-Fc density map of catalytic C144 and H41 are also shown at 1.0 σ (D) The detailed interaction between PF-07304814 and M^pro is presented in sticks, with residues involved in inhibitor binding (within 3.5 Å) highlighted. The hydrogen bond interactions are depicted as yellow dashed lines (E) A schematic interaction between PF-07304814 and M^pro is provided, with hydrogen bond interactions shown as blue dashed lines and blue boxed residues, with hydrophobic interaction shown as magenta boxed residues. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

3.3. Structural comparison of PF-07321332 with different M^pro

To illustrate the structural basis of PF-07321332 inhibition of different coronaviruses, we compared the structures of PF-07321332 in M^pro of HCoV-229E with those of SARS-CoV-2, SARS-CoV and MERS-CoV. Based on the overall structure, M^pro of HCoV-229E, SARS-CoV-2, SARS-CoV and MERS-CoV showed highly similar conformations when bound to PF-07321332 (Fig. 4 A, left). Enlarging the substrate binding sites, we found that despite the slight difference in the orientation of PF-07321332, the substrate binding sites in HCoV-229E-M^pro, SARS-CoV-2-M^pro, SARS-CoV-M^pro and MERS-CoV-M^pro still showed highly similar conformations (Fig. 4A, right).

Fig. 4.

Comparison of the binding modes between PF-07321332 and different coronavirus M^pro. (A) Comparison of overall structures among different coronavirus M^pro in complex with PF-07321332. Left: Overall structures of HCoV-229E-M^pro-PF-07321332(cyan), SARS-CoV-2-M^pro-PF-07321332 (gray, PDB ID 7VLP), SARS-CoV-M^pro-PF-07321332 (yellow, PDB ID 7VH8) and MERS-CoV-M^pro-PF-07321332 (salmon, PDB ID 7VTC). Right: An enlarged view of substrate binding pocket of different coronavirus M^pro. (B–D) The comparison of detailed interactions (within 3.5 Å) between HCoV-229E-M^pro-PF-07321332 and SARS-CoV-2-M^pro-PF-07321332 (B), SARS-CoV-M^pro-PF-07321332 (C), MERS-CoV-2-M^pro-PF-07321332(D). The involved residues are shown as cyan (HCoV-229E-M^pro), gray (SARS-CoV-2-M^pro), yellow (SARS-CoV-M^pro) and salmon (MERS-CoV-M^pro) sticks, respectively. PF-07321332 in HCoV-229E-M^pro, SARS-CoV-2-M^pro, SARS-CoV-M^pro and MERS-CoV-M^pro are shown in warmpink, gray, yellow and salmon, respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

To analyze the structural detail of PF-07321332 inhibition of different coronaviruses, we also compared the interaction (within 3.5 Å) of HCoV-229E-M^pro with those of other coronaviruses with PF-07321332, respectively (Fig. 4B, C and 4D). The superposition of HCoV-229E-M^pro with SARS-CoV-2-M^pro showed that both interaction sites with PF-07321332 were mostly conserved, despite differences in some amino acids (Fig. 4A). Phe139, Ala143, Pro188, Asn189 of HCoV-229E-M^pro involved in the interaction with PF-07321332 were not involved in the interaction between SARS-CoV-2-M^pro and PF-07321332 (within 3.5 Å). In contrast, Asn142 of SARS-CoV-2-M^pro also did not find conserved amino acids among those involved in the interaction of HCoV-229E-M^pro. Among these amino acids with differences, most of them were hydrophobic interactions. Similarly, in SARS-CoV-M^pro and MERS-CoV-M^pro, the amino acids involved in the interaction with PF-07321332 are mostly conserved, and those that are not conserved are mainly those that form hydrophobic interactions (Fig. 4C and D). In the structure superimpositions of HCoV-229E-M^pro-PF-07321332 and SARS-CoV-M^pro-PF-07321332, the less conserved amino acids were Ala143, Asn189 for HCoV-229E-M^pro and Asn142, Leu167, His172 for SARS-CoV-M^pro (Fig. 4C). In the complex superimposition of HCoV-229E-M^pro- PF-07321332 and MERS-CoV-M^pro-PF-07321332, the amino acids that are not conserved are Asn189 and Gln191 of HCoV-229E-M^pro and Leu49, Leu170, His175 and Thr183 of MERS-CoV-M^pro (Fig. 4D). Overall, the main proteases of various coronavirus were well-conserved at the site of interaction with PF-07321332.

3.4. Structural comparison of PF-07304814 with different M^pro

The complex of SASRS-CoV-2-M^pro with PF-07304814 (PDB download) contains PF-07304814 only in protomer B. Consequently, we compared different coronaviruses in the monomeric structure of the complex with PF-07304814 (Fig. 5 A). In the monomer structure, the three structural domains were displayed more clearly (Fig. 5A). Moreover, we also found that PF-07304814 showed a highly similar conformation upon binding to M^pro of different coronaviruses (Fig. 5A, left), despite a slight difference in the partial orientation of the magnified 07304814 (Fig. 5A, right). Further, we compared the details of the interaction of HCoV-229E-M^pro with M^pro of other moderately pathogenic coronaviruses and highly pathogenic coronaviruses with PF-07304814, respectively (Fig. 5B–E). The amino acids involved in the interaction with PF-07304814 were largely conserved in the M^pro of different coronaviruses, and the less conserved amino acids included not only those that formed hydrophobic interactions but also those that formed hydrogen bond interactions. The structure superposition of HCoV-229E-M^pro-PF-07321332 and SARS-CoV-2-M^pro-PF-07304814 shows that the less conserved amino acids include His41 (hydrogen bonding interaction), Asn141 (hydrogen bonding interaction), Ala143 (hydrophobic interaction), His171 (hydrophobic interaction), and Asn189 (hydrophobic interaction) of HCoV-229E-M^pro. For the structural superposition of HCoV-229E-M^pro-PF-07304814 and SARS-CoV-M^pro-PF-07304814, the less conserved amino acids were Asn141, His171, Asn189 of HCoV-229E-M^pro. In the structure superposition of HCoV-229E-M^pro-PF-07304814 and MERS-CoV-M^pro-PF-07304814, the non-conserved amino acids contain His41, His171 of HCoV-229E-M^pro. The amino acids that are less conserved in the structure superposition of HCoV-229E-M^pro-PF-07304814 and HCoV-NL63- -PF-07304814 including Ala143 of HCoV-229E-M^pro. As a whole, even though the interactions of the coronavirus M^pro with PF-07304814 were well conserved, the structural basis for the inhibition of PF-07304814 by the low pathogenic strain HCoV-229E-M^pro is more similar to that of the moderately pathogenic strain compared to the highly pathogenic strain.

Fig. 5.

Comparison of the binding modes between PF-07304814 and different coronavirus M^pro. (A) Comparison of monomeric structures among different coronavirus M^pro in complex with PF-07304814. Left: Overall structures of HCoV-229E-M^pro-PF-07304814(cyan), SARS-CoV-2-M^pro-PF-07304814 (gray, PDB ID 7VVP), SARS-CoV-M^pro-PF-07304814 (yellow, PDB ID 7WQI), MERS-CoV-M^pro-PF-07304814 (salmon, PDB ID 7WQJ) and HCoV-NL63-M^pro-PF-07304814 (marine, 7WQH). Right: An enlarged view of substrate binding pocket of different coronavirus M^pro. (B–E) The comparison of detailed interactions (within 3.5 Å) between HCoV-229E-M^pro-PF-07304814 and SARS-CoV-2-M^pro-PF-07304814 (B), SARS-CoV-M^pro-PF-07304814 (C), MERS-CoV-2-M^pro-PF-07304814(D) and HCoV-NL63-M^pro-PF-07304814 (E). The involved residues are shown as cyan (HCoV-229E-M^pro), gray (SARS-CoV-2-M^pro), yellow (SARS-CoV-M^pro), salmon (MERS-CoV-M^pro) and marine (HCoV-NL63-M^pro) sticks, respectively. PF-07304814 in HCoV-229E-M^pro, SARS-CoV-2-M^pro, SARS-CoV-M^pro, MERS-CoV-M^pro and HCoV-NL63-M^pro are shown in warmpink, gray, yellow, salmon, marine respectively. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

4. Discussion

Diseases caused by coronaviruses affect human health, and the search for antiviral treatments is ongoing. In this regard, the development of inhibitor drugs against M^pro of coronavirus has attracted great interest. Among the numerous inhibitors, PF-07321332 and PF-07304814 represent the most promising candidates with therapeutic potential, of which PF-07321332 in combination with Ritonavir has been approved for the early treatment of patients with mild to moderate COVID-19. The inhibition mechanism of PF-07321332 or PF-07304814 with M^pro in different coronaviruses is currently heavily investigated. Among them, the structures of M^pro of SARS-CoV-2, SARS-CoV, MERS-CoV, and HCoV-NL63 in complex with PF-07321332 or PF-07304814 have been reported. However, there is a deficiency of structural studies on the complexes of the low pathogenic strain M^pro with PF-07321332 and PF-07304814. Consequently, we determined the structures of two complexes, HCoV-229E-M^pro-PF-07321332 and HCoV-229E-M^pro-PF-07304814, and elaborated the structural basis for the inhibition of HCoV-229E-M^pro by PF-07321332 and PF-07304814.

The structures of the two complexes showed that both PF-07321332 and PF-07304814 could form C–S covalent bonds with HCoV-229E-M^pro at a bond length of 1.8 Å and 2.1 Å, respectively, which indicated that PF-07321332 could form C–S covalent bonds with HCoV-229E-M^pro more easily. However, Cys144 of HCoV-229E-M^pro could not form hydrogen bonds with PF-07321332, which is different from SARS-CoV-2, SARS-CoV, and MERS-CoV [26,27]. In addition, the catalytic dyad of Cys144 and His41 could be clearly found in HCoV-229E-M^pro. In the HCoV-229E-M^pro-PF-07321332 complex, His41 interacts with PF-07321332 mainly indirectly through water (Fig. 2D and E), while in the HCoV 229E-M^pro-PF-07304814, His41 could form hydrogen bonding interaction with PF-07304814 directly (Fig. 3D and E), and this direct hydrogen bonding interaction is not found in SARS-CoV-2, SARS-CoV, MERS-CoV and HCoV-NL63 [23]. In addition, structural analysis of HCoV-229E-M^pro with PF-07321332 and PF-07304814 showed that PF-07321332 occupied S1, S2, and S4 pockets, while PF-07304814 employed S1, S2, and S3 pockets of HCoV-229E-M^pro, but both were highly conserved in amino acids at the interaction sites with HCoV-229E-M^pro, especially those forming hydrogen bonds (Fig. 2, Fig. 3E).

After structural superposition of the different coronavirus M^pro complexes with the HCoV-229E-M^pro complex, we found that the binding inhibitor sites of the different coronaviruses are also conserved in HCoV-229E-M^pro (Fig. 4B-D, 5B-5E), which implies that these two inhibitors are expected to be drugs against different coronavirus-induced agents, including HCoV-229E. Furthermore, we found that the amino acids involved in the binding pocket of HCoV-229E-M^pro-PF-07304814 are most conserved in HCoV-NL63-M^pro-PF-07304814, implying that both adopt a highly consistent mechanism in binding to the inhibitors, which would provide a theoretical basis and structural basis for future treatment of diseases caused by lower and moderately pathogenic strains. Overall, our structural studies of HCoV-229E-M^pro-PF-07321332 and HCoV-229E-M^pro-PF-07304814 complement previous reports on the broad-spectrum resistance of PF-07321332 and PF-07304814, and contributes to a comprehensive understanding of the structural basis of inhibition of PF-07321332 and PF-07304814, thus providing a structural basis and key insights for drug development. Furthermore, further structure-based optimization of these two covalent inhibitors will yield effective drugs against potential future coronaviruses.

Funding

This work was supported by Jiangxi natural science foundation for distinguished young scholar (20212ACB216001), Gannan Medical University (QD201910), Jiangxi key research and development program (20203BBG73063) and Jiangxi “Double Thousand Plan (jxsq2019101064)", Shanghai Science and Technology Plan Project (21ZR1471800).

Author contributions

Conceptualization: J.L., H.Y., and Q.W.; supervision: H.Y., and Q.W.; formal analysis: Y.Z., W.W., P.Z., J.F., D.L., J.Y., J.Z., X.Y., and J.L.; investigation: Y.Z., W.W., P.Z., J.F., D.L., J.Y., J.Z., and X.Y.; Writing—original draft: Y.Z., and W.W.; Writing—review and editing: J.L., H.Y., and Q.W.; project administration: H.Y., and Q.W. All authors have read and agreed to the published version of the manuscript.

Declaration of competing interest

This study was not published elsewhere for publication. No conflict of interest exits in the submission of this manuscript, and manuscript is approved by all authors for publication.