Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease

Dr Hangchen Hu; Qian Wang; Dr Haixia Su; Dr Qiang Shao; Dr Wenfeng Zhao; Guofeng Chen; Dr Minjun Li; Dr Yechun Xu

doi:10.1002/anie.202212378

. 2022 Nov 28;61(52):e202212378. doi: 10.1002/anie.202212378

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease^**

Hangchen Hu ^1,^2,³, Qian Wang ⁴, Haixia Su ², Qiang Shao ², Wenfeng Zhao ², Guofeng Chen ^2,³, Minjun Li ^5,^✉, Yechun Xu ^1,^2,^3,^4,^✉

PMCID: PMC9874598 PMID: 36308706

Abstract

The coronavirus papain‐like protease (PL^pro) plays an important role in the proteolytic processing of viral polyproteins and the dysregulation of the host immune response, providing a promising therapeutic target. However, the development of inhibitors against severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) PL^pro is challenging owing to the restricted S1/S2 sites in the substrate binding pocket. Here we report the discovery of two activators of SARS‐CoV‐2 PL^pro and the identification of the unique residue, cysteine 270 (C270), as an allosteric and covalent regulatory site for the activators. This site is also specifically modified by glutathione, resulting in protease activation. Furthermore, a compound was found to allosterically inhibit the protease activity by covalent binding to C270. Together, these results elucidate an unrevealed molecular mechanism for allosteric modulation of SARS‐CoV‐2 PL^pro and provid a novel site for allosteric inhibitors design.

Keywords: Allosteric Modulator, Cysteine 270, Drug Design, Papain-Like Protease, SARS-CoV-2

Two allosteric and covalent activators of SARS‐CoV‐2 papain‐like protease (PL^pro) were identified to elucidate a novel site, cysteine 270 (C270), which led to the discovery of an endogenous activator and an inhibitor of the protease. This study provides a paradigm for identifying covalent and allosteric sites and offers an opportunity for the development of SARS‐CoV‐2 PL^pro inhibitors.

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Introduction

The coronavirus disease 2019 (COVID‐19) pandemic is caused by a new human coronavirus (CoV), severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2), that has led to an unbearable number of infections and deaths worldwide. CoVs share key genomic elements that provide promising therapeutic targets. ^[1] A chymotrypsin‐like cysteine protease, also called 3C‐like protease (3CL^pro) or main protease (M^pro), ^[2] together with a papain‐like protease (PL^pro), ^[3] is required to process the two viral polyproteins into mature non‐structural proteins that are essential for viral transcription and replication. In addition, SARS‐CoV‐2 PL^pro recognizes and cleaves the C‐terminal LxGG sequence of ubiquitin (Ub) and ubiquitin‐like proteins (UbLs) such as interferon‐stimulated gene product 15 and acts as a deubiquitinase (DUB) to remove Ub or UbL from host proteins.[ ⁴ , ⁵ , ⁶ , ⁷ , ⁸ ] Antiviral immunity includes broad post‐translational modifications by Ub and UbL that regulate host protein localization and stability. ^[9] The DUB activity of PL^pro is thought to induce dysregulation in the host immune response against viral infection.[ ¹⁰ , ¹¹ , ¹² , ¹³ , ¹⁴ ] Thus, targeting PL^pro is an attractive strategy for both inhibiting viral replication and preventing the disruption of the host immune response against viral infection.

There has been significant progress in the development of inhibitors targeting SARS‐CoV‐2 3CL^pro.[ ¹⁵ , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ ] The discovery of PF‐00835231 as a covalent active‐site‐directed inhibitor of SARS‐CoV 3CL^pro in 2003 is conducive to the rapid development of inhibitors of SARS‐CoV‐2 3CL^pro.[ ¹⁶ , ¹⁷ , ²⁰ , ²¹ ] At present, the most advanced inhibitor, nirmatrelvir (PF‐07321332), in combination with ritonavir, has been approved for the treatment of COVID‐19. ^[20] In comparison, the PL^pro of SARS‐CoV and SARS‐CoV‐2 presents a more challenging target, because very few potent PL^pro inhibitors have been reported with validated in vitro as well as in vivo efficacy. A key issue from the standpoint of inhibitor design is the barrier derived from the restricted binding sites (S1/S2) for recognizing two consecutive glycine residues (P1/P2) in SARS‐CoV‐2 or SARS‐CoV PL^pro substrates.[ ²² , ²³ , ²⁴ ] Although several high‐throughput screening campaigns have been performed,[ ⁴ , ²³ , ²⁵ , ²⁶ , ²⁷ , ²⁸ ] GRL0617 that was originally identified as an inhibitor of SARS‐CoV PL^pro, ^[29] remains one of the most potent PL^pro inhibitors and a major starting point for optimization.[ ⁸ , ²² , ³⁰ , ³¹ , ³² ] A comprehensive understanding of the structure–activity relationship of the protease and exploration of more ligand‐binding sites in addition to the catalytic (orthosteric) site is warranted to design new potent inhibitors.

In this study, we performed high‐throughput screening of compounds, including the Food and Drug Administration (FDA)‐approved drugs and candidates in clinical trials, using an enzymatic assay. Although we embarked on our search for new inhibitors of SARS‐CoV‐2 PL^pro, two disulfide‐containing activators were unexpectedly found, that enabled us to identify a novel binding site on SARS‐CoV‐2 PL^pro and to elucidate an unrevealed allosteric and covalent binding mechanism to regulate the activity of the protease. Inspired by this discovery, endogenous activators and the first allosteric inhibitor that covalently modified the protease at this site were also identified. These findings provide mechanistic insights into the regulation of SARS‐CoV‐2 PL^pro activity and provide a vital allosteric site for the development of novel inhibitors against SARS‐CoV‐2.

Results and Discussion

Discovery of Two Activators of SARS‐CoV‐2 PL^pro with a Novel Mechanism

To measure the proteolytic activity of the recombinant SARS‐CoV‐2 PL^pro, we carried out an enzymatic assay with a short fluorogenic substrate, RLRGG‐AMC, which was labelled with 7‐amino‐4‐methylcoumarin (AMC). ^[33] The hydrolysis of this substrate by protease resulted in the release of the AMC group and a substantial increase in the fluorescence intensity. Using this assay, ∼4 000 FDA‐approved drugs and candidate compounds in clinical trials were tested at 100 μM concentration against 50 nM SARS‐CoV‐2 PL^pro to screen inhibitors (Table S1). Intriguingly, a number of activators increased the proteolytic activity of the protease. Among them, two compounds, both containing a disulfide bond, dimesna and pyritinol, induced the activation of SARS‐CoV‐2 PL^pro by more than 50 % (Figure 1a). Moreover, both compounds significantly upregulated protease activity in a dose‐dependent manner (Figure S1). Subsequently, we determined the half maximal effective concentration (EC₅₀) and maximal effect (E_max), representing the activation potency and efficacy, respectively, of these two activators. Dimesna, a uroprotective agent, activated SARS‐CoV‐2 PL^pro with an EC₅₀ value of 1046 μM and an E_max value of 300.0 % (Figure 1a and Figure S1). The other activator, pyritinol, which is an analogue of vitamin B6 and used for the treatment of cognitive disorders, activated the protease with an EC₅₀ value of 18 μM and an E_max value of 226.5 % (Figure 1a and Figure S1). The effects of these two compounds on the Michaelis constant (K _m) and maximum reaction rate (V _max) of the fluorogenic substrate hydrolyzed by SARS‐CoV‐2 PL^pro were also determined (Table 1). Both activators barely influenced K _m but increased V _max, which indicated that their ability to enhance the reaction rate rather than the binding affinity of the substrate with the protease. Taken together, dimesna and pyritinol were found to be allosteric activators of SARS‐CoV‐2 PL^pro for the first time.

Dimesna and pyritinol upregulated the enzymatic activity of SARS‐CoV‐2 PL^pro by covalently binding to C270. a) Chemical structures, activation potency and efficacy (EC₅₀ and E_max) of dimesna and pyritinol. b–c) Time‐dependent activation of SARS‐CoV‐2 PL^pro by dimesna (b) and pyritinol (c) at various concentrations, and the calculated covalent binding kinetic parameters. d) Activation effect of dimesna and pyritinol on the wild‐type (WT) SARS‐CoV‐2 PL^pro and its four variants, C155S, C181S, C270S, and C284S mutants. Error bars represent mean±SD of three independent experiments in b–d.

Table 1.

K _m and V _max values of the fluorogenic substrate (RLRGG‐AMC) hydrolyzed by the wild‐type SARS‐CoV‐2 PL^pro in the absence or presence of activators represented by mean±SD of three independent experiments.

Activators		K _m [μM]	V _max [10⁴ RFU min⁻¹]
	(wild‐type)	566.9±36.0	14.9±2.2
Dimesna (2 mM)		574.2±59.4	42.6±4.7
Pyritinol (50 μM)		559.2±28.5	19.6±2.0
GSSG (2 mM)		463.8±13.3	25.2±1.1

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In addition, the two compounds showed time‐dependent activation of SARS‐CoV‐2 PL^pro (Figure 1b and 1c), which is in line with the model of covalent modulation. Covalent ligand binding generally involves a two‐step process, an initial reversible binding event followed by the formation of a covalent bond, which is characterized by the binding affinity (K _a for activators, K _i for inhibitors) and the rate constant of covalent bond formation (k _inact), respectively. To obtain kinetic parameters of the activators binding to SARS‐CoV‐2 PL^pro, 50 nM (final concentration) protease was incubated with various concentrations of the compound for several indicated times and the protease activity was measured after incubation (Figure 1b and 1c). The results showed that the two activators had comparable k _inact values (Figure S2a, and S2b), which might be attributed to the same reaction group, the disulfide bond. However, pyritinol had a much lower K _a value than dimesna, which suggests that more noncovalent interactions formed between pyritinol and the protease. Therefore, dimesna and pyritinol are two covalent activators of SARS‐CoV‐2 PL^pro with the same reaction group but different amounts of noncovalent contacts.

Considering that compounds containing a disulfide bond can act as covalent probes to characterize cysteine at allosteric sites,[ ³⁴ , ³⁵ , ³⁶ , ³⁷ , ³⁸ , ³⁹ ] we predicted that the activity modulation of SARS‐CoV‐2 PL^pro by dimesna and pyritinol might result from the modification of cysteine residue(s) of the protease through a disulfide exchange reaction. To test this hypothesis, four vulnerable cysteines on the surface of the protease, C155, C181, C270, and C284 were individually mutated to serine. The resulting variants of the recombinant SARS‐CoV‐2 PL^pro C155S, C181S, C270S, and C284S were expressed and purified for proteolytic activity measurement with or without the addition of the activator (Figure 1d). We found that the enzymatic activity of all variants except C270S was upregulated by the two activators, demonstrating that C270 of SARS‐CoV‐2 PL^pro was the key modification site for the activators. It has been reported that C270 has the lowest pK _a among all the cysteine residues of SARS‐CoV‐2 PL^pro except for the catalytic cysteine (C111). ^[40] Thus, C270 has a high reactivity and the tendency to undergo covalent modification. Although the catalytic C111 has the highest reactivity, it is not easily modified by two activators because it is located inside the protease and still works for the catalytic hydrolysis of the substrate. Consequently, the enzymatic activity of the protease with a single mutation of several surface cysteines revealed that C270 was modified by two activators; this phenomenon led to the upregulation in SARS‐CoV‐2 PL^pro activity, consistent with our prediction.

Glutathione Activated SARS‐CoV‐2 PL^pro through C270 Modification

The confirmation that C270 was the modulating site for two disulfide‐containing compounds to activate SARS‐CoV‐2 PL^pro turned our attention to an endogenous molecule, glutathione oxidized (GSSG), which also contains a disulfide bond and has the potential to modify the protease in this manner. To support this speculation, GSSG was tested and shown to upregulate protease activity, as anticipated (Figure 2a). In addition, such activation was eliminated by the introduction of the C270S mutant (Figure 2a), which confirmed that the activity regulation was achieved through the modification of C270. The effect of GSSG on the K _m and V _max values of SARS‐CoV‐2 PL^pro was also investigated (Table 1). Similar to the two activators mentioned above, GSSG had little influence on K _m but increased the V _max value (25.2 vs. 14.9). GSSG also showed time‐dependent activation of SARS‐CoV‐2 PL^pro, which is in line with a covalent binding mode (Figure 2b). Kinetic parameters of GSSG binding to SARS‐CoV‐2 PL^pro were calculated based on the measurement of protease activity after incubation with various concentrations of GSSG for the indicated time (Figure 2b and Figure S2c). GSSG had k _inact and K _a values similar to those of dimesna. These data demonstrate that GSSG upregulates protease activity in the same manner as dimesna and pyritinol.

GSSG modified C270 of SARS‐CoV‐2 PL^pro to enhance the enzymatic activity of the protease. a) Representative profiles for the activation of the wild‐type (red) and C270S mutant (green) of SARS‐CoV‐2 PL^pro by GSSG after 30 min of incubation. b) Time‐dependent activation of SARS‐CoV‐2 PL^pro by GSSG at various concentrations, and the calculated covalent binding kinetic parameters. c) Immunoblotting of the S‐glutathionylation of the wild‐type (WT) or C270S mutant of SARS‐CoV‐2 PL^pro with indicated treatments. d) The activity of the WT or C270S mutant of SARS‐CoV‐2 PL^pro after treatment with various ratio of GSSG to GSH (5 mM for total glutathione) for 30 min. e) Intracellular activity of the WT or C270S variant of SARS‐CoV‐2 PL^pro after transfection with different amount of plasmids. Immunoblotting evaluated the protein expression level of the WT and mutated protease in cells. Error bars represent mean±SD of three independent experiments in a, b, d, and e.

To further assess the covalent modification of C270 by GSSG, a commercial antibody capable of detecting S‐glutathionylated protein was used for immunoblotting (Figure 2c). Both the wild‐type and C270S mutant of SARS‐CoV‐2 PL^pro were incubated with GSSG, and the resulting complexes were detected by the antibody. The results showed that only the wild‐type protease incubated with GSSG and then denatured under non‐reduced conditions showed a clear band on the immunoblot (Figure 2c), providing evidence for the GSSG‐mediated S‐glutathionylation of C270. Moreover, this band was diminished under reduced conditions, which suggests the covalent linkage of GSSG to the C270 of the protease through disulfide exchange. In contrast, the C270S mutant of SARS‐CoV‐2 PL^pro treated with GSSG did not show any band (Figure 2c). Thus, it can be concluded that the modification of GSSG on the protease mainly occurred at C270 rather than at other cysteine residues.

It is worth noting that the physiological concentration of total glutathione in cells ranges from 1 to 10 mM, while the ratio of reduced to oxidized glutathione (GSH/GSSG) varies depending on different conditions and cell organelles.[ ⁴¹ , ⁴² ] Accordingly, we tested different ratios of GSH/GSSG at a total concentration of 5 mM. The enzymatic activity was upregulated at each ratio for the wild‐type SARS‐CoV‐2 PL^pro but did not change for the C270S variant (Figure 2d). This result indicates that the proteolytic activity of SARS‐CoV‐2 PL^pro could be modulated by GSH/GSSG under cellular conditions. In this context, a PL‐FlipGFP assay, ^[23] was performed to determine the intracellular activity of wild‐type SARS‐CoV‐2 PL^pro and C270S variant (Figure S3). In this assay, a fluorogenic GFP containing a cleavage site (LRGGAPTK) for SARS‐CoV‐2 PL^pro, namely PL‐FlipGFP, and a red fluorescent protein (mCherry) were constructed together as the substrates for the protease and the internal control, respectively. The fluorescence of PL‐FlipGFP was produced after cleavage by the protease (Figure S3). The ratio of GFP/mCherry fluorescent signal was used to represent the amount of the product hydrolyzed by the protease in cells. The expression levels of FLAG‐tagged proteases were also detected by immunoblotting.

The results showed that the GFP/mCherry ratios of the wild‐type and C270S variants were nearly the same (161 % vs. 163 %) when transfected with the same amount of plasmids (Figure 2e and Figure S4). However, under these conditions, the expression level of the C270S variant was much higher than that of the wild type. As the GFP/mCherry ratio implied that the amount of the hydrolyzed product resulting from the two cases was similar, the higher expression level of the C270S variant over the wild‐type reflected its lower activity over the wild‐type SARS‐CoV‐2 PL^pro in cells. Nevertheless, this situation may also be caused by insufficient substrates availability. To exclude this possibility, the amount of plasmid expressing the C270S variant was reduced to achieve an expression level similar to that of the wild‐type. Consequently, the GFP/mCherry ratio of the wild‐type was significant higher than that of the C270S variant (161 % vs. 100 %), demonstrating that more hydrolyzed products were produced by the wild‐type when the expression levels of the two proteases were similar (Figure 2e). Taken together, these data revealed higher activity of the wild‐type over the C270S variant of SARS‐CoV‐2 PL^pro in cells containing millimolar glutathione with different ratios of GSH/GSSG.

To further explore whether the intracellular activity of SARS‐CoV‐2 PL^pro was affected by different levels of glutathione, buthionine sulfoximine (BSO), an inhibitor of γ‐glutamylcysteine synthetase, was used to reduce the level of glutathione in the cells. ^[43] After treatment with BSO, GSH level significantly reduced to 5.5 % of the total glutathione in HEK293T cells (Figure S5a). Meanwhile, the intracellular activity of the wild‐type SARS‐CoV‐2 PL^pro, determined by the FlipGFP assay, was reduced (100 % vs. 90 %, Figure S5b), while that of the C270S variant was slightly increased (Figure S5c). The measured values of EC₅₀ and E_max of GSH on the purified recombinant SARS‐CoV‐2 PL^pro were 0.44 mM and 153 %, respectively (Figure S5d), indicating that SARS‐CoV‐2 PL^pro could still be activated by the remaining 5.5 % (≈0.3–0.5 mM) glutathione in cells. This may explain why the intracellular activity of SARS‐CoV‐2 PL^pro was slightly reduced after the treatment with BSO.

In addition, as shown in Figure 2d, the activity of SARS‐CoV‐2 PL^pro enhanced by increasing the intracellular ratio of GSSG/GSH. Because cells were quickly damaged under the condition of a high intracellular ratio of GSSG/GSH and the performance of PL‐FlipGFP assay requires a certain time, we applied the enzymatic assay instead of the PL‐FlipGFP assay to determine the intracellular activity of SARS‐CoV‐2 PL^pro using the cell lysate with a fluorogenic substrate (RLRGG‐AMC). The results also revealed the higher activity of the wild‐type over the C270S variant of SARS‐CoV‐2 PL^pro (193 % vs. 100 %, Figure S6) in cells. After treatment with diamide that increased the ratio of GSSG/GSH within several minutes, ^[44] the intracellular activity of SARS‐CoV‐2 PL^pro further increased (227 % vs. 193 %) but the activity of C270S variant slightly reduced (Figure S6). Therefore, the intracellular activity of SARS‐CoV‐2 PL^pro can be increased by increasing the GSSG/GSH ratio. It is, therefore, speculated that the performance of the intracellular proteolytic activity of SARS‐CoV‐2 PL^pro on viral polyproteins might be benefitted from the activation caused by endogenous glutathione and varied ratio of GSSG/GSH.

Impact of C270 Mutants on the Enzymatic Activity of SARS‐CoV‐2 PL^pro

The covalent modification of C270 by dimesna, pyritinol and glutathione leads to the upregulation in SARS‐CoV‐2 PL^pro activity, while the C270S mutant lacks such an effect, underscoring the importance of C270 for the modulation of enzymatic activity. This raises the question of whether C270 modification can inhibit protease activity. To answer this question and to aid future efforts in developing novel inhibitors targeting this site, we first explored the influence of different C270 mutants on the K _m and V _max values of the protease. Considering the different sizes, polarities, and charge states of the side chain, 11 mutants including C270S, C270V, C270L, C270E, C270Q, C270M, C270F, C270Y, C270H, C270K, and C270R, were used. Table 2 shows that none of the mutants had any significant influence on K _m as compared to the wild‐type enzyme. As for V _max, C270S, C270V, and C270L had similar values as the wild‐type. The C270E mutant with a negative charge was originally designed to resemble the modification of C270 by dimesna, but its V _max value was much lower than that resulting from the addition of dimesna (11.4 vs. 42.6) and was even lower than that of the wild‐type (11.4 vs. 14.9). Further, the V _max values of C270M (21.6), C270F (26.0), and C270Y (26.5) mutants were higher than that of the wild‐type (14.9), which may be ascribed to relatively large and electron‐rich side chains of methionine, phenylalanine, and tyrosine. In contrast, C270H, C270K, and C270R mutants, all of which contained a positively charged side chain, resulted in V _max values that were smaller than the value of the wild‐type (9.3, 6.0, 4.7 vs. 14.9), suggesting that these mutants caused a weak inhibition effect on the protease. Together, these results revealed that the substitution of C270 with amino acids containing large side chains to mimic the modification of C270 by compounds would affect enzymatic activity, although detailed explanations for these results remains to be determined. Thus, C270 is a crucial allosteric site to modulate protease activity, and covalent modification by a positively charged molecule may inhibit protease activity.

Table 2.

K _m and V _max values of SARS‐CoV‐2 PL^pro with different mutations of C270, represented by mean±SD of three independent experiments.

Wild‐type or mutants	K _m [μM]	V _max [10⁴ RFU min⁻¹]
C270 (wild‐type)	566.9±36.0	14.9±2.2
C270S	641.56±4.6	15.0±1.3
C270V	572.1±23.5	17.4±0.9
C270L	640.6±35.0	18.1±0.3
C270E	603.8±14.6	11.4±0.6
C270Q	493.1±57.2	14.7±1.3
C270M	616.4±60.9	21.6±1.9
C270F	654.9±32.7	26.0±1.1
C270Y	768.4±36.0	26.5±1.1
C270H	528.6±44.4	9.3±0.2
C270K	674.8±31.3	6.0±0.1
C270R	864.5±33.6	4.7±0.4

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Discovery of a Novel Allosteric Inhibitor of SARS‐CoV‐2 PL^pro

With the aid of all suggestions from the above results, bis[2‐(N,N‐dimethylamino)ethyl] disulfide (DMGA) that contains a disulfide bond and two positive‐charged methylamine, was found to perfectly meet the requirement for the inhibitor (Figure 3a). As anticipated, DMGA exhibited dose‐dependent inhibition of SARS‐CoV‐2 PL^pro, with an IC₅₀ value of 9.4 μM (Figure 3b). Activity measurements of the wild‐type and C270S mutant of the protease incubated with DMGA revealed that the inhibitory potency of DMGA was caused by the covalent modification of C270 (Figure 3c). DMGA also inhibited the C270S variant at high concentrations, resulting in an IC₅₀ of 577 μM (Figure S7). It is about 61‐fold of the IC₅₀ value (9.4 μM) of DMGA against the wild‐type, implying that DMGA covalently binds to C270 with high selectivity. In addition, DMGA showed a dose‐dependent decrease in the V _max value but hardly affected the K _m except at the highest concentration (40 μM) where the K _m value increased from 566.9 to 798.1 μM (Figure 3d). These results are in line with the model of non‐competitive inhibition.

DMGA allosterically inhibits SARS‐CoV‐2 PL^pro by covalently binding to C270. a) Chemical structure of DMGA. b) Representative profile for DMGA inhibiting SARS‐CoV‐2 PL^pro after 30 min of incubation. c) Different inhibitory rates of 50 μM DMGA for the wild‐type and C270S mutant of SARS‐CoV‐2 PL^pro after 30 min of incubation. d) The determination of K _m and V _max values of the substrate hydrolyzed by SARS‐CoV‐2 PL^pro in the presence of different concentrations (0–40 μM) of DMGA. e) Time‐dependent inhibition of SARS‐CoV‐2 PL^pro by DMGA at various concentrations and indicated time was determined to calculate kinetic parameters of DMGA binding to and reacting with the protease. Error bars represent mean±SD of three independent experiments in b–e.

To further validate the modification of C270 of SARS‐CoV‐2 PL^pro by DMGA, a mass spectrometry (MS) analysis was conducted. SARS‐CoV‐2 PL^pro incubated with DMGA was digested with chymotrypsin and analyzed via high‐resolution mass spectrometry which achieved a high coverage (85 %) of the SARS‐CoV‐2 PL^pro protein sequence (Table S2). Among all identified SARS‐CoV‐2 PL^pro peptides, the liquid chromatography (LC)‐MS scan suggested only one peptide modified by DMGA (Figure S8a and Table S2). Further, tandem mass spectrometry (MS/MS) sequencing of this peptide revealed that it spans residues 265–273 of SARS‐CoV‐2 PL^pro (265TGNYQC(+104)GHY273), and a mass shift of +104 Da corresponding to the exact mass of C4H11NS was verified (Figure S8b). In addition, a SARS‐CoV‐2 PL^pro peptide containing catalytic cysteine (107ADNNC₁₁₁YL113) was also detected (Table S2) but the results showed that it was not modified by DMGA (Figure S9). It is thus indicated again that DMGA is a selective covalent binder for C270 of SARS‐CoV‐2 PL^pro.

Moreover, DMGA inhibited SARS‐CoV‐2 PL^pro in a time‐dependent manner, demonstrating its covalent binding mode (Figure 3e). Kinetic parameters of DMGA binding with SARS‐CoV‐2 PL^pro were calculated based on the activity measurements of the protease incubated with various concentrations of DMGA for the indicated time (Figure 3e and Figure S2d). The resulting k _inact and K _i values were 0.030 min⁻¹ and 58 μM, respectively, which were similar to those of pyritinol (Figure 3e and Figure 1c). Together, these data indicate that DMGA allosterically inhibits SARS‐CoV‐2 PL^pro through a specific covalent modification of C270 and provide convincing evidence that C270 is a pivotal site for the design of novel allosteric protease inhibitors.

As mentioned before, the discovery of a SARS‐CoV‐2 PL^pro inhibitor is hampered by the restricted S1/S2 site for the binding of tandem glycine in substrates. Such a space restriction also prevents covalent binding of the ligand to the catalytic C111. The discovery of C270 as an allosteric site thus provides an alternative route for inhibitor design. DMGA with single‐digit micromolar potency acts as a hit for further development of potent allosteric inhibitors.

Conservation of C270 among PL^pros of CoVs and SARS‐CoV‐2 Variants

To investigate the sequence conservation of C270 among PL^pro, we performed a sequence alignment of pp1ab from seven human CoVs (Figure S10). It was revealed that C270 is unique to SARS‐CoV and SARS‐CoV‐2 PL^pro, and valine was found in the PL^pro of the other five CoVs at the equivalent position. Considering that only C270 was targeted by glutathione to increase protease activity, this unique residue might be associated with the severe consequences of SARS‐CoV and SARS‐CoV‐2 infections in humans. In addition, the unique C270 of SARS‐CoV and SARS‐CoV‐2 PL^pro provides a structural basis for the discovery of selective allosteric modulators. We further performed sequence alignment of pp1ab resulting from 15 SARS‐CoV‐2 variants of concern (Figure S11). C270 is highly conserved, indicating that these variants are unlikely to escape from inhibitors targeting C270.

Conclusion

The pandemic COVID‐19 together with the epidemics of SARS and Middle East respiratory syndrome (MERS) has raised great awareness about the increasing infection risks of highly pathogenic CoVs. This concern calls for a huge demand for the discovery of anti‐coronavirus drugs. PL^pro is a highly conserved cysteine proteinase that is indispensable for coronavirus replication and provides an attractive but challenging target for the development of broad‐spectrum antiviral drugs. Herein, we report a novel allosteric inhibitor of SARS‐CoV‐2 PL^pro with a covalent action on C270 which is a previously unrecognized site for allosteric regulation of protease activity. The mechanism of action of such a new inhibitor and structural determinants associated with its binding uniquely suited for engaging the non‐catalytic cysteine are significantly distinct from known inhibitors that bind to the catalytic site of SARS‐CoV‐2 PL^pro. Unexpectedly, the discovery of this allosteric site was based on the identification of two activators, dimesna and pyritinol, using high‐throughput screening. Moreover, this vital site is covalently modified by glutathione, the endogenous activator that may be linked to the intracellular proteolytic activity of SARS‐CoV‐2 or SARS‐CoV PL^pro. Our study highlights a promising strategy for the identification of unrecognized allosteric modulation sites based on the discovery of covalent probes and reveals an exquisite allosteric modulation mechanism for the protease, which offers a great opportunity for development of new inhibitors of SARS‐CoV PL^pro.

Conflict of interest

The authors declare no conflict of interest.

1. Supporting information

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Supporting Information

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Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 22277130, No. 21877122 and No. 32071248), the Science and Technology Commission of Shanghai Municipality (No. 20430780300 and No. TM202101H003), and the Qiusuo Outstanding Youth Project of Lingang Laboratory (No. LG‐QS‐202205‐02).

H. Hu, Q. Wang, H. Su, Q. Shao, W. Zhao, G. Chen, M. Li, Y. Xu, Angew. Chem. Int. Ed. 2022, 61, e202212378; Angew. Chem. 2022, 134, e202212378.

^**

A previous version of this manuscript has been deposited on a preprint server (https://doi.org/10.1101/2022.03.30.486313).

Contributor Information

Dr. Minjun Li, Email: liminjun@zjlab.org.cn.

Dr. Yechun Xu, Email: ycxu@simm.ac.cn.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supporting Information

Click here for additional data file.^{(2.1MB, pdf)}

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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PERMALINK

Identification of Cysteine 270 as a Novel Site for Allosteric Modulators of SARS‐CoV‐2 Papain‐Like Protease^**

Dr Hangchen Hu

Qian Wang

Dr Haixia Su

Dr Qiang Shao

Dr Wenfeng Zhao

Guofeng Chen

Dr Minjun Li

Dr Yechun Xu

Abstract

Introduction

Results and Discussion