Progress and challenges in targeting the SARS-CoV-2 papain-like protease

Abstract

SARS-CoV-2 is the causative agent of COVID-19 pandemic. The approval of vaccines and small molecule antivirals is vital in combating the pandemic. The viral polymerase inhibitors remdesivir and molnupiravir and the viral main protease inhibitor nirmatrelvir/ritonavir, have been approved by FDA. However, emergence of variants of concern/interest strains calls for additional antivirals with a novel mechanism of action. The SARS-CoV-2 papain-like protease (PL^pro) mediates the cleavage of viral polyprotein as well as modulates the host innate immune response upon viral infection, rending it a promising antiviral drug target. This perspective highlights major achievements in structure-based design and high-throughput screening of SARS-CoV-2 PL^pro inhibitors since the beginning of the pandemic. Encouraging progress includes the design of non-covalent PL^pro inhibitors with favorable pharmacokinetic properties and the first-in-class covalent PL^pro inhibitors. In addition, we offer our opinion of the knowledge gaps that need to be filled to advance PL^pro inhibitors to clinic.

Graphical Abstract

1. INTRODUCTION

Coronaviruses (CoV) are enveloped, positive-sense, and single-stranded RNA(+ssRNA) viruses. CoVs belong to the subfamily Orthocoronavirinae, family Coronaviridae, and order Nidovirales. Seven coronaviruses are known to infect humans including four common human coronaviruses HCoV-229E, HCoV-NL63, HCoV-OC43, and HCoV-HKU1 that cause mild symptoms,¹ and three coronaviruses SARS-CoV, MERS-CoV, and SARS-CoV-2 that cause severe acute respiratory tract infections.^{2, 3} Although humans around the world are commonly infected with HCoV-229E, HCoV-NL63, HCoV-OC43, or HCoV-HKU1, the infection generally only causes mild symptoms that do not require medical treatments.^{4, 5} Accordingly, no major efforts have been devoted to developing vaccines and antiviral drugs against these viruses. Nonetheless, the 21^st century witnessed several coronavirus outbreaks that raised the alarm of this virus family. In late 2002, SARS-CoV emerged in Guangdong, China, and caused approximately 8,000 cases with the fatality rate of 9.6%.⁶ In 2012, MERS-CoV emerged in Saudi Arabia and South Korea, caused approximately 2,400 cases in the following 8 years with a fatality rate of 34%.⁷ Notably, in 2019, SARS-CoV-2 emerged in Hubei, China, and quickly ramped to the coronavirus disease 2019 (COVID-19) pandemic.^{8, 9} The clinical outcomes of COVID-19 range from non-symptomatic, mild to severe respiratory tract infections, influenza-like illness, to lung injuries, organ failure, and death.¹⁰ To date, SARS-CoV-2 has spread all over the world and is the most severe pandemic in recent history. As of May 3^rd, 2022, 511 million cases and 6.23 million deaths have been reported worldwide, among which United States has 80.5 million cases and 986,298 deaths.¹¹

Given the devastating impact of COVID19 on social life, public health, and global economy, researchers around the globe are working relentlessly to develop countermeasures. This effort has led to the development of vaccines and antiviral drugs in record-breaking speed.^{12, 13} Vaccine mainly targets the viral surface spike protein and rely on the production of antibodies to block the viral entry through inhibiting the interaction between the viral spike protein and the host cell angiotensin converting enzyme 2 (ACE2) receptor.¹⁴ Three vaccines received FDA approval including two mRNA vaccines from Pfizer/BioNTech (Comirnaty) and Moderna (Spikevax), and one adenovirus-based vaccine from Johnson & Johnson/Janssen. In addition, several vaccines from China and Russia have been approved by the World Health Organization.¹⁵

For small molecule antivirals, major progress has been made in targeting the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp), the main protease (M^pro or 3CL^pro), and the papain-like protease (PL^pro).^{16, 17} The first RdRp inhibitor, remdesivir (1) (Figure 1A), was identified from a drug repurposing approach, and approved for the treatment of severe SARS-CoV-2 infection by intravenous (i.v.) administration.¹⁸ Remdesivir acts as a chain terminator during viral RNA synthesis.¹⁹ Similarly, the second RdRp inhibitor molnupiravir (2) (Figure 1A) was originally developed as an influenza antiviral and was later shown to have broad-spectrum antiviral activity against several viruses including SARS-CoV-2.^{20, 21} Molnupiravir (2) is a mutagen, and when incorporated into the RNA chain, it increases the mutation rate of the virus.²² Molnupiravir (2) is a prodrug and has the advantage of oral administration.²³ The main protease inhibitor Paxlovid developed by Pfizer is a combination of nirmatrelvir (3) (Figure 1A) and ritonavir.¹³ Nirmatrelvir (3) is a M^pro inhibitor, and ritonavir is included as a boosting agent to increase the half-life of nirmatrelvir. A similar approach that was explored in the HIV drug combination Kaletra (lopinavir+ritonavir). Ritonavir is an inhibitor of cytochrome P450 3A4 (CYP3A4) and coadministration of ritonavir is required to increase the in vivo concentration of nirmatrelvir (3) to the target therapeutic range.

Figure 1.

Chemical structures of FDA-approved COVID-19 antiviral drugs (A) and the schematic representation of the SARS-CoV and SARS-CoV-2 Open Reading Frame (B), the polyprotein replicase (C), and the recognition motifs of PL^pro (D). The genome contains two open reading frames, ORF1a and ORF1b, which are directly translated into polyprotein pp1a and pp1ab due to the ribosomal frameshift between the two ORFs. pp1a contains 11 NSPs and pp1ab contains 16 NSPs. The PL^pro is located within the NSP3. The polyproteins are processed into functional NSP units through cleavage by PL^pro and M^pro, and the cleavage sites of PL^pro are shown in (C). The substrate amino acid sequence alignment of P4-P1’ recognized by PL^pro is shown in (D).

The approval of vaccines and RdRp and M^pro inhibitors are encouraging signs to combat the COVID-19 pandemic and possibly return to the pre-pandemic normalcy.²⁴ However, the emergence of SARS-CoV-2 variants of concern (VOC) and variants of interests (VOI) poses a pressing need for additional vaccines and antiviral drugs.²⁵ Multiple studies have shown the reduced efficacy of vaccines against Omicron VOC.^{26, 27} Drug resistant mutations have been evolved against remdesivir (1) in cell culture through serial passage experiments^{28, 29} as well as in an immunocompromised patient.³⁰ In addition, the therapeutic benefits of remdesivir (1) are still under debate from several clinical trials.^{31, 32} Molnupiravir (2) has the potential risk of inducing mutations in the host, which is pending validation.^{33, 34} Molnupiravir (2) was shown to be positive in the Ames test,³⁵ which is a standard assay to measure mutagenic potential of drug candidates in bacteria. NHC (β-d-N⁴-hydroxycytidine), the active metabolite of molnupiravir (2), displayed host mutational activity in mammalian cell culture.³⁴ Multiple mutations have been identified in M^pro among the SARS-CoV-2 VOC and VOI including the Omicron M^pro P132H mutant.³⁶ Although the currently identified M^pro mutants remain sensitive to nirmatrelvir (3),^36–38 the scientific community is on high alert for future mutations such as H172Y and S144A that might lead to drug resistance.³⁹ Genetic barrier to resistance for protease inhibitors is generally moderate to low as shown by HIV and HCV protease inhibitors.⁴⁰ Resistance to Paxlovid is expected to rise with the increasing prescription. In addition, nirmatrelvir (3) is used in combination with ritonavir in clinics to prolong its half-life. Ritonavir is a potent inhibitor of the CYP3A4 isoenzyme and thus poses the risk of drug-drug interactions.⁴¹ As such, additional antivirals with a novel mechanism of action are clearly needed to combat emerging variants and drug resistant viruses. In this regard, the SARS-CoV-2 PL^pro stands out as one of the next in line high-profile drug targets.

PL^pro and the M^pro are the two essential proteases encoded by the SARS-CoV-2 genome. Both PL^pro and M^pro cleave the peptide bonds in the viral polyprotein to release functional non-structural proteins (NSPs) for viral transcription and replication. In addition, PL^pro is involved in antagonizing the host immune response upon viral infection. PL^pro has deubiquitinating and deISGylating activities, and removes ubiquitin and ISG15 modifications from host proteins, leading to suppression of innate immune response and promotion of viral replication.^42–44 The deubiquitinating and deISGylating activities of PL^pro are indispensable in antagonizing host immune response.^{45, 46} Recent studies showed that SARS-CoV-2 infection of human macrophages triggers the release of extracellular free ISG15 through the viral PL^pro, leading to the subsequent secretion of proinflammatory cytokines and chemokines, which recapitulates the cytokine storm of COVID-19.^{47, 48} This finding suggests that inhibiting the PL^pro activity might alleviate the hyper-inflammation in COVID patients. Thus, targeting PL^pro is expected to not only suppress viral replication but also restore antiviral immunity in the host.⁴⁵

There are two types of PL^pros, PL1^pro and PL2^pro.^{49, 50} The HCoV-229E, HCoV-NL63, HCoV-HKU1, and HCoV-OC43 encode both PL1^pro and PL2^pro. PL1^pro and PL2^pro have distinct substrate specificities in different coronaviruses.⁵¹ In contrast, SARS-CoV, MERS-CoV, and SARS-CoV-2 comprise only one functional PL2^pro.

PL^pro is part of the nsp3, a 215-KDa multidomain viral protein. SARS-CoV-2 PL^pro specifically recognizes a consensus cleavage motif, LXGG↓(A/K/X), which is present in between nsp1/2, nsp2/3 and nsp3/4 at the viral polyprotein as well as the C-terminal sequences of ubiquitin and ISG15 with an isopeptide bond (Figure 1B–D).

The SARS-CoV-2 PL^pro contains four domains: the thumb, palm, zinc-finger domain, and a N-terminal ubiquitin-like domain (Figure 2). The catalytic triad consists of Cys111, His272, and Asp286, which are located at the interface of the palm and thumb domains. The zinc finger motif comprises four cysteines coordinating with a zinc ion and is vital for the structural integrity and the protease activity of PL^pro. The flexible BL2 loop undergoes conformational changes from open to closed upon substrate binding (Figure 2A).⁵² This site is also the drug-binding site for GRL0617 (4) and its analogues.¹⁶ The X-ray crystal structures for the apo SARS-CoV-2 PL^pro, drug-bound form,^52–55 and complex forms with ubiquitin (Figure 2A) and ISG15 (Figure 2B) have been solved,⁵⁶ paving the way for structure-based drug design and the understanding of the virology of PL^pro.

Figure 2.

X-ray crystal structures of SARS-CoV-2 PL^pro. (A) X-ray crystal structure of SARS-CoV-2 PL^pro C111S mutant with K48-linked Ub2 (PDB: 7RBR). The BL2 loop is colored in magenta. (B) X-ray crystal structure of SARS-CoV-2 PL^pro C111S mutant with human ISG15 (PDB: 7RBS).⁵⁶

SARS-CoV-2 PL^pro shares a sequence identity of 82.9% with SARS-CoV PL^pro and to a lesser extent of 32.9% identify with the MERS-CoV PL^pro. Despite the high sequence similarity, SARS-CoV-2 PL^pro has enhanced deISGylating activity and reduced deubiquitinating activity compared to SARS-CoV PL^pro.^{45, 46, 57} PL^pro is a conserved drug target among SARS-CoV-2 variants (Figure 3). Although mutations have been identified, top high frequency mutations are all located distal from the drug binding site (Figure 3C). Nonetheless, it remains to be experimentally validated whether these mutations will alter drug sensitivity. In addition, resistance might emerge under drug selection pressure.

Figure 3.

Analysis of SARS-CoV-2 PL^pro mutations. Based on the data retrieved from GISAID (www.gisaid.org/epiflu-applications/covsurver-mutations-app), 2,487,047 sequences that contains mutations on PL^pro have been identified, which fall into 5,754 different types of mutations on various positions of PL^pro. All numbers shown are accurate as of Jan 25, 2022. (A) Cumulative frequency of SARS-CoV-2 PL^pro mutants. (B) Top 30 most common SARS-CoV-2 PL^pro mutants. Among these mutants, A145 has most frequent mutation to D with 1,131,252 occurrences (99.8% on 145); P77L with 372,993 occurrences (95.7% on 77); K232Q with 117,247 occurrences (99.1% on 232); V187A with 87,861 occurrences (97.9% on 187); and K92N with 47,110 occurrences (98.2%). (C) Mapping of top six SARS-CoV-2 PL^pro mutants to the X-ray crystal structure of PL^pro in complex with GRL0617 (4) (PDB: 7JRN). The residues are shown as spheres. The BL2 loop in the drug binding site is colored in marine, and the drug GRL0617 (4) is colored in magenta.

The knowledge accumulated through studying the SARS-CoV PL^pro provides the foundation for the understanding of the virology of SARS-CoV-2 PL^pro and the development of SARS-CoV-2 PL^pro inhibitors. For excellent reviews of the structure, function, and inhibition of SARS-CoV PL^pro, please refer to previous publications.^{16, 58–62} This perspective covers recent advances in the development of SARS-CoV-2 PL^pro inhibitors and their mechanism of action. We also discuss the knowledge gaps that need to be filled to advance PL^pro inhibitors to clinic.

It is not the objective of this perspective to enumerate all SARS-CoV-2 PL^pro inhibitors reported in the literature, instead the focus is on highlighting several well-characterized examples. Non-specific PL^pro inhibitors will also be discussed with the intention to alert the scientific community.

2. SARS-CoV-2 PL^pro ASSAYS

Vigorous pharmacological characterization is vital in triaging non-specific inhibitors at the early stage and prioritizing hits with translational potential for further development. For this, we provide a brief introduction of the commonly used assays for the pharmacological characterization of PL^pro inhibitors (Figure 4A).

Figure 4.

SARS-CoV-2 PL^pro assays. (A) General flow chart for the pharmacological characterization of PL^pro inhibitors. (B) Assay principle for the FRET-based enzymatic assay. (C) Assay principle for the cell based FlipGFP PL^pro assay. (D) Assay principle for the Protease-Glo luciferase PL^pro assay.

The gold standard assay for protease is the FRET (fluorescence resonance energy transfer)-based enzymatic assay, which is typically used as a primary assay for compound testing. In the FRET assay, a peptide corresponding to the protease substrate is designed with a fluorophore donor and a quencher at the two ends (Figure 4B). Upon cleavage by the protease, an increase in fluorescence signal is observed. However, the enzymatic assay condition varies among different labs in terms of enzyme concentration, FRET substrate sequence, pH, the addition of detergent (to rule out aggregates), bovine serum albumin (to rule out non-specific hydrophobic interactions) and reducing reagent (to prevent non-specific modification of catalytic Cys111). For this reason, the IC₅₀ values from different studies should be interpreted with caution and not be used for direct comparison. Instead, positive controls such as GRL0617 (4) need to be included as a refence to normalize the results. The assay guidance manual compiled by Eli Lilly & Company and the National Center for Advancing Translational Sciences offer detailed guidance for assay optimization, which might help limit the variations between individual labs.⁶³ In addition, counter screening against unrelated cysteine proteases should be conducted to rule out non-specific inhibitors. Furthermore, compounds that either quench the fluorophore or have overlapping absorbance/emission with the fluorophore will lead to false positive/negative results.

Our studies have shown that reducing reagents such as dithiothreitol (DTT) or glutathione are essential in the FRET enzymatic buffer to rule out promiscuous compounds that have non-specific inhibition against cystine proteases. Our recent studies of validation and invalidation of reported M^pro and PL^pro inhibitors demonstrated that the FRET IC₅₀ values obtained in the absence of reducing reagent DTT had poor correlation with the antiviral activity.^64–66 We therefore urge the scientific community to be cautious in interpreting the PL^pro assay IC₅₀ values obtained in the absence of reducing reagent.

Several binding assays are also commonly used to determine the binding affinity between inhibitors and the PL^pro, the thermal shift assay,⁵⁴ surface plasma resonance (SPR) assay⁶⁷ and isothermal titration calorimetry (ITC)^{67, 68}. Thermal shift assay measures protein stability, and ligand binding typically leads to the increase of the melting temperature T_m. Nevertheless, decrease in protein stability is also observed for certain ligand-protein interactions. Compared to the thermal shift assay, SPR is more quantitative and binding kinetics k_on, k_off, and K_D can be derived from the binding curves. ITC can determine the thermodynamic binding parameters ΔG, ΔH, and ΔS in a single experiment without a need to modify the protein. To gain molecular level understanding of the PL^pro-inhibitor interactions, co-crystal structure needs to be solved.

It is expected that the cell free enzymatic assay or binding assay results can be used to faithfully predict the cellular antiviral activity. However, SARS-CoV-2 is a biological safety level 3 (BSL-3) pathogen, which limits the number of compounds to be tested in the antiviral assay given the paucity of the resources. In this regard, there is a need for cell-based protease assay to help predict the antiviral activity at the BSL-1/2 setting. The cell-based protease assay not only reveals intracellular target engagement but also can rule out compounds that are cell membrane impermeable or cytotoxic. The FlipGFP and Protease-Glo luciferase assays are two representative cell-based protease assays that have been applied for the screening and validation of SARS-CoV-2 PL^pro inhibitors.^{54, 69} In the FlipGFP assay, cells are transfected with two plasmids, one expressing the PL^pro and another expressing the GFP reporter (Figure 4C).^70–72 The reporter plasmid encodes three proteins including the GFP β1–9 template, the β10–11 fragment, and the mCherry. The β10–11 fragment was restrained in the parallel orientation through the K5/E5 coiled coil, therefore cannot associate with the β1–9 template. Upon cleavage of the PL^pro substrate linker, β10 and β11 become antiparallel and can associate with the β1–9 template, leading to the restoration of the GFP signal. mCherry serves as an internal control to normalize the transfection efficiency. As such, the GFP/mCherry ratio correlates to the enzymatic activity of PL^pro. Results from us as well as others have shown that the FlipGFP assay is a valuable assay in characterizing the cellular M^pro and PL^pro inhibition without the need of the infectious SARS-CoV-2 virus.^{54, 64, 69, 70, 72, 73} A positive correlation between the FlipGFP IC₅₀ values and the antiviral EC values was observed for the PL^pro inhibitors,⁵⁴ 50 suggesting FlipGFP assay can be used as a surrogate assay to prioritize lead compounds for antiviral testing.

The Protease-Glo luciferase assay is designed in an analogous way as the FlipGFP assay in which the luciferase activity depends on the cleavage of the substrate linker by the protease.⁶⁴ Specifically, the firefly luciferase is engineered with a protease substrate cleavage sequence (Figure 4D). Before cleavage, firefly luciferase is in the permuted circular inactive conformation. Upon protease cleavage, a conformational change leads to the association of the two domains and the restoration of the luciferase activity. The Protease-Glo luciferase assay can be performed either in live cells or in cell lysates.^{69, 74, 75} As the readout is luminescence, the Protease-Glo luciferase assay can help rule out compounds that have fluorescence interference properties. Other cell-based assays including the GFP ER translocation assay, bioluminescence resonance energy transfer (BRET) assay, and the cell cytotoxicity assay can be similarly engineered for PL^pro.^75–77

3. SARS-CoV-2 PL^pro INHIBITORS

We group SARS-CoV-2 PL^pro inhibitors into two categories, the non-covalent inhibitors and covalent inhibitors. The non-covalent inhibitors are further divided into GRL0617 (4) analogues and non-GRL0617 inhibitors (Table 1).

Table 1.

SARS-CoV-2 PL^pro inhibitors.

Structure	Enzymatic inhibition IC50 (μM)	Antiviral EC50/CC50 (μM)	Structure	Enzymatic inhibition IC50 (μM)	Antiviral EC50/CC50 (μM)
Non-covalent SARS-CoV-2 PLpro inhibitors - GRL0617 analogues
	1.39 ± 0.2680 1.78981	3.18 ± 0.71/~50080 32.6481 > 2067		0.59 ± 0.0467 Kd = 0.963 μM (SPR)	N.T.
	0.56 ± 0.0367 Kd = 0.372 μM (SPR) PDB:7LBS	1.2 ± 0.2		0.39 ± 0.0567 Kd = 0.235 μM (SPR)	1.4 ± 0.1
	0.44 ± 0.0579 Kd = 2.60 ± 0.39 μM (SPR)	0.18 ± 0.10 CC50 > 10		2.69 ± 0.3479 PDB: 7E35	N.T.
	7.29 ± 1.03	N.T.		6.67 ± 0.55	N.T.
	0.67 ± 0.0854 PDB: 7SDR	6.62 ± 1.31 (Vero) 7.90 ± 2.40 (Caco-2 hACE2)		0.67 ± 0.1454 PDB: 7SQE	8.31 ± 2.68 (Vero) 11.99 ± 4.52 (Caca-2 hACE2)
	5.1 ± 0.752 GRL0617 (4) (2.3 ± 0.2) PDB: 7JIT	Not active		6.4 ± 0.652 PDB: 7JIV, 7JIW	Not active
	7.0 ± 0.652	Not active		12.7 ± 1.352	5.2 ± 4.2 (Vero E6)
	0.8153	< 11 μM		11 ± 368 GRL0617 (4) (2.1 ± 0.2)	N.T.
Non-covalent SARS-CoV-2 PLpro inhibitors – Non-GRL0617 analogues
	3.76 ± 1.1355 (ISG15-Rh substrate) PDB: 7OFU	0.13 (qRTPCR) 10 (CPE)		6.68 ± 1.2055 (ISG15-Rh substrate) PDB: 7OFS	1 (qRTPCR) Not active (CPE)
	3.99 ± 1.3355 (ISG15-Rh substrate) PDB: 7OFT	Not active (CPE)		1.66 (RLRGG-AMC)82 1.46 (ISG15-AMC) PDB: 7NT4	A549/ACE 2 EC50 = 86 nM, CC50 = 3.1 μM SI = 36 Vero EC50 = 64 nM, CC50 = 3.4 μM SI = 53
	0.5 (TAP-nsp123) 1.0 (TAP-nap23) 0.1 (de-ISGylation) 72 ± 1268	2.13 ± 1.16/35.5 ± 9.45 (Vero E6)		2.47 ± 0.4680 Assay condition: 50 mM HEPES, pH 7.5, 2 mM DTT PDB: 7D7L	0.17 ± 0.02/~40080
	5.63 ± 1.4580 1.33681	0.70 ± 0.09/>30080 >20081		2.21 ± 0.1080	2.26 ± 0.11/>20080
	0.5981	8.1581		1.57181	>20081
	0.2681	N.T.		0.5381	20
	0.3981	cytotoxic		0.6181	cytotoxic
	1.65 ± 0.1383 Kd = 61.0 ± 12.1 μM (BLI assay)	N.T.		7.24 ± 0.6883 (FP assay) Kd = 4.6 ± 0.29 μM (BLI assay)	N.T.
	1.784	Not active		2.284	Not active
	3.184 Mpro (IC50 = 66 μM)	Not active		0.56 ± 0.1685	N.T.
	13.1686 15.68 (DBU)	N.T. CC50 > 100 μM (Vero E6)		16.0886 17.51 (DBU)	N.T. CC50 > 100 μM (Vero E6)
Covalent SARS-CoV-2 PLpro inhibitors
	Near complete inhibition at 100 μM57 PDB: 6WUU	N.T.		Near complete inhibition at 100 μM57 PDB: 6WX4	N.T.
	0.09487 kinact/Ki=10,0 00 M−1S−1	1.1		0.23087 kinact/Ki=14,00 0 M−1S−1	Not active
	0.09887 kinact/Ki=4,80 0 M−1S−1	Cytotoxic		5.487 kinact/Ki=103 M−1S−1	34
	8.087	Not active		> 20087	Cytotoxic
	7.40 ± 0.3788	N.T.		8.63 ± 0.5588	N.T.
	0.6789 Mpro (IC50 = 1.72 μM)	0.32 (UC1074) 1.37 (UC1075)
Ebselen analogues
	2.02 ± 1.0290	N.T.		0.236 ± 0.10790	N.T.
	0.256 ± 0.03590	N.T.		0.339 ± 0.10990	N.T.
	0.263 ± 0.12190	N.T.		PLpro IC50 = 1.255 ± 0.095 μM91 Mpro IC50 = 25.69 ± 2.64 nM	N.T.
	PLpro IC50 = 0.578 ± 0.040 μM Mpro 91 IC50 = 49.55 ± 2.95 nM	N.T.		PLpro IC50 = 1.885 ± 0.098 μM Mpro 91 IC50 = 27.95 ± 5.10 nM	N.T.
	PLpro IC50 = 0.990 ± 0.058 μM Mpro 91 IC50 = 52.50 ± 4.51 nM	N.T.		PLpro IC50 = 2.067 ± 0.078 μM Mpro 91 IC50 = 15.24 ± 4.58 nM	N.T.
	PLpro IC50 = 1.038 ± 0.083 μM Mpro 91 IC50 = 37.81 ± 3.28 nM	N.T.		PLpro IC50 = 1.288 ± 0.052 μM Mpro 91 IC50 = 27.37 ± 2.35 nM	N.T.
Zinc ejectors
	7.52 ± 2.13100	N.T.		N.T.	N.T.
	N.T.	N.T.		N.T.	N.T.

Reactive warheads are colored in red; N.T. = not tested.

3.1.1. Non-covalent SARS-CoV-2 PL^pro inhibitors – GRL0617 analogues

The naphthalene containing GRL0617 (4) was a well-characterized SARS-CoV PL^pro inhibitor. It was originally developed through lead optimization based on a high-throughput screening hit.⁶² Several follow up studies have been conducted with the aim of improving the potency of enzymatic inhibition and antiviral activity as well as pharmacokinetic (PK) properties. However, no significant improvement has been achieved.^{60, 61} As the SARS-CoV-2 PL^pro is 83% identical and 90% similar to SARS-CoV PL^pro, GRL0617 (4) became a top candidate as the SARS-CoV-2 PL^pro inhibitor. Several groups independently showed the potent inhibition of SARS-CoV-2 PL^pro by GRL0617 (4).^{45, 53, 54, 68, 78} However, the moderate to weak antiviral activity of GRL0617 (4) prevents it from advancing to animal model studies.^{54, 67} Since the beginning of the COVID-19 pandemic, encouraging progress has been made in re-designing GRL0617 analogues as potent SARS-CoV-2 PL^pro inhibitors. The X-ray crystal structure of SARS-CoV-2 PL^pro in complex with GRL0617 (4) has also been solved by multiple groups,^{53, 54, 56, 57, 78} paving the way for structure-based lead optimization.

A recent elegant structure-based drug design led to the discovery of potent PL^pro inhibitors with favorable PK properties.⁶⁷ One of the major contributions of this study is the conversion of naphthalene to 2-phenylthiophene, which leads to improved PK properties. In addition, the thiophene substitution extends further into the BL2 groove (Figure 5A), and when coupled with additional substitutions on the aniline amine to engage interaction with Glu167 (Figure 5B, 5C), multiple nanomolar PL^pro inhibitors have been identified. Among the more than 100 analogues tested, compounds ZN-3–80 (5), XR8–24 (6), and XR8–23 (7) were the most potent ones with IC₅₀ values of 0.59, 0.56, and 0.39 μM, respectively (Table 1). Compounds 6 and 7 also showed a significantly improved antiviral activity against SARS-CoV-2 in A549-hACE2 cells with EC₅₀ values of 1.2 and 1.4 μM, respectively. In comparison, GRL0617 (4) was not active in the virus yield reduction antiviral assay (EC₅₀ > 20 μM). The complex structure with compound XR8–24 (6) (PDB: 7LBS) revealed several key hydrogen bonds/electrostatic interactions including the water mediated hydrogen bonds between the pyrrolidine NH⁺ and the main chain carbonyl oxygen of Tyr264 (not shown), the electrostatic interaction between the NH²⁺ from the azetidine ring and side chain carboxylate from Glu167 (Figure 5C), and the hydrogen bond between the amide NH from compound XR8–24 (6) with the Asp164 side chain carboxylate. When dosed in male C57BL/6 mice at 50 mg/kg by intraperitoneal injection (i.p.), compound XR8–23 (7) and XR8–24 (6) reached the C_max of 6130 ng/mL and 6403 ng/mL, respectively, indicating favorable in vivo bioavailability. Further optimization might lead to candidates that are suitable for the in vivo antiviral efficacy study.

Figure 5.

GRL0617-based SARS-CoV-2 PL^pro inhibitors. (A) X-ray crystal structure of SARS-CoV-2 PL^pro with GRL0617 (4) (PDB: 7JRN). (B) Design hypothesis for the 2-phenylthiophene series of PL^pro inhibitors based on GRL0617 (4). (C) X-ray crystal structure of SARS-CoV-2 PL^pro with compound XR8–24 (6) (PDB: 7LBS). (D) X-ray crystal structure of SARS-CoV-2 PL^pro with compound 9 (PDB: 7E35). (E) X-ray crystal structure of SARS-CoV-2 PL^pro with Jun9-72-2 (12) (PDB: 7SDR). (F) X-ray crystal structure of SARS-CoV-2 PL^pro with Jun9-84-3 (13) (PDB: 7SQE). Panels A and C were adapted with permission from Shen, Z.; Ratia, K.; Cooper, L.; Kong, D.; Lee, H.; Kwon, Y.; Li, Y.; Alqarni, S.; Huang, F.; Dubrovskyi, O.; Rong, L.; Thatcher, G. R. J.; Xiong, R. Design of SARS-CoV-2 PL^pro inhibitors for COVID-19 antiviral therapy leveraging binding cooperativity. J. Med. Chem. 2022, 65, 2940–2955.⁶⁷ copyright 2022, American Chemical Society).

In another study, Shan et al. reported the structure-based design of SARS-CoV-2 PL^pro inhibitors based on the GRL0617 scaffold.⁷⁹ The most potent lead compound 8 inhibited PL^pro and SARS-CoV-2 viral replication with IC₅₀ of 0.44 μM and EC₅₀ of 0.18 μM, respectively (Table 1). The K_d was 2.60 μM for compound 8 in the SPR assay, compared to the K_d of 10.79 μM for GRL0617 (4). In the counter screening against 10 deubiquitinases (DUBs) or DUB-like proteases, compound 8 was highly selective towards PL^pro and did not show significant inhibition towards a panel of host DUBs and DUB-like proteases. The X-ray crystal structure of PL^pro with an analogue 9 showed that compound 9 binds to PL^pro in a similar mode as GRL0617 (4) (Figure 5D). It is noted that compound 9 adapts different binding poses in the two monomers (Figure 5D).

Our group recently conducted a high-throughput screening against SARS-CoV-2 PL^pro using the FRET-based enzymatic assay.⁵⁴ Two closely related compounds Jun9-13-7 (10) and Jun9-13-9 (11) were identified as potent hits with IC₅₀ values of 7.9 and 6.67 μM, respectively (Table 1). Subsequent lead optimization led to the discovery of several compounds with IC₅₀ values in the sub-micromolar range including Jun9-72-2 (12) (IC₅₀ = 0.67 ± 0.08 μM) and Jun9-84-3 (13) (IC₅₀ = 0.67 ± 0.14 μM). In the cell-based FlipGFP reporter assay, Jun9-72-2 (12) and Jun9-84-3 (13) showed dose dependent inhibition with EC₅₀ values of 7.93 and 17.07 μM, respectively, suggesting both compounds are cell membrane permeable and can inhibit the intracellular protease activity of PL^pro. In agreement, both compounds had potent antiviral activity against SARS-CoV-2 in Vero E6 and Caco2-hACE2 cells (Table 1). Significantly, there is a positive correlation between the FlipGFP assay results and the antiviral assay results, validating the FlipGFP as a surrogate assay for the prediction of the antiviral activity of PL^pro inhibitors.⁵⁴ In the X-ray crystal structure of PL^pro with Jun9-72-2 (12) (PDB: 7SDR), the tertiary NH⁺ in the linker electrostatically interacts with the Asp164 carboxylate group (Figure 5E). The X-ray crystal structure of PL^pro with Jun9-84-3 (13) (PDB: 7SQE) revealed an additional hydrogen bond between the indole NH with the Glu167 side chain carboxylate (Figure 5F).

Additional GRL0617 analogues including 14, 15, 16, 17, 18, and 19 have been reported as SARS-CoV-2 PL^pro inhibitors (Table 1),^{52, 53, 68} however, no significant improvement has been made.

3.1.2. Non-covalent SARS-CoV-2 PL^pro inhibitors – non-GRL0617 inhibitors

Three phenolic compounds including methyl 3, 4-dihydroxybenzoate (HE9, 20), 4-(2-hydroxyethyl)phenol (YRL, 21), and 4-hydroxybenzaldehyde (HBA, 22) were identified as allosteric SARS-CoV-2 PL^pro inhibitors through a high-throughput X-ray crystallization.⁵⁵ The screened library contains 500 compounds from the ICCBS (International Center for Chemical and Biological Sciences) Molecular Bank. Interestingly, HE9 (20), YRL (21), and HBA (22) all bind to the ISG15/Ub-S2 binding site of PL^pro (Figure 6A), an allosteric binding pocket that has not been explored for drug design. The allosteric binding site is located about 30 Å away from the active site residue Cys111. The superimposition structures of PL^pro+inhibitors and the PL^pro+ISG15 indicate that these compounds might compete with ISG15 for the same binding site. As expected, all three compounds inhibited the deISGylating activity of PL^pro with IC₅₀ values of 3.76 ± 1.13 μM (20), 6.68 ± 1.20 μM (21), and 3.99 ± 1.33 μM (22). However, it remains unknown whether these compounds can inhibit the hydrolysis of viral polyprotein by PL^pro. HE9 (20) and YRL (21) inhibited SARS-CoV-2 replication in Vero E6 cells in the qRT-PCR assay with EC₅₀ values of 0.13 μM and 1 μM, respectively. However, the antiviral assay results for HBA (22) were not conclusive. In the cytopathic effect (CPE) assay, HE9 (20) had an EC₅₀ of 10.37 μM. In contrast, YRL (21) failed to show inhibition in the CPE assay. The discrepancy of antiviral activity in different assays suggests further validation is needed. Furthermore, these results raise the question of whether inhibiting the deISGlyation activity of PL^pro alone is sufficient for the inhibition of viral replication.

Figure 6.

Non-covalent SARS-CoV-2 PL^pro inhibitors that do not share structural similarity with GRL0617 (4). (A) X-ray crystal structures of SARS-CoV-2 PL^pro in complex with fragments HE9 (20), YRL (21), and HBA (22). (B) X-ray crystal structure of SARS-CoV-2 PL^pro in complex with proflavine (25) showing three molecules bind near the BL2 loop (PDB: 7NT4). Two molecules stack on top of each other and fit in the GRL0617 (4) binding pocket, and a third molecule binds at the backside of the BL2 loop. (C) X-ray crystal structures of SARS-CoV-2 PL^pro in complex with YM155 (27) (PDB: 7D7L). YM155 (27) binds three different sites located at the zinc-finger domain, thumb domain, and the substrate-binding pocket. Detailed interactions between YM155 and the BL2 loop region residues were shown on the right side.

A drug repurposing screening by Napolitano et al. identified acriflavine (23) as a potent inhibitor of SARS-CoV-2 PL^pro with in vivo antiviral efficacy.⁸² Acriflavine (23) is a mixture of trypaflavine (24) and proflavine (25).⁸² Acriflavine (23) inhibited PL^pro with IC₅₀ values of 1.66 and 1.46 μM when RLRGG-AMC and ISG15-AMC were used as substrates, respectively (Table 1). Acriflavine (23) also inhibited the deubiquitylating activity of PL^pro in gel-based assay, thus ruling out the potential fluorescence interference effect of acriflavine (23). In addition, acriflavine (23) did not inhibit M^pro. The X-ray crystal structure of PL^pro with proflavine (25) was determined (PDB: 7NT4), revealing two molecules of proflavine (25) bind to the S3–S5 pockets of PL^pro simultaneously (Figure 6B). The BL2 loop folds inward towards the substrate-recognition cleft, similar to the binding mode of GRL0617 (4). A third proflavine (25) molecule is located at the surface of the protein on the opposite side of the BL2 loop. Acriflavine (23) inhibited SARS-CoV-2 replication in A549-ACE2 and Vero cells with EC₅₀ values of 86 and 64 nM, respectively. However, the selectivity index was low (A549-ACE2 IS = 36; Vero SI = 53). The antiviral activity was further confirmed in human airway epithelial (HAE) cells. Acriflavine (23) also showed potent inhibition against MERS-CoV (IC₅₀ = 21 nM, SI = 162) and HCoV-OC43 (IC₅₀ = 105 nM, SI = 27), but not the alphacoronaviruses including feline infectious peritonitis virus (FIPV) and HCoV-NL63. In the in vivo SARS-CoV-2 infection model with K18-ACE2 mice, acriflavine (23) treatment by either i.p. or intramuscular (i.m.) injection significantly lowered the viral titers in the brain and the lung.

6-thioguanine (6-TG, 26) was previously reported as an inhibitor for the SARS-CoV and MERS-CoV,^{92, 93} therefore, it was hypothesized that it might also inhibit the SARS-CoV-2 PL^pro. Swaim et al. recently demonstrated that 6-TG (26) is a potent inhibitor for SARS-CoV-2 in Vero E6 cells with an EC₅₀ of 2.13 μM (Table 1).⁹⁴ Next, to confirm the intracellular inhibition of PL^pro by 6-TG (26), a TAP-tagged pp1a protein consisting of nsp1, 2, and 3 was expressed. As expected, TAP-nsp1 was the major product due to the self-cleavage pf pp1a polyprotein by PL^pro. Treatment with 6-TG (26) led to dose-dependent inhibition of the cleavage with an IC₅₀ of approximately 0.5 μM. In addition, 6-TG (26) showed potent inhibition of the deISGlyation activity of PL^pro in HEK293T cells. No in vitro enzymatic assay was performed. In addition, it was proposed that 6-TG (26) might have a secondary mechanism of action by inhibiting the viral RNA synthesis. Nonetheless, in our recently hit validation study, 6-TG (26) did not show inhibition against SARS-CoV-2 PL^pro in the enzymatic assay (IC₅₀ > 50 μM), had no binding to PL^pro in the thermal shift assay, and did not inhibit the intracellular PL^pro activity in the FlipGFP assay.⁶⁹ Therefore, the antiviral activity of 6-TG (26) may not arise from inhibiting the PL^pro.

Through screening a library of 6,000 compounds using the FRET-based enzymatic assay with the Arg-Leu-Arg-Gly-Gly-AMC substrate, Zhao et al. identified YM155 (27) (IC₅₀ = 2.47 ± 0.46 μM), cryptotanshinone (28) (IC₅₀ = 5.63 ± 1.45 μM), tanshinone I (29) (IC₅₀ = 2.21 ± 0.10 μM), and GRL0617 (4) (IC₅₀ = 1.39 ± 0.26 μM) as SARS-CoV-2 PL^pro inhibitors (Table 1).⁸⁰ All four compounds displayed potent antiviral activity against SARS-CoV-2 in Vero E6 cells with the most potent compound being YM155 (27) (EC₅₀ = 0.17 ± 0.02 μM, CC₅₀ ~ 400 μM). The structure of PL^pro in complex with YM155 (27) was solved by crystal soaking (PDB: 7D7L). Unexpectedly, YM155 (27) was found in three different binding sites including the orthosteric site, the thumb domain, and the zinc-finger domain (Figure 6C). The binding at the thumb domain is expected to inhibit the binding between PL^pro and ISG15. A conformational change was observed at the zinc-finger domain upon YM155 (27) binding, but the physiological relevant of this binding mode has not been validated.

Similarly, cryptotanshinone (28) (IC₅₀ = 1.34 μM), together with two other analogues dihydrotanshinone I (30) (IC₅₀ = 0.59 μM) and tanshinone IIA (31) (IC₅₀ = 1.57 μM), were shown as potent SARS-CoV-2 PL^pro inhibitors through a HTS (Table 1).⁸¹ In addition, four additional compounds, PKK1/Akt/Flt dual pathway inhibitor (32) (IC₅₀ = 0.26 μM), Ro 08–2750 (33) (IC₅₀ = 0.53 μM), Cdk4 inhibitor III (34) (IC₅₀ = 0.39 μM), and β-lapachone (35) (IC₅₀ = 0.61 μM) were also identified as potent PL^pro inhibitors (Table 1). Dihydrotanshinone I (30) inhibited SARS-CoV-2 with an EC₅₀ of 8.15 μM. Unexpectedly, cryptotanshinone (28) and tanshinone IIA (31) had no antiviral activity (EC₅₀ > 200 μM), despite their potent enzymatic inhibition. The antiviral result of cryptotanshinone (28) is also in controversy with the previous study which showed that cryptotanshinone (28) is a potent antiviral with an EC₅₀ of 0.7 μM.⁸⁰ Further validation is needed to test the antiviral activity of cryptotanshinone (28) against SARS-CoV-2 in multiple cell lines.

Xu et al. recently reported the discovery of tanshinone IIA sulfonate (36) and chloroxine (37) as SARS-CoV-2 PL^pro inhibitors from a drug repurposing screening.⁸³ Tanshinone IIA sulfonate (36) was identified in the fluorogenic assay using the ALKGG-AMC substrate with an IC₅₀ of 1.65 μM (Table 1). Chloroxine (37) was discovered in the fluorescence polarization-based assay using the fluorescein 5-isothiocyanate (FITC) labeled ISG15 with an IC₅₀ of 7.24 μM. Tanshinone IIA sulfonate (36) and chloroxine (37) also showed binding to PL^pro in the biolayer interferometry and thermal shift assays. The antiviral activity against SARS-CoV-2 was not reported.

We performed hit validations for YM155 (27), cryptotanshinone (28), tanshinone I (29), dihydrotanshinone I (30), and tanshinone IIA (31).⁶⁹ Our study found that YM155 (27) (IC₅₀ = 20.13 μM), cryptotanshinone (28) (IC₅₀ = 52.24 μM), tanshinone I (29) (IC₅₀ = 18.58 μM), dihydrotanshinone I (30) (IC₅₀ = 33.01 μM), and tanshinone IIA (31) (IC₅₀ = 15.30 μM) had much higher IC₅₀ values against SARS-CoV-2 PL^pro in the FRET assay compared to the previous reports. The intracellular PL^pro inhibition by YM155 (27) and cryptotanshinone (28) in the FlipGFP assay was not conclusive due to cell cytotoxicity, while tanshinone I (29), dihydrotanshinone I (30), and tanshinone IIA (31) had no intracellular PL^pro inhibition at non-toxic concentrations. Collectively, our results suggest that YM155 (27), cryptotanshinone (28), tanshinone I (29), dihydrotanshinone I (30), and tanshinone IIA (31) are weak PL^pro inhibitors and tanshinone I (29), dihydrotanshinone I (30), and tanshinone IIA (31) lack intracellular target engagement.

In agreement with our results, Brewitz et al. applied mass spectrometry assay to monitor PL^pro-mediated cleavage of the nsp 2/3 substrate.⁹⁵ Among the list of compounds tested, YM155 (27), tanshinone I (29), tanshinone IIA sulfonate sodium (36) were not active (IC₅₀ > 50 μM), while cryptotanshinone (28) showed moderate activity with an IC₅₀ of 19.4 μM.

Through virtual screening of a library of naphthoquinoidal compounds followed by enzymatic assay validation, Santos et al. identified three compounds 38, 39, and 40 as potent SARS-CoV-2 PL^pro inhibitors with IC₅₀ values of 1.7 μM, 2.2 μM, and 3.1 μM, respectively (Table 1).⁸⁴ Among the three hits, compound 40 had moderate inhibition against M^pro with an IC₅₀ of 66 μM, therefore it was considered as a dual inhibitor for further optimization. MD simulations predicted that compound 39 binds non-covalently to the S3 and S4 subsites in PL^pro. However, the detailed mechanism of action remains to be characterized. When tested in the antiviral assay against SARS-CoV-2 in two different cell lines Vero E6 and HeLa-ACE2, none of the identified M^pro and PL^pro inhibitors had antiviral activity, suggesting these naphthoquinoidal compounds might have off-target effects. It is noted that no reducing agent such as dithiothreitol (DTT) was added in the M^pro enzymatic assay, however, 0.1 mM DTT was included in the PL^pro assay. Therefore, the observed PL^pro inhibition might not be due to non-specific modification of the PL^pro C111 residue. Further validation studies are warranted to confirm their enzymatic inhibition.

Cho et al. reported SJB2–043 (41) as a SARS-CoV-2 PL^pro inhibitor with an apparent IC₅₀ of 0.56 μM.⁸⁵ However, no complete inhibition was achieved at high drug concentration. Therefore, it remains to be validated whether SJB2–043 (41) is a specific PL^pro inhibitor.

Commercial mouth rinses are known to inactivate SARS-CoV-2,^{96, 97} but the detailed mechanism remains elusive. Lewis et al. tested the active ingredients of mouth rinses against the SARS-CoV-2 M^pro and PL^pro.⁸⁶ Although none of the compounds were active against M^pro, two compounds, aloin A (42) and aloin B (43), inhibited PL^pro with IC₅₀ values of 13.16 and 16.08 μM, respectively in the enzymatic assay. Aloin A (42) and B (43) also inhibited the deubiquitinating activity of PL^pro with IC₅₀ values of 15.68 and 17.51 μM. Molecular dynamics simulations suggest that aloin A (42) and B (43) bind to the GRL0617 (4) binding site and mainly interact with Glu167, Tyr268, and Glu269.

3.2.1. Specific covalent PL^pro inhibitors

The cleavage of PL^pro substrate occurs after the second glycine in the Leu-X-Gly-Gly sequence.⁵⁷ As a result, the binding pockets for the S2 and S1 subsites are absent, which leaves the S4 and S3 subsites for inhibitor binding. Accordingly, to develop covalent inhibitor to react with the catalytic C111, a linker is needed to conjugate the S4/S3 pocket binder with a reactive warhead.^{53, 57}

A positional scanning was conducted to identify the optimal substrate of SARS-CoV-2 PL^pro.⁵⁷ A total of 19 natural and 109 nonproteinogenic amino acids were screened at each position. It was found that the P2 and P4 positions have high preference for glycine and hydrophobic residues, respectively, while the P3 position can tolerate both charged residues including Phe(guan), Dap, Dab, Arg, Lys, Orn, and hArg and hydrophobic residues including hTyr, Phe(F5), Cha, Met, Met(O), Met(O)₂, D-hPhe. Leveraging this information, two covalent inhibitors VIR250 (44) (Ac-Abu(Bth)-Dap-Gly-Gly-VME) and VIR251 (45) (Ac-hTyr-Dap-Gly-Gly-VME) were designed by incorporating the optimal P3 and P4 substitutions with the vinylmethyl ester (VME) reactive warhead (Table 1). VIR250 (44) and VIR251 (45) showed dose-dependent inhibition against both SARS-CoV-2 and SARS-CoV PL^pros, however the IC₅₀ values were not quantified. The X-ray crystal structures of SARS-CoV-2 PL^pro in complex with VIR250 (44) (PDB: 6WUU) and VIR251 (45) (PDB: 6WX4) were solved (Figure 7), revealing covalent thioether linkage of the C111 thiol and the β carbon of the vinyl group. Although no antiviral assay results were reported, this is an elegant rational design that led to the first covalent SARS-CoV-2 PL^pro inhibitors.

Figure 7.

X-ray crystal structures of SARS-CoV-2 PL^pro in complex with peptidomimetic covalent inhibitors VIR250 (44) (PDB: 6WUU) (A) and VIR251 (45) (PDB: 6WX4) (B).

Sanders et al. recently reported the rational design of the first-in-class drug-like covalent SARS-CoV-2 PL^pro inhibitors.⁸⁷ An N, N’-diacetylhydrazine linker was designed as a mimetic of the Gly-Gly to conjugate the GRL0617 methyl group with different reactive warheads. A series of commonly used cysteine reactive warheads including fumarate methyl ester, chloroacetamide, propiolamide, cyanoacetamide, and α-cyanoacrylamide have been exploited. Among the designed covalent PL^pro inhibitors, compounds 46 and 47 with the fumarate methyl ester, and compound 48 with the propiolamide showed significantly improved potency with IC₅₀ values of 0.094, 0.230, and 0.098 μM, respectively (Table 1). Compound 49 with the chloroacetamide and compound 50 with the cyanoacetamide were less active and the IC₅₀ values were 5.4 and 8.0 μM, respectively. In contrast, compound 51 with the α-cyanoacrylamide was not active (IC₅₀ > 200 μM). As expected, covalent protein adduct with inhibitors were observed for compounds 46, 47, 48, 49, and 50 in electrospray ionization (ESI) mass spectrometry. The X-ray crystal structure of PL^pro with compound 46 was solved at 3.10 Å resolution (PDB: not released), showing a covalent adduct between the C111 thiol and the C1 of compound 46. The N, N’-diacetylhydrazine linker forms four hydrogen bonds with Gly163 and Gly271, highlighting the importance of this rationally designed linker. In SARS-CoV-2 infected Vero E6 cells, compound 46 had an EC₅₀ of 1.1 μM, which is comparable to the potency of remdesivir (EC₅₀ = 0.74 μM). Surprisingly, compound 47 had insignificant cytoprotective effects, despite its potent enzymatic inhibition. Compound 48 was cytotoxic; therefore, its antiviral activity was not conclusive. Similar to GRL0617 (4), compound 46 also inhibited the deubiquitinating and the deISGylating activities with IC₅₀ values of 76 and 39 nM, respectively. Selectivity screening against a panel of DUBs showed that compound 46 is highly selective and no inhibition was observed at up to 30 μM. In vitro pharmacokinetic profiling showed that compound 46 is stable in human liver S9 and microsomes with T_1/2 of 60 and 50 mins, respectively. This study represents the first rational design of drug-like covalent PL^pro inhibitors with potent antiviral activity, and the X-ray crystal structure is invaluable in guiding the lead optimization.

Liu et al. reported the design of peptide-drug conjugates (PDCs) as covalent inhibitors of SARS-CoV-2 PL^pro.⁸⁸ The PDCs consist of GRL0617 and cyclic sulfonium-containing peptides derived from PL^pro substrate Leu-Arg-Gly-Gly (Table 1). The sulfonium serves as a warhead and is designed to react with the C111. Among the examined PDCs, EM-C (52) and EC-M (53) were the most potent against SARS-CoV-2 PL^pro with IC₅₀ values of 7.40 ± 0.37 and 8.63 ± 0.55 μM, respectively (Table 1). Both conjugates were cell membrane permeable and inhibited the deISGylating activity of PL^pro. In-gel digestion of the PL^pro+PDC mixture followed by MS/MS analysis confirmed that C111 is the enriched conjugation site. No antiviral assay results were presented. Although the results presented convincingly demonstrated the covalent labeling of PL^pro C111, it remains unknown about their binding mode. The EC-M (52) and EM-C (53) PDCs contain the GRL0617 and the Leu-Arg dipeptide sequence, both of which are S3 and S4 subsite binders. It is not clear why the design contains duplicate binding elements. The x-ray crystal structure might solve the puzzle.

A tryptophane containing dipeptide, compound 54, was recently reported as a dual inhibitor of SARS-CoV-2 M^pro and PL^pro.⁸⁹ Compound 54 inhibited M^pro and PL^pro with IC₅₀ values of 1.72 and 0.67 μM, respectively, while had no binding to the viral spike protein (K_D > 25 μM). In the antiviral assay, compound 54 inhibited two SARS-CoV-2 clinical isolates UC-1074 and UC-1075 with EC₅₀ values of 0.32 and 1.37 μM, respectively. Given the lack of structural similarities between M^pro and PL^pro, coupled with the high reactivity of α-chloroacetamide warhead in 54, it remains to be investigated whether the inhibition of M^pro and PL^pro by compound 54 is specific. Nevertheless, the potent antiviral activity of compound 54 is encouraging, which warrants further optimization.

3.2.2. Non-specific covalent PL^pro inhibitors – Ebselen analogues

Given the broad-spectrum antiviral activity of ebselen against several viruses, WeglarzTomczak- et al. explored ebselen and its analogues as SARS-CoV-2 PL^pro inhibitors.⁹⁰ Ebselen (55) inhibited PL^pro with an IC₅₀ of 2.02 ± 1.02 μM, and dialysis experiment showed that no enzymatic activity was recovered, suggesting irreversible inhibition. Subsequently, a library of analogues was designed, among which two ebselen derivatives 56 (IC₅₀ = 236 ± 107 nM) and 57 (IC₅₀ = 256 ±35 nM), and two diselenide orthologs 58 (IC₅₀ = 339 ±109 nM) and 59 (IC₅₀ = 263 ± 121 nM), had improved enzymatic inhibition against SARS-CoV-2 PL^pro compared to ebselen (55) (IC₅₀ = 2.02 ± 1.02 μM) (Table 1). In this study, 2 mM DTT was added in the enzymatic assay buffer. However, our previous studies showed that ebselen (55) only inhibited SARS-CoV-2 PL^pro in the absence of DTT but not with DTT.⁶⁵ This discrepancy needs to be further validated.

In another study, a similar strategy has been exploited for the development of dual inhibitors targeting both SARS-CoV-2 M^pro and PL^pro based on the ebselen scaffold.⁹¹ Among the 23 ebselen analogs, seven showed dual inhibition with the M^pro IC₅₀ values in the nanomolar range and the PL^pro IC₅₀ values in the single digit to submicromolar range (60-66, Table 1). No reducing reagent was added in either the M^pro or the PL^pro enzymatic assay. The antiviral activity of the potent hits was not reported. Nonetheless, ebselen (55) was previously reported to inhibit SARS-CoV-2 replication with an EC₅₀ value of 4.67 μM in the plaque assay, albeit the proposed mechanism of action is through M^pro inhibition.⁹⁸

The inconsistent PL^pro enzymatic inhibitory activity of ebselen (55) and its analogues from several groups, coupled with their antiviral activity against SARS-CoV-2, suggest that further characterizations are needed to confirm their cellular PL^pro target engagement and additional targets that might contribute to the antiviral activity.

3.2.3. Non-specific covalent PL^pro inhibitors – Zinc ejector

PL^pro contains a zinc-binding domain in which the zinc ion is coordinated by four conserved cysteine residues Cys₁₈₉, Cys₁₉₂, Cys₂₂₄, Cys₂₂₆. The Zinc-binding domain is essential for the structural integrity and hence the enzymatic activity of PL^pro. As such, the cysteine rich zinc-binding domain (ZBD) was also proposed as a putative drug target.⁹⁹

Disulfiram (67), ebselen (55), together with 5,5’ -dithiobis(2-nitrobenzoic acid) (DTNB, 68), 2,2’ -dithiodipyridine (69), and 2,2’ -dithiobis(benzothiazole) (70) were found to eject zinc from PL^pro as shown by the increase in fluorescence emission signal from the zinc-specific fluorophore, FluoZin-3.¹⁰⁰ Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrum further confirmed the covalent adduct formation between disulfiram and ebselen with PL^pro and nsp10. LC-MS/MS experiment mapped the ebselen and disulfiram conjugation sites to C189 and C192, both of which are involved in zinc chelation in the ZBD of PL^pro. In the FRET-based enzymatic assay, disulfiram (67) and ebselen (55) inhibited PL^pro with IC₅₀ values of 7.52 and 2.36 μM, respectively. It is noted that the enzymatic inhibition might be a combined effect of targeting both the catalytic C111 and the cysteines in the ZBD. Combination experiment showed that ebselen and disulfiram had synergistic antiviral effect when combined with hydroxychloroquine. This study suggested that clinically safe zinc-ejectors could potentially target the conserved ZBD in multiple viral proteins and could potentially be exploited as broad-spectrum antiviral drug candidates. Following studies from the same group further showed that disulfiram (67) and ebselen (55) are zinc-ejectors of the SARS-CoV-2 nsp13 and nsp14 and consequently inhibit nsp13 ATPase and nsp14 exoribonuclease activities.¹⁰¹ The antiviral activity of ebselen (55) and disulfiram (67) against SARS-CoV-2 was synergistic with remdesivir.

As discussed above, ebselen analogs have also been extensively exploited as M^pro and PL^pro inhibitors by targeting the active site cysteine.^{102, 103} Combined with the zinc ejecting property, the antiviral activity of ebselen (55) and its derivatives might be due to its polypharmacology in targeting the ZBD, PL^pro, M^pro, and others.

4. Perspectives of targeting the SARS-CoV-2 PL^pro

The COVID-19 pandemic is a timely call for the immediate need of antivirals. As the SARS-CoV and MERS-CoV epidemics subsided, the interest of developing coronavirus inhibitors unfortunately waned, and no significant efforts were devoted to optimizing the hits identified from early high-throughput screening campaigns. Nevertheless, the COVID-19 pandemic reignited the interest in PL^pro drug discovery and the past two years have seen encouraging progress in the field. Although drug repurposing largely failed to identify potent and selective PL^pro inhibitors, rational design based on the X-ray crystal structures led to major breakthroughs including the design of 2-phenylthiophene PL^pro inhibitors with favorable PK properties and the fist-in-class covalent PL^pro inhibitors since the pandemic. In light of this encouraging progress, we hereby share our opinions in the further development of SARS-CoV-2 PL^pro inhibitors and hope to clarify some of the confusions in the field based on our experience.

First, there is a need to broaden the antiviral spectrum of PL^pro inhibitors to target MERS-CoV. The BL2 loop located at the drug binding site is poorly conserved among SARS-CoV and MERS-CoV,¹⁰⁴ explaining the lack of activity of GRL0617 (4) series of compounds against MERS-CoV PL^pro. No potent and specific MERS-CoV PL^pro inhibitors have been reported till now. In search of PL^pro inhibitors with a broader spectrum of antiviral activity, it is worthwhile to include MERS-CoV PL^pro in the secondary assays. It might be possible to identify allosteric inhibitors with dual inhibitions against both SARS-CoV-2 PL^pro and MERS-CoV PL^pro. Alternatively, PL^pro inhibitors can be developed specifically for SARS-CoV-2 and SARS-CoV, and MERS-CoV PL^pro inhibitors can be pursued separately.

Second, structurally disparate PL^pro inhibitors are needed to advance PL^pro inhibitors to clinic. Compared to PL^pro, M^pro is a more amenable drug target and structurally disparate inhibitors have been identified from HTS as potent M^pro inhibitors. In contrast, several recent HTS failed to identify additional potent and selective SARS-CoV-2 PL^pro inhibitors other than GRL0617 analogues.^{67, 85} GRL0617 (4) contains the naphthalene ring, which is a known metabolic labile group, and a possible toxicophore.¹⁰⁵ Therefore, it might present a challenge in PK optimization. To increase the chances of success, additional structurally disparate PL^pro inhibitors are needed as backups. The recent elegant design of 2-phenylthiophene and the covalent PL^pro inhibitors are prominent examples in this direction.^{67, 87}

Third, target selectivity needs to be addressed at the early stage of development. Although there is a lack of sequence or structural similarity between PL^pro and human DUBs, both PL^pro and human DUBs bind ubiquitin at the extended C-terminus with the consensus sequence Leu-X-Gly-Gly, raising the potential concern of the off-target effects of PL^pro inhibitors against human DUBs.¹⁰⁶ Consequently, it is important to conduct counter screening of PL^pro inhibitors against a panel related human DUBs to avoid potential toxicity. Along this line, counter screening should also be conducted with other cysteine proteases like the M^pro, cathepsin L, calpains and etc to rule out promiscuous inhibitors that non-specifically inhibit unrelated proteases.

Fourth, be aware of promiscuous inhibitors and compounds with polypharmacology. Promiscuous compounds are defined as compounds that lack a defined mechanism of action or compounds that showed inconsistent results in different assays. PL^pro is a cysteine protease that is prone to non-specific inhibition by redox cycling compounds (quinone, arylsulfonamide, tolyl-hydrazide, etc)^{107, 108}, alkylating reagents, and other pan-assay interference compounds (PAINS).^109–111 In addition, compounds such as acriflavine and YM155 are cationic amphiphilic drugs (CADs), which could cause phospholipidosis and disturb endosome/lysosome functions. This effect may explain the improved antiviral potency over biochemical potency. In this regard, the antiviral activity of acriflavine and YM155 might be a combined effect of PL^pro inhibition and endosome/lysosome disruption. Furthermore, it is better to perform the antiviral assays in different cell lines, especially in physiologically relevant cell lines such as Calu3 or normal human airway epithelial cells. This eliminates the cell-type dependent antiviral activity of certain compounds.

Fifth, for translational drug discovery, we need to differentiate chemical probes from drug candidates. Compounds such as ebselen and disulfiram having non-specific inhibition against PL^pro and M^pro as well as other unrelated cysteine proteases should not be classified as PL^pro inhibitors. Nevertheless, this does not indicate that these promiscuous compounds should not be further pursued as SARS-CoV-2 antivirals. Instead, they should be defined as chemical probes for mechanistic studies. The aforementioned cell-based protease assays such as the FlipGFP and Protease-Glo luciferase assays are valuable tools to help rule out promiscuous compounds like ebselen and disulfiram and delineate the cellular target engagement of the specific PL^pro inhibitors.

In summary, despite the encouraging progress in the past two years, there is still a long journey to advance PL^pro inhibitors to clinic. No rationally designed drug-like PL^pro inhibitors have been shown to have in vivo antiviral efficacy against SARS-CoV-2 infection in animal models yet. In addition to the RdRp and M^pro inhibitors, PL^pro inhibitors are expected to enrich our armamentarium in flighting the current COVID-19 pandemic and future unforeseeable coronavirus outbreaks. Combination experiments need to be planned to characterize the combination therapy potential of PL^pro inhibitors with RdRp or M^pro inhibitors. Furthermore, the knowledge accumulated in developing SARS-CoV-2 PL^pro inhibitors can be similarly applied to MERS-CoV PL^pro.

ABBREVIATIONS USED

ACE2

angiotensin converting enzyme 2

BRET

bioluminescence resonance energy transfer

BSL-3

biological safety level 3

COVID-19

coronavirus disease 2019

DTT

dithiothreitol

DUBs

deubiquitinases

ESI

electrospray ionization

FITC

fluorescein 5-isothiocyanate

FRET

fluorescence resonance energy transfer

HBA

4-hydroxybenzaldehyde

HE9

methyl 3, 4-dihydroxybenzoate

i.m.

intramuscular

i.p.

intraperitoneal

ITC

isothermal titration calorimetry

i.v.

intravenous

MALDI-TOF

matrix-assisted laser desorption/ionization-time of flight

M^pro

main protease

NSP

non-structural protein

PAINS

pan-assay interference compounds

PDC

peptide-drug conjugate

PK

pharmacokinetic

PL^pro

papain-like protease

RdRp

RNA-dependent RNA polymerase

SPR

surface plasma resonance

6-TG

6-thioguanine

VOC

variants of concern

VOI

variants of interests

YRL

4-(2-hydroxyethyl)phenol

ZBD

zinc-binding domain

Biographies

Jun Wang earned his Ph.D. degree from the University of Pennsylvania in 2010. He continued the postdoctoral training with Dr. William F. DeGrado at the University of California, San Francisco. In 2014 he started an independent career as an assistant professor at the college of pharmacy at the University of Arizona. He was promoted to associate professor in 2020. In 2022, he relocated his lab to the Ernest Mario School of Pharmacy at Rutgers University. His current research interests include antiviral drug discovery, assay development, drug resistance, and combination therapy. Dr. Wang serves as the associate editor and editorial board member for multiple journals including Journal of Medical Virology, Acta Pharmaceutica Sinica B, Medicinal Research Reviews, and European Journal of Pharmaceutical Sciences.

Haozhou Tan earned his BS degree from the Hunan Agricultural University (2016, Changsha, China) and the master’s degree from Northeastern University (2018, Boston, Massachusetts). In 2020, he joined Dr. Jun Wang Lab as graduate student at the College of Pharmacy at the University of Arizona. In 2022, he relocated to the Ernest Mario School of Pharmacy at the Rutgers University with his PI Dr. Wang. His current study focuses on antiviral drug research for Influenza and coronavirus.

Yanmei Hu earned her Ph.D. degree from the University of Arizona in August 2021. She continued the postdoctoral training with Dr. Jun Wang and relocated with the Wang laboratory to the Ernest Mario School of Pharmacy at the Rutgers University in 2022. Her current research interests include antiviral drug discovery, assay development, drug resistance mechanism study, and combination therapy targeting enteroviruses and coronaviruses.

Prakash Jadhav obtained the M.Sc. in Chemistry from Pune University, India in 2011, and then joined Dr. D. S. Reddy’s research group as a research assistant in CSIR-National Chemical Laboratory, Pune. He received his Ph.D. in 2019 from the National Tsing-Hua University, Taiwan, under the supervision of Dr. R. S. Liu and continued his postdoctoral studies at the same research group. After completion of his postdoctoral work in the group of Dr. S. C. Hung at the Genomics Research Center, Academia Sinica, Taiwan, he joined Dr. Jun Wang group as a postdoctoral researcher in January 2022. His research interests include the design of SARS-CoV-2 PL^pro inhibitors.

Bin Tan received his bachelor’s and master’s degrees from the China Pharmaceutical University in 2018 and 2020, respectively. After graduation, he worked for one year as a research assistant in Dr. Jing Xu’s group in Southern University of Science and Technology. He is now a first-year PhD student in Dr. Jun Wang’s lab at Rutgers University and his research interests include drug discovery targeting SARS-CoV-2 M^pro and PL^pro.