Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2

Article abstract of DOI:10.1016/j.chembiol.2021.04.020

The COVID-19 pandemic has been disastrous to society and effective drugs are urgently needed. The papain-like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is indispensable for viral replication and represents a putative target for pharmacological intervention. In this work, we describe the development of a potent and selective SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate cleavage and immunosuppressive function imparted by PLpro, but also markedly mitigates SCoV2 replication in human cells, with a submicromolar IC₅₀. We further present a convenient and sensitive activity probe, 7, and complementary assays to readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. In addition, we disclose the co-crystal structure of SCoV2 PLpro in complex with a prototype inhibitor, which illuminates their detailed binding mode. Overall, these findings provide promising leads and important tools for drug discovery aiming to target SCoV2 PLpro.

Full text of DOI:10.1016/j.chembiol.2021.04.020

Article
Development of potent and selective inhibitors
targeting the papain-like protease of SARS-CoV-2
Graphical abstract
Authors
Hengyue Shan, Jianping Liu,
Jiali Shen, ..., Ying Li, Lifeng Pan, Li Tan
Correspondence
zhangzheng1975@aliyun.com (Z.Z.),
liying@sioc.ac.cn (Y.L.),
panlf@sioc.ac.cn (L.P.),
tanli@sioc.ac.cn (L.T.)
In brief
PLpro represents a putative druggable
target for SCoV2. Utilizing multiple
approaches, including fluorogenic
activity probes, HTS, co-crystallization,
structure-based design, and other
optimized cellular assays, Shan et al.
successfully develop a potent and
selective inhibitor of SCoV2 PLpro.
Highlights
d
Development of a sensitive and affordable PLpro
activity probe
d
Identification of potential SCoV2 PLpro inhibitors
through HTS
d
d
Co-crystal structure determination and lead optimization
Characterization of a submicromolar inhibitor of SCoV2
Shan et al., 2021, Cell Chemical Biology 28, 1–11
June 17, 2021 ª 2021 Elsevier Ltd.
https://doi.org/10.1016/j.chembiol.2021.04.020
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Article
Development of potent and selective inhibitors
targeting the papain-like protease of SARS-CoV-2
Hengyue Shan,1,2,6 Jianping Liu,3,6 Jiali Shen,1,2,6 Jialin Dai,1,6 Gang Xu,4,6 Kuankuan Lu,1,2 Chao Han,1,2 Yaru Wang,2,3
1,
Xiaolong Xu,2,3 Yilun Tong,1,2 Huaijiang Xiang,1 Zhiyuan Ai,1 Guanglei Zhuang,5 Junhao Hu,1 Zheng Zhang,4, Ying Li,
*
*
1,7,
Lifeng Pan,2,3, and Li Tan
*
*
1Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences,
Shanghai 201210, China
2University of Chinese Academy of Sciences, Beijing 100049, China
3State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of
Sciences, Shanghai 200032, China
4Institute of Hepatology, National Clinical Research Center for Infectious Disease, Shenzhen Third People’s Hospital, The Second Affiliated
Hospital, School of Medicine, Southern University of Science and Technology, Shenzhen, Guangdong Province 518112, China
5State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong
University, Shanghai 200240, China
6These authors contributed equally
7Lead contact
*Correspondence: zhangzheng1975@aliyun.com (Z.Z.), liying@sioc.ac.cn (Y.L.), panlf@sioc.ac.cn (L.P.), tanli@sioc.ac.cn (L.T.)
https://doi.org/10.1016/j.chembiol.2021.04.020
SUMMARY
The COVID-19 pandemic has been disastrous to society and effective drugs are urgently needed. The papain-
like protease domain (PLpro) of SARS-CoV-2 (SCoV2) is indispensable for viral replication and represents a
putative target for pharmacological intervention. In this work, we describe the development of a potent and
selective SCoV2 PLpro inhibitor, 19. The inhibitor not only effectively blocks substrate cleavage and immu-
nosuppressive function imparted by PLpro, but also markedly mitigates SCoV2 replication in human cells,
with a submicromolar IC50. We further present a convenient and sensitive activity probe, 7, and complemen-
tary assays to readily evaluate SCoV2 PLpro inhibitors in vitro or in cells. In addition, we disclose the co-crys-
tal structure of SCoV2 PLpro in complex with a prototype inhibitor, which illuminates their detailed binding
mode. Overall, these findings provide promising leads and important tools for drug discovery aiming to target
SCoV2 PLpro.
INTRODUCTION
pensable enzymatic domains of SCoV2, such as RdRP, the main
protease (Mpro/3CLpro), and the papain-like protease (PLpro),
The pandemic of coronavirus disease 2019 (COVID-19) has been are considered druggable targets (Li and De Clercq, 2020).
ravaging for over a year, causing mounting infections and mil- Indeed, RdRP inhibitors, e.g., Remdesivir, have already been
lions of deaths, as well as incalculable devastation of the global approved, while Mpro inhibitors have advanced into clinical trials
economy (Morens and Fauci, 2020). To overcome COVID-19, re- (NCT04535167; NCT04627532). On the other hand, potent and
searchers worldwide are racing to discover effective drugs. Up selective inhibitors of SCoV2 PLpro remain to be discovered.
to now, several vaccines have been approved, and the US
SCoV2 PLpro shares 83% sequence identity as well as pro-
FDA has granted Remdesivir, an antiviral agent targeting RNA teolytic activity with SCoV PLpro. It can process nonstructural
polymerase (RdRP), approval for treatment of COVID-19. How- proteins, in particular, nsps 1–3, that are essential for virus
ever, the agents currently applied in the clinic will most likely replication, and can cleave K48-linked ubiquitin (Ub) chains or
face anticipated drug resistance arising from the evolving virus interferon-stimulated gene 15 (ISG15) modifications on host
(Thomson et al., 2021). Thus, developing more therapeutic stra- cell proteins to counteract the immune response during viral
tegies and drug candidates for COVID-19 treatment is an urgent infection (Figure 1A) (Li and De Clercq, 2020; Ratia et al., 2006;
demand.
Shin et al., 2020). Previously, selective inhibitors had been devel-
The pathogen of COVID-19 is a novel coronavirus called se- oped for SCoV PLpro, which could restrain SCoV replication at
vere acute respiratory syndrome coronavirus 2 (SARS-CoV-2/ high concentrations (Baez-Santos et al., 2014; Ratia et al.,
SCoV2), a close relative of SARS-CoV (SCoV), which emerged 2008). Some of these reported inhibitors were tested against
in 2002 (Zhou et al., 2020). Based on their high genetic homology SCoV2 and exhibited only weak to moderate antiviral effects in
and precedent studies on SCoV, certain nonstructural but indis- cells (Freitas et al., 2020; Klemm et al., 2020; Shin et al., 2020).
Cell Chemical Biology 28, 1–11, June 17, 2021 ª 2021 Elsevier Ltd.
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Figure 1. Development of peptidomimetic activity probes for SCoV2 PLpro
(A) A cartoon summarizing the enzymatic functions of SCoV2 PLpro. As a part of nsp3, PLpro is responsible for the cleavage of viral nsp1–3 proteins, and can
cleave K48-linked Ub chains or ISGylation in host cells.
(B) A diagram showing the cleavage-site residue sequences of PLpro substrates and the formulas of peptidomimetic fluorogenic probes.
(C) The chemical structure of 7 and a cartoon depicting its fluorogenic mechanism in the presence of PLpro.
(D) Bar graph showing relative sensitivity of each probe to SCoV2 PLpro. Data are presented as the mean SD (n = 3).
(E) Optimization of the enzymatic activity assays by titration of SCoV2 PLpro and probes. Data are presented as the mean SD (n = 3).
(F) Comparison of the enzymatic activities between SCoV2 PLproWT and PLproC112S (1 mM) with 5 or 7 (2.5 mM) as substrate. Data are presented as the mean SD
(n = 3).
(G) Michaelis-Menten curves for 5 and 7 with 1 mM SCoV2 PLpro. Data are presented as the mean SD (n = 3).
Of note, given the functional similarity between PLpro and hu-
Herein, we present the discovery of a potent and selective
man deubiquitinating enzymes (DUBs), competitive inhibitors SCoV2 PLpro inhibitor, i.e., 19, which efficiently suppressed viral
of PLpro might potentially hit off-targets in infected cells. Indeed, replication with a submicromolar IC50. In addition, several opti-
two peptidomimetic covalent PLpro inhibitors reported recently mized approaches are described to facilitate the identification
exhibited in vitro reactivity toward both SCoV2 and a DUB (Rut and optimization of SCoV2 PLpro inhibitors, including drug
et al., 2020).
design based on the co-crystal structure of SCoV2 PLpro in
2
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complex with an inhibitor; development of enzymatic activity as- inhibition (~50%). Among these compounds, the SCoV PLpro in-
says that allow quick, large-scale, and affordable high- hibitor GRL0617 (Ratia et al., 2008), recently reported to inhibit
throughput screening (HTS); and implementation of a virus-free SCoV2 PLpro in vitro (Freitas et al., 2020; Shin et al., 2020), stood
experimental system ready to assess cellular potency of lead out, with over 80% inhibition of SCoV2 PLpro in our assays, as
compounds.
expected.
Using GRL0617 as a positive control, the SCoV2 PLpro enzy-
matic activity assay was further optimized to suit HTS, with a Z0-
factor of 0.633 determined by 100 wells per control group (Zhang
et al., 1999). Then we performed a primary screen on 35,360
diverse compounds, including lead-like fragments, FDA-
RESULTS
Development of peptidomimetic activity probes for
SCoV2 PLpro
As PLpro and DUBs share conserved target cleavage sites, we approved drugs, and small molecules with reported biological
screened several reported DUB substrates to develop an enzy- activities. This HTS was repeated and resulted in a moderate
matic activity assay for SCoV2 PLpro (Figures 1B, 1C, and S1). hit rate of 0.037% with the Z-score threshold set at ꢀ8, repre-
The purified recombinant SCoV2 PLpro protein exhibited robust senting a strong negative effect (Zhang, 2007). A total of 13 com-
activity in cleaving K48-linked diUb, but weak or no activity pounds were selected for further validation (Figures 2C and 2D
against K63-linked or linear diUb, respectively (Figure S1). and Table S1).
SCoV2 PLpro could readily cleave a Ub-rhodamine 110 conju-
Of the 13 hits, 9 reproducibly inhibited SCoV2 PLpro, with at
gate (Ub-Rho/1) (Hassiepen et al., 2007) and release the fluoro- least 25% inhibitory activity, and 7 exhibited over 50% inhibition,
phore (Rho) from its C terminus. We demonstrated that it could including an essential nutrient, vitamin B12, and inhibitors re-
likewise release the fluorogenic 7-amido-4-methylcoumarin ported for other targets, such as avasimibe and tiplaxtinin. An
(AMC) from the peptidomimetic probes 2 and 3, which harbored antibiotic commonly used in hair shampoo, zinc pyrithione, and
a conserved RLRGG sequence homologous to the C-terminal an electrophilic a-chloroketone, compound 8, showed potency
pentapeptide of Ub (Figures 1B and 1D) (Renatus et al., 2006).
comparable to that of GRL0617. Given its high hydrolytic activity
To better mimic the isopeptide bond of SCoV2 PLpro sub- but low selectivity toward Ub chain linkages (Ritorto et al., 2014),
strates, we synthesized a hexapeptide probe (5) with a fluoro- the deubiquitinating protease USP2 was used in a counter-
genic 7-hydroxy-3-carboxycoumarin (HCC) conjugated to its N screen to assess the selectivity of all these hits, and the results
terminus, and a 4-(dimethylaminoazo) benzene-4-carboxylic indicated GRL0617 to be more selective than others (Figure 2D).
acid (DABCYL) to its C-terminal lysine (Figures 1B and S2). The GRL0617, which had already been optimized for SCoV PLpro,
maximal absorption of the HCC fluorophore in 5 is around generally showed superior potency, selectivity, and molecular
410 nm, and its emission peak is at 450 nm. In the absence of complexity than other hits. These findings are consistent with
SCoV2 PLpro, the emission of HCC upon excitation will mostly previously reported HTS results (Smith et al., 2020) and support
be quenched by the proximate DABCYL subunit in 5 through a that GRL0617 serves as a better lead for SCoV2 PLpro.
fluorescence resonance energy transfer (FRET) mechanism. In
the presence of SCoV2 PLpro, 5 will be cleaved and the resultant Determination of the co-crystal structure of the SCoV2
fluorogenic pentapeptide (50) will no longer be quenched (Figures PLpro-12 complex
1C and S2). Compared with previous probes, 5 exhibited Encouraged by the potential of GRL0617, we synthesized and
improved sensitivity to SCoV2 PLpro with lower background tested a series of reported SCoV PLpro inhibitors (11–13) that
signal. We then replaced the N-terminal lysine of 5 with a methi- partially resemble GRL0617 with a shared naphthyl subunit
onine to mimic the linear diUb (6), or with an aliphatic chain (7), (Baez-Santos et al., 2014). These inhibitors exhibited even better
and observed that 6 was slightly less sensitive, whereas 7 was potency than GRL0617 in our assay based on 7, with IC50 values
as sensitive as 5 (Figures 1B–1D).
ranging between 2.6 and 4.3 mM (Figures 3A and S3). To study
To optimize the SCoV2 PLpro enzymatic activity assays with 5 the pharmacophore of this series of inhibitors and to facilitate
and 7, we titrated each probe and confirmed that 5 or 7 at 2.5 mM the following chemical optimization, we attempted to co-crystal-
was efficient enough (Figure 1E). Then we set probes at 2.5 mM lize SCoV2 PLpro with these inhibitors (11–13). We first deter-
and titrated SCoV2 PLpro, and confirmed 1 mM as an efficient mined the high-resolution apo-form structure of the catalytically
concentration for the enzyme. Under the optimized conditions, inactive mutant, SCoV2 PLproC112S (Figure S4A). More impor-
both 5 and 7 exhibited excellent sensitivity to wild-type (WT) tantly, the co-crystal structure of the SCoV2 PLproC112S-12
˚
SCoV2 PLpro, but not to the catalytically inactive mutant, complex was solved at 2.4 A resolution as a dimer. Although
SCoV2 PLproC112S (Figure 1F). However, the Km value for 7 is the overall Rfree (0.326) and Rwork (0.272) values for the co-crystal
3-fold lower than that for 5 (Figure 1G), suggesting that 7 repre- are relatively high due to the anisotropy of diffraction data,
˚
sents a better substrate for SCoV2 PLpro.
particularly in the resolution range of 3.15–2.4 A, the electron-
density map of the binding site shows good quality, and 12
can be well traced in the map (Figures 3B and 3C and Table S2).
Based on the co-crystal structure, 12 adopts a U-shape
Screening for SCoV2 PLpro inhibitors
Having established the enzymatic activity assay with 7, we
initially screened 25 reported inhibitors of DUB or DUB-like pro- conformation to occupy a pocket between the S1 site and the
teases for their inhibitory activity against SCoV2 PLpro at 10 mM catalytic site of SCoV2 PLpro, consequently blocking the N-ter-
(Figures 2A and 2B and Table S1). Most molecules were weak (% minal moiety of its substrates from accessing the S1 site (Fig-
30% inhibition) or inactive against SCoV2 PLpro, except for a ure 3B). Notably, the 2mFo-DFc electron-density maps indicated
UCHL3 inhibitor, TCID (Liu et al., 2003), which showed moderate distinct binding modes for each unit of the asymmetrically
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Figure 2. Screening for SCoV2 PLpro inhibitors
(A) A bar graph showing inhibition of SCoV2 PLpro by reported DUB or DUB-like protease inhibitors (10 mM) in an enzymatic assay based on 7. Data are presented
as the mean SD (n = 3).
(B) Chemical structures of exemplary compounds showing inhibition of SCoV2 PLpro in (A) or (C).
(C) HTS assay for SCoV2 PLpro inhibitor discovery based on probe 7. Screening data for a representative library of 9,071 small molecules are shown. Compounds
with Z scores below ꢀ8 in both experiments were designated as hits for PLpro (red dots).
(D) A bar graph showing inhibition of SCoV2 PLpro or USP2 by selected hits (10 mM) in enzymatic assays based on 7. Data are presented as the mean SD (n = 3).
dimerized complex (Figure 3C). Nevertheless, further analyses (Klemm et al., 2020; Shin et al., 2020). Collectively, 12 efficiently
were based on the complex structure of chain A only, given its exploits the switchable Tyr269 to inhibit SCoV2 PLpro.
better quality of electron density compared with chain B. Upon
closer examination, 12 interacts with the side chains of multiple Optimization of SCoV2 PLpro inhibitors
surrounding residues, including Leu163, Pro248, Pro249, Based on the co-crystal structure, we performed structure-activ-
Tyr265, Tyr269, and Gln270, through van der Waals forces. In ity relationship (SAR) studies to optimize the SCoV2 PLpro inhib-
particular, the phenyl ring of Tyr269 simultaneously engages itors. Given that derivatization of the naphthyl group is reportedly
with all three hydrophobic rings of 12, acting as a latch of the unfavorable (Baez-Santos et al., 2014), and as the piperidyl ring
binding pocket. In addition, the carbonyl of Tyr269 forms an H is tightly surrounded by Tyr269, Asp165, and other residues, we
bond with the backbone amide of 12. Moreover, the electrostatic focused our efforts on the substitution of benzyl group (Figures 3
interaction between the negatively charged Asp165 and the and S3). First, inspired by 11, we added a 5-fluorine to 12 to
positively charged tertiary amine of 12 further stabilizes the com- strengthen the interaction with Gln270, and the resultant 14
plex (Figures 3C and 3D).
showed improved potency. Meanwhile, as the substituted
Upon binding to SCoV2 PLpro, 12 pushes the side chain of benzyl group of 12 is exposed to the solution, we postulated
Leu163 away from its binding pocket, but induces the side chain that hydrophilic substituents might stabilize the complex. How-
of Tyr269 to bend down steeply toward it (Figures 3D and S4A). ever, replacing the hydrophobic benzyl ring of 12 with a
Likewise, GRL0617 induces similar conformational changes in substituted piperidine ring (15) resulted in potency decrease.
Leu163 and Tyr269, but to a lesser extent (PDB: 7IJR) (Osipiuk To introduce hydrophilic substitutions without disturbing the hy-
et al., 2021). In contrast, GRL0617 forms an H bond with the drophobic interaction between 12 and Gln270 and Leu163, we
backbone nitrogen of Gln270, and induces significant conforma- decided to modify the acetamide group, which seems to pro-
tional change in it (Figure 3D). Binding with Ub or ISG15 also in- trude toward the solvent in the co-crystal, rather than the whole
duces the side chain of Tyr269 to bend down; however, it bends benzyl ring. As expected, both 16 and 17, each introduced with a
toward Pro248 rather than the binding pocket (Figure S4B) hydrophilic tertiary amine, showed improved potency compared
4
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Figure 3. Determination of the co-crystal structure of the SCoV2 PLpro-12 complex and optimization of inhibitors
(A) Structure-activity relationship of 11 and analogs. Each compound was titrated in enzymatic assays based on 7 or Ub-Rho. Data are presented as the mean
SD (n = 3), assays were repeated at least three times with similar results.
(B) Surface structure of SCoV2 PLpro (chain A) in complex with 12 (purple sticks) (PDB: 7E35) showing that 12 resides in a channel in front of the catalytic site in the
PLpro domain.
(C) Cartoon structures of the binding pockets of 12 (purple sticks) in two asymmetric SCoV2 PLpro units (green/azure ribbons for chains A/B). The key residues
interacting with 12 are shown as sticks, and the 2mFo-DFc omit maps are shown as blue meshes.
(D) Superimposition and comparison of the binding sites of SCoV2 PLpro-12 (green/purple sticks, PDB: 7E35), SCoV2 PLpro-GRL0617 (gray/yellow sticks, PDB:
7JIR) (Osipiuk et al., 2021), and unbound SCoV2 PLpro (gray sticks, PDB: 7D6H).
with 12 and 15, while 17 showed a submicromolar IC50. Finally, studies to elucidate the outstanding potency of 19 based on
we added 5-fluorine to 16 and 17 to generate 18 and 19 with the co-crystal structure of SCoV2 PLpro-12 (Figures 4A–4C).
further improved potency. All these analogs were also tested in The docking results predicted a binding mode for 19, which is
a SCoV2 PLpro activity assay based on Ub-Rho, and their rela- partially similar to that of 12, but includes additional interactions
tive activities were overall consistent with those based on 7 (Fig- for 19. Based on this mode, the substituted piperazine group and
ures 3A and S3). the 5-fluorine of 19 interact with the side chains of Tyr269 and
Given the unsuccessful efforts to co-crystalize SCoV2 PLpro Gln270, respectively, mainly through van der Waals forces.
with 19 or other analogs, we performed molecular docking Compared with 12, which interacts with Tyr269 in three
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Figure 4. Inhibitor 19 is a potent and selective SCoV2 PLpro inhibitor
(A) Chemical structures of 12 and 19.
(B and C) Inhibitor 19 was docked into the binding pocket of 12 in SCoV2 PLpro (chain A, PDB: 7E35). Superimposition of 19 (purple sticks) and 12 (azure sticks)
indicated additional interactions between 19 and Tyr269-Gln 270 (green sticks in B) of SCoV2 PLpro (surface in C).
(D) SPR analysis of 19 binding to SCoV2 PLpro; GRL0617 was used as a control. Kd and SD were calculated based on three independent experiments.
(E) A bar graph showing the inhibition of 11 DUBs or DUB-like proteases by 19 (10 mM) (n = 3). Data are presented as the mean SD (n = 3).
orthogonal directions, 19 almost encircles Tyr269 with interac- these 10 proteases were significantly inhibited by 19 at 10 mM
tions in four orthogonal directions, which provides a plausible (Figure 4E). Cumulatively, the results show that 19 is a potent
explanation for its significantly improved potency (Figure 4C).
To precisely characterize the affinity of 19 for SCoV2 PLpro,
and selective inhibitor of SCoV2 PLpro in vitro.
we performed surface plasma resonance (SPR) binding analysis Inhibitor 19 and analogs effectively inhibited SCoV2
of 19 in comparison with GRL0617. Based on the SPR results, 19 PLpro in live cells
bound to SCoV2 PLpro with a Kd of 2.6 mM, while that of To investigate if 19 and other analogs maintain their potency in
GRL0617 was 10.8 mM (Figure 4D). To evaluate its selectivity, live cells, we designed a cellular assay to assess the enzymatic
19 was profiled against 10 DUBs or DUB-like proteases, activity of SCoV2 PLpro (Figure 5A). We found that
including USP14 and OTUB1, which reportedly favor K48-linked SCoV2 nsp1-nsp2 protein expressed in 293T cells is rather sta-
Ub chains as substrates. Compared with SCoV2 PLpro, none of ble despite endogenous human DUBs, but can be readily
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Figure 5. Inhibitor 19 and analogs inhibited SCoV2 PLpro and viral replication in live cells
(A) Inhibitors 17, 18, and 19 inhibited the cleavage of recombinant nsp1-nsp2 by SCoV2 PLpro in a dose-dependent manner. 293T cells were pretreated with each
inhibitor at the indicated concentrations and transfected with FLAG-tagged nsp1-nsp2 and SCoV2 PLpro for 24 h, followed by western blots.
(B) Inhibitors 17, 18, and 19 significantly recovered the activation level of NF-kB perturbed by SCoV2 PLpro. NF-kB-luc-reporting 293T cells were transfected with
SCoV2 PLproWT or PLproC112S for 18 h in the presence of inhibitors (10 mM) and then stimulated by TNF-a for 6 h and measured for their NF-kB activity by the
luciferase reporting system. Data are presented as the mean SD (n = 3, ***p < 0.001).
(C) Inhibitors 17, 18, and 19 significantly recovered the level of ISGylation perturbed by SCoV2 PLpro. A549 cells stably expressing SCoV2 PLproWT or PLproC112S
were preincubated with inhibitors for 6 h and then stimulated by IFN-a for 24 h and subjected to western blots.
(D) Inhibitors 15–17 at 5 mM abolished SCoV2 replication in hACE2-HeLa cells. hACE2-HeLa cells were infected with SCoV2 (0.3 MOI) for 1 h, then washed out
with PBS, and cultured with normal medium and the indicated inhibitors (5 mM) for an additional 48 h, followed by western blots.
(E) Inhibitor 19 inhibited the replication of SCoV2 in human cells in a dose-dependent manner. hACE2-HeLa cells were cultured with SCoV2 for 1 h, then washed
out with PBS, and cultured with normal medium and the indicated inhibitors for another 48 h, followed by immunostaining of spike protein (red) and nuclei (blue).
Assays were repeated at least three times with similar results, and representative images are shown.
(F) Inhibition of SCoV2 replication curves based on the quantification of fluorescence, and the calculated IC50, CC50, and therapeutic indexes (CC50/IC50) for 17–
19. Data are presented as the mean SD (n = 3).
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cleaved into two parts by the co-expressed SCoV2 PLpro pro- expensive for HTS. AMC-based peptidomimetic probes like 2
tein. However, such cleavage should be perturbed by SCoV2 or 3 are moderately sensitive and much more cost effective.
PLpro inhibitors. And to our satisfaction, 17, 18, and 19 at However, they often interfere with the screened fluorescent
10 mM all significantly inhibited SCoV2 PLpro in 293T cells.
compounds with overlapping spectrums. In contrast, our
As PLpro putatively impairs the immune response of host FRET-based fluorogenic probe 7 exhibited excellent sensitivity
cells, to further evaluate our inhibitors for their cellular efficacy, with minimal background for SCoV2 PLpro and showed high
we checked their modulatory effects on the immune signaling reliability and low cost in HTS.
regulated by K48-linked Ub chains or ISGylation in live cells.
With probe 7 we screened reported inhibitors of DUBs or other
Based on a luciferase-reporting assay, we confirmed that proteases, performed a pilot HTS, and successfully identified
expression of SCoV2 PLproWT notably decreased NF-kB activa- several hits for SCoV2 PLpro. Some of these hits demonstrated
tion in 293T cells. In contrast, expression of catalytically inactive encouraging chemical properties for further development. For
PLproC112S led to a much weaker effect, and 17, 18, or 19 at example, avasimibe and tiplaxtinin could be repurposed and
10 mM could significantly recover the activation level of NF-kB optimized for SCoV2 PLpro, whereas zinc pyrithione and 8 might
(Figure 5B). We also confirmed that expression of SCoV2 PLpro potentially serve as covalent fragments in the design of covalent
significantly decreased the overall ISGylation induced by inter- inhibitors of SCoV2 PLpro. Of particular importance, a series of
feron a (IFN-a) in A549 cells, which could be efficiently restored SCoV PLpro inhibitors, including GRL0617 and 11–13, were
by all three inhibitors (Figure 5C). These results further corrobo- identified as the best leads for SCoV2 PLpro in the current study.
rated that 19 and analogs are effective SCoV2 PLpro inhibitors in Thus, we determined the co-crystal structure of the 12-SCoV2
human cells.
PLpro complex, which complemented well the recently reported
structures of SCoV2 PLpro complexes (Fu et al., 2021; Gao et al.,
2021; Osipiuk et al., 2021) and greatly facilitated our SAR
studies. An optimized analog, 19, proved to be a potent and se-
Inhibitor 19 and analogs effectively mitigate the
replication of SCoV2 in cells
As GRL0617 and other reported inhibitors exhibited sufficient lective inhibitor of SCoV2 PLpro in vitro.
antiviral effects only at rather high concentrations, we sought
We have utilized several cellular assays in combination to
to test our inhibitors in hACE2-HeLa cells (Zhou et al., 2020) for evaluate our inhibitors in live cells. These assays are based
their antiviral potency. As the nucleocapsid protein (NP) is a reli- on the cleavage of nsp proteins or IGSylation by SCoV2
able biomarker for indicating the replication rate of SCoV2 in PLpro, or based on its viral suppression of the host immune
cells, we checked the NP level in infected hACE2-HeLa cells response. Indeed, 19 and other analogs exhibited robust inhi-
treated with each inhibitor. Based on western blot analysis, the bition of SCoV2 PLpro in these assays. Furthermore, we
reported inhibitors at 5 mM only moderately or weakly decreased demonstrate that 19 is a remarkably more effective antiviral
the NP level, with 11 as the strongest. In contrast, our optimized agent, compared with the reported analogs, and can mitigate
analogs exhibited obviously improved potency, and 15, 16, or 17 the replication of SCoV2 in human cells with submicromolar
almost completely diminished the expression of NP in infected IC50 values. Based on the preliminary evaluation, 19 exhibited
cells (Figure 5D).
moderate metabolic stability in mouse liver microsomes or
Immunostaining of the spike protein demonstrated that SCoV2 plasma with half-lives of 28.0 or over 120 min, respectively
viruses replicated effectively in hACE2-HeLa cells, which conse- (Figure S5C), and is warranted to be further assessed with an-
quently induced severe cell death (Figure 5E). Consistently, in- imal models in the future.
hibitors 15–19 alleviated both replication of SCoV2 and the death
In summary, we report the development of 19 as a potent and
of hACE2-HeLa cells in a dose-dependent manner, while 19 selective inhibitor of SCoV2 PLpro and demonstrate a repertoire
could significantly inhibit SCoV2 replication even at 400 nM (Fig- of complementary approaches to facilitate drug discovery for
ures 5E and S5B). On the other hand, at 10 mM, neither 17 nor 19 SCoV2. Our sensitive and affordable activity probe, 7, could
showed detectable cytotoxicity in hACE2-HeLa cells, whereas considerably expand the capacity of HTS and accelerate the
18 did reduce the cell viability to nearly 60% compared with identification of new hits; the virus-free cellular assays utilized
the control (Figure S5A). Based on the quantification of immuno- in this work are feasible and can be very helpful in the early stage
fluorescence, the IC50 values for 17–19 all fell within the nanomo- of development with limited resources, such as P3 laboratories;
lar range, yet 19 remained the best, with a IC50 of 182 nM and a the detailed binding mode of 12-SCoV2 PLpro, revealed by a co-
therapeutic index (CC50/IC50) over 55 (Figure 5F). Together, crystal structure, will certainly assist inhibitor design and optimi-
these results show that 19 can effectively and selectively inhibit zation; and last, 19 has provided an optimized pharmacophore
the replication of SCoV2 in human cells.
and a promising lead for further developments. Collectively,
these findings represent a small but solid milestone in the
arduous and prolonged battle against COVID-19.
DISCUSSION
HTS could be an efficient approach to providing promising Limitations of the study
pharmacophores for the development of SCoV2 PLpro inhibi- Despite the favorable potency and selectivity of 19 in cultured
tors. However, its success rate depends not only on the capac- cells, further evaluation of the efficacy and tolerability of 19 in vivo
ity and diversity of screened chemical libraries, but also on the is necessary to establish its translational relevance to COVID-19.
robustness, convenience, and cost efficiency of the HTS as- On the other hand, probe 7 may be incapable of screening for in-
says. Among the reported fluorogenic activity probes for hibitors that target other allosteric sites of SCoV2 PLpro. Thus, it
DUBs and SCoV PLpro, Ub-Rho is very sensitive, but too should be considered as a complement to, rather than a
8
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B SARS-CoV2 infection and evaluation of antiviral ac-
tivities
substitute for, Ub-based probes. In addition, our efforts to co-
crystallize SCoV2 PLpro bound to 19 were unsuccessful, and
other advanced technologies, e.g., cryogenic electron micro-
scopy, could help solve this obstacle and facilitate further opti-
mization of 19.
B Metabolic stability studies
B Chemical synthesis
d QUANTIFICATION AND STATISTICAL ANALYSIS
SUPPLEMENTAL INFORMATION
SIGNIFICANCE
Supplemental information can be found online at https://doi.org/10.1016/j.
chembiol.2021.04.020.
COVID-19 has struck our society as a great calamity, and the
need for effective drugs is more urgent than ever. PLpro is
one of the most promising druggable targets of SCoV2; how-
ever, current small-molecule inhibitors of SCoV2 PLpro
exhibit only weak to moderate inhibition of SCoV2 replica-
tion, precluding their further clinical translation. This study
reports the development of potent and selective inhibitors
of SCoV2 PLpro that effectively inhibit replication of SCoV2
ACKNOWLEDGMENTS
We thank Dr. Sara J. Buhrlage (Harvard) for her generous help with the re-
ported DUB and DUB-like protease inhibitors, Dr. Sheng-Ce Tao (Shanghai
Jiao Tong University) for the cDNA of SCoV2 PLpro, Dr. Zairong Zhang
(IRCBC, CAS) for DUB plasmids, Dr. Deju Ye (Nanjing University) for help
with the development of fluorogenic probes, and Dr. Zhaoyin Wang (IRCBC,
CAS) for help with the metabolic stability studies. This work was supported
by grants from the National Natural Science Foundation of China (21778066
to L.T., 31801163 to Y.L., 21822705 to J.L. and L.P., 32071297 to J.L., and
21621002 to L.P.), Shanghai Municipal Science and Technology Commission
(18ZR1448400 to Y.L., 20XD1425200 to L.P., 18JC1420500 and Major Project
2019SHZDZX02 to L.T.), and the Strategic Priority Research Program of the
Chinese Academy of Sciences (XDB39030500 to L.T. and XDB20000000
to L.P.).
and the death of infected cells, with a submicromolar IC50
.
We also describe repertoire of complementary ap-
a
proaches to aid in drug development, including a fluoro-
genic probe with better sensitivity than reported peptidomi-
metic probes, which is more affordable than Ub-based
probes and compatible with high-throughput screening to
identify SCoV2 PLpro inhibitors. Several virus-free cellular
assays are optimized to assess the enzymatic activity of
SCoV2 PLpro, which can be very useful to evaluate the po-
tency of candidate inhibitors in live cells outside of P3
labs. Structural studies reveal the detailed binding mode of
the pharmacophore to be optimized, and our lead com-
pounds effectively inhibit replication of SCoV2 and the death
AUTHOR CONTRIBUTIONS
Conceptualization, L.T., L.P., and Y.L.; methodology, S.H., J.L., J.S., G.X., and
Y.L.; software, L.P. and Y.L.; formal analysis, H.S., J.L., J.S., Y.T., H.X., Y.L.,
and L.T.; investigation, H.S., J.L., J.S., J.D., G.X., K.L., C.H., Y.W., X.X., Y.T.,
and Z.A.; resources, L.P. and Z.Z.; writing – original draft, L.T., Y.L., and
G.Z.; supervision, L.T., L.P., Y.L., Z.Z., and J.H.; funding acquisition, L.T.,
L.P., and Y.L.
of infected cells with submicromolar IC50
.
DECLARATION OF INTERESTS
STAR+METHODS
The authors declare no competing interest.
Detailed methods are provided in the online version of this paper
and include the following:
Received: November 29, 2020
Revised: March 2, 2021
Accepted: April 23, 2021
Published: May 12, 2021
d KEY RESOURCES TABLE
d RESOURSE AVAILABILITY
B Lead contact
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Zhou, P., Yang, X.L., Wang, X.G., Hu, B., Zhang, L., Zhang, W., Si, H.R., Zhu,
Y., Li, B., Huang, C.L., et al. (2020). A pneumonia outbreak associated with a
new coronavirus of probable bat origin. Nature 579, 270–273.
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STAR+METHODS
KEY RESOURCES TABLE
REAGENT or RESOURCE
Antibodies
SOURCE
IDENTIFIER
Monoclonal ANTI-FLAGꢀ M2-Peroxidase
(HRP) antibody produced in mouse
Sigma-Aldrich
Cat# A8592; RRID: AB_439702
b-Tubulin Mouse MAb
Transgen
Cat# HC101
ISG15 Recombinant Rabbit MAb
SARS-CoV / SARS-CoV-2 Spike Rabbit PAb
b-Actin Mouse MAb
Invitrogen
Cat# 703131; RRID: AB_2784562
Cat# 40150-T62
SinoBiological
Transgen
Cat# HC201
SARS-CoV-2 nucleocapsid protein Rabbit MAb
SinoBiological
Cat# 40143-R019
Bacterial and virus strains
E.coli BL21 (DE3)
Lifeng Pan’s laboratory, Shanghai
Institute of Organic Chemistry
BL21 (DE3)
SZ02
SARS-CoV2 (SZ02)
Shenzhen Third People’s Hospital
Chemicals, peptides, and recombinant proteins
Recombinant Human Ubiquitin
Rhodamine 110 Protein, CF
BostonBiochem
Cat# U-555
Probe 2-7
SARS-CoV2-PLproWT
Li Tan’s laboratory
This paper
N/A
N/A
SARS-CoV2-PLproC112S
This paper
N/A
Recombinant Human FLAG-USP36 Protein, CF
Recombinant Human His6 USP14
Recombination Human His6 USP8
Recombination Human His6 USP7
Recombinant Human His6 USP2
Recombination Human UCH-L1/PGP9.5
BostonBiochem
Li Tan’s laboratory
BostonBiochem
BostonBiochem
Li Tan’s laboratory
BostonBiochem
BostonBiochem
Cat# E-628-025
N/A
Cat# E-520
Cat# E-519
N/A
Cat# E-340
Cat# E-700
Recombination Human His6 SUMO-Specific
Peptidase 1/SENP1 Catalytic Domain
Recombinant Human His6 OTUB1
Recombination Human Ataxin-3 Like
Li Tan’s laboratory
BostonBiochem
Novus Biologicals
N/A
Cat# E-343
Cat# NBP2-22825
Recombination Human AMSH/STAMBP
protein
Recombinant Human TNFa
Recombinant human Interferon alpha
K48-linked diUb
K63-linked diUb
M1-linked diUb
Aloxistatin
Novoprotein
Cat# C008
Sigma-Aldrich
Cat# SRP4595
N/A
This paper
This paper
N/A
This paper
N/A
Selleck (Zang et al., 2020)
Selleck (Ward et al., 2020)
MCE (Mori et al., 2002)
MCE (Colombo et al., 2010)
MCE (Colombo et al., 2010)
Selleck (Wadhawan et al., 2014)
MCE (Ratia et al., 2008)
MCE (Luo et al., 2017)
MCE
CAS 88321-09-9
CAS 1009817-63-3
CAS 19542-67-7
CAS 924296-18-4
CAS 924296-19-5
CAS 66701-25-5
CAS 1093070-16-6
CAS 265129-71-3
CAS 924296-39-9
CAS 1247825-37-1
b-AP15
BAY11-7082
DUB-IN-1
DUB-IN-2
E-64
GRL0617
GW7647
HBX41108
HY-13487
MCE (Weinstock et al., 2012)
(Continued on next page)
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Continued
REAGENT or RESOURCE
SOURCE
IDENTIFIER
HY-17541A
IU1
MCE
NA
Selleck (Lee et al., 2010)
MCE (Liu et al., 2003)
CAS 314245-33-5
CAS 668467-91-2
CAS 1373215-15-6
CAS 70476-82-3
CAS 1572414-83-5
CAS 157654-67-6
CAS 603139-19-1
CAS 1247819-59-5
CAS 2062-78-4
CAS 418805-02-4
NA
LDN57444
LY-3000328
Mitoxantrone
ML323
MCE (Jadhav et al., 2014)
Selleck (Takeuchi et al., 1992)
MCE (Liang et al., 2014)
MCE (Nicholson et al., 2008)
Selleck (Gauthier et al., 2008)
MCE (Altun et al., 2011)
MCE (Ybema et al., 1994)
Selleck (Yang et al., 2007)
MCE (Mistry et al., 2013)
Selleck (Shao et al., 2014)
Selleck (Liu et al., 2003)
Selleck (Bartholomeusz et al., 2007)
Selleck (Cencic et al., 2011)
Selleck (Tardif et al., 2004)
Selleck (Vidya Balasubramanian, 2014)
Selleck (Akwabi-Ameyaw et al., 2008)
MCE (Ding et al., 2013)
Selleck (Hennan et al., 2008)
Selleck (Abulwerdi et al., 2014)
Selleck (Banerjee, 2003)
Selleck (Finnerty et al., 1992)
Selleck (Ermolayeva adn Sanders, 1995)
ChemDiv
NSC632839
Odanacatib
P22077
Pimozide
PYR-41
SJB3-019A
Spautin-1
TCID
CAS 1262888-28-7
CAS 30675-13-9
CAS 856243-80-6
CAS 328998-25-0
CAS 166518-60-1
CAS 1450655-76-1
CAS 278779-30-9
CAS 1229705-06-9
CAS 393105-53-8
CAS 518303-20-3
CAS 68-19-9
WP1130
4E1Rcat
Avasimibe
CPI-169
GW4064
RG-7388
Tiplaxtinin
UMI-77
Vitamin B12
Zafirlukast
Zinc Pyrithiene
8
CAS 107753-78-6
CAS 13463-41-7
www.chemdiv.com
www.chemdiv.com
www.chemdiv.com
9
ChemDiv
10
ChemDiv
Critical commercial assays
SPR analysis
GE Healthcare
Transgene
Cat# BR-1005-30
Cat# FR201-02
Cat# G7570
NF-kB activity luciferase-reporting system.
CellTiter-Glo luminescent cell viability assays
Microsomal stability assay
Promega
Corningꢀ Gentestꢁ
Cat# 452701
Deposited data
SCoV2 Plpro apo structure
SCoV2 PLpro/12 structure
SCoV2 PLpro/GRL0617 structure
SCoV2 PLpro/Ub structure
SCoV2 PLpro/ISG15 structure
This paper
PDB ID 7D6H
PDB ID 7E35
PDB ID 7JIR
PDB ID 6XAA
PDB ID 6YVA
This paper
Osipiuk et al., 2021
Klemm et al., 2020
Shin et al., 2020
Experimental models: cell lines
Human: 293T
Human: SCoV2 PLproWT A549
Human: SCoV2 PLproC112S A549
Human: hACE2-HeLa
Li Tan’s laboratory
This paper
N/A
N/A
N/A
N/A
This paper
Vitalstar
Recombinant DNA
pET32a-6xHis-3C-PLproWT
pET32a-6xHis-3C-PlproC112S
pET32a-6xHis-Ub2
This paper
This paper
This paper
N/A
N/A
N/A
(Continued on next page)
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Continued
REAGENT or RESOURCE
pCDNA3.1-NSP1-NSP2-FLAG
pCDNA3.1-3xFLAG- PLproWT
pCDNA3.1-3xFLAG- PlproC112S
Lenti-3xFLAG- PLproWT
SOURCE
IDENTIFIER
N/A
This paper
This paper
This paper
This paper
This paper
N/A
N/A
N/A
Lenti-3xFLAG- PlproC112S
N/A
Software and algorithms
GraphPad Prism
Graphpad Software Inc
https://www.graphpad.com/scientific-
software/prism/
¨
Schrodinger Glide software
Friesner et al., 2006
National Institutes of Health
Kabsch, 2010
https://www.schrodinger.com/products/glide
https://imagej.nih.gov/ij/
ImageJ
XDS program
autoPROC suite
Phaser
https://xds.mr.mpg.de
Vonrhein et al., 2011
McCoy et al., 2007
https://www.globalphasing.com/autoproc
https://www.phaser.cimr.cam.ac.uk/index.
php/ Phaser_Crystallographic_Software
Coot
Emsley et al., 2010
https://www2.mrc-lmb.cam.ac.uk/personal/
pemsley/coot/
Phenix
Liebschner et al., 2019
Williams et al., 2018
Schro¨ dinger, Inc
https://www.phenix-online.org
https//molprobity.manchester.ac.uk
http://www.pymol.org
MolProbity
PyMol
RESOURSE AVAILABILITY
Lead contact
Further information and requests for resources and reagents should be directed to and will be fulfilled by Lead Contact Li Tan (tanli@
sioc.ac.cn)
Materiala availability
Plasmid, Probe 2-7 and all compounds can be requested through contacting the Lead Contact.
Data and code availability
The accession numbers for the crystal structures reported in this paper are PDB: 7D6H and PDB: 7E35.
EXPERIMENTAL MODEL AND SUBJECT DETAILS
Cell lines and cell culture
HEK293T / SCoV2 PLproWT A549 / SCoV2 PLproC112S A549
HEK293T (human embryonic kidney cells; hypotriploid; female ; fetal) were obtained from Junhao Hu’s laboratory and were not
further authenticated. A549 expressing WT SCoV2 PLpro or C112S mutant SCoV2 PLpro were constructed in this paper. hACE2-
Hela cells were purchased from Vitalstar. Cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) supplemented
with 10% (v/v) fetal bovine serum (FBS, Gibco), 50 units/mL penicillin, 50 units/mL streptomycin. Cells were incubated in 5% CO2 at
37 oC. Cell lines were passaged every 72 hours with trypsin and not allowed to reach confluency.
293T cells / A549 lung adenocarcinoma cells
293T cells (female) and A549 lung adenocarcinoma cells (male) were obtained from ATCC. hACE2-HeLa cells (female) were obtained
from Vitalstar. All the cells were cultured in Dulbecco Modified Eagle medium (DMEM) with 10% FBS and 100 U/mL penicillin-strep-
tomycin at 37ꢁC/5% CO2 in a humidified environment.
METHOD DETAILS
Protein expression and purification
DNA fragment that encodes full-length PLpro of SCoV2 was synthesized and cloned into a modified pET32a vector which fuses a
6✕His tag and an HRV 3C protease cutting sequence to the N-terminal of PLpro. C112S point mutation was introduced by the stan-
dard PCR-based mutagenesis method and verified by Sanger sequencing.
PLpro wild-type and C112S mutant proteins were expressed in E. coli BL21(DE3). Specifically, when the OD600 of bacteria culture
reached about 0.8, zinc chloride was supplemented to the culture with a final concentration of 0.1 mM and protein expression was
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induced with 0.1 mM IPTG at 16 ꢁC for 18 hours. The bacterial cells were pelleted by centrifugation and resuspended in binding buffer
(50 mM Tris-HCl at pH 7.9, 500 mM NaCl and 5 mM imidazole) and disrupted with a high-pressure homogenizer FB-110S (Shanghai
Litu Machinery Equipment Engineering Co., Ltd.). Supernatant of the lysate after centrifugation at 16,000g for 30 min was subjected
to His-tag affinity purification with Ni2+-NTA agarose (GE Healthcare). The N-terminal 6✕His tag was cut by homemade HRV 3C pro-
tease and removed by size-exclusion chromatography on an HiLoad Superdex 26/60 column (GE healthcare) equilibrated in a buffer
containing 20 mM Tris-HCl at pH 7.5, 100 mM NaCl and 1 mM DTT.
The wild-type ubiquitin and its mutants (K48R, K63R or Ub-D77 which contains a glutamate following the last G76 residue of Ub)
were expressed as 6✕His-tagged recombinant proteins in E. coli BL21(DE3) and purified in a similar manner as SCoV2 PLpro.
Preparation of Di-Ubiquitin proteins
K48- and K63-linked di-ubiquitin were synthesized according to published protocols (Hofmann and Pickart, 2001; Piotrowski et al.,
1997). For M1-linked di-ubiquitin preparation, two tandem repeats of coding sequence for human ubiquitin were cloned into a
modified pET32a vector which fuses a 6✕His tag and an HRV 3C protease cutting sequence to the N-terminal of insert. The M1-linked
di-ubiquitin was expressed and purified in a similar way as mono-ubiquitin.
Protein crystallization and structural determination
Crystallization of apo-form SCov-2 PLproC112S was set up in hanging-drop manner at 16 oC by mixing fresh protein (10 or 15 mg/mL in
buffer containing 20 mM Tris-HCl at pH 7.5, 100 mM NaCl and 1 mM DTT) with equal volume of reservoir solution (0.1 M acetate buffer
pH 4.5, 0.8 M NaH2PO4/1.2M K2HPO4) and incubated at 16 ꢁC.
For the crystal growth of PLproC112S/compound complex, PLproC112S protein (10 mg/mL) was incubated with 3 mM compound 12
at 4 ꢁC overnight and then used to set up crystallization in sitting-drops by mixing protein/compound with equal volume of reservoir
solution which contains 0.1 M sodium citrate tribasic dihydrate at pH 5.5 and 16% (v/v) PEG 8000.
After about 10 days, both apo-form and complex crystals appeared and were harvested in reservoir solution supplemented with
25% (v/v) glycerol as cryo-protectant and flash frozen in liquid nitrogen. Diffraction datasets were collected at the beamline BL17U1
of the Shanghai Synchrotron Radiation Facility (SSRF). The diffraction data sets were processed with XDS program and the auto-
PROC suite (Kabsch, 2010; Tickle et al., 2018; Vonrhein et al., 2011).
The initial phase for the structure of both apo-form and complex form were solved by molecular replacement using the PDB entry
6W9C as the search model with program Phaser (McCoy et al., 2007). The structural model was further manually re-built in Coot and
refined with Phenix (Emsley et al., 2010; Liebschner et al., 2019). The qualities of final models were validated by MolProbity and listed
in Table S2 (Williams et al., 2018). All the structural figures were prepared in program PyMol.
Molecular docking study
¨
The docking study of 19 was performed based on coordinates of SCoV2 PLpro-12 (Chain A, PDB 7E35) and Schrodinger Glide soft-
ware (Friesner et al., 2006). Meanwhile, 12 as a control was docked back with same parameters. GlideScores simulate binding free
energies, more negative values represent tighter binders, and scores of -10 or lower usually represent very good binding. In this
study, the GlideScore for the top-ranking predicted binding mode of 19 was -11.814, in comparison, that for for 12 was -10.851.
Absorption and emission spectra
Absorption and emission spectra were recorded by a Enspire Multimode plate reader (Perkin Elmer). Solutions of probes (20 mM,
20 mM Tris-HCl (pH 8.0), 100 mM NaCl, 5 mM 2-Hydroxy-1-ethanethiol) were added to the wells. The absorption was recorded be-
tween 300-600 nm, and then the Emission was recorded between 420-650 nm at an excitation of 405 nm.
Kinetic assays
Kinetic assay was performed in assay buffer (20 mM Tris-HCl, 100 mM NaCl, 5 mM 2-hydroxy-1-ethanethiol, pH 7.8) with 5 mM 2-
hydroxy-1-ethanethiol as the reducing agent to eliminate cysteine-reactive compounds. 15 min was selected as the initial condition
to compare these probes with different lex and lem (probe 1: lex = 485 nm; lem = 535nm; probe 2: lex =345 nm; lem = 445nm; probe 3:
lex =330 nm; lem = 500nm; probe 5/6/7: lex = 405 nm; lem = 450nm). PLproC112S was used to determine whether signal generation
was dependent on enzyme activity. Enzyme concentration (PLpro 1 mM) and incubation time with substrate were optimized to yield a
linear response in a 60-mintime frame (probe 5, 2.5mM), and a 30-mintime frame (probe 7, 2.5 mM) for HTS Screening. The enzyme
activity was monitored using the Enspire Multimode Plate Reader (Perkin Elmer).
KM value determination
The assay was performed in a 40 mL reaction volume using a black flat-bottom 384-well plates (Greiner Bio-One, #781076). We used
the substrate after cleaving the quencher to make the standard curve of substrate concentration and signal value. Initial velocity of
enzymatic reaction was plotted over time within the linear range in various concentrations of enzyme and substrates. According to the
Michaelis equation, the initial velocity and substrate concentration were plotted to get the KM value:
Vmax½Sꢂ
=
V0
KM + ½Sꢂ
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Where V0 is initial velocity of enzymatic reaction, Vmax is the reaction rate when the enzyme is saturated with the substrate, ½Sꢂ is sub-
strate concentration, and KM is Michaelis constant.
HTS screening
A compound library consisting of 35,360 structurally diverse small molecules was purchased from Chemdiv and maintained as 5 mg/
ml stock solutions dissolved in DMSO and stored at -20ꢁC. The automated liquid dispensing of primary screen was performed on
Echo 550 (Labcyte) and Viafill (Integra). Fluorescence values were measured on Enspire Multimode Plate Reader (PerkinElmer).
All assays were performed in duplicate at room temperature, using black flat-bottom 384-well plates (Greiner Bio-One, #784076)
containing a final reaction volume of 20 mL. The assays were assembled as follows: 20 nL compounds (final concentration 5 mg/
ml) was dispensed into wells and then incubated with 10 mL of 2 mM PLpro (final concentration 1 mM) in Buffer A (20 mM Tris-HCl,
100 mM NaCl, 5 mM 2-hydroxy-1-ethanethiol, pH 7.8) for 30 min. Reactions were initiated with 10 mL of 5 mM probe 5 (final concen-
tration 2.5 mM) in Buffer A, shaken vigorously for 10 sec, and then incubated for 60 min, before measuring fluorescence emission
intensity (lex = 405 nm; lem = 450 nm). Each 384-well plate contained 11 positive control wells (20 nL of DMSO replacing 20 nL of
inhibitor in DMSO), 12 negative control wells (assay components lacking PLpro) and 1 positive inhibitor control well (20 nL of
10 mM GRL0617). Compounds that showed Z-scores of less than -8 were cherry picked for further analysis.
IC50 value determination
IC50 values for all inhibitors were determined in 384-well format. The final enzyme concentrations were 1 mM and 0.5 mM for probe 7
and Ub-Rho, respectively, and the final substrate concentrations were 2.5 mM and 0.5 mM in a 30 min time frame and a 5 min time
frame. The assays were performed at room temperature, and the enzyme activity was monitored as described above.
SPR analysis
SPR experiment was performed by Biacore T200 (GE Healthcare) at 20 ꢁC. PBS-P buffer was filtered through a 0.22 mm membrane
filter and degassed prior to use. First, PLpro protein was immobilized on the CM5 chip surface. PLpro protein was diluted to 50 mg/mL
using sodium acetate solution at pH 5.5. The diluted protein flowed at 10 mL/min and was soon covalently immobilized to a flow cell of
CM5 sensor chip via primary amine group, using standard Amine Coupling Kit. The final level of immobilized protein was 13537 RU. A
flow cell without coupling proteins was used as a blank reference. Small molecules were diluted in PBS-P buffer in a series of con-
centrations (1.25-125 mM for GRL0617, 0.24-125 mM for 19) and flowed over the immobilized PLpro or blank cell at 30 mL/min with
90 s contact time and 120 s dissociation time. The equilibrium dissociation constant (Kd) was used to evaluate the binding activity of
each compound. Considering the influence of non-specific binding, we used non-specific binding function to fit the curve in Graph-
Pad Prism. Then subtract the non-specific contribution from the original data, and we got the actual specific binding response. Kd
was then calculated this response (R) and the corresponding concentration ([S]) with the formula below.
Rmax½Sꢂ
R =
½Sꢂ + Kd
Inhibitor specificity assay
To verify the specificity of inhibitor, Ub-Rho was used as substrate of PLpro and the release of Rhodamine 110 was measured by
increase of fluorescence intensity (Ex./Em. 485/535 nm) on 384-well microplate reader (Enspire, PerkinElmer). Compound 19
(10 mM) was incubated with 10 mL different DUBs (suitable concentration) for 1 h at RT respectively and reaction was initiated by addi-
tion of 10 mL Ub-Rho (1 mM) to the well. The reaction was maintained at 37 ꢁC until the appropriate fluorescence intensity value was
reached. The experiment was repeated three times.
Nsp1-Nsp2 cleavage assay
4 x 105 HEK293T cells per well were seeded onto 12-well plates a day before pretreated with compounds for 1 h, and then transiently
transfected with 500 ng nsp1-nsp2-flag and 250 ng w/o Flag-SCoV2-PLpro plasmid. 24h after transfection, cell lysates were
collected and analyzed for nsp1-nsp2-flag cleavage by immunoblot analysis.
NF-kB activity luciferase-reporting assay
1x105 HEK293T cells per well were seeded onto 96-well plates and transiently transfected with 0.02 mg p65 reporter construct
bearing firefly luciferase, 0.01 mg construct bearing renilla luciferase downstream of CMV promoter and w/o Flag-SCoV2 PLpro
plasmid. 18 h after seeding, HEK293T cells were stimulated with TNF-a (200 ng/mL) for 6 h and then firefly and renilla luciferase ac-
tivity was determined with TransDetect double-luciferase reporter assay kit (Transgene, FR201-02) according to the manufacturers’
instruction.
DeISGylation assay
A549 cells stably expressing of PLproWT or PLproC112S mutant were pretreated with compounds (10 mM) for 6 h, and treated with
1.25 nM IFN-a for 24 h to induce ISGylation. Cells were lysed with lysis buffer (100 mM Tris-HCl (pH 8.0), 1% (v/v) SDS), boiled at
ꢁ
95 C for 10 min and protein concentration was measured with BCA assay (Thermo Fischer). 10 mg cell lysates were loaded onto
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13.5% Tris-Glycin PAGE gel, transferred to nitrocellulose using a semi-dry transfer apparatus (0.2 A for 90 mins), and blocked for
30 mins at RT. The membrane was incubated with anti-ISG15 (1:1000 in 3% BSA, Invitrogen #703131) at 4 ꢁC overnight and incu-
bated with secondary antibody (1:5000) for 1 h at RT.
SARS-CoV2 infection and evaluation of antiviral activities
A clinically isolated SARS-CoV2 (SZ02) was propagated in Vero-E6 cells, viral titer was determined by 50% tissue culture infective
dose (TCID50). All the infection experiments were performed in a biosafety level-3 (BLS-3) laboratory. To evaluate the antiviral efficacy
of these inhibitors, hACE2-Hela cells were cultured in 24-well plate (1 x 105 cells/well) or in 96-well plate (1 x 104 cells/well) overnight,
and then infected with 0.3 MOI virus for 2 h. The supernatant was removed and the cells were washed 3 times with PBS, then cultured
with fresh medium plus inhibitors at indicated doses. At 48 h post infection, cells were collected with 2X SDS sample buffer for immu-
noblotting with antibody against SCoV2 NP (Sino Biological 40143-R019), or fixed with 4% PFA and immunostained with anti-Spike
antibody (Sinobiologica, 40150-T62). The nuclei were stained with DAPI (Beyotime, China). The images were recorded with fluores-
cence microscopy.
To evaluate the cytotoxicity of the inhibitors, 2 x 103 cells/well hACE2-HeLa cells cultured in 384-well plate were treated with each
inhibitor at indicated concentrations for 72 h, then subjected to viability assays (CellTiter-Glo, Promega) followed with measuring and
quantification.
Metabolic stability studies
Inhibitor 19 or Verapamil (control) and mouse liver microsomes (MLM) were primarily incubated in a thermostat water bath maintained
at 37 ꢁC for 5 min (250 mL, containing 1.5 mM compound and 750 ng/mL MLM in PBS). The primary incubations were divided into 7
tubes with 30 uL which was added into 15 mL NADPH (3 mM) or 15 mL PBS, and incubated in a thermostat water bath maintained at 37
ꢁC. At various time points, e.g., 0, 5, 10, 20, 30, 60 min (all above with 15 mL NADPH), 60 min (with 15 mL PBS), incubations were
quenched with 90 mL ice-cold acetonitrile containing 100 ng/mL IS. These mixtures were centrifuged at 10,000 rpm for 10 min,
the liquid supernatants were transferred to clean tubes. After diluted with water (1:1), the incubations were analyzed by LC-MS.
Inhibitor 19 (0.25 mM in PBS) and mouse plasma were incubated in a thermostat water bath maintained at 37 ꢁC (200 mL, containing
8.35 mM compound). At various time points, e.g., 0, 15, 30, 60, 30, 120 min, 30 mL incubations were quenched with 120 mL ice-cold
acetonitrile containing 100 ng/mL IS. These mixtures were centrifuged at 10,000 rpm for 10 min, the liquid supernatants were trans-
ferred to clean tubes. After diluted with water (1:1), the incubations were analyzed by LC-MS.
Chemical synthesis
General synthetic procedures
Unless otherwise noted, reagents and solvents were obtained from commercial suppliers and were used without further purification.
Reactions were run in roundbottom flasks or glass vials and stirred with Teflon-coated magnetic stir bars. Solvent evaporation was
€
performed on rotary evaporator (Buchi) under reduced pressure. Reactions were monitored by thin layer chromatography or LC-MS
(Waters). Acquity H-Class UPLC/MS with QDa mass spectrometer using water + 0.05% formic acid (solvent A) and acetonitrile +
0.05% formic acid (solvent B). Preparative HPLC was performed on a Waters Sunfire C18 column (19 x 100 mm, 5mM) using a
gradient of 10-95% acetonitrile in water containing 0.05% trifluoroacetic acid (TFA) over 10 min (15 min run time) at a flow rate of
1
15 mL/min. 1H/13C NMR spectra were recorded at ambient temperature on Bruker DMX 400 (400 MHz for H NMR and 101 MHz
for 13C NMR) instruments in the specified deuterated solvents. Observed proton absorptions were reported as d units of parts per
million (ppm) relative to tetramethylsilane (d 0.0). Multiplicities were reported: s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet
of doublets) and m (multiplet). Coupling constants were reported as a J value in Hertz (Hz).
Abbreviations used in this section: THF = tetrahydrofuran; DCM = dichloromethane; DMF = N,N-dimethylformamide; DMSO =
dimethyl sulfoxide; DCE = 1,2-dichloroethane; DIPEA = diisopropylethylamine; EA = ethyl acetate; TFA = trifluoroacetic acid;
HCl = hydrochloric acid; RT = room temperature.
Reaction schemes
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Reagents and conditions: (a) Ms2O, DIEA, DCM, 40 min, 0 ꢁC; (b) ethyl isonipecotate, DCM, RT, 16 h; (c) 1 M NaOH, MeOH, RT, 16
h; (d) EDCI, HOBt, DIEA, DCM, RT, 16 h.
Reagents and conditions: (a) triphosgene, DCM, RT; (b) methylpiperazine, RT, 16 h; (c) dimethylglycine, HATU, DIEA, DCM, RT, 10
h; (d) Acetic anhydride, THF, RT, 2 h; (f) H2, Raney Ni, MeOH (NH3), RT, 16 h.
Ethyl (R)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxylate (21). (S)-1-(naphthalen-1-yl) ethan-1-ol (500 mg, 2.9 mmol) was dis-
solved in dry DCM (14.5 mL) at 0 ꢁC. With continuous stirring at 0 ꢁC, DIEA (2 mL, 11.6 mmol) was added, and Ms2O (758 mg,
4.35 mmol) was added dropwise. The solution was stirred for 40 min at 0 ꢁC, at which point ethyl piperidine-4-carboxylate
(0.32 mL, 2.03 mmol) was added. The mixture was then stirred for 16 h at room temperature. The reaction mixture was quenched
with water and extracted with CH2Cl2, washed with 10% aqueous Na2CO3. The organic layer was then dried (Na2SO4), filtered,
and removed in vacuo. The residue was purified by silica gel column chromatography to furnish compound 21 in 54% yield (slightly
yellow oil). 1H NMR (400 MHz, DMSO-d6) d 8.46 – 8.40 (m, 1H), 7.93 – 7.88 (m, 1H), 7.79 (dd, J = 8.1, 1.3 Hz, 1H), 7.54 – 7.44 (m, 4H),
4.15 (q, J = 6.7 Hz, 1H), 4.06 – 4.01 (m, 2H), 2.96 (d, J = 11.3 Hz, 1H), 2.76 – 2.68 (m, 1H), 2.25 (ddt, J = 11.2, 8.0, 4.0 Hz, 1H), 2.08 (tdd,
J = 11.2, 5.5, 2.8 Hz, 2H), 1.84 – 1.77 (m, 1H), 1.74 – 1.67 (m, 1H), 1.55 – 1.45 (m, 2H), 1.39 (d, J = 6.7 Hz, 3H), 1.15 (t, J = 7.1 Hz, 3H).
(R)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxylic acid (22). 21 (300 mg, 0.96 mmol) was dissolved in methanol (15 mL), fol-
lowed by the addition of 1 N sodium hydroxide (2 mL, 2 mmol) in one portion. The resulting mixture was stirred for 16 h at room tem-
perature, then concentrated in vacuo, and dissolved in small amount of water. The solution was treated with AcOH dropwise until the
solution was acidic by pH paper. The residue was purified by reversed phase column chromatography to furnish compound 22 in
98% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 8.38 (d, J = 8.5 Hz, 1H), 8.07 – 8.03 (m, 2H), 7.88 (dt, J = 7.4, 2.1 Hz, 1H),
7.73 – 7.57 (m, 4H), 5.46 – 5.38 (m, 1H), 3.90 (d, J = 12.2 Hz, 2H), 3.10 (s, 1H), 2.90 (q, J = 11.2, 10.7 Hz, 1H), 2.44 (s, 1H), 2.13 (d,
J = 14.2 Hz, 1H), 1.94 (d, J = 14.1 Hz, 1H), 1.83 (d, J = 13.0 Hz, 1H), 1.75 (d, J = 6.7 Hz, 3H), 1.64 (d, J = 11.7 Hz, 1H).
N-(3-(aminomethyl)-5-fluorophenyl) acetamide (14c). 3-amino-5-fluorobenzonitrile (500 mg, 4 mmol) was dissolved in THF (3 mL),
followed by the addition of acetic anhydride (0.58 mL, 6 mmol) in one portion. The resulting mixture was stirred for 2 h at room tem-
perature, then concentrated in vacuo. The residue was purified by silica gel column chromatography to furnish compound 14a in 92%
yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 10.46 (s, 1H), 7.80 – 7.76 (m, 2H), 7.50 (ddd, J = 8.4, 2.4, 1.4 Hz, 1H), 2.09 (s, 3H).
To a solution of 14a (300 mg, 1.68 mmol) in saturated ammonia in MeOH, Raney-Ni (30 mg, 10%) was added, and the mixture was
stirred under H2 atmosphere at room temperature for 16 h. The reaction was filtered through a celite pad and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 14c in 98% yield
(slightly yellow oil).
N-(3-(aminomethyl) phenyl)-2-(dimethylamino) acetamide (16c). To a solution of dimethylglycine (600 mg, 5.82 mmol), EDCI (1.12 g,
5.82 mmol), and HOBt (0.79 g, 5.82 mmol) in dry DCM (20 mL) were added a solution of DIEA (4.2 mL, 24.2 mmol) and 3-aminoben-
zonitrile (573 mg, .4.85 mmol) in dry CH2Cl2 (20 ml) at 0ꢁC under argon atmosphere, and it was allowed to stir for 16 h at room tem-
perature. The reaction mixture was quenched with water and extracted with CH2Cl2. The organic layers were dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 16a in
36% yield (slightly yellow oil). 1H NMR (400 MHz, Chloroform-d) d 9.31 (s, 1H), 8.03 – 7.97 (m, 1H), 7.81 (ddd, J = 8.0, 2.2, 1.4 Hz,
1H), 7.48 – 7.41 (m, 1H), 7.38 (dt, J = 7.7, 1.4 Hz, 1H), 3.10 (s, 2H), 2.39 (s, 6H).
e7 Cell Chemical Biology 28, 1–11.e1–e9, June 17, 2021

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To a solution of 16a (200 mg, 0.98 mmol) in saturated ammonia in MeOH, Raney-Ni (20 mg, 10%) was added, and the mixture was
stirred under H2 atmosphere at room temperature for 16 h. The reaction was filtered through a celite pad and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 16c in 98% yield
(slightly yellow oil).
N-(3-(aminomethyl) phenyl)-4-methylpiperazine-1-carboxamide (17c). A solution of 3-isocyanatobenzonitrile (100 mg, 0.69 mmol)
and 1-methylpiperazine (76 mg, 0.76 mmol) in diethylether (1.7 ml) prepared at room temperature, and was stirred at the same tem-
perature. The reaction was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to
furnish compound 17b in 83% yield (colorless oil). 1H NMR (400 MHz, DMSO-d6) d 8.84 (s, 1H), 7.93 (t, J = 1.9 Hz, 1H), 7.74 (ddd, J =
8.4, 2.3, 1.2 Hz, 1H), 7.44 (t, J = 7.9 Hz, 1H), 7.37 (dt, J = 7.6, 1.4 Hz, 1H), 3.45 (t, J = 5.0 Hz, 4H), 2.32 (t, J = 5.0 Hz, 4H), 2.20 (s, 3H).
To a solution of 17b (140 mg, 0.57 mmol) in saturated ammonia in MeOH, Raney-Ni (14 mg, 10%) was added, and the mixture was
stirred under H2 atmosphere at room temperature for 16 h. The reaction was filtered through a celite pad and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 17c in 98% yield
(slightly yellow oil).
N-(3-(aminomethyl)-5-fluorophenyl)-2-(dimethylamino) acetamide (18c). To a solution of dimethylglycine (500 mg, 3.67 mmol), and
HATU (1.67 g, 4.4 mmol) in dry DCM (20 mL) were added a solution of DIEA (3.2 mL, 18.3 mmol) and 3-amino-5-fluorobenzonitrile
(454 mg, 4.4 mmol) in dry CH2Cl2 (17 ml) at 0ꢁC under argon atmosphere, and it was allowed to stir for 16 h at room temperature.
The reaction mixture was quenched with water and extracted with CH2Cl2. The organic layers were dried over Na2SO4 and concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 18a in 37% yield
(white solid).
To a solution of 18a (110 mg, 0.5 mmol) in saturated ammonia in MeOH, Raney-Ni (11 mg, 10%) was added, and the mixture was
stirred under H2 atmosphere at room temperature for 16 h. The reaction was filtered through a celite pad and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 18c in 98% yield
(slightly yellow oil).
N-(3-(aminomethyl)-5-fluorophenyl)-4-methylpiperazine-1-carboxamide (19c). 3-amino-5-fluorobenzonitrile (500 mg, 3.67 mmol)
was dissolved in CH2Cl2 (10 ml), and added dropwise in portions to a solution of triphosgene (546 mg, 1.84 mmol) in CH2Cl2
(20 ml). The reaction was stirred at room temperature for 6 h, and was concentrated under reduced pressure to get the compound
19a.
A solution of 19a (3.67 mmol) and 1-methylpiperazine (407 mg, 3.67 mmol) in CH2Cl2 (44 ml) prepared at room temperature, and
was stirred at the same temperature for 16 h. The reaction was concentrated under reduced pressure. The residue was purified by
silica gel column chromatography to furnish compound 19c in 52% yield (slightly yellow solid).
To a solution of 19c (200 mg, 0.76 mmol) in saturated ammonia in MeOH, Raney-Ni (20 mg, 10%) was added, and the mixture was
stirred under H2 atmosphere at room temperature for 16 h. The reaction was filtered through a celite pad and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound 19c in 98% yield
(slightly yellow oil).
General Amide Coupling Method. To a solution of (R)-1-(1(naphthalen-1-yl)ethyl) piperidine-4-carboxylic acid (0.05 g,
0.18 mmol), EDCI (0.04 g, 0.21 mmol), and HOBt (0.03 g, 0.21 mmol) in dry DCM (1 mL) were added a solution of DIEA (0.154 mL,
0.88 mmol) and amine (0.21 mmol) in dry CH2Cl2 at 0ꢁC under argon atmosphere, and it was allowed to stir for 16 h at room temper-
ature. The reaction mixture was quenched with water and extracted with CH2Cl2. The organic layers were dried over Na2SO4 and
concentrated under reduced pressure. The residue was purified by silica gel column chromatography to furnish compound.
(R)-N-(3-fluorobenzyl)-1-(1-(naphthalen-1-yl)ethyl)piperidine-4-carboxamide (11). The title compound was obtained as described in
the general procedure in 53% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 8.46 (d, J = 8.2 Hz, 1H), 8.30 (t, J = 6.0 Hz, 1H), 7.91
(dd, J = 7.2, 2.4 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.55 – 7.43 (m, 4H), 7.33 (td, J = 7.9, 6.1 Hz, 1H), 7.07 – 6.96 (m, 3H), 4.24 (d, J = 6.0 Hz,
2H), 4.15 (q, J = 6.5 Hz, 1H), 3.07 (d, J = 11.2 Hz, 1H), 2.79 (d, J = 11.4 Hz, 1H), 2.18 – 2.12 (m, 1H), 2.05 – 1.97 (m, 2H), 1.72 (d, J =
12.7 Hz, 1H), 1.63 – 1.52 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H).
(R)-N-(3-acetamidobenzyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (12). The title compound was obtained as
described in the general procedure in 52% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 9.87 (s, 1H), 8.46 (d, J = 8.1 Hz,
1H), 8.24 (t, J = 5.9 Hz, 1H), 7.94 – 7.88 (m, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.56 – 7.40 (m, 6H), 7.19 (t, J = 7.8 Hz, 1H), 6.86 (d, J =
7.6 Hz, 1H), 4.19 (d, J = 5.9 Hz, 2H), 4.14 (d, J = 6.7 Hz, 1H), 3.07 (d, J = 10.9 Hz, 1H), 2.79 (d, J = 11.3 Hz, 1H), 2.13 (d, J =
6.6 Hz, 1H), 2.01 (s, 5H), 1.73 (d, J = 12.7 Hz, 1H), 1.57 (d, J = 12.2 Hz, 3H), 1.40 (d, J = 6.6 Hz, 3H).
(R)-N-((2-methoxypyridin-4-yl) methyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (13). The title compound was ob-
1
tained as described in the general procedure in 66% yield (white solid). H NMR (400 MHz, DMSO-d6) d 8.46 (d, J = 8.1 Hz, 1H),
8.32 (t, J = 6.0 Hz, 1H), 8.05 (d, J = 5.2 Hz, 1H), 7.91 (dd, J = 7.9, 1.8 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.56 – 7.44 (m, 4H), 6.80
(dd, J = 5.3, 1.4 Hz, 1H), 6.59 – 6.56 (m, 1H), 4.20 (d, J = 6.0 Hz, 2H), 4.15 (q, J = 6.6 Hz, 1H), 3.81 (s, 3H), 3.07 (d, J = 11.2 Hz,
1H), 2.79 (d, J = 11.4 Hz, 1H), 2.17 (ddd, J = 11.6, 7.5, 4.1 Hz, 1H), 2.02 (tdd, J = 8.5, 5.6, 2.9 Hz, 2H), 1.73 (d, J = 12.9 Hz, 1H),
1.63 – 1.50 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H).
(R)-N-(3-acetamido-5-fluorobenzyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (14). The title compound was obtained
as described in the general procedure in 41% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 10.09 (s, 1H), 8.46 (d, J = 8.1 Hz, 1H),
Cell Chemical Biology 28, 1–11.e1–e9, June 17, 2021 e8

Please cite this article in press as: Shan et al., Development of potent and selective inhibitors targeting the papain-like protease of SARS-CoV-2, Cell
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8.30 (d, J = 6.0 Hz, 1H), 7.91 (d, J = 7.2 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.50 (m, 5H), 7.11 (s, 1H), 6.68 (d, J = 9.4 Hz, 1H), 4.19 (d, J =
5.9 Hz, 2H), 4.15 (d, J = 6.5 Hz, 1H), 3.08 (d, J = 10.9 Hz, 1H), 2.80 (d, J = 11.2 Hz, 1H), 2.13 (d, J = 9.2 Hz, 1H), 2.02 (d, J = 1.1 Hz, 5H),
1.74 (d, J = 13.0 Hz, 1H), 1.58 (m, 3H), 1.40 (d, J = 6.5 Hz, 3H).
(R)-N-((1-methylpiperidin-4-yl) methyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (15). The title compound was ob-
1
tained as described in the general procedure in 58% yield (white solid). H NMR (400 MHz, DMSO-d6) d 8.45 (d, J = 8.0 Hz, 1H),
7.91 (dd, J = 7.2, 2.3 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.68 (t, J = 5.7 Hz, 1H), 7.55 – 7.43 (m, 4H), 4.13 (q, J = 6.7 Hz, 1H), 3.05
(d, J = 11.1 Hz, 1H), 2.89 (t, J = 6.3 Hz, 2H), 2.73 (d, J = 12.5 Hz, 3H), 2.14 (s, 2H), 2.09 (s, 3H), 2.02 – 1.94 (m, 2H), 1.81 (t, J =
11.6 Hz, 2H), 1.65 (d, J = 12.4 Hz, 1H), 1.55 (s, 1H), 1.54 – 1.50 (m, 3H), 1.39 (d, J = 6.6 Hz, 3H), 1.33 – 1.26 (m, 1H), 1.13 – 1.03
(m, 2H).
(R)-N-(3-(2-(dimethylamino) acetamido) benzyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (16). The title compound
1
was obtained as described in the general procedure in 54% yield (white solid). H NMR (400 MHz, DMSO-d6) d 9.70 (s, 1H), 8.46
(d, J = 8.1 Hz, 1H), 8.25 (t, J = 6.0 Hz, 1H), 7.94 – 7.89 (m, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.56 – 7.45 (m, 6H), 7.21 (t, J = 7.8 Hz,
1H), 6.89 (dt, J = 7.7, 1.4 Hz, 1H), 4.19 (d, J = 5.9 Hz, 2H), 4.15 (s, 1H), 3.09 (s, 3H), 2.79 (d, J = 11.1 Hz, 1H), 2.28 (s, 6H), 2.15 (s,
1H), 2.01 (s, 2H), 1.73 (d, J = 12.5 Hz, 1H), 1.58 (d, J = 13.0 Hz, 3H), 1.41 (d, J = 6.6 Hz, 3H).
(R)-4-methyl-N-(3-((1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamido) methyl) phenyl) piperazine-1-carboxamide (17). The ti-
tle compound was obtained as described in the general procedure in 43% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 8.46 (d,
J = 6.0 Hz, 2H), 8.20 (t, J = 6.0 Hz, 1H), 7.91 (dd, J = 7.9, 1.7 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.56 – 7.44 (m, 4H), 7.29 (dt, J = 7.1, 1.8 Hz,
2H), 7.16 – 7.10 (m, 1H), 6.77 (dt, J = 7.5, 1.4 Hz, 1H), 4.17 (d, J = 5.9 Hz, 2H), 4.14 (d, J = 6.5 Hz, 1H), 3.40 (d, J = 10.1 Hz, 3H), 3.07 (d,
J = 10.9 Hz, 1H), 2.78 (d, J = 11.2 Hz, 1H), 2.28 (t, J = 5.0 Hz, 4H), 2.18 (s, 3H), 2.09 (s, 3H), 1.99 (dd, J = 10.6, 6.7 Hz, 2H), 1.72 (d, J =
12.8 Hz, 1H), 1.57 (d, J = 11.3 Hz, 2H), 1.40 (d, J = 6.6 Hz, 3H).
(R)-N-(3-(2-(dimethylamino) acetamido)-5-fluorobenzyl)-1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamide (18). The title com-
pound was obtained as described in the general procedure in 55% yield (white solid). 1H NMR (400 MHz, DMSO-d6) d 9.88 (s, 1H),
8.46 (d, J = 8.0 Hz, 1H), 8.28 (t, J = 6.0 Hz, 1H), 7.91 (dd, J = 7.9, 1.7 Hz, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.56 – 7.44 (m, 5H), 7.30 (d, J =
1.7 Hz, 1H), 6.69 (dd, J = 9.5, 2.2 Hz, 1H), 4.19 (d, J = 6.0 Hz, 2H), 4.15 (d, J = 6.7 Hz, 1H), 3.05 (s, 3H), 2.79 (d, J = 11.3 Hz, 1H), 2.24 (s,
6H), 2.18 – 2.12 (m, 1H), 2.06 – 1.98 (m, 2H), 1.73 (d, J = 12.6 Hz, 1H), 1.63 – 1.52 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H).
(R)-N-(3-fluoro-5-((1-(1-(naphthalen-1-yl) ethyl) piperidine-4-carboxamido) methyl) phenyl)-4-methylpiperazine-1-carboxamide
(19). The title compound was obtained as described in the general procedure in 32% yield (white solid). 1H NMR (400 MHz,
DMSO-d6) d 8.68 (s, 1H), 8.48 – 8.44 (m, 1H), 8.25 (t, J = 6.0 Hz, 1H), 7.93 – 7.89 (m, 1H), 7.80 (d, J = 8.1 Hz, 1H), 7.55 – 7.45 (m,
4H), 7.28 (dt, J = 11.9, 2.3 Hz, 1H), 7.11 (t, J = 1.7 Hz, 1H), 6.56 (ddd, J = 9.5, 2.5, 1.4 Hz, 1H), 4.16 (d, J = 6.0 Hz, 2H), 4.14 (s,
1H), 3.41 (t, J = 5.0 Hz, 4H), 3.07 (d, J = 11.2 Hz, 1H), 2.79 (d, J = 11.1 Hz, 1H), 2.29 (t, J = 5.0 Hz, 4H), 2.19 (s, 3H), 2.15 (d, J =
5.9 Hz, 1H), 2.01 (s, 2H), 1.73 (d, J = 12.9 Hz, 1H), 1.55 (m, 3H), 1.40 (d, J = 6.6 Hz, 3H).
QUANTIFICATION AND STATISTICAL ANALYSIS
All statistical analyses and curves were produced using GraphPad Prism 8. All the enzymatic activity assay were performed in trip-
licate and quantified data were presented as mean SD.
For microscopy experiments, fluorescence intensities were quantified with ImageJ software. Data was presented as mean SD of
three experimental replicates.
IC50 values were calculated by means of non-linear regression using Graphpad Prism 8 and were shown as data points represent-
ing as mean SD.
e9 Cell Chemical Biology 28, 1–11.e1–e9, June 17, 2021