Naoya Kitamura1, Michael Dominic Sacco2, Chunlong Ma1, Yanmei Hu1, Julia Alma Townsend3, Xiangzhi Meng4, Fushun Zhang4, Xiujun Zhang2, Mandy Ba1, Tommy Szeto1, Adis Kukuljac2, Michael Thomas Marty3, David Schultz5, Sara Cherry6, Yan Xiang4, Yu Chen2, Jun Wang1. 1. Department of Pharmacology and Toxicology, College of Pharmacy, The University of Arizona, Tucson, Arizona 85721, United States. 2. Department of Molecular Medicine, Morsani College of Medicine, University of South Florida, Tampa, Florida 33612, United States. 3. Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721, United States. 4. Department of Microbiology, Immunology and Molecular Genetics, University of Texas Health Science Center at San Antonio, San Antonio, Texas 78229, United States. 5. Department of Biochemistry and Biophysics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States. 6. Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104, United States.
Abstract
The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.
The main protease (Mpro) of SARS-CoV-2 is a validated antiviral drug target. Several Mpro inhibitors have been reported with potent enzymatic inhibition and cellular antiviral activity, including GC376, boceprevir, calpain inhibitors II, and XII, with each containing a reactive warhead that covalently modifies the catalytic Cys145. Coupling structure-based drug design with the one-pot Ugi four-component reaction, we discovered one of the most potent noncovalent inhibitors, 23R (Jun8-76-3A) that is structurally distinct from the canonical Mpro inhibitor GC376. Significantly, 23R is highly selective compared with covalent inhibitors such as GC376, especially toward host proteases. The cocrystal structure of SARS-CoV-2 Mpro with 23R revealed a previously unexplored binding site located in between the S2 and S4 pockets. Overall, this study discovered 23R, one of the most potent and selective noncovalent SARS-CoV-2 Mpro inhibitors reported to date, and a novel binding pocket in Mpro that can be explored for inhibitor design.
The COVID-19 pandemic
had a significant impact on the global economy
and public health, and there is an urgent need for therapeutic interventions.
The viral polymerase inhibitor remdesivir gained FDA approval on October
22nd, 2020. The combination therapy of remdesivir with the Janus kinase
(JAK) inhibitor baricitinib also received FDA emergency use authorization.[1] Among the other drug targets being pursued at
preclinical and clinical stages,[2] the viral
main protease (Mpro), also called 3-chymotrypsin-like protease
(3CLpro), is one of the most extensively explored high
profile antiviral drug targets.[3] Mpro is a cysteine protease encoded in the viral polyprotein
as nonstructural protein 5 (Nsp5) that cleaves the viral polyproteins
pp1a and pp1ab at more than 11 sites. Despite its multiple proteolytic
sites, Mpro was shown to have a high substrate specificity
of glutamine at the P1 position.[4] As such,
the majority of the reported Mpro inhibitors were designed
to contain a 2-pyrrolidone at the P1 substitution as a mimetic of
the glutamine in the substrate.[5] Most advanced
Mpro inhibitors including PF-07304814,[6]GC376,[7,8]6j,[9]MI-09, and MI-30(10) all belong to this category (Figure ). PF-07304814, an α-hydroxyl ketone prodrug, is being developed by Pfizer
and has optimal pharmacokinetic properties and recently entered human
clinical trials.[6]GC376 has in vivo antiviral efficacy in treating cats infected with
lethal feline infectious peritonitis virus.[11,12] Recently, the GC376 analog 6j was shown
to protect mice from MERS-CoV infection.[9]MI-09 and MI-30 were shown to protect
mice from lethal SARS-CoV-2 infection.[10] These promising results highlight the translational potential of
Mpro inhibitors as potent SARS-CoV-2 antivirals and validate
Mpro as an antiviral drug target for coronaviruses.
Figure 1
Promising SARS-CoV-2
Mpro inhibitors reported in the
literature with translational potential.
Promising SARS-CoV-2
Mpro inhibitors reported in the
literature with translational potential.Drug discovery is a lengthy process involving iterative cycles
of design, synthesis, and pharmacological characterization. In the
event of the COVID-19 pandemic, an expedited approach with a fast
turnover of this development cycle is highly desired. Using SARS-CoV-2
Mpro as a drug target, we report herein a fast-track drug
discovery approach by coupling structure-based drug design and the
Ugi four-component reaction (Ugi-4CR) methodology. The design was
based on the superimposed structures of SARS-CoV or SARS-CoV-2 Mpro in complex with inhibitors including GC376, calpain inhibitor XII, and ML188 (R).[7,8,13] The lead compound 23R from this study is the most potent noncovalent SARS-CoV-2 Mpro inhibitor reported to date in terms of enzymatic inhibition
and cellular antiviral activity. The target selectivity of the designed
inhibitors was profiled against a panel of viral proteases and host
proteases, and the noncovalent inhibitor 23R was found
to be highly selective compared to the covalent inhibitor GC376. An X-ray crystal structure of SARS-CoV-2 Mpro in complex
with 23R was solved, revealing a drug-induced conformational
change and a previously unexplored binding site in between the S2
and S4 pockets. Overall, this study led to the discovery of the noncovalent
Mpro inhibitor 23R with potent enzymatic inhibition
and in vitro cellular antiviral activity with a novel
mechanism of action.
Results and Discussion
Rational Design of Noncovalent
SARS-CoV-2 Mpro Inhibitors
Among the noncanonical
SARS-CoV-2 Mpro inhibitors we
recently discovered, calpain inhibitor XII has an unexpected
binding mode showing an inverted conformation in the active site (Figure A).[8] Instead of projecting the norvaline and leucine side chains
into the S1 and S2 pockets as one would expect from its chemical structure,
the pyridinyl substitution snuggly fits in the S1 pocket and forms
a hydrogen bond with the H163 imidazole (Figure A). This hydrogen bond is essential, as replacing
the pyridine with benzene led to an analog UAWJ257 with
a significant loss of enzymatic inhibition.[8] Examining the X-ray crystal structures of SARS-CoV and SARS-CoV-2
Mpro in the PDB database revealed another compound ML188 (R),[13] which shares a similar
binding mode with calpain inhibitor XII. ML188
(R) is a noncovalent SARS-CoV Mpro inhibitor derived
from a high-throughput screening hit.[13] The pyridinyl from ML188 (R) similarly fits in the
S1 pocket and forms a hydrogen bond with the H163 side chain imidazole
(Figure B). In addition,
the furyl oxygen and its amide oxygen both form a hydrogen bond with
the G143 main chain amide amine. ML188 (R) was reported
to inhibit the SARS-CoV Mpro with an IC50 value
of 1.5 ± 0.3 μM and the SARS-CoV viral replication in Vero
E6 cells with an EC50 value of 12.9 μM.[13] Several follow up studies have been conducted
to optimize the enzymatic inhibition and cellular antiviral activity
of this series of compounds; however, no significant improvement has
been made.[14,15]
Figure 2
Structure of Mpro with inhibitors.
(A) X-ray crystal
structure of SARS-CoV-2 Mpro in complex with calpain
inhibitor XII (PDB: 6XFN). (B) X-ray crystal structure of SARS-Co-V Mpro in complex with ML188 (R) (PDB: 3V3M). Hydrogen bonds
are shown in dashed lines.
Structure of Mpro with inhibitors.
(A) X-ray crystal
structure of SARS-CoV-2 Mpro in complex with calpain
inhibitor XII (PDB: 6XFN). (B) X-ray crystal structure of SARS-Co-V Mpro in complex with ML188 (R) (PDB: 3V3M). Hydrogen bonds
are shown in dashed lines.The similar binding mode of ML188 (R) with calpain
inhibitor XII, coupled with the convenient synthesis through
the one pot Ugi-4CR, inspired us to design noncovalent SARS-CoV-2
Mpro inhibitors based on the ML188 (R) scaffold.
Specifically, we leverage our understanding of the Mpro inhibition mechanism based on the X-ray crystal structures of SARS-CoV-2
Mpro with multiple inhibitors to guide the lead optimization
(Figure A–D).[7,8] Overlaying the X-ray crystal structures of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and the SARS-CoV Mpro H41A
mutant + the peptide substrate (PDB: 2Q6G) revealed that the furyl, 4-tert-butylphenyl, pyridinyl, and tert-butyl of ML188 (R) fit in the S1′, S2, S1, and S3 pockets, respectively
(Figure A, D). Therefore,
the furyl, 4-tert-butylphenyl, pyridinyl, and tert-butyl substitutions in ML188 (R) were
defined as P1′, P2, P1, and P3, respectively. Next, overlaying
the structure of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2 Mpro + GC376 (PDB: 6WTT) suggested that
the tert-butyl at the P3 substitution of ML188
(R) can be extended to fit in the S4 pocket (Figure B, D). Previous structure–activity
relationship studies of GC376 indicate that P4 substitution
is important, while P3 substitution does not contribute significantly
to the binding affinity, as it is solvent exposed.[3,8,9,16] Similarly,
the overlaying structures of SARS-CoV Mpro + ML188
(R) (PDB: 3V3M) and SARS-CoV-2 Mpro + UAWJ247 (PDB: 6XBH) suggested that
the 4-tert-butyl at the P2 substitution of ML188 (R) can be replaced by phenyl to occupy the extra space
in the S2 pocket (Figure C, D). Overall, binding site analysis suggests that extending
the P2 and P3 substitutions of ML188 (R) might lead to
better shape complementarity with the SARS-CoV-2 Mpro (Figure E). In practice,
we adopted a stepwise optimization procedure in which the P3 and P2
substitutions were optimized individually in step 1 and then the optimal
P2/P3 substitutions were combined in step 2 (Figure E).
Figure 3
Design rationale for the noncovalent SARS-CoV-2
Mpro inhibitors. (A) Superimposed X-ray crystal structures
of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M, green) and SARS-CoV Mpro H41A
mutant + peptide substrate (PDB: 2Q6G, yellow with backbone shown as ribbon
representation). (B) Superimposed X-ray crystal structures of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2
Mpro + GC376 (PDB: 6WTT). (C) Superimposed X-ray crystal structures
of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2
Mpro + UAWJ247 (PDB: 6XBH). (D) Chemical structures
of ML188 (R), peptide substrate VLQS, GC376, and UAWJ247. (E) Stepwise optimization of ML188
(R) toward potent noncovalent SARS-CoV-2 Mpro inhibitor.
Design rationale for the noncovalent SARS-CoV-2
Mpro inhibitors. (A) Superimposed X-ray crystal structures
of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M, green) and SARS-CoV Mpro H41A
mutant + peptide substrate (PDB: 2Q6G, yellow with backbone shown as ribbon
representation). (B) Superimposed X-ray crystal structures of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2
Mpro + GC376 (PDB: 6WTT). (C) Superimposed X-ray crystal structures
of SARS-CoV Mpro + ML188 (R) (PDB: 3V3M) and SARS-CoV-2
Mpro + UAWJ247 (PDB: 6XBH). (D) Chemical structures
of ML188 (R), peptide substrate VLQS, GC376, and UAWJ247. (E) Stepwise optimization of ML188
(R) toward potent noncovalent SARS-CoV-2 Mpro inhibitor.Guided by the design rationale elucidated above,
a focused library
of ML188 analogs was designed and synthesized (Figure ). As the P1′
furyl and P1 pyridinyl both form a critical hydrogen bond with the
Mpro (Figure A, B), the P1′ and P1 substitutions were kept with minimal
variations for the design of noncovalent inhibitors (Figure C). All designed compounds
were synthesized using the one pot Ugi four-component reaction and
tested as enantiomer/diastereomer mixtures (Figure C). To circumvent the need of relying on
an expensive chiral HPLC column for the separation of enantiomers,
we strategically introduced the chiral isocyanide so that the diastereomer
product mixture can be separated by convenient silica gel column or
reverse phase HPLC column purification.[17]
Figure 4
Design
and synthesis of a focused library of noncovalent SARS-CoV-2
Mpro inhibitors. (A) X-ray crystal structure of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M). (B) Binding interactions
of ML188 (R) with SARS-CoV Mpro. (C) Synthesis
of ML188 analogs using the Ugi four-component reaction.
Design
and synthesis of a focused library of noncovalent SARS-CoV-2
Mpro inhibitors. (A) X-ray crystal structure of SARS-CoV
Mpro + ML188 (R) (PDB: 3V3M). (B) Binding interactions
of ML188 (R) with SARS-CoV Mpro. (C) Synthesis
of ML188 analogs using the Ugi four-component reaction.
Structure–Activity Relationship Studies
of Noncovalent
SARS-CoV-2 Mpro Inhibitors
In total, 39 compounds
were synthesized (Figure A–E) and all compounds were initially tested as a mixture
of enantiomers or diastereomers in the FRET-based enzymatic assay
against SARS-CoV-2 Mpro at 20 μM (Figure F). Compounds showing more
than 50% inhibition at 20 μM were further titrated to determine
the IC50 values. Next, compounds with IC50 values
lower than 5 μM were selected for cellular cytotoxicity profiling
in Vero E6 cells, the cell line which was used for the SARS-CoV-2
antiviral assay. The purpose was to prioritize lead candidates for
the in vitro cellular antiviral assay with infectious
SARS-CoV-2. As shown in Figure , the majority of the designed compounds showed more than
50% inhibition when tested at 20 μM. Specifically, Figure A lists compounds
with P4 variations. As a reference, ML188 (1) (racemic mixture) inhibits SARS-CoV-2 Mpro with an IC50 value of 10.96 ± 1.58 μM. It was found that compounds 2, 3, 5, 6, 7, 8, 10, and 13 had improved
enzymatic inhibition compared to ML188 (1). These results suggest that (a) isopropyl (2), cyclopropyl
(3), cyclopentyl (5), cyclohexyl (6), and phenyl (7 and 8) are the
more favorable substitutions at the P3 position than tert-butyl and (b) compound 13 with the (S)-α-methylbenzyl substitution at the P3 position had improved
potency, which suggests that extending the substitutions to the S4
pocket might improve the enzymatic inhibition (Figure B). Given the advantage of convenient separation
of diastereomers over enantiomers, we therefore decided to fix the
P3/P4 substitution as α-methylbenzyl substitution during the
P2 optimization (Figure B). All compounds in Figure B were designed to have extended substitutions at the 4-position
of benzyl to occupy the extra space in the S2 pocket (Figure C). Consistent with the design
hypothesis, several compounds including 14, 17, 18, 19, 20, 21, and 23 had significantly improved enzymatic inhibition
(IC50 < 3 μM) compared to compound 13. Replacing the tert-butyl in compound 13 with the bulkier trimethylsilyl led to compound 14 with
a 2.9-fold increase in Mpro inhibition. Cyclohexyl (17), thienyl (19), pyrrolyl (20),
pyridinyl (21), and phenyl (23) were found
to be the most favorable substitutions at the S2 pocket. Compound 16 with piperidyl substitution had similar potency as compound 13, while compound 15 with O-tert butyl was less active. Further extending the substitution to benzyl
led to compound 22 that was inactive, suggesting biphenyl
might be the longest substitution that can be accommodated at the
S2 pocket.
Figure 5
Structures of noncovalent and covalent SARS-CoV-2 Mpro inhibitors and the enzymatic inhibition against SARS-CoV-2 Mpro. (A) Noncovalent analogs with P3/P4 modifications. (B)
Noncovalent analogs with P2 modifications. (C) Noncovalent analogs
with P1′ modifications. (D) Noncovalent analogs with P1 modifications.
(E) Noncovalent analogs with combined P1′, P1, P2, and P3/P4
modifications. Compounds with potent enzymatic inhibition (IC50 < 5 μM) but moderate to high cellular cytotoxicity
(CC50 < 100 μM) are labeled in red. Compounds
with both potent enzymatic inhibition (IC50 < 5 μM)
and low cellular cytotoxicity (CC50 > 100 μM)
are
labeled in blue. (F) Percentage enzymatic activity of SARS-CoV-2 Mpro in the presence of the designed compounds at 20 μM
concentration.
Structures of noncovalent and covalent SARS-CoV-2 Mpro inhibitors and the enzymatic inhibition against SARS-CoV-2 Mpro. (A) Noncovalent analogs with P3/P4 modifications. (B)
Noncovalent analogs with P2 modifications. (C) Noncovalent analogs
with P1′ modifications. (D) Noncovalent analogs with P1 modifications.
(E) Noncovalent analogs with combined P1′, P1, P2, and P3/P4
modifications. Compounds with potent enzymatic inhibition (IC50 < 5 μM) but moderate to high cellular cytotoxicity
(CC50 < 100 μM) are labeled in red. Compounds
with both potent enzymatic inhibition (IC50 < 5 μM)
and low cellular cytotoxicity (CC50 > 100 μM)
are
labeled in blue. (F) Percentage enzymatic activity of SARS-CoV-2 Mpro in the presence of the designed compounds at 20 μM
concentration.The P1′ and P1 substitutions
(Figure C, D) were
chosen to retain the critical
hydrogen bonds in ML188 (Figure A). It was found that imidazole (24) was tolerated at the P1′ position (IC50 = 0.96
± 0.09 μM), followed by isoxazole (25) (IC50 = 2.47 ± 0.27 μM) and oxazole (26) (IC50 = 4.97 ± 0.78 μM). Pyrazine (27) was tolerated at the P1 position (IC50 = 4.93
± 0.79 μM); however, imidazole (28) and pyrimidine
(29) were not preferred (IC50 > 20 μM).Next, the above identified favorable P1′, P2, P1, and P3/P4
substitutions were combined and the designed compounds were shown
in Figure E. Compounds 36, 37, and 38 were the most potent
leads with IC50 values of 0.81 ± 0.24, 0.67 ±
0.15, and 0.28 ± 0.07 μM, respectively. Compounds 39 and 40 were also highly active with IC50 values of 1.48 ± 0.56 and 1.05 ± 0.09 μM,
respectively.Among the active compounds with IC50 values lower than
5 μM, compounds 3, 6, 14, 17, 18, 19, 26, 34, and 36 had moderate to high cellular
cytotoxicity in Vero E6 cells (Figure A–E red), while compounds 5, 20, 21, 23, 24, 25, 27, 31, 32, 37, 38, 39, and 40 were
well tolerated and the CC50 values were greater than 100
μM (Figure A–E
blue).
Cellular Antiviral Activity of Noncovalent SARS-CoV-2 Mpro Inhibitors
Next, compounds with potent enzymatic
inhibition (IC50 ≤ 1 μM) and low cellular
cytotoxicity (CC50 > 100 μM) were prioritized
for
the cellular antiviral assay with infectious SARS-CoV-2 in Vero E6
cells using the immunofluorescence assay as the primary assay (Figure A). ML188 (1) was included as a control. It was found that ML188 (1) was inactive in the antiviral assay
(EC50 > 20 μM), probably due to its incomplete
inhibition
of the Mpro in the cellular content. Gratifyingly, compounds 20, 23, 37, 38, and 40 all had potent cellular antiviral activity with EC50 values ranging from 0.82 to 4.54 μM. Compound 24 was less active (EC50 = 13.06 ± 2.30 μM),
possibly due to the poor cellular membrane permeability.
Figure 6
Enzymatic inhibition,
cellular antiviral activity, and selectivity
index of noncovalent Mpro inhibitors. (A) Antiviral activity
and selectivity index of noncovalent SARS-CoV-2 Mpro inhibitors.
(Selection criteria IC50 < 1 μM, CC50 > 100 μM). Antiviral assay was performed using the SARS-CoV-2
USA-WA1/2020 isolate in Vero E6 cells with an MOI of 0.05. (B) Chemical
structures of the two diastereomers 23R and 23S. The absolute stereochemistry of compound 23R was determined
in the cocrystal structure of this diastereomer with SARS-CoV-2 Mpro (PDB: 7KX5). (C and D) Antiviral activity of 23R against SARS-CoV-2
in Calu-3 cells. (C) Raw data of the percentage of immunofluorescence
positive cells with different concentrations of 23R.
(D) Antiviral potency and cytotoxicity plots. N.T. = not tested. N.A.
= not applicable.
Enzymatic inhibition,
cellular antiviral activity, and selectivity
index of noncovalent Mpro inhibitors. (A) Antiviral activity
and selectivity index of noncovalent SARS-CoV-2 Mpro inhibitors.
(Selection criteria IC50 < 1 μM, CC50 > 100 μM). Antiviral assay was performed using the SARS-CoV-2
USA-WA1/2020 isolate in Vero E6 cells with an MOI of 0.05. (B) Chemical
structures of the two diastereomers 23R and 23S. The absolute stereochemistry of compound 23R was determined
in the cocrystal structure of this diastereomer with SARS-CoV-2 Mpro (PDB: 7KX5). (C and D) Antiviral activity of 23R against SARS-CoV-2
in Calu-3 cells. (C) Raw data of the percentage of immunofluorescence
positive cells with different concentrations of 23R.
(D) Antiviral potency and cytotoxicity plots. N.T. = not tested. N.A.
= not applicable.Given the potent antiviral
activity and high selectivity index
of these potent lead compounds, we then selected the noncovalent inhibitor 23 for further characterization. The two diastereomers of 23 were separated by reverse phase HPLC (Figure B). Both diastereomers were
tested in the FRET-based enzymatic assay. GC376 was included
as a positive control. It was found that 23R is the active
diastereomer with an IC50 value of 0.20 ± 0.01 μM,
while the 23S diastereomer was not active (IC50 > 20 μM) (Figure A). The stereochemistry of 23R was determined
by the cocrystal structure with SARS-CoV-2 Mpro as described
in the following section. Compared with the parent compound ML188 (1), the optimized lead 23R had more than a 54-fold increase in enzymatic inhibition against
SARS-CoV-2 Mpro. Compound 23R also showed
comparable potency against SARS-CoV Mpro with an IC50 value of 0.27 ± 0.03 μM. Neither ML188 (1) nor 23R inhibited the SARS-CoV-2 papain-like
protease (PLpro) (IC50 > 20 μM) (Figure A), suggesting the
inhibition of SARS-CoV-2 Mpro by 23R is specific.Next, the antiviral activity of 23R was tested against
SARS-CoV-2 (USA-WA1/2020 isolate) in Vero E6 cells using the immunofluorescence
assay. It was found that compound 23R had an EC50 value of 1.27 μM (Figure A), which was similar to the antiviral potency of the
covalent inhibitor GC376 (EC50 = 1.51 μM).
Compound 23R was also not cytotoxic to Vero E6 cells
at up to 100 μM. In contrast, the parent compound ML188 (1) had no detectable antiviral activity when tested
at up to 20 μM. To further confirm the antiviral activity of
compound 23R, we performed a secondary antiviral assay
in the human lung epithelial Calu-3 cell line, which endogenously
expresses TMPRSS2 and is widely used as a physiological relevant cell
line for SARS-CoV-2 infection. It was found that compound 23R inhibited SARS-CoV-2 (USA-WA1/2020 isolate) replication in Calu-3
cells with an EC50 value of 3.03 μM, and it was not
cytotoxic at up to 100 μM (Figure C, D).
Profiling the Target Selectivity
of 23R against
Other Viral Cysteine Proteases and Host Proteases
One of
the major challenges facing cysteine protease inhibitors is the target
selectivity.[18,19] It was recently reported that GC376 inhibits host cathepsins B and L in addition to SARS-CoV-2
Mpro,[20] and GC376 analogs compound_1 and zyy16 that contained
the aldehyde and cyanohydrin warheads, respectively, inhibit both
calpain 1 and cathepsin K.[21] These results
raised the concern of the selectivity of this class of covalent inhibitors.
To profile the target selectivity of the lead compounds developed
in this study, we selected compound 23R as a representative
example of the noncovalent inhibitor. ML188 (1) and GC376 were included as controls. The compounds
were tested against a panel of viral cysteine proteases including
SARS-CoV Mpro, MERS-CoV Mpro, HCoV-OC43 Mpro, EV-A71 3Cpro, EV-D68 3Cpro, EV-A71
2Apro, EV-D68 2Apro, and SARS-CoV-2 PLpro, as well as host cysteine proteases calpain 1, cathepsin K, cathepsin
L, and serine protease trypsin. It was found that GC376 inhibited all the Mpro and 3Cpro tested including
SARS-CoV-2 Mpro, SARS-CoV Mpro, MERS-CoV Mpro, HCoV-OC43 Mpro, EV-A71 3Cpro, and
EV-D68 3Cpro (IC50 ≤ 0.16 μM),
but not the unrelated EV-A71 2Apro, EV-D68 2Apro, and SARS-CoV-2 PLpro (IC50 > 50 μM)
(Figure A and Table S1). In addition, GC376 also
inhibited host cysteine proteases calpain 1, cathepsin K, and cathepsin
L (IC50 ≤ 0.074 μM) but not the serine protease
trypsin (IC50 > 50 μM) (Figure B and Table S1). In contrast, the noncovalent inhibitor 23R developed
in this study only selectively inhibited SARS-CoV-2 Mpro and SARS-CoV Mpro with high potency (IC50 ≤
0.27 μM) but not other viral proteases tested, which was similar
to ML188 (1) (Figure A and Table S1). Compound 23R showed weak inhibition against calpain
1 and cathepsin L with IC50 values of 6.00 μM and
10.5 μM, respectively, corresponding to selectivity indexes
of 40 and 70 with regard to SARS-CoV-2 Mpro inhibition
(Figure B and Table S1).
Figure 7
Selectivity of noncovalent and covalent
SARS-CoV-2 Mpro inhibitors shown in a color map. (A) Selectivity
against viral cysteine
proteases. (B) Selectivity against host proteases. The values plotted
were the IC50 values from the FRET-based enzymatic assay.
Selectivity of noncovalent and covalent
SARS-CoV-2 Mpro inhibitors shown in a color map. (A) Selectivity
against viral cysteine
proteases. (B) Selectivity against host proteases. The values plotted
were the IC50 values from the FRET-based enzymatic assay.
Mechanism of Action of Noncovalent Inhibitor 23R in Inhibiting SARS-CoV-2 Mpro
The
mechanism
of action was characterized using native mass spectrometry, the thermal
shift-binding assay, and enzymatic kinetic studies (Figure ). In the native mass spectrometry
binding assay, compound 23R showed dose-dependent binding
to SARS-CoV-2 Mpro, similar to the positive control GC376, with a binding stoichiometry of one drug per monomer
(Figure A). Similarly,
compound 23R showed dose-dependent stabilization of the
SARS-CoV-2 Mpro in the thermal shift binding assay with
an apparent Kd value of 9.43 μM, a 9.3-fold decrease
compared to ML188 (1) (Figure B). In the enzymatic kinetic
studies, 23R was shown to be a noncovalent inhibitor
with a Ki value of 0.07 μM (Figure C, D top and middle panels). In comparison,
the Ki for the parent compound ML188 (1) is 2.29 μM.
The Lineweaver–Burk or double-reciprocal plot with different
compound concentrations yielded an intercept at the Y-axis, suggesting that 23R is a competitive inhibitor
similar to ML188 (1) (Figure C, D bottom panel). Overall, the enzymatic
kinetic studies confirmed that compound 23R is a noncovalent
inhibitor of SARS-CoV-2 Mpro.
Figure 8
Characterization of the
mechanism of action of 23R to SARS-CoV-2 Mpro using native mass spectrometry, thermal
shift assay, and enzyme kinetic studies. (A) Binding of 23R to SARS-CoV-2 Mpro using native mass spectrometry. Native
mass spectra with the inset deconvolved spectra revealing ligand binding
with 10 μM or 30 μM GC376 added (middle panel)
and 10 μM and 30 μM 23R (bottom panel) with
4 mM DTT added. The peaks are annotated with the blue circle as the
dimer, the green down triangle as the dimer with one ligand bound,
and the purple up triangle as the dimer with two ligands bound. (B)
Dose-dependent melting temperature (Tm) shift in the thermal shift assay. 3 μM SARS-CoV-2 Mpro protein was incubated with various concentrations of ML188 or 23R in the presence of 4 mM DTT. Measured Tm was plotted against compound concentration with one-site
binding function in Prism 8. (C and D) Enzymatic kinetic assay with ML188 and compound 23R. Kinetic parameters in
the presence of various concentrations of ML188 or 23R were globally fitted with a Michaelis–Menten function
in prism 8 (top panels); double-reciprocal plots are shown in the
right panels. The middle panels show the Morrison plots of compounds ML188 and 23R with 20 μM FRET substrate
used.
Characterization of the
mechanism of action of 23R to SARS-CoV-2 Mpro using native mass spectrometry, thermal
shift assay, and enzyme kinetic studies. (A) Binding of 23R to SARS-CoV-2 Mpro using native mass spectrometry. Native
mass spectra with the inset deconvolved spectra revealing ligand binding
with 10 μM or 30 μM GC376 added (middle panel)
and 10 μM and 30 μM 23R (bottom panel) with
4 mM DTT added. The peaks are annotated with the blue circle as the
dimer, the green down triangle as the dimer with one ligand bound,
and the purple up triangle as the dimer with two ligands bound. (B)
Dose-dependent melting temperature (Tm) shift in the thermal shift assay. 3 μM SARS-CoV-2 Mpro protein was incubated with various concentrations of ML188 or 23R in the presence of 4 mM DTT. Measured Tm was plotted against compound concentration with one-site
binding function in Prism 8. (C and D) Enzymatic kinetic assay with ML188 and compound 23R. Kinetic parameters in
the presence of various concentrations of ML188 or 23R were globally fitted with a Michaelis–Menten function
in prism 8 (top panels); double-reciprocal plots are shown in the
right panels. The middle panels show the Morrison plots of compounds ML188 and 23R with 20 μM FRET substrate
used.
X-ray Crystal Structure
of SARS-CoV-2 Mpro with 23R
Using
X-ray crystallography, we successfully
determined the binding pose of 23R with SARS-CoV-2 Mpro at 2.6 Å resolution (Figure A, Table S2).
Electron density reveals the body of 23R extends throughout
the substrate binding channel, with side chains occupying S1′,
S1, S2, and a previously unexplored subpocket in between S2 and S4.
The binding pose is similar to the previously solved structure of
SARS-CoV Mpro with ML188 (R) (PDB: 3V3M),[13] consistent with the similarities between the two compounds
and between the two proteins (Figure B). The furyl moiety of 23R binds to a
portion of the P1′ site, which normally accommodates small
hydrophobic residues. While the furylamide carbonyl group of 23R does not insert into the oxyanion hole, it does form a
bifurcated hydrogen bond with the apical residue of this oxyanion
hole, Gly143. However, the furan ring oxygen is likely a weaker hydrogen
bond acceptor than the amide oxygen, and it lies outside of the plane
of Gly143’s amide NH. Directly attached to the furylamide moiety
is a P2 biphenyl group and a P1 pyridinyl ring. The P2 biphenyl group
projects directly into the S2 pocket, which prefers hydrophobic residues
such as leucine and phenylalanine. As expected, the P1 pyridinyl ring
occupies the S1 pocket, which is known for its strict preference for
glutamine. While most Mpro inhibitors bear a pyrrolidinone
glutamine mimetic at the P1 position, we determined that more hydrophobic
residues can also bind to the S1 site and that hydrogen bond formation
with His163 is critical for inhibition.[8] In this instance, the pyridinyl ring of 23R is nearly
superimposable with the same moiety from calpain inhibitor XII (Figure C) forming
a close (2.9 Å) hydrogen bond with His163. An amide bond connecting
the pyridinyl ring to the α-methylbenzyl moiety forms a hydrogen
bond with the main chain of Glu166. The benzyl ring of the α-methylbenzyl
moiety is partially positioned in both the S2 and S4 pockets, a novel
binding pose that has not been observed with existing Mpro inhibitors. Normally, a substituent at this position would be expected
to flip away from the enzyme core toward the solvent-exposed S3 pocket,
which explains why P3 substitutions have little to no influence on
the enzymatic inhibition.[4] However, the
chirality and hydrophobic nature of the benzyl ring in 23R cause it to project toward the core near the S2 pocket, forcing
Gln189 to rotate outward (Figure D). This conformation is reinforced by π-stacking
interactions with the first phenyl of the biphenyl substituent. Notably,
the binding pose of 23R features continuous intramolecular
π-stacking, where the phenyl is sandwiched by furan and benzyl
groups, potentially contributing to its potent inhibition of Mpro. Meanwhile, the S4 pocket remains largely unoccupied by 23R, leaving room for further improvement. In summary, the
X-ray crystal structure of SARS-CoV-2 Mpro in complex with 23R revealed two interesting structural features: (1) The
P2 biphenyl is probably the longest substitution that can be accommodated
in the S2 pocket, which is consistent with our design hypothesis.
(2) The benzyl group from the terminal α-methylbenzyl fits in
a pocket in between the S2 and S4 pockets, and this is a ligand-induced
binding site that has not been previously explored. Although this
is unexpected from the design perspective, this novel binding mode
suggests that the new binding pocket in between S2 and S4 can be explored
for inhibitor design.
Figure 9
X-ray crystal structure of SARS-CoV-2 Mpro in
complex
with 23R. (A) X-ray crystal structure of SARS-CoV-2 Mpro in complex with 23R (PDB: 7KX5). (B) Superimposed
structures of SARS-CoV-2 Mpro + 23R (PDB: 7KX5) and SARS-CoV Mpro + ML188 (R) (PDB: 3V3M). (C) Superimposed structures of SARS-CoV-2
Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + calpain inhibitor XII (PDB: 6XFN). (D) Superimposed structures of SARS-CoV-2
Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + GC376 (PDB: 6WTT).
X-ray crystal structure of SARS-CoV-2 Mpro in
complex
with 23R. (A) X-ray crystal structure of SARS-CoV-2 Mpro in complex with 23R (PDB: 7KX5). (B) Superimposed
structures of SARS-CoV-2 Mpro + 23R (PDB: 7KX5) and SARS-CoV Mpro + ML188 (R) (PDB: 3V3M). (C) Superimposed structures of SARS-CoV-2
Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + calpain inhibitor XII (PDB: 6XFN). (D) Superimposed structures of SARS-CoV-2
Mpro + 23R (PDB: 7KX5) and SARS-CoV-2 Mpro + GC376 (PDB: 6WTT).
Conclusion
The
viral Mpro is a high profile antiviral drug target,
and several Mpro inhibitors are now in animal model studies
and human clinical trials.[6] Among the known
Mpro inhibitors, the majority are covalent inhibitors such
as GC376 analogs that contain a pyrrolidone in the P1
position as a glutamine mimetic. Several structurally distinct compounds
including ebselen, disulfiram, carmofur, PX-12, tideglusib, and shiknonin
were claimed as Mpro inhibitors[22,23] but were later proved to be promiscuous nonspecific cysteine protease
inhibitors.[24,25] In addition, noncovalent inhibitors
such as ML188 (R) were developed and validated as SARS-CoV
Mpro inhibitors.[5,13] Several follow up studies
have been conducted to optimize the enzymatic potency of this series
of compounds against SARS-CoV Mpro and SARS-CoV-2 Mpro. However, no significant improvement has been made, and ML188 (R) remains the only noncovalent inhibitor with moderate
antiviral activity against SARS-CoV (EC50 = 12.9 ±
0.7 μM).[14,15] Nevertheless, given the sequence
and structural similarities between SARS-CoV and SARS-CoV-2 Mpro, and the similar binding mode of ML188 (R) and calpain inhibitor XII, we hypothesize that ML188 (R) is a promising scaffold for structure-based drug
design. In this study, we developed an expedited drug discovery approach
for the design of noncovalent SARS-CoV-2 Mpro inhibitors.
The design approach couples the superimposed X-ray crystal structures
with the one-pot Ugi 4CR synthetic methodology. We were able to improve
the enzymatic inhibition potency of ML188 (1) by 54-fold from a focused library of 39 noncovalent inhibitors.
This is a significant advantage compared to covalent inhibitors such
as GC376, which involves at least a five-step synthesis.
In addition, by introducing the chiral isocyanide, the diastereomer
product can be conveniently separated by either silica gel column
or reverse phase HPLC column, bypassing the need for an expensive
chiral HPLC column. Target selectivity profiling showed that the noncovalent
inhibitor 23R only selectively inhibits SARS-CoV-2 and
SARS-CoV Mpro, but not other viral proteases and host proteases
including calpain 1, cathepsins L and K, and trypsin. In contrast,
the covalent Mpro inhibitor GC376 is not selective
and inhibits host cysteine proteases, which might result in potential
side effects. Furthermore, the X-ray crystal structure of SARS-CoV-2
Mpro in complex with 23R reveals a ligand-induced
binding pocket in between S2 and S4 sites that can be explored for
drug design. Overall, using the expedited drug discovery approach,
this study revealed a promising noncovalent Mpro inhibitor 23R with a well characterized mechanism of action and potent
cellular antiviral activity for further development.
Materials and Methods
Cell Lines and Viruses
VERO E6 cells
(ATCC, CRL-1586)
were cultured in Dulbecco’s modified Eagle’s medium
(DMEM), supplemented with 5% heat inactivated FBS in a 37 °C
incubator with 5% CO2. SARS-CoV-2, isolate USA-WA1/2020
(NR-52281), was obtained through BEI Resources and propagated once
on VERO E6 cells before it was used for this study. Studies involving
SARS-CoV-2 were performed at the UTHSCSA biosafety level-3 laboratory
by personnel wearing powered air purifying respirators.
Protein Expression
and Purification
SARS CoV-2 main
protease (Mpro or 3CL) gene from strain BetaCoV/Wuhan/WIV04/2019
and SARS-CoV main protease from strain CDC#200301157 in the pET29a(+)
vector with E. coli codon optimization were ordered
from GenScript (Piscataway, NJ). The Mpro gene was then
subcloned into pE-SUMO vector as described previously.[7,8] The expression and purification of SARS-CoV and SARS-CoV-2 Mpro with unmodified N- and C-termini were detailed in our previous
publication.[8]The expression and
purification of SARS CoV-2 papain-like protease (PLpro)
were also described in our previous publications.[7,8,24]The expression and purification of
EV-A71 2Apro and
3Cpro, EV-D68 2Apro and 3Cpro were
described in our previous publications.[24,26]Cathepsin
K and Cathepsin L were purchased from EMD Millipore (catalog
#. 219461 and 219402, respectively).Calpain 1 and trypsin were
purchased from Sigma-Aldrich (catalog
#. C6108 and T6763, respectively).
Peptide Synthesis
The SARS-CoV-2 Mpro FRET
substrate Dabcyl-KTSAVLQ/SGFRKME(Edans) was synthesized as described
before.[7] The SARS-CoV-2 PLpro, EV-A71 2Apro and 3Cpro, and EV-D68 2Apro and 3Cpro FRET substrates were listed in our
previous publication[24] and were synthesized
by solid-phase synthesis through iterative cycles of coupling and
deprotection using the previously optimized procedure.[27]
General Chemical Methods
All chemicals
were purchased
from commercial vendors and used without further purification unless
otherwise noted. 1H and 13C NMR spectra were
recorded on a Bruker-400 or -500 NMR spectrometer. Chemical shifts
are reported in parts per million referenced with respect to residual
solvent (CD3OD) 3.31 ppm, (DMSO-d6) 2.50 ppm, and (CDCl3) 7.26 ppm or from internal
standard tetramethylsilane (TMS) 0.00 ppm. The following abbreviations
were used in reporting spectra: s, singlet; d, doublet; t, triplet;
q, quartet; m, multiplet; dd, doublet of doublets; ddd, doublet of
doublet of doublets. All reactions were carried out under N2 atmosphere, unless otherwise stated. HPLC-grade solvents were used
for all reactions. Flash column chromatography was performed using
silica gel (230–400 mesh, Merck). High resolution mass spectra
were obtained using an OrbitrapTM for all the compounds, obtained
in an Ion Cyclotron Resonance (ICR) spectrometer. The purity was assessed
by using Shimadzu UPLC with Shimdazu C18-AQ column (4.6 × 150
mm P/N #227-30767-05) at a flow rate of 1 mL/min; λ = 254 and
220 nm; mobile phase A, 0.1% trifluoroacetic acid in H2O, and mobile phase B, 0.1% trifluoroacetic acid in 90% CH3CN and 10% H2O. The gradients are 0–2 min 10% B,
2–15 min 10%–100% B, 15–18 min, 100% B, 18.1–20
min 10% B. All compounds submitted for testing were confirmed to be
>95.0% purity by HPLC traces. All final products were characterized
by proton and carbon NMR, HPLC, and HRMS.
General Procedure for Ugi-4CR
Reaction
Ug-4CR reaction was performed according to the reported
procedure
with modifications.[13] Amine (1.0 equiv)
and aldehyde (1.0 equiv) were mixed in methanol (10 mL) and stirred
at room temperature for 30 min. Then carboxylic acid (1.0 equiv) and
isocyanide (1.0 equiv) were added sequentially and the resulting mixture
was stirred at room temperature overnight. After that, the solvent
was removed under reduced pressure and the crude product was purified
with flash silica gel chromatography (methanol in dichloromethane
1–5% or acetone in hexane 30–80%).
General
Procedures for the Synthesis of Compounds 24,
28, 33, 35, and 37 by TFA Deprotection
To a solution of N-trityl-protected Ugi-4CR compound in dichloromethane
(5 mL) was added TFA (1 mL). The mixture was stirred at room temperature
for 2 h, and the solvent was removed under reduced pressure. The crude
mixture was diluted in dichloromethane and purified by silica gel
flash column chromatography (ammonia 10% methanol in dichloromethane
10–15%) to give the final product.
Procedure
for the Synthesis of Compound 18 by Suzuki–Miyamura
Cross-coupling
To solution of 2-[N-(4-bromophenyl)-1-(furan-2-yl)formamido]-N-[(1S)-1-phenylethyl]-2-(pyridin-3-yl)acetamide
(1 mmol) and furan-2-boronic acid (1 mmol) in 1,4-dioxane in a microwave
reaction vial was added an aqueous solution of K2CO3 (4 mmol). The resulting solution was purged with N2 for 5 min. The catalyst, Pd(PPh3)4 (0.1 mmol),
was added in one portion. The vial was capped and heated to 140 °C
for 30 min with microwave irradiation. After cooling down to room
temperature, the reaction solution was diluted with dichloromethane
and extracted with water, followed by brine. The organic layer was
dried over MgSO4, filtrated, and concentrated under reduced
pressure. The crude product was purified by silica gel flash column
chromatography (methanol in dichloromethane 1–5%) to give the
final product.
Procedure for the Synthesis of 4-(Thiophen-2-yl)aniline
by Suzuki–Miyamura
Cross-coupling
The starting material 4-(thiophen-2-yl)aniline
used for the synthesis
of compound 19 was prepared using the following procedure.
To a suspension of thiophene-2-boronic acid (1.0 mmol) and sodium
carbonate (1.0 mmol) in toluene/methanol (4:1, 40 mL) was added 4-bromoaniline
(1.0 mmol). The resulting solution was purged with N2 for
10 min. The catalyst, Pd(PPh3)4 (0.1 mmol),
was added in one portion. The reaction mixture was stirred for 16
h at 100 °C. After cooling to room temperature, the reaction
mixture was diluted with ethyl acetate (100 mL) and washed with water
(100 mL). The organic layer was dried over MgSO4, filtrated,
and concentrated under reduced pressure. The crude product was purified
by silica gel flash column chromatography (ethyl acetate in hexane
20–40%) to give the final product.4-(Thiophen-2-yl)aniline.
White solid. 31% yield. 1H NMR (500 MHz, CDCl3) δ 7.43–7.38 (m, 2H), 7.17–7.12 (m, 2H), 7.01
(dd, J = 5.0, 3.6 Hz, 1H), 6.69–6.63 (m, 2H),
3.68 (s, 2H). 13C NMR (126 MHz, CDCl3) δ
146.10, 145.11, 127.93, 127.25, 125.24, 123.17, 121.36, 115.39.The isocyanide used for the synthesis of compound 40 was prepared and used in the method below:
Procedure
for Synthesis of N-(3-Phenylpropyl)formamide
3-Phenyl
propylamine (2 mmol) in a microwave reaction vial was
mixed with ethyl formate (6 mL). The vial was capped and heated to
70 °C overnight. After reaction residual ethyl formate was evaporated
under reduced pressure. The crude product was purified by silica gel
flash column chromatography (methanol in dichloromethane 1–3%)
to give the product N-(3-phenylpropyl)formamide. Oil, 98% yield. 1H NMR (500 MHz, CDCl3) δ 8.15–7.96
(m, 1H), 7.33–7.24 (m, 2H), 7.24–7.12 (m, 3H), 6.18–5.73
(m, 1H), 3.35–3.17 (m, 2H), 2.69–2.60 (m, 2H), 1.89–1.78
(m, 2H). 13C NMR (126 MHz, CDCl3) δ 170.33,
164.88, 161.44, 141.51, 141.27, 140.65, 128.67, 128.55, 128.52, 128.42,
128.41, 126.32, 126.13, 126.07, 41.15, 39.39, 37.84, 33.36, 33.22,
32.60, 32.54, 31.17, 31.16, 23.31.
Procedure
for the Synthesis of Compound 40
To solution
of N-(3-phenylpropyl)formamide (2 mmol) in dichloromethane
(50 mL) was added triethylamine (4.8 mmol). The solution was cooled
to approximately −10 °C using an ethanol ice bath. Triphosgene
(0.4 mmol) was added to the stirring mixture. The resulting mixture
was stirred at −10 °C for 10 min. In a separate 250 mL
round-bottomed flask, 4-aminobiphneyl (2 mmol) and 3-pyridinecarboxaldehyde
(2 mmol) were mixed in methanol (100 mL) and stirred at room temperature
for 30 min. Then carboxylic acid (2 mmol) and the resulting isocyanide
solution (1.0 equiv) were added sequentially, and the resulting mixture
was stirred at room temperature overnight. After that, the solvent
was removed under reduced pressure and the crude product was purified
with flash silica gel chromatography (methanol in dichloromethane
1–5%) to give the final product.
Prior to
analysis, the protein
was buffer exchanged into 0.2 M ammonium acetate (pH 6.8) and diluted
to 10 μM. DTT was dissolved in water and prepared at a 400 mM
stock. Each ligand was dissolved in ethanol and diluted to 10×
stock concentrations. The final mixture was prepared by adding 4 μL
of protein, 0.5 μL of DTT stock, and 0.5 μL of ligand
stock for a final concentration of 4 mM DTT and 70 μM protein.
The final ligand concentrations were 10 μM and 30 μM.
The mixtures were then incubated for 10 min at room temperature prior
to analysis. Each sample was mixed and analyzed in triplicate.Native mass spectrometry (MS) was performed using a Q-Exactive HF
quadrupole-Orbitrap mass spectrometer with the Ultra-High Mass Range
research modifications (Thermo Fisher Scientific). Samples were ionized
using nanoelectrospray ionization in positive ion mode using 1.0 kV
capillary voltage at a 150 °C capillary temperature. The samples
were all analyzed with a 1,000–25,000 m/z range, the resolution set to 30,000, and a trapping gas
pressure set to 3. Between 10 and 50 V of source fragmentation was
applied to all samples to aid in desolvation. Data were deconvolved
and analyzed with UniDec.[28]
Enzymatic
Assays
The main protease (Mpro) enzymatic assays
were carried out in Mpro reaction buffer
containing 20 mM HEPES pH 6.5, 120 mM NaCl, 0.4 mM EDTA, 20% glycerol,
and 4 mM DTT, and the SARS-CoV-2 papain-like protease (PLpro) enzymatic assays were carried out in PLPro reaction
buffer containing 50 mM HEPES, pH7.5, 0.01% triton-100, and 5 mM DTT.
The percentage of inhibition and enzymatic IC50 values
were calculated as previously described.[7,8] Briefly, the
assay was performed in 96-well plates with 100 μL of 100 nM
Mpro protein or 200 nM PLPro protein in their
respective reaction buffers. Then 1 μL testing compound at various
concentrations was added to each well and incubated at 30 °C
for 30 min. The enzymatic reaction was initiated by adding 1 μL
of 1 mM corresponding FRET substrate (the final substrate concentration
is 10 μM). The reaction was monitored in a Cytation 5 image
reader with filters for excitation at 360/40 nm and emission at 460/40
nm at 30 °C for 1 h. The initial velocity of the enzymatic reaction
with and without testing compounds was calculated by linear regression
for the first 15 min of the kinetic progress curve.For the
Morrison plot, 10 μL of 100 nM SARS-CoV-2 Mpro protein
was added to 190 μL of Mpro reaction buffer containing
testing compound and the FRET substrate, and the reaction was monitored
for 2 h. The final FRET substrate concentration in this assay is 20
μM. Detailed curve fitting and KI determination were
described previously.[7,8]For Michaelis–Menten
and Lineweaver–Burk plots, assay
was carried out as follows: 50 μL of 50 μM Mpro protein was added to 50 μL of reaction buffer containing testing
compound and various concentrations of FRET substrate to initiate
the enzyme reaction. The initial velocity of the enzymatic reaction
with and without testing compounds was calculated by linear regression
for the first 15 min of the kinetic progress curve, plotted against
substrate concentrations in Prism 8 with the Michaelis–Menten
equation.Cathepsin K and cathepsin L enzymatic assay was carried
out as
follows: cathepsin K or cathepsin L was activated by incubating in
reaction buffer [20 mM sodium acetate, 1 mM EDTA, and 5 mM DTT (pH
5.5)] for 30 min at 30 °C. Upon activation, the assay was assembled
in 96-well plates with 100 μL of cathepsin K at 200 pM or cathepsin
L at 300 pM in reaction buffer. Then, 1 μL of testing compound
at various concentrations was added to each well and incubated at
30 °C for 30 min. The enzymatic reaction was initiated by adding
1 μL of FRET substrate Z-Phe-Arg-AMC (BACHEM, catalog #. 4003379.0050)
(the final substrate concentration is about 5 μM for cathepsin
K and 1 μM for cathepsin L). The reaction was monitored in a
Cytation 5 image reader with filters for excitation at 360/40 nm and
emission at 460/40 nm at 30 °C for 1 h. The IC50 values
were calculated as described in the previous section.Calpain
1 enzymatic assay was carried out as follows: 1 μL
of 1 μg/μL calpain 1 protein was added to 100 μL
of calpain I reaction buffer [50 mM HEPES, 50 mM NaCl, and 10 mM DTT
and 5 mM CaCl2 (pH 7.5)]; the enzymatic reaction was iniated
by adding 1 μL of 4 mM N-succinyl-Leu-Leu-Val-Tyr-7-amido-4-methylcoumarin
substrate (Sigma-Aldrich catalog #. S6510). The reaction was monitored
with a Cytation 5 image reader with filters for excitation at 360/40
nm and emission at 460/40 nm at 30 °C for 1 h. The IC50 values were calculated as described in the previous section.Trypsin enzymatic assay was carried out in a 50 μL volume
containing 100 nM trypsin in 50 mM HEPES (pH7.6) reaction buffer,
100 μM Bz-Arg-AMC·HCl (BACHEM, catalog no. 4002540.0050),
and serial concentrations of test compounds in duplicate. The reaction
was monitored with a Cytation 5 image reader with filters for excitation
at 360/40 nm and emission at 460/40 nm at 30 °C for 1 h. The
IC50 values were calculated as described in the previous
section.
Differential Scanning Fluorimetry (DSF)
The thermal
shift binding assay (TSA) was carried out using a Thermal Fisher QuantStudio
5 Real-Time PCR System as described previously.[7,8,29] Briefly, 3 μM SARS-CoV-2 Mpro protein in Mpro reaction buffer was incubated with various
concentrations of compound ML188 or 23R at
30 °C for 30 min. 1X SYPRO orange dye was added, and the fluorescence
of the well was monitored under a temperature gradient range from
20 to 90 °C with a 0.05 °C/s incremental step. Measured Tm was plotted against compound concentration
with one-site binding function in Prism 8.
Cytotoxicity Measurement
Evaluation of the cytotoxicity
of compounds was carried out using the neutral red uptake assay.[30,31] Briefly, 80,000 cells/mL of the tested cell lines was dispensed
into 96-well cell culture plates at 100 μL/well. Twenty-four
hours later, the growth medium was removed and washed with 150 μL
PBS buffer. 200 μL of fresh serum-free medium containing serial
diluted compounds was added to each well. After incubating for 5 days
at 37 °C, the medium was removed and replaced with 100 μL
DMEM medium containing 40 μg/mL neutral red and incubated for
2–4 h at 37 °C. The amount of neutral red taken up was
determined by measuring the absorbance at 540 nm using a Multiskan
FC Microplate Photometer (Fisher Scientific). The CC50 values
were calculated from best-fit dose response curves with variable slope
in Prism 8.
Immunofluorescence Assay
Antiviral
immunofluorescence
assay was carried out as previously described.[8] Briefly, Vero E6 cells in 96-well plates (Corning) were infected
with SARS-CoV-2 (USA-WA1/2020 isolate) at MOI of 0.05 in DMEM supplemented
with 1% FBS. Immediately before the viral inoculation, the tested
compounds in a 3-fold dilution concentration series were also added
to the wells in triplicate. The infection proceeded for 48 h without
the removal of the viruses or the compounds. The cells were then fixed
with 4% paraformaldehyde, permeabilized with 0.1% Triton-100, blocked
with DMEM containing 10% FBS, and stained with a rabbit monoclonal
antibody against SARS-CoV-2 NP (GeneTex, GTX635679) and an Alexa Fluor
488-conjugated goat antimouse secondary antibody (ThermoFisher Scientific).
Hoechst 33342 was added in the final step to counterstain the nuclei.
Fluorescence images of approximately ten thousand cells were acquired
per well with a 10× objective in a Cytation 5 (BioTek). The total
number of cells, as indicated by the nuclei staining, and the fraction
of the infected cells, as indicated by the NP staining, was quantified
with the cellular analysis module of the Gen5 software (BioTek).
Antiviral Assay in Calu-3 Cells
Calu-3 cells (ATCC,
HTB-55) grown in Minimal Eagles Medium supplemented with 1% nonessential
amino acids, 1% penicillin/streptomycin, and 10% FBS are plated in
384 well plates. The next day, 50 nL of drug suspended in DMSO is
added as an 8-pt dose response with 3-fold dilutions between test
concentrations in triplicate, starting at 40 μM final concentration.
The negative control (DMSO, n = 32) and positive control (10 μM
Remdesivir, n = 32) are included on each assay plate. Calu3 cells
are pretreated with controls and test drugs (in triplicate) for 2
h prior to infection. In BSL3 containment, SARS-CoV-2 (isolate USA-WA1/2020)
diluted in serum free growth medium is added to plates to achieve
an MOI = 0.5. Cells are incubated continuously with drugs and SARS-CoV-2
for 48 h. Cells are fixed and then immunstained with anti-dsRNA (J2),
and nuclei are counterstained with Hoechst 33342 for automated microscopy.
Automated image analysis quantifies the number of cells per well (toxicity)
and the percentage of infected cells (dsRNA+ cells/cell number) per
well. SARS-CoV-2 infection at each drug concentration was normalized
to aggregated DMSO plate control wells and expressed as percentage-of-control
(POC = % Infection sample/Avg % Infection DMSO cont). A nonlinear regression curve fit analysis (GraphPad Prism 8) of
POC infection and cell viability versus the log10 transformed
concentration values to calculate EC50 values for infection
and CC50 values for cell viability. Selectivity index (SI)
was calculated as a ratio of drug’s CC50 and EC50 values (SI = CC50/IC50).
Mpro Crystallization and Structure Determination
23R was added to 20 mg/mL SARS-CoV-2 Mpro to a final concentration
of 1.75 mM and incubated overnight at 4°C.
This mixture was then diluted 4-fold with protein stock buffer (20
mM Tris pH 7.5, 200 mM NaCl, 1 mM DTT) and then spun down at 13,000g for 1 min to remove precipitate. Crystals were grown by
mixing the protein-inhibitor sample with an equal volume of crystallization
buffer (20% PEG 3350, 0.2 M NaF) in a vapor diffusion, hanging drop
apparatus. Crystals were then transferred to a drop with crystallization
buffer containing 5 mM 23R for 1 h, followed by a brief
soaking in a cryoprotectant solution of 30% PEG 3350 and 15% glycerol
with 2 mM 23R. Crystals were then flash frozen in liquid
nitrogen for X-ray diffraction.X-ray diffraction data for the
SARS-CoV-2 Mpro structures were collected on the SBC 19-ID
beamline at the Advanced Photon Source (APS) in Argonne, IL, and processed
with the HKL3000 software suite. The CCP4 versions of MOLREP[32] was used for molecular replacement using a previously
solved SARS-CoV-2 Mpro structure, 6YB7. Structural refinement
was performed using REFMAC5[33] and COOT.[34] The crystallographic statistics is shown in Supporting Information Table S2. The complex
structure for SARS-CoV-2 Mpro with 23R has
been deposited in the Protein Data Bank with the accession ID of 7KX5 (SARS-CoV-2 MPro + Jun8-76-3A).
Authors: Lorenzo Cianni; Christian Wolfgang Feldmann; Erik Gilberg; Michael Gütschow; Luiz Juliano; Andrei Leitão; Jürgen Bajorath; Carlos A Montanari Journal: J Med Chem Date: 2019-08-15 Impact factor: 7.446
Authors: Michael T Marty; Andrew J Baldwin; Erik G Marklund; Georg K A Hochberg; Justin L P Benesch; Carol V Robinson Journal: Anal Chem Date: 2015-04-01 Impact factor: 6.986
Authors: Garib N Murshudov; Pavol Skubák; Andrey A Lebedev; Navraj S Pannu; Roberto A Steiner; Robert A Nicholls; Martyn D Winn; Fei Long; Alexei A Vagin Journal: Acta Crystallogr D Biol Crystallogr Date: 2011-03-18
Authors: Kas Steuten; Heeyoung Kim; John C Widen; Brett M Babin; Ouma Onguka; Scott Lovell; Oguz Bolgi; Berati Cerikan; Christopher J Neufeldt; Mirko Cortese; Ryan K Muir; John M Bennett; Ruth Geiss-Friedlander; Christoph Peters; Ralf Bartenschlager; Matthew Bogyo Journal: ACS Infect Dis Date: 2021-02-11 Impact factor: 5.084
Authors: Zachary A Gurard-Levin; Cheng Liu; Andreas Jekle; Ruchika Jaisinghani; Suping Ren; Koen Vandyck; Dirk Jochmans; Pieter Leyssen; Johan Neyts; Lawrence M Blatt; Leonid Beigelman; Julian A Symons; Pierre Raboisson; Michael D Scholle; Jerome Deval Journal: Antiviral Res Date: 2020-09-05 Impact factor: 5.970
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