Thanigaimalai Pillaiyar1, Philipp Flury1, Nadine Krüger2, Haixia Su3, Laura Schäkel4, Elany Barbosa Da Silva5, Olga Eppler1, Thales Kronenberger1, Tianqing Nie3, Stephanie Luedtke5, Cheila Rocha2, Katharina Sylvester4, Marvin R I Petry4, James H McKerrow5, Antti Poso1,6, Stefan Pöhlmann2,7, Michael Gütschow4, Anthony J O'Donoghue5, Yechun Xu3, Christa E Müller4, Stefan A Laufer1. 1. Institute of Pharmacy, Pharmaceutical/Medicinal Chemistry and Tübingen Center for Academic Drug Discovery, Eberhard Karls University Tübingen, Auf der Morgenstelle 8, 72076 Tübingen, Germany. Cluster of Excellence iFIT (EXC 2180) "Image-Guided & Functionally Instructed Tumor Therapies", University of Tübingen, Tübingen 72076, Germany. 2. Infection Biology Unit, German Primate Center, Leibniz Institute for Primate Research Göttingen, Kellnerweg 4, Göttingen 37077, Germany. 3. CAS Key Laboratory of Receptor Research, and Stake Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, China. 4. PharmaCenter Bonn, Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry, University of Bonn, An der Immenburg 4, Bonn D-53121, Germany. 5. Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, La Jolla, California 92093, United States. 6. School of Pharmacy, Faculty of Health Sciences, University of Eastern Finland, Kuopio 70211, Finland. 7. Faculty of Biology and Psychology, University Göttingen,Göttingen 37073, Germany.
Abstract
The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M-1 s-1 (Ki = 0.0141 μM) and 27,200 M-1 s-1 (Ki = 0.0332 μM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.
The main protease (Mpro, 3CLpro) of SARS-CoV-2 is an attractive target in coronaviruses because of its crucial involvement in viral replication and transcription. Here, we report on the design, synthesis, and structure-activity relationships of novel small-molecule thioesters as SARS-CoV-2 Mpro inhibitors. Compounds 3w and 3x exhibited excellent SARS-CoV-2 Mpro inhibition with kinac/Ki of 58,700 M-1 s-1 (Ki = 0.0141 μM) and 27,200 M-1 s-1 (Ki = 0.0332 μM), respectively. In Calu-3 and Vero76 cells, compounds 3h, 3i, 3l, 3r, 3v, 3w, and 3x displayed antiviral activity in the nanomolar range without host cell toxicity. Co-crystallization of 3w and 3af with SARS-CoV-2 Mpro was accomplished, and the X-ray structures showed covalent binding with the catalytic Cys145 residue of the protease. The potent SARS-CoV-2 Mpro inhibitors also inhibited the Mpro of other beta-coronaviruses, including SARS-CoV-1 and MERS-CoV, indicating that they might be useful to treat a broader range of coronaviral infections.
The etiological agent of the current human coronavirus disease 2019 (COVID-19) is the
severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). It is a global health concern
due to its ability to rapidly transmit from person to person and evade human immune
surveillance.[1−4] As of 25 February 2022, SARS-CoV-2 has infected around 430 million
individuals globally, resulting in approximately 6 million deaths.[5] The
severe disease generally occurs in the elderly or those with pre-existing medical
conditions. There are more than 20 different SARS-CoV-2 vaccines in use, all of which have
been shown to be effective. However, because vaccine hesitancy persists globally and
breakthrough infections are prevalent in vaccinated people, efficient antiviral therapeutics
are required. Furthermore, SARS-CoV-2 is constantly evolving, and vaccination effectiveness
will continue to diminish as mutations accumulate.[6,7] Because of their faster transmission capacity and higher
mortality,[8,9] the
B.1.617.2 (Delta) and B.1.1.529 (Omicron) variants have emerged as prominent in the current
pandemic.[8−10] According to new findings,
available COVID-19 immunizations are less effective against the Delta and Omicron variants,
and persons who have been vaccinated are still at risk of infection.[11,12]The only direct-acting antivirals approved for emergency use are remdesivir and
nirmatrelvir.[13−15] Remdesivir is an
RNA-dependent RNA polymerase (RdRp) inhibitor that is given intravenously to individuals
with COVID-19 infection.[13,14] Nirmatrelvir is a peptidomimetic Mpro inhibitor
commercialized under the brand name Paxlovid in combination with ritonavir.[15]SARS-CoV-2 is one of many beta-coronaviruses in the Coronaviridae family, also including
SARS-CoV-1 and Middle East respiratory syndrome coronavirus (MERS-CoV) that were responsible
for the SARS and MERS epidemics in 2003 and 2012, respectively.[16−18]The RNA genome sequence of the SARS-CoV-2 virus offers an ideal starting point for drug
discovery and the development of efficient COVID-19 treatments.[19,20] Coronaviruses are enveloped,
single-stranded, nonsegmented positive-sense RNA viruses featuring the largest viral RNA
genomes known to date. The genome of SARS-CoV-2 encodes two large polyproteins: pp1a
(∼450 kDa) and pp1ab (∼750 kDa). These are extensively processed by two viral
proteases, the papain-like protease (PLpro) and the main protease
(Mpro, also called 3CLpro), producing 16 nonstructural proteins
(NSPs), including the RNA-dependent RNA polymerase (RdRp), that are involved in viral
replication and transcription.[21,22]Due to the indispensable role of Mpro in viral replication and its high
conservation among related pathogenic coronaviruses such as MERS-CoV and SARS-CoV-1, it
represents a promising target for the development of broad-spectrum antiviral
therapy.[16,17,23,24] Moreover, Mpro is unique in
recognizing the substrate specificity and cleaves at the glutamine position. Because human
proteases lack this property, it may be possible to develop selective protease inhibitors
that are not toxic to humans. Only one human protease, kallikrein-3, has a comparable
substrate specificity as Mpro for cleavage on the C-terminal side of glutamine.
Because kallikrein-3 is a serine protease expressed solely in the prostate gland,[25] inhibitors affecting the cysteine protease Mpro are unlikely to
target it, and if they do, inhibition will be restricted to the prostate gland.So far, a variety of SARS-CoV-2 Mpro inhibitors have been reported that can be
classified into covalently and noncovalently acting agents.[26−31] The
active-site Mpro contains a catalytic dyad where Cys145 functions as a
nucleophile and His41 serves as a catalytic general base. Covalent inhibitors, including
small molecules and peptidomimetics, bear different warhead groups and act through a
reaction with the active-site cysteine, producing a covalent bond with the
inhibitor.[32−36] In continuous
efforts to develop SARS-CoV-1 and SARS-CoV-2 therapeutics, we and others have developed a
wide variety of substrate-derived peptidomimetic and nonpeptidic small-molecule inhibitors
targeting the main protease.[23,24,28−38] In
particular, a series of novel chloropyridyl ester derivatives (I and
II, see Figure ) displayed potent
SARS-CoV-2 Mpro inhibitory activities.[37,39] The positions of an ester moiety and other substituents on
the indole ring affected SARS-CoV-2 Mpro inhibitory potency and antiviral
activity.[37,39] The
indole carboxylate scaffold is thought to be important for binding to the Mpro
active site. The unique benzothiazolyl ketone derivative (III, see Figure ), which was first reported as a SARS-CoV-1
Mpro inhibitor,[24] also has a significant antiviral and
SARS-CoV-2 Mpro activity.[38]
Figure 1
Structures of SARS-CoV-2 RdRp and Mpro inhibitors.
Structures of SARS-CoV-2 RdRp and Mpro inhibitors.In the present study, a virtual screening of the Tübingen Kinase Inhibitor
Collection (TüKIC), containing >10,000 proprietary kinase inhibitors, was
performed. Based on the results from this screen, we report the design and synthesis of a
novel class of thioesters and discuss their structure–activity relationships. The
compounds were tested for inhibition at recombinant SARS-CoV-2 Mpro employing a
recently developed robust assay,[37] and enzyme inhibition kinetics were
measured to elucidate the compounds’ mechanism of inhibition. For potent inhibitors,
antiviral activity was determined. Moreover, the co-crystallization of selected compounds
with SARS-CoV-2 Mpro was performed to obtain insights into their molecular
interactions with the target. The SARS-CoV-2 Mpro inhibitors were additionally
tested against Mpro from other pathogenic beta-coronaviruses, namely, SARS-CoV-1
and MERS-CoV, which they also inhibited with high potency.
Results and Discussion
Virtual Screening
The 3D structure of SARS-CoV-2 Mpro in association with multiple inhibitors is
accessible for in silico screening to discover novel lead structures. We virtually
screened our in-house TüKIC library against Mpro. This collection
contains over 10,000 compounds that target a nucleophilic cysteine residue in a variety of
kinases. Starting from the Mpro 3D structure,[40] we employed
a pipeline combining docking with short molecular dynamics (MD) simulations of 200 ns to
yield 15 selected hit molecules, which were subsequently tested for their SARS-CoV-2
Mpro inhibitory activity (Supporting information, Table S1).All compounds were initially screened at 10 μM against SARS-CoV-2 Mpro,
and three compounds, inhibiting Mpro by 50% or more, were chosen to determine
concentration-dependent inhibition. Inhibitors IV and V showed
moderate potency against Mpro, while VI had a
Ki value of 6.5 μM (Figure ). Its proposed binding mode suggests that the
imidazo[4,5-d]pyrrolo[2,3-b]pyridine unit occupies the
S2 pocket, displaying a stable Π–Π interaction with His41. The
structure-dependent activity analysis of these three inhibitors suggests that the
acrylonitrile warhead was slightly preferred for Mpro inhibition. Additionally,
these inhibitors are selective JAK-3 inhibitors with very high potencies.[41] The inhibition of JAK-3 is a promising approach for the treatment of
COVID-19 as it reduces the inflammatory/cytokine storm that is one of the major factors
for the organ damage that leads to death.[42] Indeed, many JAK inhibitors
are already in advanced clinical trials for the treatment of COVID-19.[43] Therefore, inhibitors targeting the viral main protease and JAK would not only have
direct antiviral effects but also beneficially suppress the overproduction of cytokines
induced by viral infection.
Figure 2
SARS-CoV-2 Mpro inhibitory activity (IC50) of hit compounds
identified by high-throughput screening. The proposed warhead or reactive group of the
inhibitors is highlighted in red.
SARS-CoV-2 Mpro inhibitory activity (IC50) of hit compounds
identified by high-throughput screening. The proposed warhead or reactive group of the
inhibitors is highlighted in red.Some disulfides including IPA-3 and JX-06 were identified as hit molecules with
IC50 values of 2.35 and 4.03 μM (Figure ). IPA-3 was reported as a non-ATP competitive, allosteric, and
reversible covalent inhibitor of dysregulated p21-activated kinase 1 (Pak1),[44] which has been linked to oncogenesis. In previous studies by us and
others, disulfiram was reported as a SARS-CoV-2 Mpro inhibitor with
IC50 values ranging from 7 to 9 μM.[37,45] JX-06, a morpholine derivative, was discovered
as a hit molecule with a lower IC50 value than disulfiram. JX-06 was the first
covalent 3-phosphoinositide-dependent protein kinase-1 (PDK1) inhibitor forming a
reversible mixed disulfide with a conserved cysteine residue of the target.[46] We also discovered that a maleimide derivative of indole-2-carboxamide
(LN5535) inhibited Mpro with an IC50 of 2.08 μM.The active site of Mpro is divided into four subsites (S1′, S1, S2, and
S3), each of which accommodates four fragments (P1′, P1, P2, and P3) of
peptidomimetic inhibitors.[23,24] However, particularly for irreversible small-molecule inhibitors, the
efficacy is frequently determined by the strength of the warhead group even when the four
sites are not fully occupied.[23,24] The co-crystal structure of Mpro in a complex with an
indole chloropyridyl ester derivative showed the acylation of the active site Cys145 to
form a thioester intermediate with the indole moiety oriented toward the S2 pocket (Figure a).[39]
Figure 3
(A) Mpro active site (PDB ID: 7RC0) showing the catalytic Cys145 (yellow) and the subsites S1′,
S1, S2, and S3. (B) Design of new thioesters by combining IPA-3 and LN5535.
(A) Mpro active site (PDB ID: 7RC0) showing the catalytic Cys145 (yellow) and the subsites S1′,
S1, S2, and S3. (B) Design of new thioesters by combining IPA-3 and LN5535.To design a novel class of inhibitors, we docked our two screening hits, IPA-3 and
LN5535, at the Mpro active site (PDB ID: 7RC0) and observed that they share a similar orientation to the indole
chloropyridyl ester derivative. Subsequently, we designed a novel class of thioesters by
inserting variable cyclic systems from IPA-3 and the (hetero)aryl thiols from LN5535, as
shown in Figure B. Subsequently, we designed a
novel class of thioesters by inserting variable cyclic systems from IPA-3 and the
(hetero)aryl thiols from LN5535, as shown in Figure B. We proposed that the designed new inhibitors may have a similar binding mode
to that of the indole chloropyridyl ester derivatives.[39] A series of
(hetero)aromatic thioesters were developed with different thiolate residues to be
accommodated in the S1′ pocket. The electronic properties of (hetero)aromatic
P1′ moieties may alter the reactivity of the thioester toward Cys145. As P2
fragments, a broad range of mono-cyclic or bi-cyclic rings with various physiochemical
characteristics was investigated. This resulted in 40 novel thioesters that were evaluated
against SARS-CoV-2 Mpro.
Chemistry
Our priority was to find the best P2 scaffold (Scheme ). We expected indole to be optimal for inhibiting Mpro based on
previous reports.[37,39]
Several indole carboxylic acids, 1a–f, were first treated
with POCl3 in dichloromethane (DCM) in the presence of pyridine, and the in
situ generated carboxylic acid chlorides were subsequently reacted with thiophenols
(2a–b) or 1,3,4-thiadiazole-2-thiol derivatives
(2c–d) to yield compounds
3a–i in good yields (47–80%). Compounds
3j–n (53–73%) were obtained from differently
substituted 1,3,4-thiadiazolyl-2-thiols (2e–h) or
5-methyl-1,3,4-oxadiazolyl-2-thiol (2i). The reaction of
5-methyl-4H-1,2,4-triazolyl-3-thiol (2j) with
1a produced S,N-diacylated product
3o in 45% yield. Simultaneously, we explored the role of the indole unit by
replacing it with a variety of furan, pyrrole, and naphthalene substructures. The relevant
carboxylic acids 1g–l were reacted with the thiols
2d or 2e, producing a series of final products,
3p–v, in yields ranging from 58 to 82%.
Scheme 1
Synthesis of Thioesters 3a-v
Reagents and conditions: (a) POCl3, pyridine, DCM, 12 h,
55–82%.
Synthesis of Thioesters 3a-v
Reagents and conditions: (a) POCl3, pyridine, DCM, 12 h,
55–82%.Next, the combination of P2 and P1′ moieties was investigated (Scheme ). The P2 indole was kept constant, as in our previous
strategy. The thioesters 3w–z were obtained
(48–84%) by reacting 1a or 1l with three
pyrimidine-2-thiols (2k–m) under the same reaction
conditions. Alternatively, pyrimidine-2-thiols were maintained and combined with various
carboxylic acids (1i–r) to produce the products
3aa–ag, most of which contained building block
2k. The reaction of several bicyclic carboxylic acids (1a,
1g, 1i, 1s, 1n, 1o, and
1q) with the bicyclic benzo[d]thiazolyl-2-thiol
(2n) yielded the final compounds 3ah–3an
in good yields of 56–80%.
Scheme 2
Synthesis of Thioesters 3w–z and
3aa–an
Reagents and conditions: (a) POCl3, pyridine, DCM, 12 h,
48–84%.
Synthesis of Thioesters 3w–z and
3aa–an
Reagents and conditions: (a) POCl3, pyridine, DCM, 12 h,
48–84%.
SARS-CoV-2 Mpro Inhibition Assay
SARS-CoV-2 Mpro was expressed and purified as previously described.[37] SARS-CoV-2 Mpro activity assays were performed using a recently
developed fluorogenic substrate (Boc-Abu-Tle-Leu-Gln-AMC),[38] and
compounds were initially screened at a high concentration of 10 μM. For compounds
showing Mpro inhibition by at least 50%, full concentration–inhibition
curves were determined with at least eight different inhibitor concentrations, and the
respective product formation rates were observed during the first 10 min of the enzymatic
reaction. IC50 values were calculated by nonlinear regression analysis. For
inhibitors that showed a time-dependent inhibition, the second-order rate constant
kinac/Ki was determined by
monitoring the effects of five different inhibitor concentrations on the product formation
rate for 60 min (see Table ).
Table 1
Structures and Activities of Thioesters as SARS-CoV-2 Mpro
Inhibitors
Inhibitors showed time-dependent inhibition. Progress curves in the presence of
five different inhibitor concentrations were followed over 60 minutes and analyzed
by nonlinear regression using the equation [P] =
vi × (1 –
exp(−kobs ×
t)/kobs + d), where
[P] is the product concentration, vi
is the initial rate, kobs is the observed first-order
rate constant, and d is the offset.
The second-order rate constant,
kinac/Ki, was determined
by plotting kobs versus [I] and
nonlinear regression using the equation kobs =
(kinac ×
[I])/([I] + Ki
× (1 + [S]/Km)). The deviation of each data point
from the calculated nonlinear regression was less than 10%.
n.d.: not determined.
Compounds displayed time-dependent inhibition, but progress curves bent upward,
which indicated that the inhibitor was covalently bound to the enzyme and was slowly
released, hence restoring the enzyme activity. Consequently, the inhibitor
concentration decreased over time due to protease-catalyzed cleavage. Such compounds
were only characterized by their IC50 values.
Evaluation was not possible as progress curves were linear. The compound might have
inhibited the enzyme noncovalently as kobs did not
increase with increasing inhibitor concentration.
Inhibitors showed time-dependent inhibition. Progress curves in the presence of
five different inhibitor concentrations were followed over 60 minutes and analyzed
by nonlinear regression using the equation [P] =
vi × (1 –
exp(−kobs ×
t)/kobs + d), where
[P] is the product concentration, vi
is the initial rate, kobs is the observed first-order
rate constant, and d is the offset.The second-order rate constant,
kinac/Ki, was determined
by plotting kobs versus [I] and
nonlinear regression using the equation kobs =
(kinac ×
[I])/([I] + Ki
× (1 + [S]/Km)). The deviation of each data point
from the calculated nonlinear regression was less than 10%.n.d.: not determined.Compounds displayed time-dependent inhibition, but progress curves bent upward,
which indicated that the inhibitor was covalently bound to the enzyme and was slowly
released, hence restoring the enzyme activity. Consequently, the inhibitor
concentration decreased over time due to protease-catalyzed cleavage. Such compounds
were only characterized by their IC50 values.Evaluation was not possible as progress curves were linear. The compound might have
inhibited the enzyme noncovalently as kobs did not
increase with increasing inhibitor concentration.
Structure–Activity Relationships
The indole-2-thioesters with 4-chlorobenzene (3a) and 2,4-dichlorobenzene
(3b) did not show any inhibition of SARS-CoV-2 Mpro.
Surprisingly, replacing the aryl ring with a heteroaromatic ring system such as
1,3,4-thiadiazole in 3c (IC50 0.143 μM) resulted in potent
inhibitory activity against Mpro. This inhibitor was kinetically characterized
as an irreversible SARS-CoV-2 Mpro inhibitor, and the second-order rate
constant of inactivation,
kinac/Ki, was 10,700
M–1 s–1 (Ki = 0.0744
μM) (3c, see Figure S1).5-Methyl substitution of the 1,3,4-thiadiazole ring slightly reduced the inhibitor
potency (3d, IC50 0.624 μM). A positioning scan of the
1,3,4-thiadiazole thioester residue connected to the indole core was performed to study
the chemical space of the indole thioesters. When compared to inhibitor 3d,
which bears the thioester moiety at position 2 of the indole residue, all of the
regioisomeric thioesters (3e–h) displayed an increased
inhibitory activity against Mpro, with the thioester moiety at position 6 being
the most potent of all isomeric inhibitors (3g, IC50 0.0547
μM). The kinac/Ki for
inhibitors 3d and 3e were not calculated due to considerable
Mpro reactivation within 1 h; for 3f
(Ki = 0.393 μM), 3g
(Ki = 0.186 μM), and 3h
(Ki = 0.250 μM), they are 3369, 5850, and 3960
M–1 s–1, respectively. Methylation of the indole
nitrogen resulted in a 2-fold increase in inhibitory activity (3i,
IC50 0.349 μM) compared to 3d, along with a
kinac/Ki value of 7170
M–1 s–1 (Ki = 0.295
μM), demonstrating that the free NH is not necessary (Table ).Next, the 5-methyl group on the 1,3,4-thiadiazole was exchanged for different moieties
such as 5-CF3, 5-SCH3, 5-OCH3, and
5-OC2H5 using 3d as a lead inhibitor. The
incorporation of 5-SMe (3k, IC50 0.0914 μM) and
5-OCH3 (3l) improved the potency of 3d by up to
6-fold, whereas the 5-CF3 (3j) and 5-OC2H5
(3m) replacements were disadvantageous. Inhibitors 3k
(Ki = 0.0430 μM) and 3l
(Ki = 0.0844 μM) have
kinac/Ki values of 11,800
(3k, see Figure S2) and 7970 M–1 s–1,
respectively.The replacement of 5-methyl-1,3,4-thiadiazole with 5-methyl-1,3,4-oxadiazole reduced the
inhibitory activity slightly, resulting in compound 3n (IC50 1.22
μM), which, however, displayed a sustained inhibition reaching a
kinac/Ki value of 3880
M–1 s–1 (Ki = 0.0773
μM). Surprisingly, the 5-methyl-4H-1,2,4-triazole-derived double
acylated compound 3o had higher inhibitory potency than 3d,
suggesting that there is room in the active site of the Mpro for the additional
indole-3-carbonyl residue. 3o has a
kinac/Ki value of 12,200
M–1 s–1 (Ki = 0.0773
μM) (see Figure S3).The bioisosteric substitution of the indole moiety in 3d by a benzofuran
ring resulted in a more potent inhibitor, 3p, with a
kinac/Ki value of 5190
M–1 s–1 (Ki = 0.0357
μM). Next, we continued modifying the benzofuran ring of 3p. For
example, the replacement of benzofuran by 5-bromofuran marginally reduced the affinity
(3q), whereas 5-phenylfuran (3r, IC50 0.0344
μM) and 5-(phenylethynyl)furan derivatives (3s, IC50 0.250
μM) showed stronger inhibitory activity than 3p; in particular,
compound 3r had a 14-fold higher potency. The
kinac/Ki values were not
determined for 3q and 3r, but for 3s, it was found
to be 5200 M–1 s–1 (Ki =
0.0344 μM). The inhibitory activity of the trifluoromethyl analog of 3t
dropped, demonstrating once again that a 5-CF3-1,3,4-thiadiazole thioester was
not beneficial for Mpro inhibition. The furan ring in 3r was
exchanged for pyrrole, resulting in the inhibitor 3u, which had a lower
potency but still showed inhibitory activity in the low micromolar range. The
kinac/Ki value for
3u is 5070 M–1 s–1
(Ki = 0.605 μM). Potency was somewhat increased by
replacing the indole ring in 3d by a naphthalene ring system. The resulting
inhibitor, 3v, had an IC50 value of 0.454 μM. The
kinac/Ki value for
3v is 6960 M–1 s–1
(Ki = 0.0211 μM).In the next attempt, we addressed the ring size of the thiolate portion. The replacement
of 1,3,4-thiadiazole in 3d with pyrimidine led to the most potent inhibitor,
3w, of the present study. It has an IC50 value of 0.0114
μM and a kinac/Ki value of
58,700 M–1 s–1 (Ki =
0.0141 μM) (3w, see Figure S4), making it a suitable scaffold for further development. The
introduction of one methyl group into the 4-position of the pyrimidine ring marginally
reduced inhibitory activity (3x, IC50 0.0876 μM
(kinac/Ki = 27,200
M–1 s–1, Ki = 0.0332
μM), see Figure S5), while 4,6-dimethyl substitution decreased it significantly
(3y, IC50 2.03 μM). These findings imply that the
thioester with an unsubstituted pyrimidine is the best leaving group for covalent
Mpro inhibition of the present series.With these new warheads, we explored another part of the molecules. Initially, we
incorporated a 6-ethoxy group into the indole, resulting in 3z that had no
inhibitory activity. When indole was exchanged for 5-phenylfuran resulting in inhibitor
3aa, Mpro inhibition was equipotent to that of 3w
with an IC50 value of 0.0181 μM, but noticeable Mpro
reactivation within 1 h was observed. Compound 3ab, in which
5-(phenylethynyl)furan was combined with a 6-dimethylpyrimidine thioester group, displayed
stronger Mpro inhibitory activity (IC50 of 0.288 μM) than
3y; however,
kinac/Ki could not be
calculated.The replacement of indole by benzothiophene (3ac, IC50 0.0155
μM), 5-phenylthiophene (3ad, IC50 0.0137 μM), or
5-(4-Br-phenyl)thiophene (3ae, IC50 0.0144
μM) resulted in outstanding Mpro inhibitory potency comparable to that of
indole derivative 3w, but
kinac/Ki values could not be
calculated owing to Mpro reactivation. The quinolone system (3af)
likewise demonstrated efficacy, with an IC50 value of 0.0206 μM.
Surprisingly, the pyrimidine thioester of naproxen, a nonsteroidal anti-inflammatory drug,
inhibited Mpro with an IC50 of 0.551 μM and a
kinac/Ki value of 33,900
M–1 s–1 (Ki = 0.021
μM) (3ag, Ki = 0.021 μM, see
Figure S6).Finally, we installed a benzothiazole (in 3ah,
Ki = 0.0726 μM, see Figure S7) in place of a pyrimidine (in 3w), achieving an
IC50 of 0.117 μM along with a high
kinac/Ki value of 14,200
M–1 s–1 (Ki = 0.0726
μM) (3ah, see Figure S7), indicating that this bicyclic thioester is also well suitable.
Following this discovery, the Mpro inhibitory activity of a number of bicyclic
benzothiazole thioesters was studied. Inhibition was lost when indole was replaced with
benzofuran. However, the introduction of 5-phenylfuran (3aj, IC50
0.166 μM), 5-(4-Br-phenyl)furan (3ak, IC50
0.255 μM), benzothiophene (3al, IC50 0.190 μM),
5-phenylthiophene (3am, IC50 0.125 μM), or quinoline
(3an, IC50 0.089 μM) resulted in a similar inhibitory
activity as compound 3ah, although none of these modified compounds
outperformed 3ah. The concentration–inhibition curves for selected
compounds are shown in Figure .
Figure 4
Concentration-dependent inhibition of SARS-CoV-2 Mpro by the best
inhibitors of the present series: 3ag (IC50 0.551 ± 0.092
μM), 3i (IC50 0.349 ± 0.123 μM),
3c (IC50 0.143 ± 0.021 μM), 3l
(IC50 0.147 ± 0.044 μM), 3ah (IC50
0.117 ± 0.048 μM), 3k (IC50 0.0914 ± 0.0163
μM), 3x (IC50 0.0876 ± 0.0109 μM), and
3w (IC50 0.0114 ± 0.0028 μM).
Concentration-dependent inhibition of SARS-CoV-2 Mpro by the best
inhibitors of the present series: 3ag (IC50 0.551 ± 0.092
μM), 3i (IC50 0.349 ± 0.123 μM),
3c (IC50 0.143 ± 0.021 μM), 3l
(IC50 0.147 ± 0.044 μM), 3ah (IC50
0.117 ± 0.048 μM), 3k (IC50 0.0914 ± 0.0163
μM), 3x (IC50 0.0876 ± 0.0109 μM), and
3w (IC50 0.0114 ± 0.0028 μM).
Cytotoxicity and Antiviral Activity against SARS-CoV-2
Cytotoxicity
Selected Mpro inhibitors were further investigated for their antiviral
activity in Calu-3 and Vero 76 cells. It is worth mentioning that the cytotoxicity of
test compounds reduces cell virion production, which is commonly misunderstood as
antiviral activity. Therefore, we first carefully checked the cytotoxicity of
Mpro inhibitors in both cell lines at a high concentration of 10 μM.
As outlined in Figure S8, none of the tested compounds exerted cytotoxicity.
Antiviral Activity
Based on the promising inhibitory activity of the thioesters against SARS-CoV-2
Mpro, we evaluated the antiviral efficacy of selected potent inhibitors
against SARS-CoV-2 in vitro. Calu-3 and Vero 76 cells were infected with SARS-CoV-2 to
evaluate each of these Mpro inhibitors. The Calu-3 cell line, which is
derived from human lung, is the most extensively used cellular model for assessing
antiviral efficacy against respiratory pathogens in vitro. Whereas in Calu-3 cells,
SARS-CoV-2 enters the cells in a TMPRSS2-dependent manner, Vero cells barely express
TMPRSS2 and viral entry is mediated by cathepsins.[47]Cells were incubated with 10 μM of each inhibitor 1 h prior to infection and 24 h
post-infection (p.i.) at a multiplicity of infection (MOI) of 0.01. Viral titers of
Calu-3 and Vero 76 cell culture supernatants were evaluated by titration on Vero E6
cells 24 h p.i. and are given as plaque-forming units (PFU) per milliliter (see
Figure S9). As positive controls, we used remdesivir and I,
an established SARS-Mpro inhibitor.[37,39]In Figures S10 and S11, the viral titers of SARS-CoV-2 derived from Calu-3 or
Vero 76 cells incubated with 10-fold serial dilutions (10–0.001 μM) of each
inhibitor or DMSO (solvent control) 1 h prior and 24 h post infection (p.i.) are shown.
Based on these results, dose-dependent inhibition curves were generated for all active
inhibitors that exhibited EC50 values ranging from 0.0383 to 4.05 μM in
Calu-3 (Figure , see Figure S12 for compounds 3f, 3g,
3aa, 3ae, and 3af) and 0.00458 to 0.642
μM in Vero 76 cells (Figure ),
respectively.
Figure 5
EC50 values of Mpro inhibitors in Calu-3 cells. Lung-derived
human Calu-3 cells were incubated with 10-fold serial dilutions (10–0.001
μM) of each inhibitor or DMSO (solvent control) for 1 h followed by infection
with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. After virus
inoculation, cells were further incubated with the respective inhibitors for 24 h.
Supernatants were harvested, and viral titers were determined by titration on Vero
E6 cells. For normalization, viral titers of DMSO-treated cells were set as 0%
inhibition. Means ± SDs from three biological replicates are presented. Red
numbers indicate individual EC50 values of the respective inhibitors.
Figure 6
EC50 values of Mpro inhibitors in Vero 76 cells. Vero 76
cells were incubated with 10-fold serial dilutions (10–0.001 μM) of
each inhibitor or DMSO (solvent control) for 1 h followed by infection with
SARS-CoV-2 at an MOI of 0.01. After virus inoculation, cells were further incubated
with the respective inhibitors for 24 h. Supernatants were harvested, and viral
titers were determined by titration on Vero E6 cells. For normalization, viral
titers of DMSO-treated cells were set as 0% inhibition. Means ± SDs from three
biological replicates are presented. Red numbers indicate individual EC50
values of the respective inhibitors.
EC50 values of Mpro inhibitors in Calu-3 cells. Lung-derived
human Calu-3 cells were incubated with 10-fold serial dilutions (10–0.001
μM) of each inhibitor or DMSO (solvent control) for 1 h followed by infection
with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01. After virus
inoculation, cells were further incubated with the respective inhibitors for 24 h.
Supernatants were harvested, and viral titers were determined by titration on Vero
E6 cells. For normalization, viral titers of DMSO-treated cells were set as 0%
inhibition. Means ± SDs from three biological replicates are presented. Red
numbers indicate individual EC50 values of the respective inhibitors.EC50 values of Mpro inhibitors in Vero 76 cells. Vero 76
cells were incubated with 10-fold serial dilutions (10–0.001 μM) of
each inhibitor or DMSO (solvent control) for 1 h followed by infection with
SARS-CoV-2 at an MOI of 0.01. After virus inoculation, cells were further incubated
with the respective inhibitors for 24 h. Supernatants were harvested, and viral
titers were determined by titration on Vero E6 cells. For normalization, viral
titers of DMSO-treated cells were set as 0% inhibition. Means ± SDs from three
biological replicates are presented. Red numbers indicate individual EC50
values of the respective inhibitors.In Calu-3 cells, the compounds 3v (EC50 0.0450 μM) and
3l (EC50 0.0383 μM) showed excellent antiviral activity
(Figure ). Compounds 3w,
3ah, and 3r showed antiviral activity in the submicromolar
range (0.100–0.137 μM). The pyrimidine derivative 3w, one of
the most potent Mpro inhibitors in the enzymatic assay, had an
EC50 of 0.110 μM, which is around 10 times less potent than its
Mpro inhibitory activity. The analogous benzothiazole derivative
3ah had similar Mpro inhibitory (IC50 0.117
μM) and antiviral activities (EC50 of 0.137 μM). With an
EC50 of 0.100 μM, 3r showed very good antiviral
activity. The antiviral activities of 3c, 3i, 3x,
3h, and 3e were also submicromolar, with EC50
values ranging from 0.367 to 0.734 μM. The antiviral activities of compounds
3f, 3g, 3aa, 3ae, and
3af were lower, in the micromolar range, with EC50 values of
1.21–4.05 μM. In our testing system, control compounds like remdesivir and
I possessed EC50 values of 0.0796 and 0.0285 μM,
respectively.In Vero 76 cells, compounds 3c, 3i, and 3x were
confirmed to act as anti-SARS-CoV-2 agents (see Figure S11). With an EC50 value of 0.00458 μM,
3i was the most potent compound identified in this study. The antiviral
activity of 3c was maintained in Vero 76 cells with an EC50 of
0.641 μM, whereas the activity of 3x was higher when measured in Vero
76 cells as compared to Calu-3 cells, showing an EC50 of 0.185 μM.
Remdesivir and I, tested in the same system as control compounds, exhibited
EC50 values of 0.0026 and 0.00958 μM, respectively. All other
compounds that showed activity as antiviral agents in Calu-3 cells
(3f-h, 3l, 3r, 3v,
3w, 3aa, 3ae, 3af, and
3ah) did not display antiviral activity in Vero 76 cells at the highest
test concentration of 10 μM. This might be attributed to the poor compound
permeability in Vero cells or high expression of the efflux transporter P-glycoprotein;
many recent reports have revealed such variances.[48−50] Concentration–response curves for 3c,
3i, 3x, remdesivir, and I in Vero76 cells are
shown in Figure .
Broad-Spectrum Effect against SARS-CoV-1 Mpro and MERS-CoV
Mpro
Mpro sequences for SARS-CoV-2, SARS-CoV-1, and MERS-CoV are known to have a
high similarity in the catalytic domain, which is responsible for protein cleavage. Due to
the promising Mpro inhibition and antiviral activity against SARS-CoV-2, we
investigated the inhibitory activities of selected compounds on the main proteases of the
human-pathogenic coronaviruses SARS-CoV-1 and MERS-CoV as well. Compounds were tested
against recombinant SARS-CoV-1 Mpro and MERS-CoV Mpro at a high
concentration of 10 μM. The classical fluorescence resonance transfer (FRET) assay
was used.[51−53] Concentration-dependent
inhibition curves for compounds that achieved at least 50% inhibition against the relevant
Mpro enzyme were determined to calculate IC50 values (see Table for IC50 values and Figures S13 and S14 for concentration–inhibition curves).
GC373(54) (see Table ) was employed as a positive control for both Mpro inhibitory
activities.
Table 2
Inhibitory Activities of Selected SARS-CoV-Mpro Inhibitors against
Recombinant SARS-CoV-1 Mpro and MERS-CoV Mpro
compd. no.
SARS-CoV-2 Mpro
SARS-CoV-1 Mpro
MERS-CoV Mpro
IC50 (μM)
IC50 (μM)a or %
inhibition at 10 μM
IC50 (μM)a or %
inhibition at 10 μM
3c
0.143 ± 0.021
3.21 ± 0.24
0.535 ± 0.032
3e
0.0600 ± 0.0257
n.d.b,c
0.486 ± 0.223
3f
0.0723 ± 0.0221
7.35 ± 0.93
0.945 ± 0.045
3g
0.0547 ± 0.0073
n.d.c
0.271 ± 0.157
3h
0.159 ± 0.038
2.15 ± 0.14
0.196 ± 0.001
3i
0.349 ± 0.123
n.d.b
0.223 ± 0.051
3k
0.0914 ± 0.0163
n.d.
1.13 ± 0.03
3l
0.147 ± 0.044
n.d.
0.503 ± 0.051
3s
0.250 ± 0.076
2.76 ± 0.10
0.378 ± 0.214
3v
0.454 ± 0.011
1.65 ± 0.18
0.526 ± 0.016
3w
0.0114 ± 0.0028
0.06 ± 0.00
0.303 ± 0.059
3x
0.0876 ± 0.0109
1.52 ± 0.01
1.31 ± 0.28
3aa
0.0181 ± 0.0039
n.d.c
0.0790 ± 0.0280
3ae
0.0144 ± 0.0020
n.d.b
n.d.
3af
0.0206 ± 0.0006
0.253 ± 0.163
0.613 ± 0.155
3ah
0.117 ± 0.048
n.d.
n.d.
GC373d
n.d.
0.0446 ± 0.0020
0.0780 ± 0.0170
IC50 values represent the average of two independent experiments
determined in triplicate. Errors are given by the ratio between the standard
deviation and the square root of the number of measurements.
n.d.: not determined.
Compounds 3e, 3g, and 3aa displayed high
fluorescence in the SARS-CoV-1 Mpro assay, interfering with the
measurement of the enzymatic activity readout and hindering the evaluation of their
potency in our assay.
IC50 values represent the average of two independent experiments
determined in triplicate. Errors are given by the ratio between the standard
deviation and the square root of the number of measurements.n.d.: not determined.Compounds 3e, 3g, and 3aa displayed high
fluorescence in the SARS-CoV-1 Mpro assay, interfering with the
measurement of the enzymatic activity readout and hindering the evaluation of their
potency in our assay.GC373 =
((2S)-2-((S)-2-(((benzyloxy)carbonyl)amino)-4-methylpentanamido)-3-(2-oxopyrrolidin-3-yl)propanal).At 10 μM, 3c, 3h, 3s, 3v,
3w, 3x, and 3af displayed more than 50%
inhibition of SARS-CoV-1 Mpro. Among all of the examined compounds,
3w demonstrated the highest inhibitory activity, with an IC50
value of 0.0613 μM. 3af had submicromolar inhibitory activity
(IC50 0.253 μM), whereas 3c (IC50 3.21
μM), 3f (IC50 7.35 μM), 3h
(IC50 2.15 μM), 3s (IC50 2.76 μM),
3v (IC50 1.65 μM), and 3y (IC50
1.52 μM) displayed inhibitory activities in the micromolar range. Compounds
3f, 3k, 3l, 3ae, and
3ah did not inhibit SARS-CoV Mpro, although they did inhibit
SARS-CoV-2 Mpro. This is possibly due to the difference in shape and size
between the two Mpro enzymes.[55]
Concentration–response curves for 3v (IC50 1.65 μM),
3w (IC50 0.0614 μM), 3x (IC50 1.52
μM), and 3af (IC50 0.253 μM) are shown in Figure S13.We identified many compounds to inhibit the MERS-CoV Mpro activity in the
submicromolar to nanomolar range (see Table ).
With an IC50 value of 0.0792 μM, compound 3aa was discovered
to be one of the most effective representatives. The rank order of potency at MERS-CoV
Mpro is as follows: 3aa > 3h >
3i > 3g > 3w > 3s >
3e > 3l ∼ 3v ∼ 3c
∼ 3af > 3f > 3k ∼
3x. MERS-CoV Mpro was not inhibited by compounds
3ah and 3ae. Concentration–inhibition curves for
3c, 3e, 3f–i,
3k, 3l, 3s, 3v, 3w,
3x, 3aa, and 3af are shown in Figure S14.Thus, these thioesters represent a novel class of broad-spectrum inhibitors that target
Mpro of SARS-CoV-1, SARS-CoV-2, and MERS-CoV, with the highest potency
against SARS-CoV-2 Mpro followed by MERS-CoV Mpro and SARS-CoV-1
Mpro.
X-ray Structures of SARS-CoV-2 Mpro Bound with Inhibitors
To understand the binding interactions of the inhibitors with the protease, co-crystal
structures of SARS-CoV-2 Mpro in complex with the broad-spectrum
Mpro inhibitors 3w and 3af were determined at a
resolution of 2.3, and 1.5 Å, respectively (Figure ). Table S2 summarizes the X-ray data and refinement statistics for both
complexes. Figure shows that the
protease-catalyzed cleavage of the inhibitors produced a thioester-type
enzyme–inhibitor complex. The remaining fragment of the compounds was covalently
bound to the catalytic Cys145, while the pyrimidine thiolate acted as a leaving group and
departed from the active site. The indole ring of compound 3w and the
quinoline ring of 3af mainly formed hydrophobic interactions with surrounding
residues including His41, Met165, Asp187, Arg188, and Gln189 (Figure
A,B). The catalytic His41 together with Met165 functioned as
a clamp to catch the aromatic indole and quinoline ring of the inhibitors. In the complex
structure of 3w with SARS-CoV-2 Mpro, Met49 also contributed to
the hydrophobic interactions with the indole ring, while this interaction was absent in
the complex structure of 3af due to the unclear electron density was missing
in the loop where Met49 is located. The superposition of the two complex structures
revealed that the quinoline of 3af was shifted toward His41 compared to the
position of the indole ring of 3w, leading to a cooperative movement of His41
and of Met165. Such a movement of the quinoline ring might sterically clash with Met49 and
thus cause a disorder of the loop containing Met49. Overall, the co-crystal structures
provide a mechanistic insight into the inhibition of SARS-CoV-2 Mpro by two
pyrimidine thioesters in which the catalytic Cys145 is acylated by the indole-carbonyl or
quinolone-carbonyl group, which are well positioned in a relatively hydrophobic binding
pocket.
Figure 7
Binding modes of (A) 3w (PDB ID: 7X6K) and (B) 3af (PDB ID: 7X6J) with SARS-CoV-2 Mpro
revealed by co-crystal structures. The protease is shown in gray cartoon,
3w in green sticks, 3af in yellow sticks, and the
surrounding residues in light gray sticks. (C) A superposition of 3w
(green) and 3af (yellow) bound with the protease shows the different
conformational perturbations at the active site caused by the binding of the
inhibitors.
Binding modes of (A) 3w (PDB ID: 7X6K) and (B) 3af (PDB ID: 7X6J) with SARS-CoV-2 Mpro
revealed by co-crystal structures. The protease is shown in gray cartoon,
3w in green sticks, 3af in yellow sticks, and the
surrounding residues in light gray sticks. (C) A superposition of 3w
(green) and 3af (yellow) bound with the protease shows the different
conformational perturbations at the active site caused by the binding of the
inhibitors.
Molecular Modeling of 3w and 3af with SARS-1-CoV
Mpro and MERS-CoV Mpro
Docking studies of compounds 3w and 3af into Mpro of
SARS-CoV-1 and MERS-CoV followed by short molecular dynamics (MD) simulations (5 ×
200 ns) reveal a stable π–π interaction with His41 (31–58% of
the analyzed simulation time) for both proteins and a specific hydrogen bond interaction
between MERS-CoV Mpro-Q167 and the nitrogen of the indole ring for
3w, pointing toward the solvent. Several polar contacts with the S2 pocket
support the binding mode: M49, M165, and H41 (numbering of SARS-CoV-1 Mpro). By
comparison, 3af stabilizes a water molecule within the site, with its extra
H-bond acceptor group. It is important to highlight that during our simulations, a
classical force-field model was adopted that disregards potential changes in the
ionization state of side chains. This is especially relevant for His41 that could further
display a π-charge interaction with either the indole or the quinoline ring,
yielding a more stable interaction (see Figure S15).
Glutathione Stability
We tested the stability of selected potent Mpro thioester inhibitors against
excess glutathione (GSH) as a physiological nucleophile to identify the inherent activity
toward thiols under physiological conditions. These experiments revealed that inhibitors
are stable with low inherent activity under the assay conditions (5 mM GSH at pH 7.4) (see
a representative example, 3an, in Figure S16). This indicates that the Mpro inhibition of
thioesters is dependent not on their chemical reactivity toward GSH but rather on the
specific reactivity toward catalytic Cys145 at the active site of Mpro, as
shown by the X-ray structures of SARS-CoV-2 Mpro bound with inhibitors in Figure .
Conclusions
The Mpro of SARS-CoV-2 is an important target since it is involved in the
coronavirus life cycle. We developed, synthesized, and tested novel low-molecular-weight
thioesters that act as inhibitors of Mpro of SARS-CoV-2, the virus that is
causing the current COVID-19 pandemic. A number of compounds showed low nanomolar
Mpro inhibitory activity with an irreversible inhibition mechanism. The
co-crystal structure of Mpro with 3a and 3af, two of
the most potent inhibitors, validated the proposed binding mechanism. In cell-based
experiments, several compounds exhibited excellent antiviral activity. The SARS-CoV-2
Mpro inhibitors were additionally shown to inhibit Mpro of the
related pathogenic coronaviruses SARS-CoV-1 and MERS-CoV, indicating that they are potential
candidates for further development as broad-spectrum anti-coronaviral agents.
Experimental Section
General Experimental Procedures
All commercially available reagents and solvents were used as received. Reactions
sensitive to air or moisture were performed under an atmosphere of argon and/or in
anhydrous solvents. Anhydrous solvents were purchased from Acros Organics (AcroSeal).
Unless stated otherwise, extracts were dried over sodium sulfate prior to filtration.
Thin-layer chromatography was performed on TLC Silica Gel 60F254 aluminum sheets provided
by Merck, with detection at 254 and 366 nm. Flash chromatography was carried out on an
Interchim PuriFlash XS420 flash chromatography system and Grace Davison Davisil LC60A
20e45 mm silica or Merck Geduran Si60 63–200 μm. Mass spectra were recorded
on an Advion DCMS interface (ESI voltage: 3.50 kV, capillary voltage: 187 V, source
voltage: 44 V, capillary temperature: 250 °C, desolvation gas temperature: 250
°C, gas flow rate: 5 L/min N2), with elution of the spots with MeOH.
High-resolution mass spectrometry (HRMS) for the final compound was measured by the mass
spectrometry department, Institute of Organic Chemistry, Eberhard Karls University
Tuebingen, on a Bruker maXis 4G ESI-TOF instrument. The instrument was run in ESI+ mode,
and the settings were as follows: nebulizer gas of 1.2 bar, gas flow of 6.0 L/min, source
temperature of 200 °C, capillary voltage of 4500 V, endplate offset of −500 V,
and m/z range from 80 to 1000. NMR spectra were measured
on Bruker Avance 400 or 600 NMR spectrometers. The spectra were calibrated on the
deuterated solvents, and chemical shifts (d) are stated relative to
tetramethylsilane in ppm. The solvent used was deuterated chloroform
(d3) and deuterated DMSO (d6).
Melting points were measured on a melting point apparatus (Mettler Toledo MP70, hand
method, temperature from 60 to 360 °C, heating rate 20.0 °C/min) and are
uncorrected.The purity of the final compound was determined via high-performance liquid
chromatography (HPLC) using an Agilent 1100 Series LC system (Agilent Technologies, Santa
Clara, CA) with a Phenomenex Kinetex C8 100A column (150 mm × 4.6 mm, 2.6 μm)
(Phenomenex Inc., Torrance, CA), and detection was performed with a UV DAD at a wavelength
of 254 nm. Elution was carried out with the following gradients: 0.01 M KH2PO4 (pH 2.32)
(solvent A) and MeOH (solvent B). Method A was follows: 0 min, 40% B/60% A; 9 min, 95%
B/5% A; 10 min, 95% B/5% A; 11 min, 40% B/60% A; and 16 min, 40% B/60% A, with a flow of
0.5 mL/min. Method B was follows: 0 min, 40% B/60% A; 15 min, 85% B/15% A; 20 min, 85%
B/15% A; 22 min, 40% B/60% A; and 28 min, 40% B/60% A, with a flow 0.5 mL/min. The final
compounds showed a purity of >95% according to the peak areas.
Synthesis of Thioesters 3a–z and
3aa–an
POCl3 (1.37 mmol, 1.1 equiv) was added to a solution of pyridine (1.37 mmol,
1.1 equiv) and corresponding carboxylic acids (1a–s, 1.25
mmol, 1.0 equiv) in dry DCM (10 mL), and the solution was stirred for 10–15 min at
room temperature to generate carboxylic acid chlorides in situ. The appropriate thiols
(2a–n) were added first followed by pyridine (2.0 mmol,
1.6 equiv). TLC with UV detection was used to monitor the reaction. The mixture was put
into ice water and extracted with DCM (2 × 25 mL) when the reaction was completed
after 12 h. After washing with 1 N HCl (20 mL) and 30–40% NaOH solutions, the
combined organic layers were brined (20 mL). The solution was then dried over
Na2SO4, filtered, and evaporated until it was completely dry. The
resulting residue was purified by silica gel chromatography using 10–20% EtOAc in
hexane to afford the desired products.
S-(4-Chlorophenyl)-1H-indole-2-carbothioate
(3a)
The product 3a was synthesized from the reaction of indole-2-carboxylic
acid (1a, 201 mg, 1.25 mmol) and 4-chlorobenzenethiol (2a,
180.7 mg, 1.25 mmol) in the presence of pyridine (0.11 mL, 1.1 equiv; 0.161 mL, 1.6
equiv). Light yellow solid; yield 78% (279 mg); Mp: 230–232 °C.
1H NMR (400 MHz, DMSO-d6) δ 12.11 (s, 1H,
NH), 7.74 (d, J = 8.0 Hz, 1H), 7.59 (s, 4H), 7.51–7.45 (m, 2H),
7.35–7.30 (m, 1H), 7.16–7.10 (m, H). 13C NMR (101 MHz,
DMSO-d6) δ 180.66, 138.39, 137.18, 135.27, 133.23,
129.89, 127.13, 126.42, 125.97, 123.12, 121.23, 113.32, 109.14. HRMS (ESI-TOF)
m/z for (C15H10ClNOS [M +
Na]+) calcd 310.0069, found 310.0067. HPLC tR =
9.581 min.
We did this by virtually screening our in-house TüKIC library against the
Mpro using a combination of docking and short MD simulations.
Three-dimensional ligand structures were generated with LigPrep (implemented in Maestro
2020v4), using Epik to predict their protonation in pH 7.0 ± 2.0 and generating
tautomers and diastereoisomers. The OPLS3e force-field was employed for structure
generation.The SARS-CoV-2 Mpro protein structure was previously prepared from the PDB
ID: 5R82(40,56) using the Protein Wizard
Preparation tool, with standard options, and MD simulations for equilibration as
described. Molecular docking was carried out with Glide SP (version 9.1).[57] Grids were centered at the central point of the active site residues
G143, C145, M49, and H41. The box was 12 Å long in each direction. The top 500 hits
were visually inspected by observing the interaction with relevant residues within P1
and P2. Whenever mentioned, covalent docking as performed using CovDock[58] using the C145 as the anchor and nucleophilic addition to the double
bond as the reaction type and generating up to 10 poses for each ligand. Poses were
selected according to the docking score and relevant interactions. Selected poses
underwent short MD simulations (200 ns, data not shown). Ligands that remained within
the pocket during the simulated time were further selected for experimental testing.
Potential Binding Mode of Compounds in MERS-CoV and SARS-CoV-1 Main Proteases
The ligands 3w and 3af were covalently docked to the MERS and
SARS-CoV-1 Mpro models to study their binding mode. 3D structural models
Mpro from MERS (PDB ID: 4YLU), SARS-Cov-1 (7LMJ), and SARS-CoV-2 were generated, filling the missing residues in the
C-terminal portion, using Prime.[59,60] The generated models then underwent preparation using PrepWizard
(Maestro 2021.4).
MD Simulations and Trajectory Analyses
The minimized structures were submitted to MD simulation for further refinement.
Selected docking poses were further validated by MD simulation, where ligand stability
within the proposed pocket and its interactions were evaluated. MD simulations were
carried out using the Desmond engine[61] with the OPLS4
force-field.[62] The simulated system encompassed the
protein–ligand complex, a predefined water model (TIP3P)[63] as
a solvent, and counterions (Na+ or Cl– adjusted to
neutralize the overall system charge). The system was treated in a cubic box with
periodic boundary conditions specifying the shape and the size of the box as 13 Å
distance from the box edges to any atom of the protein. RESPA integrator time steps of 2
fs for bonded and near and 6 fs for far were applied. Short-range Coulombic interactions
were performed using a time step of 1 fs and a cutoff value of 9.0 Å, whereas
long-range Coulombic interactions were handled using the Smooth Particle Mesh Ewald
(PME) method.[64]
Desmond Relaxation Protocol
Simulations were run in NPT ensemble, with a temperature of 310 K
(Nosé–Hoover thermostat) and pressure of 1.01325 bar
(Martyna–Tobias–Klein barostat).Results of simulations, in the form of trajectory and interaction data and RMSD and
RMSF values, are available on the Zenodo repository (code: 10.5281/zenodo.6303511). MD trajectories
were visualized, and figures were produced using PyMOl v.2.5 (Schrödinger LCC,
New York, NY, USA). For each ligand, simulations at least five independent 200 ns
replicas were carried out. Protein–ligand interactions were determined using
the Simulation Event Analysis pipeline implemented in Maestro (Maestro v2021.4) with
standard settings.
SARS-CoV-2 Mpro Assay
The SARS-CoV-2 main protease (Mpro) was expressed and purified according to
a previously published procedure.[37] In brief, a codon-optimized cDNA
sequence encoding the Mpro enzyme (accession no.: MN908947.3, ORF1ab
polyprotein residues 3264–3569) with an N-terminal Mpro autocleavage
site and a C-terminal His10 tag linked via an HRV 3C protease cleavage site
was inserted into the bacterial expression vector pGEX-6P-1. After transformation of
BL21 Escherichia coli bacteria (NEB, Ipswich, USA), cells were grown in
an LB medium supplemented with ampicillin (100 μg/mL). Subsequently, IPTG (final
concentration 1 mM) was added to induce recombinant gene expression until an
OD600 of 0.5 was reached. After growing the bacteria at 30 °C for
3–4 h, cells were sedimented at 4000g and 4 °C for 10 min.
Cells were then resuspended in cold PBS supplemented with 5 mM imidazole (pH 7.4).
Subsequently, the cells were lyzed on ice using a Sonoplus HD2070 (Bandelin, Berlin,
Germany) and centrifuged at 48,000g and 4 °C for 30 min in collect
the supernatant. The His-tagged Mpro enzyme was purified using HisPur Ni-NTA
spin columns (Thermo Fisher Scientific, Waltham, USA). For the native Mpro
enzyme, the His tag was removed by an HRV 3C protease (Merck, Darmstadt, Germany)
overnight at 4 °C. Subsequent disposal of the GST-tagged HRV 3C protease was
performed by a Glutathione Spin Column (Thermo Fisher Scientific, Waltham, USA). The
collected flow through contained the native Mpro enzyme that was subsequently
used for pharmacological assays. For the initial inhibition experiments at a single
concentration, crude cell extracts containing the Mpro enzyme were used. For
the subsequent pharmacological characterization of hit compounds to determine
IC50, Ki, and
kinac/Ki values, we employed
the purified His-tagged Mpro.SARS-CoV-2 Mpro activity assays were performed as previously
established.[37] Frozen recombinant His-tagged SARS-CoV-2
Mpro was thawed and immediately used for the enzyme assays that were
performed on a Pherastar FSX plate reader (BMG Labtech, Offenburg, Germany) at 37
°C with an excitation wavelength of 360 nm and an emission wavelength of 460 nm.
Black half area 96-well plates with a flat bottom were obtained from Greiner Bio-One
(Kremsmünster, Austria). The assay buffer contained 50 mM
3-(N-morpholino)propanesulfonic acid (MOPS, pH 7.2), 10 mM NaCl, 1 mM
EDTA, and 0.01% Triton X-100. The fluorogenic substrate Boc-Abu-Tle-Leu-Gln-AMC[37] diluted in the assay buffer was added to the test compounds, and the
mixture was preincubated at 37 °C for 5 min. The proteolytic cleavage of the
substrate was started by addition of the enzyme (e.g., 400 ng His-tagged Mpro
diluted in the assay buffer), and the fluorescence increase was followed for 10 or 60
min, respectively. The final protein concentration of SARS-CoV-2 Mpro was
adjusted to a slope increase of approximately 2000/min within the initial 10 min. The
final substrate concentration was 50 μM, which approximately corresponds to its
Km value.[37] The final DMSO
concentration was 4%. The product formation rate of the uninhibited control was set at
100%. For the determination of concentration–inhibition curves, at least eight
different inhibitor concentrations were investigated to observe the respective product
formation rates during the first 10 min. IC50 values were calculated by
nonlinear regression. For inhibitors showing time-dependent inhibition, the second-order
rate constant kinac/Ki was
determined by monitoring the effects of five different inhibitor concentrations on the
product formation rate for 60 min, and the data were analyzed by nonlinear regression
using the equation [P] = (vi × (1
– exp(−kobs ×
t)/kobs + d)), where
[P] is the product concentration, vi is
the initial rate, kobs is the observed first-order rate
constant, and d is the offset. Subsequently,
kobs was plotted versus the inhibitor concentration
[I], and a nonlinear regression using the equation
kobs = (kinac ×
[I])/([I] + Ki × (1
+ [S]/Km)) was performed. The deviation of
each data point from the calculated nonlinear regression was less than 10%. Data were
analyzed using GraphPad Prism 8.0.
Assay against Recombinant SARS-CoV-1 Mpro and MERS-CoV
Mpro
Recombinant SARS-CoV-1 Mpro and MERS-CoV Mpro were purchased from R&D Systems.
Proteolytic activity was determined using 10 μM
MCA-AVLQSGFR-K(DNP)-K-NH2 for SARS-CoV-1 Mpro and 10 μM
Ac-Abu-Tle-Leu-Gln-ACC for MERS-CoV Mpro. Fluorescence was monitored in a Synergy HTX
(Biotek) plate reader using excitation/emission wavelengths of 320/400 nm for
MCA-AVLQSGFR-K(DNP)-K-NH2 and 360/460 nm for Ac-Abu-Tle-Leu-Gln-ACC. All
assays were performed in 384-well black plates with a 15 min preincubation of the
compounds with the enzyme. The screen was performed with 10 μM of inhibitors.
Compounds that inhibited enzyme activity by 50% or more in the initial screen had their
dose–response curve determined. For each compound, two independent experiments
were performed, each in triplicate and monitored for 2 h. The percentage of inhibition
was calculated by comparison to a DMSO control. The protease inhibitor GC373 was used as
a positive control. Assays were performed at 25 °C in a final volume of 30 μL
per well of 50 mM HEPES (pH 7.5), 150 mM sodium chloride, 1 mM EDTA, and 0.01% Tween 20
in the presence of 50 nM enzyme and 10 μM substrate.[51,52,65] The
half-maximal inhibitory concentration (IC50) was determined by nonlinear
regression analysis of the velocity vs inhibitor concentration plot using GraphPad Prism
6.[66] Eleven inhibitor concentrations were used to generate each
curve.
Cytotoxicity
Cell Cultures
Calu-3 cells (human lung, ATCC Cat# HTB-55) were maintained in Dulbecco’s
modified Eagle medium (DMEM)/F-12 supplemented with 10% FCS and 10 mM sodium pyruvate.
Vero E6 cells (African green monkey kidney, ATCC cat. #CRL-1586) were maintained in DMEM
supplemented with 5% FCS. All cell lines were incubated at 37 °C and 5%
CO2 in a humidified atmosphere.
Cell Vitality Assay
To determine cell vitality of Calu-3 cells treated with inhibitors, the CellTiter-Glo
Luminescent Cell Viability Assay Kit (Promega) was used. Cells were grown in 96-well
plates until reaching 50–60% confluency before they were incubated with DMSO
(solvent control) or Mpro inhibitors at a concentration of 10 μM for 24
h. Next, cell culture supernatants were removed, and 50 μL of the CellTiter-Glo
substrate was added to each well and incubated for 30 min on a rocking platform.
Finally, samples were transferred into white 96-well plates, and luminescence was
measured using a Hidex Sense plate luminometer (Hidex).All work with infectious SARS-CoV-2 was conducted under BSL-3 conditions at the German
Primate Centre, Göttingen/Germany. Calu-3 cells were grown in 48-well plates
until reaching approx. 70% confluency. Cells were incubated with 10-fold serial
dilutions (10–0.001 μM) of Mpro inhibitors for 1 h at 37 °C
prior infection. Next, the inhibitor containing the cell culture medium was removed, and
cells were infected with SARS-CoV-2 isolate NK, Pango lineage B.1.513, at an MOI of 0.01
in an inoculum volume of 400 μL for 1 h at 37 °C. At 1 h post infection
(p.i.), the inoculum was removed, and cells were washed with PBS three times and further
incubated in a cell culture medium containing the respective inhibitor for 24 h.
Virus-containing supernatants were harvested and stored at −80 °C until
further usage.To determine viral titers, confluent grown Vero E6 cells were inoculated for 1 h at 37
°C with 10-fold serial dilutions of virus-containing supernatants. Next, the
inoculum was removed, and cells were washed once with PBS before they were overlaid with
1% plaque agarose (Biozym) dissolved in Eagle’s minimal essential medium without
phenol red (Lonza) and further incubated. At 48 h p.i., virus-induced plaques were
counted, and viral titers were determined as plaque forming units (PFU)/mL.
Crystallography Procedure
The cDNA of SARS-CoV-2 Mpro (GenBank: MN908947.3) with the N-terminal SUMO
tag was cloned into the pET-15b vector. The plasmid was transformed into BL21 (DE3)
cells for protein expression. The expressed protein was purified by a Ni-NTA column (GE
Healthcare) and cleaved by the SUMO specific peptidase 2 (SENP2) to remove the SUMO tag.
The resulting protein sample was further purified by Q-Sepharose followed by a
size-exclusion chromatography (GE Healthcare). The eluted protein samples were stored in
a solution (10 mM Tris, pH 7.5).The purified protease was concentrated to 7 mg/mL for crystallization. To obtain
complex structures, the protein was incubated with 4 mM 3w and
3af for 1 h before crystallization condition screening. Crystals of the
complexes were obtained under the condition of 10–22% PEG6000, 100 mM MES, pH
5.75–6.25, and 3% DMSO. Crystals were flash frozen in liquid nitrogen in the
presence of the reservoir solution supplemented with 20% glycerol. X-ray diffraction
data were collected at beamline BL02U1 at the Shanghai Synchrotron Radiation
Facility.[70] The data were processed with HKL3000 software
packages.[71] The complex structures were solved by molecular
replacement using the program PHASER[72] with a search model of PDB
code 6M2N.[70]
The model was built using Coot[71] and refined with XYZ
(reciprocal-space), individual B factors, TLS parameters, and occupancies implemented in
the program PHENIX.[72] The refined structures were deposited to the
Protein Data Bank with accession codes listed in Table S2 in the Supporting Information. The complete statistics as well as
the quality of the solved structures are also shown in Table S2.
Glutathione (GSH) Stability Assay
The GSH stability assay for compounds was performed according to the reported
procedures.[73,74]
A suitable HPLC/MS method was prepared, and the HPLC method was adjusted to inject a
sample every 10 min. To achieve a final concentration of 10 μM inhibitor and 5 mM
GSH in a final volume of 1 mL, 1 μL of the inhibitor stock solution (10 mM) was
added to 949 μL of the phosphate buffer (pH 7.4) as well as 50 μL of the
stock solution (100 mM) GSH. The solution was directly pipetted into a HPLC vial and
vortexed thoroughly. The temperature of the autosampler was set to 30 °C. Ten to 30
repeated measurements were performed. The reaction of the inhibitor with GSH was
monitored by measuring the decreasing area under the curve (AUC) of each compound.
Authors: Chao Lu; Chuanjie Wu; Delaram Ghoreishi; Wei Chen; Lingle Wang; Wolfgang Damm; Gregory A Ross; Markus K Dahlgren; Ellery Russell; Christopher D Von Bargen; Robert Abel; Richard A Friesner; Edward D Harder Journal: J Chem Theory Comput Date: 2021-06-07 Impact factor: 6.006
Authors: Markus Hoffmann; Nadine Krüger; Sebastian Schulz; Anne Cossmann; Cheila Rocha; Amy Kempf; Inga Nehlmeier; Luise Graichen; Anna-Sophie Moldenhauer; Martin S Winkler; Martin Lier; Alexandra Dopfer-Jablonka; Hans-Martin Jäck; Georg M N Behrens; Stefan Pöhlmann Journal: Cell Date: 2021-12-24 Impact factor: 41.582
Authors: Wayne Vuong; Conrad Fischer; Muhammad Bashir Khan; Marco J van Belkum; Tess Lamer; Kurtis D Willoughby; Jimmy Lu; Elena Arutyunova; Michael A Joyce; Holly A Saffran; Justin A Shields; Howard S Young; James A Nieman; D Lorne Tyrrell; M Joanne Lemieux; John C Vederas Journal: Eur J Med Chem Date: 2021-05-30 Impact factor: 7.088
Authors: Maren de Vries; Adil S Mohamed; Rachel A Prescott; Ana M Valero-Jimenez; Ludovic Desvignes; Rebecca O'Connor; Claire Steppan; Joseph C Devlin; Ellie Ivanova; Alberto Herrera; Austin Schinlever; Paige Loose; Kelly Ruggles; Sergei B Koralov; Annaliesa S Anderson; Joseph Binder; Meike Dittmann Journal: J Virol Date: 2021-02-23 Impact factor: 5.103
Figure 1
Figure 2
Figure 3
Scheme 1
Scheme 2
Figure 4
Figure 5
Figure 6
Figure 7