Targeting the Main Protease of SARS-CoV-2: From the Establishment of High Throughput Screening to the Design of Tailored Inhibitors

Article abstract of DOI:10.1002/anie.202016961

The main protease of SARS-CoV-2 (M^pro), the causative agent of COVID-19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M^pro. Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in-depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M^pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M^pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k_inac/K_i=37 500 m^?1 s^?1, K_i=24.0 nm) and pyridyl ester 17 (k_inac/K_i=29 100 m^?1 s^?1, K_i=10.0 nm), promising drug candidates for further development have been discovered.

Full text of DOI:10.1002/anie.202016961

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 10423–10429
International Edition: doi.org/10.1002/anie.202016961
German Edition: doi.org/10.1002/ange.202016961
SARS-CoV-2 Protease Inhibitors
Targeting the Main Protease of SARS-CoV-2: From the Establishment
of High Throughput Screening to the Design of Tailored Inhibitors
Julian Breidenbach+, Carina Lemke+, Thanigaimalai Pillaiyar+, Laura Schäkel+,
Ghazl Al Hamwi, Miriam Diett, Robin Gedschold, Nina Geiger, Vittoria Lopez,
Salahuddin Mirza, Vigneshwaran Namasivayam, Anke C. Schiedel, Katharina Sylvester,
Dominik Thimm, Christin Vielmuth, Lan Phuong Vu, Maria Zyulina, Jochen Bodem,
Michael Gütschow,* and Christa E. Müller*
Abstract: The main protease of SARS-CoV-2 (Mpro), the
causative agent of COVID-19, constitutes a significant drug
target. A new fluorogenic substrate was kinetically compared
to an internally quenched fluorescent peptide and shown to be
ideally suitable for high throughput screening with recombi-
nantly expressed Mpro. Two classes of protease inhibitors,
azanitriles and pyridyl esters, were identified, optimized and
subjected to in-depth biochemical characterization. Tailored
peptides equipped with the unique azanitrile warhead exhibited
concomitant inhibition of Mpro and cathepsin L, a protease
relevant for viral cell entry. Pyridyl indole esters were analyzed
by a positional scanning. Our focused approach towards Mpro
inhibitors proved to be superior to virtual screening. With two
irreversible inhibitors, azanitrile 8 (kinac/Ki = 37500 mÀ1 sÀ1,
Ki = 24.0 nm) and pyridyl ester 17 (kinac/Ki = 29100 mÀ1 sÀ1,
Ki = 10.0 nm), promising drug candidates for further develop-
ment have been discovered.
2 Mpro (and a second papain-like protease, PLpro) is critical for
the assembly of the viral replication–transcription complex,
the release of functional viral proteins and thus for the
replication of SARS-CoV-2.[1]
SARS-CoV-2 Mpro has a strong preference for homodi-
merization.[2] Each protomer consists of three domains, out of
which the chymotrypsin-like domains I and II form a cleft
harboring the substrate binding site.[3] Mpro is a cysteine
protease with a noncanonical catalytic dyad composed of
Cys145 residing in the S1’ site and His41 whose imidazole
serves as a hydrogen bond donor to a structural water
molecule. Cysteine proteases catalyze the hydrolysis of
peptide bonds in the course of an acyl transfer mechanism.
In the first step, accompanied by the release of the first
product, an S-acyl enzyme is formed as a result of the attack
of the active site Cys145 on the carbonyl carbon of the scissile
peptide bond. This covalent mechanism can be utilized for the
design of inhibitors bearing an electrophilic warhead, suscep-
tible for the nucleophilic attack of the catalytic cysteine.[3–5]
Mpro is highly conserved (96% amino acid sequence
identity) between SARS-CoV-2 and SARS-CoV-1, a related
virus that caused severe acute respiratory syndromes world-
wide in 2003. Due to the structural and functional similarities
of both proteases, promising attempts towards new SARS-
CoV-2 Mpro inhibitors have been based on previous com-
Introduction
Since December 2019, the outbreak of the current
unprecedented coronavirus disease (COVID-19) has led to
a global crisis with increasing morbidity. Severe acute
respiratory syndrome coronavirus 2 (SARS-CoV-2), the
pathogen of the pandemic, is a (+)ss-RNA virus that shares
the typical gene array of coronaviruses. It encodes two
overlapping polyproteins from which the main protease
(Mpro), also designated 3C-like protease (3CLpro), is excised
by autocleavage. The subsequent processing by SARS-CoV-
pounds targeting SARS-CoV-1 Mpro [4,6]
Examples include a-
.
ketoamide inhibitors which served as a starting point to
expedite the identification of SARS-CoV-2 Mpro inhibitors.[3,7]
Mpro has a unique substrate specificity mainly recognizing the
[*] J. Breidenbach,[+] C. Lemke,[+] Dr. T. Pillaiyar,[+] L. Schäkel,[+]
G. Al Hamwi, M. Diett, R. Gedschold, V. Lopez, S. Mirza,
Dr. V. Namasivayam, Dr. A. C. Schiedel, K. Sylvester, Dr. D. Thimm,
C. Vielmuth, L. Phuong Vu, M. Zyulina, Prof. Dr. M. Gütschow,
Prof. Dr. C. E. Müller
Dr. T. Pillaiyar[+]
Present address: Pharmaceutical Institute
Pharmaceutical Chemistry
Eberhard-Karls-University Tübingen
Auf der Morgenstelle 8, 72076 Tübingen (Germany)
[+] These authors contributed equally to this work.
Pharmaceutical Institute, Pharmaceutical & Medicinal Chemistry
University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
E-mails:
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016961.
E-mail: guetschow@uni-bonn.de
ꢀ 2021 The Authors. Angewandte Chemie International Edition
published by Wiley-VCH GmbH. This is an open access article under
the terms of the Creative Commons Attribution Non-Commercial
License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited and is not used
for commercial purposes.
christa.mueller@uni-bonn.de
N. Geiger, Prof. Dr. J. Bodem
Institute for Virology and Immunobiology
Julius-Maximilians-University Würzburg
Versbacher Strasse 7, 97078 Würzburg (Germany)
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substrate residues ranging from P4 to P1’ and possessing
a preference for glutamine at the P1 position. Such a feature is
absent in human proteases suggesting the feasibility of
developing selective protease inhibitors without toxicity in
patients. Considering the seriousness of COVID-19 and its
implications on public life in lockdown mode and the
economic fallout across the globe, there is an urgent unmet
medical need to develop clinically effective SARS-CoV-2-
moiety. Its structure was based on the unique preference of
Mpro for glutamine at the P1 position and the optimized P4-to-
P2 sequence as previously determined using a positional
scanning combinatorial library of natural and unnatural
amino acids.[16] The synthesis of the resulting substrate, Boc-
Abu-Tle-Leu-Gln-AMC, is depicted in Scheme S1. Very
recently, a similar substrate was used for the development
of activity-based probes for SARS-CoV-2 Mpro [17]
.
specific drugs, for which the viral proteases, Mpro and PLpro
,
By means of both substrates, Dabcyl-EDANS and Boc-
Abu-Tle-Leu-Gln-AMC, we established and optimized con-
ditions for HTS assays with respect to the choice of buffer
(Figure S2), the concentration of DMSO (Figure S3), as well
as the correlation of Mpro concentration and product forma-
tion rate (Figure S4), and of the substrate concentration and
gain of fluorescence upon complete cleavage (Figure S5).
Expectedly and advantageously, product formation with the
novel substrate Boc-Abu-Tle-Leu-Gln-AMC resulted in an
improved readout (Figure S5). Under the established assay
conditions (pH 7.2, 4% DMSO), Km values of 60.6 Æ 3.6 mm
for Dabcyl-EDANS (literature values 28.2 mm at pH 6.5;
74.4 mm at pH 7.3)[10,12] and 48.2 Æ 5.6 mm for Boc-Abu-Tle-
Leu-Gln-AMC have been determined (Figure S6; see Fig-
ure S7 for corresponding data with the purified native
protease). Dabcyl-EDANS exhibited a 10-fold higher specif-
icity constant of 5800 mÀ1 sÀ1 (literature values 3426 mÀ1 sÀ1,
represent attractive targets to combat viral replication and
pathogenesis.[8]
In this study, we developed a new SARS-CoV-2 Mpro assay
for quantitative high-throughput screening (HTS) and com-
pared it to the current standard assay. Focused libraries were
compiled taking inhibitors of related cysteine proteases and
cysteine-reactive compounds into account. As a second
approach, virtual screening of a proprietary compound
library[9] comprising 23000 unique molecules was performed.
We identified several candidate compounds and utilized the
HTS results as a basis for structure-based design, synthesis,
and biochemical characterization of highly potent inhibitors
for SARS-CoV-2 Mpro
.
Results and Discussion
5624 mÀ1 sÀ1)[3,10]
than
Boc-Abu-Tle-Leu-Gln-AMC
As a first step, we established a bacterial expression
system for SARS-CoV-2 Mpro to be used in our biochemical
assays. BL21 E. coli cells were transformed with a DNA
construct encoding the protease with an N-terminally fused
Mpro cleavage site and a C-terminal His10 tag linked via an
HRV 3C protease cleavage site. During bacterial expression,
Mpro autocatalytically cleaved the fusion protein, thereby
generating the native Mpro N-terminus. The His tag was
employed to purify the enzyme and was subsequently cleaved
off using an HRV 3C protease. After elimination of the latter
protease utilizing its GST tag, the purified, native Mpro was
obtained.
(604 mÀ1 sÀ1). Hence, the extended structure of Dabcyl-
EDANS resulted in an accelerated turnover.
Boc-Abu-Tle-Leu-Gln-AMC was cleaved by Mpro much
more efficiently than by cathepsin L, B, and trypsin, although
these proteases have been employed at a concentration
sufficient to convert three other selected substrates with very
high rates (Figure 1). In contrast, Dabcyl-EDANS was also
hydrolyzed by trypsin, presumably after one of the basic
amino acids of this substrate. HEK cell lysate of an
appropriate protein concentration degraded the five sub-
strates to a limited extent; a significant cleavage of Boc-Abu-
To monitor the proteolytic activity of His-tagged Mpro, we
applied an internally quenched fluorescent peptide substrate,
Dabcyl-Lys-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-
Met-Glu(EDANS)-NH2 (Figure S1, Supporting Informa-
tion).[3] In the intact peptide, the quencher 4-((4-(dimethyl-
amino)phenyl)azo)benzoic acid (Dabcyl) absorbs emission
energy from the fluorophore, 5-((2-aminoethyl)amino)naph-
thalene-1-sulfonic acid (EDANS), which is disrupted by Mpro
-
catalyzed cleavage of the peptide bond between the P1 amino
acid glutamine and the P1’ amino acid serine resulting in
a fluorescence signal. This substrate, referred to as “Dabcyl-
EDANS”, has recently been established for SARS-CoV-2
Mpro [3,10–13]
It has been reported that a shorter, internally
.
quenched fluorescent peptide substrate MCA-Ala-Val-Leu-
Gln-Ser-Gly-Phe-Arg-Lys(Dnp)-Lys-NH2 equipped with 7-
methoxy-coumarin-4-yl-acetic acid (MCA) as fluorophore
and the 2,4-dinitrophenyl (Dnp) quencher can also be used to
Figure 1. Conversion of fluorogenic substrates by His-tagged SARS-
CoV-2 main protease (Mpro), lysate obtained from human embryonic
kidney (HEK) cells, HEK cell lysate spiked with Mpro, human
cathepsin L (cat L), human cathepsin B (cat B), bovine trypsin, and in
the absence of enzymes (FU, fluorescence units). The product
formation was monitored for 10 min at 378C with an initial substrate
concentration of 50 mm in all cases. Each enzyme was used at the
same concentration in all respective experiments.
monitor SARS-CoV-2 Mpro [4,14,15]
Both internally quenched
.
substrates share a P4-to-P4’ consensus sequence.
We designed a second type of fluorogenic substrate
containing a C-terminal 7-amino-4-methylcoumarin (AMC)
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Tle-Leu-Gln-AMC was not observed. Addition of Mpro to the
lysate resulted in a pronounced Boc-Abu-Tle-Leu-Gln-AMC
and Dabcyl-EDANS cleavage only. At this stage, we consid-
ered Boc-Abu-Tle-Leu-Gln-AMC suitable to monitor Mpro
activity for our HTS campaign on the search for inhibitors of
this promising anti-SARS-CoV-2 target. Moreover, it can be
expected to be an adequate substrate for measuring Mpro
activity in a cellular environment, superior to the current,
less selective standard substrate. The kinetic parameters of
Mpro inhibition by selected inhibitors identified with Boc-
Abu-Tle-Leu-Gln-AMC were found to be comparable to
those from experiments with Dabcyl-EDANS. Two reported
inhibitors of Mpro, that is, disulfiram and ebselen, were initially
investigated, and enzyme inhibition was confirmed (Ta-
ble S2). We obtained similar IC50 values for disulfiram and
somewhat higher ones for ebselen in comparison to the study
of Jin et al., performed under different assay conditions.[14]
The first two in-house libraries (Table 1) were compiled
with respect to a potential electrophilic reactivity towards the
active-site cysteine nucleophile. Chloroacetamide derivatives
(e.g. 1 and 2, Figure 2) were identified as irreversible Mpro
inhibitors, and the second-order rate constants of inactivation,
kinac/Ki, were determined to assess their potency (Table S1).
Some Michael acceptors of the second library were weak
irreversible Mpro inhibitors (Table S1), but the peptidic vinyl
sulfones and acrylates of this library, known to inhibit other
cysteine proteases,[18] were inactive. The third library was
composed of compounds with a primary carboxamide, lactam,
or imide moiety turning them into potential glutamine
mimetics. However, hit compounds were not identified in
this series.
Figure 2. Selected, confirmed SARS-CoV-2 Mpro inhibitors identified by
HTS. Compounds 1 and 2 were identified in the library of in-house
chloroalkyl derivatives (entry 1 in Table 1), compound 9 in the Patho-
gen Box library (entry 6 in Table 1), and compound 10 in the virtually
generated library (entry 8 in Table 1).
inhibitors of human cysteine cathepsins,[20] as activity-based
probes for organelle-specific delivery to lysosomal targets,
and as PET-imaging agents for tumor-associated cathepsin
activity.[21]
Since none of the azadipeptide nitriles contained the
glutamine side chain and considering the respective primary
specificity of SARS-CoV-2 Mpro, we designed two peptidomi-
metics with this structural feature (Scheme 1). The synthetic
route to compounds 3 and 4 involved the preparation of Cbz-
protected leucine- and phenylalanine-derived hydrazides with
methylated internal nitrogen to avoid heterocyclization of the
envisaged products.[20] The precursor for the glutamine side
chain was introduced as dimethyl acetal whose primary
carboxamide moiety was generated from the corresponding
methyl ester. Both fragments underwent condensation under
acidic conditions. The resulting hydrazones were successfully
hydrogenated with the dimethylamine–borane complex.[22]
The following conversion with cyanogen bromide yielded
the final azadipeptide nitriles 3 and 4 with aza-glutamine in P1
and leucine or phenylalanine in P2 position, comprising the
first examples of this chemotype with a functionalized P1 side
chain.
The majority of the 186 members of the fourth library
(Table 1) were peptide nitriles with a cyano group at the a-
carbon of the P1 amino acid, in place of the scissile peptide
bond, which constitute prototypic inhibitors for cysteine
proteases.[19] We discovered several hits in this library, mainly
azapeptide nitriles, in which the cyano group was connected
to a nitrogen replacing the P1 CaH unit (Table S1). The
chemotype of azadipeptide nitriles had emerged as efficient
Table 1: Focused libraries for small-molecule Mpro inhibitors[a]
Similar to the well-established dipeptide nitriles, azadi-
peptide nitriles undergo a covalent interaction with the target
proteases by forming stabilized isothiosemicarbazide ad-
ducts.[20] Accordingly, 3 and 4 showed time-dependent inhib-
ition (Figure 3) and were analyzed as pseudo-irreversible
inhibitors of Mpro whose second-order rate constants of
inactivation kinac/Ki are listed in Table 2 (see also Table S1).
Both peptidic inhibitors were then structurally extended by
the successive introduction of one or two amino acids as
additional substrate specificity features of Mpro. Hence, we
employed l-tert-leucine and l-2-aminobutyric acid as P3 and
P4 building blocks (Scheme 1). The subsequent dimethyl-
amine-borane-promoted reduction and the final electrophilic
cyanation yielded 5 and 6, as well as 7 and 8, the first
azatripeptide and azatetrapeptide nitriles described so far.
Compounds 6, 7, and 8 exhibited strong inhibitory potency
against SARS-CoV-2 Mpro with kinac/Ki values of more than
20000 mÀ1 sÀ1 (Table 2, see also Figure S9). Phenylalanine in
library
c[b]
[mm]
number of
test compds
number
of hits[c]
in-house chloroalkyl derivatives
in-house Michael acceptors
in-house glutamine analogs
in-house carbonitriles
natural product library
Pathogen Box
50
50
50
50
10
50
10
10
29
69
33
186
143
400
78
7
5
0
17
2
2
in-house indoles
virtually generated library
0
1
140
[a] Residual activity of Mpro was measured with 50 mm Boc-Abu-Tle-Leu-
Gln-AMC in 50 mm 3-(N-morpholino)propanesulfonic acid (MOPS)
buffer, pH 7.2, 4% DMSO at 378C. Reactions were monitored
(lex =360 nm, lem =460 nm) for 10 min and the progress curves were
analyzed by linear regression. [b] Test concentration depended on
compound solubilities and expected potencies. [c] Compounds which
showed >50% inhibition at the indicated concentration in duplicate
measurements were considered as hits.
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Figure 3. Mpro-catalyzed hydrolysis of 50 mm (=1.03”Km) of Boc-Abu-
Tle-Leu-Gln-AMC in the absence (black) or presence of increasing
concentrations of inhibitor 4 (from top to bottom: 0.6 mm, 1.2 mm,
1.8 mm, 2.4 mm, 3.0 mm). Inset: A plot of first-order rate constants
versus the inhibitor concentrations and non-linear regression gave
a kinac/Ki value of 1150 mÀ1 sÀ1
.
Table 2: Mpro inhibition by azapeptide nitriles with aza-glutamine in P1
position[a]
compd
P4
P3
P2
kinac/Ki
[mÀ1 sÀ1
]
3
4
5
6
7
8
–
–
–
–
Cbz-Abu
Cbz-Abu
–
–
Cbz-Tle
Cbz-Tle
Tle
Cbz-Leu
Cbz-Phe
Leu
Phe
Leu
489
1150
2060
36000
21400
37500
Tle
Phe
[a] Progress curves in the presence of five different inhibitor concen-
trations and 50 mm Boc-Abu-Tle-Leu-Gln-AMC in 50 mm MOPS buffer,
pH 7.2, 4% DMSO at 378C were monitored for 60 min and analyzed by
non-linear regression using the equation [P]=vi ”(1Àexp(Àkobs ”t)/
k
obs +d. Values kinac/Ki were determined by non-linear regression using
the equation kobs =(kinac ”[I])/([I]+Ki ”(1+[S]/Km)). Deviation of each
data point from the calculated non-linear regression was less than 10%.
Out of a natural product library of 143 members, two
compounds were initially identified as hits (Table S1), but
could not be confirmed by subsequent concentration-depen-
dent inhibition curves. Testing of the Pathogen Box (a
collection of anti-Malaria agents)[24] yielded an Mpro inhibiting
bis-benzguanidine (compound 9, Figure 2). Moreover, we
performed a virtual screening (for details, see Supporting
Information) of our PharmaCenter Bonn compound library,
which provided the active 1H-imidazo[1,2-f]purine derivative
10 as a single hit (Figure 2). In contrast, preparation and
structural optimization of reported SARS-CoV-1 Mpro inhib-
itors was highly successful, highlighted by the class of pyridyl
indole esters.[25]
Such esters preferentially contained a chloro-substituted
pyridinolic group and were also active against the related
hepatitis A virus 3C and human rhinovirus 3C proteinases.[26]
This structural feature was a prerequisite for SARS-CoV-
2 Mpro inhibition, since a library of different indoles[27] did not
offer any hit (Table 1). To explore the chemical space of
indole esters, a positional scanning of the ester substituent at
Scheme 1. Synthesis of the azapeptide nitriles 3–8.
P2 was more advantageous than leucine, explicitly in case of
the azatripeptides (5 versus 6). The two azatetrapeptide
nitriles 7 and 8 showed similar Ki values (23.5 nm and
24.0 nm), but 8 had a faster inactivation step (Table S1).
The structural expansion (3 and 4 versus 5 and 6 versus 7
and 8) resulted in improved biological activity confirming that
such compounds gain their potency not only from covalent
binding,[23] but also from specific noncovalent interactions of
the P1–P4 residues with the active-site subsites. To further
rationalize the impressive activity of
8
(kinac/Ki =
37500 mÀ1 sÀ1, Ki = 24.0 nm), we performed a covalent docking
using the X-ray crystal structure of the SARS-CoV-2 Mpro in
complex with a peptidic acrylate inhibitor (Figures 4 and
S15).[14]
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The replacement of chlorine by bromine led to a further
improvement (13, Y= Cl versus 18, Y= Br). Strikingly, the
introduction of a chloro substituent at position 5 (compare 11,
R5 = H versus 17, R5 = Cl) provided the most potent inhibitor
of this series, the new indole ester 17 (kinac/Ki = 29100 mÀ1 sÀ1,
Ki = 10.0 nm). The observed time-dependent inhibition indi-
cated that these indole esters act by acylation of the active-site
cysteine (see also Figure S16), in accordance with literature
data.[25,26] While the majority of indole esters (11–19), could
be characterized as efficient pseudo-irreversible inhibitors,
some (Table S1) showed enzyme reactivation within a period
of one hour (e.g. Figure S11) due to hydrolytic cleavage of the
acyl-enzyme formed. Bioisosteric replacement of the indole
moiety (X = NH, Table 3) by a benzofuran ring system (X =
O, Table 3) in 20 led to an inhibitor with high affinity (IC50 =
5.41 nm) but pronounced Mpro reactivation. We analyzed the
structure–activity relationships of irreversibly acting indole
esters with the same 5-chloropyridin-3-olate leaving group
(X = NH, Y= Cl; 11–17, 19). Their inhibitory potency did not
correlate with the susceptibility to nucleophilic attack, which
could be derived from the acidity of the corresponding acids
(Figure S12).[28] This finding clearly revealed the importance
of specific protease–inhibitor interactions for Mpro inhibition
by indole esters.
Figure 4. Predicted binding pose of the azapeptide nitrile 8 (orange) in
the active site of SARS-CoV-2 Mpro with relevant amino acids (green).
The model was obtained based on a reported enzyme–inhibitor
complex (PDB ID: 6LU7).[14] The covalent bond between the sulfur of
the active site Cys145 and the cyano carbon of the warhead generated
an isothiosemicarbazide-type enzyme-inhibitor adduct. The P1 gluta-
mine side chain resides in the S1 pocket. The aromatic ring of the P2
phenylalanine is positioned in the hydrophobic S2 pocket (His41,
Met49, Met165) in proximity to the Gln189 side chain. The P3 tert-
leucine is oriented towards the solvent. The P4 aminobutyric acid and
the N-terminal, Cbz-capped part of the inhibitor are oriented towards
the S3/S4 region. Hydrogen bond interactions are shown in yellow
dotted lines, for details see Figure S15. The binding mode of 8 is
consonant with X-ray crystal structures of other peptidic inhibitors in
The potent pyridyl indolecarboxylates with their molec-
ular weight of less than 300 gmolÀ1 can even be regarded as
fragments. Nevertheless, some of them are amazingly potent
Mpro inhibitors with IC50 values in the low nanomolar
concentration range (Table S1) along with kinac/Ki values of
up to almost 30000 mÀ1 sÀ1. Thus, these compounds have high
ligand efficiency, which makes them excellent scaffolds for
drug development. Very recently, the indole ester 13, which
displayed intermediate Mpro inhibitory potency, and the
related 5-chloropyridin-3-yl dihydroindole-4-carboxylate
were demonstrated to be active against the infectivity and
replication of SARS-CoV-2 in Vero E6 cells,[29] indicating the
high therapeutic potential of this class of compounds.
Selected members of both classes of extraordinarily
potent Mpro inhibitors, azapeptide nitriles 4, 6, and 8, and
halopyridyl esters 16, 17, and 20, were additionally
complex with Mpro [4,10,14]
.
the indole core was conducted (Table 3). For this purpose, the
complete set of 1H-indolecarboxylic acids was converted by
uronium salt-mediated reactions with halo-substituted
pyridin-3-ols to yield 11–18 (Schemes S3 and S4). These
esters were kinetically analyzed as irreversible inhibitors of
Mpro (Table 3; see also Table S1 and Figure S10). All the
isomeric esters 11–16 exhibited strong Mpro inhibitory activity;
their second-order rate constants of inactivation differed by
one order of magnitude, and the ester moiety at position 7
(compound 16) resulted in the strongest Mpro inactivation.
investigated for their potency in Mpro-containing cell
Table 3: Mpro inhibition by 5-halopyridin-3-yl 1H-indole-carboxylates[a,b]
lysates. In fact, all of the investigated inhibitors
displayed similarly high Mpro inhibition in HEK and
human lung epithelial A549 cell lysates as in buffer
containing the purified enzyme, showing virtually
complete inhibition at 1 mm (6, 8, 16, 17, 20) or at
compd
X
R2
R3
R4
R5
R6
R7
Y
kinac/Ki
10 mm (4), respectively (Figure S14). These results
[mÀ1 sÀ1
]
indicate that the new Mpro inhibitors retain full
activity in a cellular context.
11
12
13
14
15
16
17
18
19
20
NH
NH
NH
NH
NH
NH
NH
NH
NH
O
R
H
H
H
H
H
R
H
R
H
H
R
H
H
H
H
R
H
H
H
R
H
H
Cl
H
R
H
H
H
H
R
H
H
H
H
H
H
H
H
H
H
R
H
H
H
H
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Br
Cl
Cl
7600
6090
14800
3200
For cell entry, coronaviral spike proteins bind to
cellular receptors and are primed by host cell
proteases. Cathepsin L, a human lysosomal cysteine
protease, contributes to the proteolytic activation of
the SARS-CoV-2 spike protein in the endosomal–
lysosomal compartment.[30] Hence, it was suggested
to develop dual inhibitors which target the viral
protease and the host cathepsin L.[11] We therefore
investigated the potential of selected compounds as
dual targeting inhibitors (Table S3). While the indole
H
H
H
H
H
H
4400
20200
29100
24000
5620
H
-(CH2)4-
R
H
H
H
H
n.d.[c]
[a] Syntheses are described in Supporting Information. [b] For kinetic analysis, see
Table 2. [c] n.d.: not determined. IC50 =5.41 nm.
Angew. Chem. Int. Ed. 2021, 60, 10423 –10429 ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org 10427

Angewandte
Research Articles
Chemie
Cen, S. Hu, X. Yang, J. Wang, X. Liu, G. Xiao, H. Jiang, Z. Rao,
L. K. Zhang, Y. Xu, H. Yang, H. Liu, Science 2020, 368, 1331 –
1335.
esters (e.g. 13 and 17) did not inhibit cathepsin L (and the
related enzyme cathepsin B) indicating selectivity of this
chemotype for viral cysteine proteases, our azapeptide nitriles
6, 7, and 8 exhibited strong anti-cathepsin L activity with Ki
values of 53 nm, 22 nm and 120 nm, respectively, (Table S3),
thus turning them into particularly interesting cysteine
protease inhibitors, acting at different stages of virus replica-
tion.
[5] a) T. Pillaiyar, M. Manickam, V. Namasivayam, Y. Hayashi, S. H.
Jung, J. Med. Chem. 2016, 59, 6595 –6628; b) M. Gehringer, S. A.
Laufer, J. Med. Chem. 2019, 62, 5673 –5724.
[6] T. Pillaiyar, S. Meenakshisundaram, M. Manickam, Drug Dis-
covery Today 2020, 25, 668 –688.
[7] L. Zhang, D. Lin, Y. Kusov, Y. Nian, Q. Ma, J. Wang, A.
von Brunn, P. Leyssen, K. Lanko, J. Neyts, A. de Wilde, E. J.
Snijder, H. Liu, R. Hilgenfeld, J. Med. Chem. 2020, 63, 4562 –
4578.
[8] a) C. Capasso, A. Nocentini, C. T. Supuran, Expert Opin. Ther.
Pat. 2020, https://doi.org/10.1080/13543776.2021.1857726; b) A.
Welker, C. Kersten, C. Müller, R. Madhugiri, C. Zimmer, P.
Müller, R. Zimmermann, S. Hammerschmidt, H. Maus, J.
Ziebuhr, C. Sotriffer, T. Schirmeister, ChemMedChem 2021,
16, 340 –354; c) A. I. Petushkova, A. A. Zamyatnin, Jr., Phar-
maceuticals 2020, 13, 277.
[9] https://www.pharmchem1.uni-bonn.de/www-en/pharmchem1-
en/mueller-laboratory/compound-library.
[10] C. Ma, M. D. Sacco, B. Hurst, J. A. Townsend, Y. Hu, T. Szeto, X.
Zhang, B. Tarbet, M. T. Marty, Y. Chen, J. Wang, Cell Res. 2020,
30, 678 –692.
[11] M. D. Sacco, C. Ma, P. Lagarias, A. Gao, J. A. Townsend, X.
Meng, P. Dube, X. Zhang, Y. Hu, N. Kitamura, B. Hurst, B.
Tarbet, M. T. Marty, A. Kolocouris, Y. Xiang, Y. Chen, J. Wang,
Sci. Adv. 2020, 6, eabe0751.
[12] W. Zhu, M. Xu, C. Z. Chen, H. Guo, M. Shen, X. Hu, P. Shinn, C.
Klumpp-Thomas, S. G. Michael, W. Zheng, ACS Pharmacol.
Transl. Sci. 2020, 3, 1008 –1016.
Conclusion
Since SARS-CoV-2 Mpro plays an irreplaceable role in the
life cycle of the virus, its inhibition by low-molecular weight
drug candidates has been recognized as a promising strategy
against COVID-19. We developed a new, readily available
specific substrate that is directly applicable for HTS and can
even be used in cell lysates to test for Mpro activity. This report
highlights the successful discovery of highly potent Mpro
inhibitors out of several focused libraries. Based on the
identification of appropriate substructures, two types of
molecules have been assembled, peptidomimetic azatripep-
tide and azatetrapeptide nitriles as well as non-peptidic
halopyridinyl 1H-indole-carboxylates. These rationally de-
signed inhibitors are promising candidates for further devel-
opment with the ultimate goal of attaining antiviral drugs
against COVID-19.
[13] a) R. L. Hoffman, R. S. Kania, M. A. Brothers, J. F. Davies,
R. A. Ferre, K. S. Gajiwala, M. He, R. J. Hogan, K. Kozminski,
L. Y. Li, J. W. Lockner, J. Lou, M. T. Marra, L. J. Mitchell, Jr.,
B. W. Murray, J. A. Nieman, S. Noell, S. P. Planken, T. Rowe, K.
Ryan, G. J. Smith 3rd, J. E. Solowiej, C. M. Steppan, B. Taggart,
J. Med. Chem. 2020, 63, 12725 –12747; b) M. M. Ghahreman-
pour, J. Tirado-Rives, M. Deshmukh, J. A. Ippolito, C. H. Zhang,
I. Cabeza de Vaca, M. E. Liosi, K. S. Anderson, W. L. Jorgensen,
ACS Med. Chem. Lett. 2020, 11, 2526 –2533.
[14] Z. Jin, X. Du, Y. Xu, Y. Deng, M. Liu, Y. Zhao, B. Zhang, X. Li,
L. Zhang, C. Peng, Y. Duan, J. Yu, L. Wang, K. Yang, F. Liu, R.
Jiang, X. Yang, T. You, X. Liu, X. Yang, F. Bai, H. Liu, X. Liu,
L. W. Guddat, W. Xu, G. Xiao, C. Qin, Z. Shi, H. Jiang, Z. Rao,
H. Yang, Nature 2020, 582, 289 –293.
Acknowledgements
The authors are grateful to the BMBF for funding our
graduate research school BIGS DrugS, and to Medicines for
Malaria Venture (MMV) for providing the Pathogen Box for
screening. Open access funding enabled and organized by
Projekt DEAL.
Conflict of interest
[15] a) C. Coelho, G. Gallo, C. B. Campos, L. Hardy, M. Würtele,
PLoS One 2020, 15, e0240079; b) Z. Li, X. Li, Y. Y. Huang, Y.
Wu, R. Liu, L. Zhou, Y. Lin, D. Wu, L. Zhang, H. Liu, X. Xu, K.
Yu, Y. Zhang, J. Cui, C. G. Zhan, X. Wang, Hai-B. Luo, Proc.
Natl. Acad. Sci. USA 2020, 117, 27381 –27387.
[16] W. Rut, K. Groborz, L. Zhang, X. Sun, M. Zmudzinski, B.
Pawlik, X. Wang, D. Jochmans, J. Neyts, W. Młynarski, R.
Hilgenfeld, M. Drag, Nat. Chem. Biol. 2021, 17, 222 –228.
[17] M. A. T. van de Plassche, M. Barniol-Xicota, S. H. L. Verhelst,
ChemBioChem 2020, 21, 3383 –3388.
[18] a) C. Breuer, C. Lemke, J. Schmitz, U. Bartz, M. Gütschow,
Bioorg. Med. Chem. Lett. 2018, 28, 2008 –2012; b) M. D.
Mertens, J. Schmitz, M. Horn, N. Furtmann, J. Bajorath, M.
Mares, M. Gütschow, ChemBioChem 2014, 15, 955 –959.
[19] a) M. Frizler, M. Stirnberg, M. T. Sisay, M. Gütschow, Curr. Top.
Med. Chem. 2010, 10, 294 –322; b) L. Cianni, C. W. Feldmann, E.
Gilberg, M. Gütschow, L. Juliano, A. Leit¼o, J. Bajorath, C. A.
Montanari, J. Med. Chem. 2019, 62, 10497 –10525.
The authors declare no conflict of interest.
Keywords: azapeptide nitriles ·fluorogenic substrates ·
high throughput screening ·pyridinyl 1H-indole-carboxylates ·
SARS-CoV-2 main protease
[1] a) Y. Chen, Q. Liu, D. Guo, J. Med. Virol. 2020, 92, 418 –423;
b) A. E. Gorbalenya, S. C. Baker, R. S. Baric, R. J. de Groot, C.
Drosten, A. A. Gulyaeva, B. L. Haagmans, C. Lauber, A. M.
Leontovich, B. W. Neuman, D. Penzar, S. Perlman, L. L. Poon,
D. V. Samborskiy, I. A. Sidorov, I. Sola, J. Ziebuhr, Nat. Micro-
biol. 2020, 5, 536 –544.
[2] T. J. El-Baba, C. A. Lutomski, A. L. Kantsadi, T. R. Malla, T.
John, V. Mikhailov, J. R. Bolla, C. J. Schofield, N. Zitzmann, I.
Vakonakis, C. V. Robinson, Angew. Chem. Int. Ed. 2020, 59,
23544 –23548; Angew. Chem. 2020, 132, 23750 –23754.
[20] a) R. Lçser, M. Frizler, K. Schilling, M. Gütschow, Angew.
Chem. Int. Ed. 2008, 47, 4331 –4334; Angew. Chem. 2008, 120,
4403 –4406; b) M. Frizler, F. Lohr, M. Lülsdorff, M. Gütschow,
Chem. Eur. J. 2011, 17, 11419 –11423; c) M. Frizler, J. Schmitz,
[3] L. Zhang, D. Lin, X. Sun, U. Curth, C. Drosten, L. Sauerhering,
S. Becker, K. Rox, R. Hilgenfeld, Science 2020, 368, 409 –412.
[4] W. Dai, B. Zhang, X. M. Jiang, H. Su, J. Li, Y. Zhao, X. Xie, Z.
Jin, J. Peng, F. Liu, C. Li, Y. Li, F. Bai, H. Wang, X. Cheng, X.
10428 www.angewandte.org ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH Angew. Chem. Int. Ed. 2021, 60, 10423 –10429

Angewandte
Research Articles
Chemie
A. C. Schulz-Fincke, M. Gütschow, J. Med. Chem. 2012, 55,
Kostenis, Q. Zhao, M. Kçse, V. Namasivayam, C. E. Müller, J.
5982 –5986.
Med. Chem. 2018, 61, 8136 –8154.
[28] S. L. Keenan, K. P. Peterson, K. Peterson, K. Jacobson, J. Chem.
Educ. 2008, 85, 558 –560.
[21] a) P. Y. Yang, M. Wang, L. Li, H. Wu, C. Y. He, S. Q. Yao, Chem.
Eur. J. 2012, 18, 6528 –6541; b) Y. Loh, H. Shi, M. Hu, S. Q. Yao,
Chem. Commun. 2010, 46, 8407 –8409; c) R. Lçser, R. Berg-
mann, M. Frizler, B. Mosch, L. Dombrowski, M. Kuchar, J.
Steinbach, M. Gütschow, J. Pietzsch, ChemMedChem 2013, 8,
1330 –1344.
[22] M. E. Casarini, F. Ghelfi, E. Libertini, U. M. Pagnoni, A. F.
Parsons, Tetrahedron 2002, 58, 7925 –7932.
[23] M. Frizler, F. Lohr, N. Furtmann, J. Kläs, M. Gütschow, J. Med.
Chem. 2011, 54, 396 –400.
[24] S. Duffy, M. L. Sykes, A. J. Jones, T. B. Shelper, M. Simpson, R.
Lang, S. A. Poulsen, B. E. Sleebs, V. M. Avery, Antimicrob.
Agents Chemother. 2017, 61, e00379-17.
[25] a) J. Zhang, H. I. Pettersson, C. Huitema, C. Niu, J. Yin, M. N.
James, L. D. Eltis, J. C. Vederas, J. Med. Chem. 2007, 50, 1850 –
1864; b) A. K. Ghosh, G. Gong, V. Grum-Tokars, D. C. Mul-
hearn, S. C. Baker, M. Coughlin, B. S. Prabhakar, K. Sleeman,
M. E. Johnson, A. D. Mesecar, Bioorg. Med. Chem. Lett. 2008,
18, 5684 –5688.
[26] a) C. Huitema, J. Zhang, J. Yin, M. N. James, J. C. Vederas, L. D.
Eltis, Bioorg. Med. Chem. 2008, 16, 5761 –5777; b) I. Im, E. S.
Lee, S. J. Choi, J. Y. Lee, Y. C. Kim, Bioorg. Med. Chem. Lett.
2009, 19, 3632 –3636.
[29] a) S. I. Hattori, N. Higshi-Kuwata, J. Raghavaiah, D. Das, H.
Bulut, D. A. Davis, Y. Takamatsu, K. Matsuda, N. Takamune, N.
Kishimoto, T. Okamura, S. Misumi, R. Yarchoan, K. Maeda,
A. K. Ghosh, H. Mitsuya, mBio 2020, 11, e01833-20; b) S. I.
Hattori, N. Higashi-Kuwata, H. Hayashi, S. R. Allu, J. Ragha-
vaiah, H. Bulut, D. Das, B. J. Anson, E. K. Lendy, Y. Takamatsu,
N. Takamune, N. Kishimoto, K. Murayama, K. Hasegawa, M. Li,
D. A. Davis, E. N. Kodama, R. Yarchoan, A. Wlodawer, S.
Misumi, A. D. Mesecar, A. K. Ghosh, H. Mitsuya, Nat. Com-
mun. 2021, 12, 668.
[30] a) M. Hoffmann, H. Kleine-Weber, S. Schroeder, N. Krüger, T.
Herrler, S. Erichsen, T. S. Schiergens, G. Herrler, N. H. Wu, A.
Nitsche, M. A. Müller, C. Drosten, S. Pçhlmann, Cell 2020, 181,
271 –280; b) J. Shang, Y. Wan, C. Luo, G. Ye, Q. Geng, A.
Auerbach, F. Li, Proc. Natl. Acad. Sci. USA 2020, 117, 11727 –
11734; c) T. Liu, S. Luo, P. Libby, G. P. Shi, Pharmacol. Ther.
2020, 213, 107587; d) S. P. Alexander, J. F. Armstrong, A. P.
Davenport, J. A. Davies, E. Faccenda, S. D. Harding, F. Levi-
Schaffer, J. J. Maguire, A. J. Pawson, C. Southan, M. Spedding,
Br. J. Pharmacol. 2020, 177, 4942 –4966.
[27] a) T. Pillaiyar, E. Gorska, G. Schnakenburg, C. E. Müller, J. Org.
Chem. 2018, 83, 9902 –9913; b) Y. Baqi, T. Pillaiyar, A.
Abdelrahman, O. Kaufmann, S. Alshaibani, M. Rafehi, S.
Ghasimi, R. Akkari, K. Ritter, K. Simon, A. Spinrath, E.
Manuscript received: December 21, 2020
Revised manuscript received: February 8, 2021
Accepted manuscript online: March 3, 2021
Version of record online: March 24, 2021
Angew. Chem. Int. Ed. 2021, 60, 10423 –10429 ꢀ 2021 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH www.angewandte.org 10429