Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies

Article abstract of DOI:10.1016/j.ejmech.2021.113584

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (M^pro) to cleave viral proteins. Consequently, M^pro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of M^pro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-M^pro complexes reveal that an alternative binding pocket in M^pro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na⁺ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for M^pro inhibitor design.

Full text of DOI:10.1016/j.ejmech.2021.113584

European Journal of Medicinal Chemistry 222 (2021) 113584
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug
GC376: Structural enhancements, increased solubility, and micellar
studies
,
Wayne Vuong a, Conrad Fischer a 1, Muhammad Bashir Khan b, Marco J. van Belkum a,
,
, d
Tess Lamer a, Kurtis D. Willoughby a, Jimmy Lu b d, Elena Arutyunova b
,
Michael A. Joyce c d, Holly A. Saffran c d, Justin A. Shields c d, Howard S. Young b,
,
,
,
James A. Nieman c e, D. Lorne Tyrrell c d, M. Joanne Lemieux b d, John C. Vederas a
,
,
,
, *
a Department of Chemistry, University of Alberta, Edmonton AB, T6G 2G2, Canada
b Department of Biochemistry, Membrane Protein Disease Research Group, University of Alberta, Edmonton AB, T6G 2R3, Canada
c Department of Medical Microbiology and Immunology, University of Alberta, Edmonton AB, T6G 2R3, Canada
d Li Ka Shing Institute of Virology, University of Alberta, Edmonton AB, T6G 2E1, Canada
e Li Ka Shing Applied Virology Institute, University of Alberta, Edmonton AB, T6G 2E1, Canada
a r t i c l e i n f o
a b s t r a c t
Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave
viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated
that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective in-
hibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives
with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro
complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alter-
native binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376
show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher con-
centrations, a property not previously reported. Replacement of its Naþ counter ion with choline greatly
increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for
Mpro inhibitor design.
Article history:
Received 15 March 2021
Received in revised form
3 May 2021
Accepted 22 May 2021
Available online 30 May 2021
Keywords:
COVID-19
Main protease
Protease inhibitor
Crystallography
Structure-guided design
GC376 analogs
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
SARS therapeutics was discontinued. Consequently, there is an ur-
gent need for anti-viral drug development for acute COVID-19 pa-
The end of 2019 saw the beginnings of what would quickly
become a global pandemic in the form of COVID-19 (coronavirus
disease 2019), caused by severe acute respiratory syndrome coro-
navirus 2 (SARS-CoV-2). Bearing a high degree of genetic similarity
to SARS-CoV that was responsible for an outbreak in 2002e2004
[1,2], it has developed into a widespread disease with >70 million
cases and >1.6 million deaths worldwide as of December 2020,
with numbers continuing to rise [3]. Due to the success of public
health measures in containing SARS-CoV, development of anti-
tients. Furthermore, in spite of vaccines being deployed for use,
current information regarding the duration of immunity, effec-
tiveness against mutant strains as well as possibility of reinfection
remains limited.
SARS-CoV and SARS-CoV-2 are positive strand RNA viruses that
exploit host cellular machinery to translate genetic information
into viral proteins required for replication. As part of this process,
two overlapping polyproteins are encoded: pp1a and pp1ab, which
are then cleaved into the individual active proteins necessary for
viral transcription and replication [4]. Two proteases are respon-
sible for executing these vital steps: Mpro (also called 3CLpro) and a
papain like protease (PLpro) that cleave 11 and 3 times, respectively.
The 11 proteolytic cleavage sites of Mpro are usually at conserved
Leu Gln | Ser Ala Gly sequences. Due to the critical role of this
* Corresponding author.
E-mail address: john.vederas@ualberta.ca (J.C. Vederas).
Present address: Department of Physical Sciences, Barry University, 11300 NE
1
2nd Ave., Miami Shores FL 33161, USA.
https://doi.org/10.1016/j.ejmech.2021.113584
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
enzyme in viral replication, Mpro is an attractive target for antiviral
agents [5,6]. Mpro is a cysteine protease, a class of enzymes for
which a large variety of inhibitors exist [7,8].
13C-labelled GC373 and GC376 confirm that in aqueous media the
former exists as a hydrated aldehyde and is a mixture of stereo-
isomers due to epimerization at the
a-carbon of the glutamine
Potent Mpro inhibitors may be viable drugs if certain criteria are
fulfilled, namely good bioavailability, low toxicity and ample solu-
bility [8]. As a result of these requirements, a number of seemingly
promising cysteine protease inhibitors have failed when examined
as possible drug candidates. A prominent example of this is
rupintrivir, targeted as a treatment for rhinoviruses [9]. As an un-
saturated ester Michael acceptor, it exhibited low bioavailability in
clinical trials [10], probably partly due to the irreversible nature of
the covalent bond formed upon attack by sulfur nucleophiles. This
lack of reversibility can result in permanent off-target binding to
other thiol-containing biomolecules present in mammalian cells,
leading to destruction or sequestration of the inhibitor. Addition-
ally, off-target binding to untargeted thiols could lead to toxicity or
host immune response [8]. Hence, inhibitors possessing warheads
that bind reversibly and preferentially to the target cysteine pro-
tease are desirable. Peptide aldehydes present an attractive solu-
tion as they are able to bind reversibly to thiol-containing target
molecules, forming a hemithioacetal in the process [11,12]. In
addition, they can be readily derivatized to more water-soluble
bisulfite adducts for administration as prodrugs [13]. Subsequent
reversion of the bisulfite adduct to the parent aldehyde under
physiological conditions then restores the active form of the in-
hibitor [6].
Early studies by Wolfenden and coworkers on peptide alde-
hydes as protease inhibitors [11], prompted us to examine them as
potent inhibitors of viral cysteine proteases [12]. In 2016, Kim et al.
discovered GC376 as a drug active against the feline coronavirus
(FCoV) responsible for feline infectious peritonitis (FIP) [14]. GC376
is a bisulfite adduct prodrug of the corresponding parent aldehyde,
GC373, which strongly inhibits the Mpro of FCoV. The effectiveness
of this compound was demonstrated when cats exhibiting FIP were
cured upon administration of this drug [14]. Once symptoms are
present, untreated FIP has close to a one hundred percent fatality
rate in cats. The utility of this drug is made more remarkable due to
the well-tolerated nature of GC376. Further studies have demon-
strated its activity against the Mpro of a number of other corona-
viruses, including those of minks and ferrets [14e16]. Recently, we
showed that GC376 and GC373 were also potent inhibitors of the
SARS-CoV-2 Mpro and that these drugs were able to block virus
replication in cell culture, indicating that they are good candidates
as antivirals for the treatment of COVID-19 [6]. Independently,
different groups published similar observations [17e22], with
further follow-up studies subsequently demonstrating efficacy in
transgenic mouse models, strongly supporting the translational
potential of GC376 and related analogs for human use [23e25].
Additionally, we also experimentally demonstrated the reversible
nature of GC376 [26]. During 2020, a number of promising potent
reversible inhibitors of SARS-CoV-2 Mpro have been uncovered,
analog. The formation of bisulfite adduct GC376 leads to additional
stereoisomers. These spontaneously convert to the aldehyde, and
only a single isomer is seen bound to the SARS-CoV-2 Mpro enzyme.
It was also found that versions of GC376 with alternative cations led
to an increase in drug solubility, potentially allowing for smaller
volumes during drug administration. Finally, solubility testing and
NMR experiments demonstrated the formation of colloidal aggre-
gates by GC376, which may enable administration of small volumes
of highly concentrated drug.
2. Results
2.1. Structure guided development of Mpro inhibitors using
independent variations in P2 and P3
Development of improved inhibitors began with sampling var-
iations in the P2 and P3 sites of GC376 in order to assess which
types of combinations would facilitate the greatest increase in
binding to Mpro (Fig. 1). The cyclic glutamine analog in the P1 site of
the inhibitor was maintained due to the numerous favorable in-
teractions of this residue observed with the S1 pocket of Mpro, as
determined from our previous study [6]. The aldehyde warhead
was also conserved due to the ability to easily and favorably form
the bisulfite prodrug in a single step, combined with the proven
efficacy of this group, as was shown with GC376 to treat FIP in fe-
lines [14].
Based on our previous crystal structures, the S2 pocket of SARS-
CoV-2 Mpro responsible for binding of the leucine moiety in GC376
was largely hydrophobic [6], and so lipophilic variants of the
leucine isopropyl group in the P2 site of the inhibitor were chosen
such as cyclopropyl (1a), cyclohexyl (1b), and phenyl (1c) (Table 1).
Replacement with a phenyl group (1c) yielded only moderate im-
provements in IC50 values using a FRET substrate-enzyme kinetic
assay, while the greatest improvement was found when a cyclo-
propyl group (1a) was utilized. Replacement with a cyclohexyl
group (1b) led to a decrease in performance of the resultant in-
hibitor, as indicated by increased IC50 values (Table 1). These con-
clusions were further verified via co-crystallization of these
inhibitors with Mpro and the observation that the cyclopropyl group
of 1a was able to insert deeper into the S2 site of the protease,
which may explain the increase in binding affinity (Fig. 2 and
Fig. 3A).
Variations in the P3 site of the inhibitor were also made based
on our previous work on co-crystallization of Mpro with the parent
aldehyde GC373 [6]. We noticed that the location in Mpro at which
the carboxybenzyl (Cbz) group binds (designated as S3) may not be
the optimal position. A deeper pocket (designated as S4) can be
observed in the vicinity of the Cbz group that may better accom-
modate the P3 position of the inhibitor, contrasting S3 which ap-
pears as a small concave depression pointing towards the surface of
Mpro (Fig. 2). Therefore, we based further modifications of the P3
position of the inhibitor on two factors: enhancing favorable dipole
interactions and inducing strategic conformations of the bound
inhibitor.
In order to make improvements at the P3 position based on
enhancing dipole interactions, we synthesized a number of struc-
tural variants containing polar groups at the benzyl ring of the Cbz
group. One variant included a 3-fluorobenzyl group (1d) to evaluate
the possibility of H-bond formation. Another replaced the benzyl
group with a 3-chlorophenylethyl group (1e) to evaluate the effect
of increased length and flexibility via the introduction of an addi-
including
a-ketoamides [2], and a-hydroxymethyl and a-acylox-
ymethylketones [27].
We now report efforts to increase the effectiveness of GC376
analogs against SARS-CoV-2. Improvements by modification of the
chemical structure of GC376 resulted in a number of compounds
with improved binding characteristics and nanomolar inhibition of
SARS-CoV-2 Mpro. The structure-activity relationship (SAR) study
was accomplished with systematic optimization of inhibitor
structures via detailed assessments of these new compounds co-
crystallized with SARS-CoV-2 Mpro and subsequent in vitro testing
with recombinant protease as well as with mammalian cell lines.
Furthermore, co-crystallization studies revealed an alternative
mode of binding for these new inhibitors compared to GC376,
facilitating a better fit into the active site of Mpro. NMR studies with
tional methylene. Selection of
a 4-methoxyindole (1f) for
2

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
Fig. 1. Developmental workflow of SARS-CoV-2 Mpro inhibitors. The process of inhibitor design and modification carried out in this publication is depicted. Evaluation of in-
hibitors was carried out at each step by FRET studies, crystallography, and viral assays.
evaluation was based on previously reported high affinity of this
functionality in binding to SARS-CoV Mpro [28]. The second
parameter, inducing advantageous conformations, was evaluated
via stereospecific installation of a methyl group (1g and 1h) at the
methylene position of the Cbz group in the parent compound. This
methyl group may be able to induce an insertion of the phenyl
group into the nearby S4 position of Mpro. FRET substrate-enzyme
kinetic assays showed that all of these modifications at the P3
position lead to improved IC50 values over GC373, indicating that
both of the aforementioned factors played a role in the binding and
interactions found at that position (Table 1). The greatest increases
in binding affinity resulted from modification of the benzyl group to
a 4-methoxyindole variant (1f), followed by a 3-fluorobenzyl (1d)
and 3-chlorophenylethyl variant (1e). These were then followed by
installation of a methyl group, with the (S) absolute configuration
(1g) outperforming the (R) variant (1h). As noted in Table 1, 1h does
not exhibit substantially better IC50 values than the parent com-
pound, providing evidence that the absolute stereochemistry at this
position plays a modest role in the degree of binding improvement.
Taken together, it appears that while both electronic and steric
factors play a role, dipole-dipole interactions have a larger effect on
improvements in IC50 than that of initial conformation.
2.2. Combination of most potent P2/P3 modifications
After identifying a number of variations in the P2 and P3 site
that lead to improved inhibitor binding to Mpro, we combined these
modifications to synthesize analogs with potentially improved
properties. The optimal modification for the P2 site of the inhibitor
was a cyclopropyl moiety, and we therefore combined this with
variations at the P3 site. Four additional analogs were obtained (1i,
1j, 1k, and 1l), as documented in Table 2 and Fig. S1. Surprisingly, it
appeared that there was little or no improvement in IC50 values
with these analogs as compared to their singly modified variants
(1d, 1e, 1f, 1g, Table 1).
In order to improve selectivity, we further transformed a num-
ber of these analogs into the bisulfite adduct prodrug versions. In
our previous work we found that transformation of the parent
aldehyde to the bisulfite adduct resulted in improved IC50 and EC50
values [6], likely a result of increased hydrophilicity and generally
improved solubility. The most potent analog from the singular P2
and P3 rounds of modification (1a and 1f) as well as the four
combination analogs (1id1l) were transformed to the sodium
bisulfite adducts in order to see whether this improved binding
would take place in this instance as well (Table 3). Interestingly,
singularly modified compounds 1a and 1f displayed slightly
3

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
Table 1
Singly modified aldehyde derivatives of GC373. GC373 and aldehyde derivative structures containing single modifications at either the P2 (1ad1c) or P3 (1dd1h) positions
are shown, along with SARS-CoV-2 Mpro IC50 and SARS-CoV-2 antiviral EC50 values, both with and without co-administration of efflux inhibitor CP-100356 (þCP). Cytotoxicity
assays showed CC50 values of >200 mM for all compounds shown. ND indicates that value was not determined.
Entry
Structure
IC50
(
mM)
EC50
(
mM)
EC50
(
m
M) þCP
GC373 [6]
0.40 0.05
0.05 0.01
0.61 0.15
1.5 0.3
1.1 0.1
1.2 0.4
0.32 0.05
0.32 0.09
ND
1a (PDB 7LCS)
1b
1c
0.23 0.10
>10
ND
1d
1e
0.13 0.04
0.15 0.05
1.4 0.5
ND
ND
1.47 0.8
1f (PDB 7LDL)
1g (PDB 7LCS)
1h
0.06 0.01
0.27 0.09
0.35 0.11
0.9 0.1
1.1 0.5
1.4 0.7
0.25 0.01
ND
ND
increased IC50 values upon transformation to bisulfite adducts 2a
and 2b. However, doubly modified compounds 2c, 2d, 2e, and 2f
(Table 3, Fig. S2) showed the expected decreases in IC50 values as
compared to the parent aldehydes (1i, 1j, 1k, and 1l, Table 2). As
such, it appears that in many cases, bisulfite adducts of the parent
aldehydes indeed lead to improved IC50 values. Of note is that each
combination analog as a bisulfite adduct displayed a higher binding
affinity for Mpro compared to GC376, ranging from a 2.5e5.0-fold
improvement. Testing of variants of GC376 in which the Naþ
cation was replaced with a Kþ cation (3a) or choline (3b) was also
conducted, showing very similar IC50 values to that of the parent
Na þ form (Table 3, Fig. S3), and demonstrating retained effective-
ness combined with increased aqueous solubility (discussed
below).
2.3. Structural analysis by X-Ray crystallography
Fig. 2. Binding sites of SARS-CoV-2 Mpro catalytic cleft. The active cysteine-145
residue (C145, yellow) as well as side-pockets S1’, S1, S2, S4 and surface depression
S3 are highlighted.
In order to better understand interactions of our new inhibitors
with the Mpro active site, crystal structures were concurrently
4

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
Fig. 3. Crystal structures and schematic depictions of inhibitors bound to SARS-CoV-2 Mpro. (A) 1a bound to the active site of SARS-CoV-2 Mpro. The benzyl ring at the P3
position of the inhibitor can be seen interacting with the S3 surface depression, similarly to the binding mode of GC373 (PDB 7LCS). (B) 1i bound to the active site of SARS-CoV-2
Mpro. The presence of a fluorine atom at the 3-position on the benzyl ring directs the aromatic moiety into the S4 binding pocket (PDB 7LCO). (C) 1f bound to the active site of SARS-
5

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
obtained throughout the inhibitor development process (Table S1).
As previously demonstrated [6], these new structures confirm the
formation of a covalent bond between the aldehyde warhead and
the active site cysteine of Mpro (Cys145, CeS bond lengths
1.8e2.0 Å). The carbinol hydroxyl maintains favorable hydrogen
bonds with the amide NH from both Cys145 and Gly143 of the
protease with (1a, 1f, 1g) or without (1i) the coordination of an
additional water molecule (Fig. 3Ad3D). In accordance with the
parent GC373, the lactam carbonyl of the Gln surrogate forms a
strong hydrogen bond with the side chain of His163 in the S1 site in
all four structures (Fig. 3Ad3D). Meanwhile, the lactam NH is also
engaged in hydrogen bonds with the side chain oxygen of Glu166
and the backbone oxygen of Phe140, as well as interactions with
the N-terminal serine from the other Mpro protomer [26]. Similarly
to the Leu-containing analogs 1f and 1g, the cyclopropylalanine-
(1a) appearing to perform the best. Replicating this, lone P3 site
modifications also led to a number of compounds with universally
improved EC50 values, with the best performers being 1f and 1g
(Table 1). Combining the best-performing modifications at each site
in the molecule then furnished a number of doubly modified in-
hibitors (1id1l), with two (1j and 1k) demonstrating a further
increase in potency and analogous decrease in EC50 values (Table 2).
The corresponding sodium bisulfite adducts were derived from the
most potent compound from each singly modified group (2a and 2b
from 1a and 1f, respectively). While 2a exhibited comparable EC50
values to GC376, indicating similar potential as a drug candidate, 2b
displayed a distinctly higher EC50 value, hinting at possible efflux
effects (Table 3). Sodium bisulfite adducts that combined a cyclo-
propyl group at the P2 position along with P3 substitutions
(2cd2f), had lower EC50 values in two out of four cases, 2c and 2d.
Based on a recent report in the literature, it was noted that related
compounds may be susceptible to cellular efflux, leading to
increased EC50 values [30]. In order to determine if this was the
case, the EC50 values for a number of our candidates (1a, 1f, 1id1l,
and 2ad2f) were reevaluated in the presence of CP-100356, a P-gp
efflux inhibitor (Table 1, Table 2, and Table 3). When administered
together with CP-100356, all but three (1l, 2b, and 2f) of the tested
derivatives performed on par or better than the analogous parent
GC373 or GC376. Furthermore, the presence of an indole moiety
may contribute to efflux, as noted by the comparatively high initial
EC50 values of 2b and 2e coupled with a significant decrease upon
administration of CP-100356, though not to the same extent as
reported for related substances [30]. This drop was less significant
for the remaining analogs, especially for 2c and 2d (Table 3), indi-
cating that while there are some cellular efflux effects present,
these new compounds possess the advantage of not requiring the
co-administration of efflux inhibitors to be effective, thereby
potentially simplifying pharmaceutical development. The results
indicate 2c and 2d are the most promising inhibitors reported
herein (Fig. 4), exhibiting both IC50 and EC50 values that are supe-
rior to those of GC376, even in the absence of efflux inhibitors.
bearing compounds 1a and 1i (Fig. 3A and B) maintain close CeH
…
p
contacts between His41 and the cyclopropyl group, filling the
S2 pocket in a more compact fashion than the parent GC373-Mpro
structure (PDB 6WTM) where the P2 position does not extend as
deeply into the S2 binding pocket [6]. An interesting feature was
observed upon examination of the P3 substituent. In the case of a
bare Cbz group (i.e. GC373 and 1a), the P3 residue assumes a tilted
conformation pointing towards S3 and the solvent exposed surface
of the protease (Fig. 3A). In contrast, substitution of Cbz by a 3-
fluorobenzyl group (1i, Fig. 3B) or a 4-methoxyindole group (1f,
Fig. 3C), or by installation of an (S)-methyl group at the benzyl
methylene (1g, Fig. 3D), forces a relocation of the P3 substituent
into the S4 pocket, allowing for additional interactions with that
side of the protease. Consequently, in the crystal structure of 1g, the
SeMe group points towards S3 (protease surface), thus defining the
attached methylene group as a bifurcation point in inhibitor design
(Fig. 3D). Such a space-filling expansion towards both S3 (protease
surface) and S4 (binding pocket) has so far not been reported and
suggests a promising approach to seal the entrance of the catalytic
cleft with a hydrophobic lid. Simultaneously, the N-terminal head
group is shown to maintain interactions with Ala188 and Pro168. Of
additional note, substitution of the Cbz group with the 4-
methoxyindole amide moiety in 1f (Fig. 3C) leads to a tighter fit
in the S4 pocket of Mpro and does not require the coordination of
any additional solvent molecules, contrasting a related SARS-CoV-2
Mpro inhibitor structure wherein an indole required the presence of
DMSO (PDB 6M0K) [29]. In addition to the NH…O hydrogen bond
between the backbone of Glu166 and the terminal inhibitor amide
carbonyl O atom, another hydrogen bond between the backbone
carbonyl O atom of Glu166 and the indole NH stabilizes the in-
hibitor in its position.
Replacement of the indole with
a 3-fluorobenzyl or 3-
chlorophenylethyl group provides the added benefit of increased
synthetic yields, contrasting previously described indole-
containing inhibitors [2,30], which may facilitate reaction scale-
up during process development.
2.5. NMR studies with 13C-labelled GC373 and GC376: formation of
a dynamic equilibrium of stereoisomeric mixtures in aqueous media
As the mechanism of GC373 and GC376 involves binding of a
defined stereoisomer [6], we investigated the properties of GC373
and isomer distribution of both GC373 and GC376 in aqueous me-
dia. Versions of GC373 and GC376 having a13C-label at the carbonyl
carbon (Fig. S4) were synthesized. An initial HSQC NMR experiment
of GC373 in CDCl3 showed a single prominent signal in the alde-
hyde region corresponding to the 13C-label (Fig. S5A). A second
HSQC NMR experiment wherein GC373 was dissolved in aqueous
solution (0.5% DMSO‑d6 in D2O) showed that the single aldehyde
peak disappeared. It was replaced by 2 signals at ppm values cor-
responding to formation of two hydrate diastereomers (Fig. S5B)
[31], indicating that GC373 epimerizes rapidly and exists almost
entirely as the hydrated form in aqueous media [6]. Loss of ste-
2.4. Assessment of inhibitory activity and cytotoxicity: plaque
reduction and viral assays
While IC50 values provide a drug potency in vitro and aid in
inhibitor design, EC50 values provide information regarding the
drug-potential of the molecule in a living system. We therefore
tested our compounds in a viral plaque reduction assay using Vero
E6 mammalian cells. In order to assess possible cytotoxic effects,
the compounds were also tested using a CellTiter-Glo assay in the
same cell line and were found to display therapeutic indices of
>200. All inhibitor variants containing lone P2 site modifications
(1ad1c), with the exception of 1c, exhibited lower EC50 values as
compared to the parent GC373, with the cyclopropyl modification
reochemical integrity at the
aqueous media is facile for aldehydes of amino acids. Comparison of
a
-carbon adjacent to the 13C-label in
CoV-2 Mpro. Substitution of the benzyloxy group with a 4-methoxyindole prompts binding of the P3 portion of the inhibitor to the S4 pocket of the enzyme. This is a result of
favorable interactions between the indole nitrogen and the carbonyl of Glu166 as well as that of the methoxy group with the binding pocket itself (PDB 7LDL). (D) 1g bound to the
active site of SARS-CoV-2 Mpro. The phenyl ring of (S)-methyl benzyl group can be seen being directed towards the S4 site of the enzyme via the presence of the additional methyl
group (PDB 7LCT).
6

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
Table 2
Doubly modified aldehyde derivatives of GC373. GC373 and aldehyde derivatives structures containing modifications at both the P2 and P3 positions are shown, along with
SARS-CoV-2 Mpro IC50 and SARS-CoV-2 antiviral EC50 values, both with and without co-administration of efflux inhibitor CP-100356 (þCP). Cytotoxicity assays showed CC50
values of >200
m
M for all compounds shown.
Structure
Entry
IC50
(m
M)
EC50
(
mM)
EC50
(
m
M) þCP
1i (PDB 7LCO)
0.14 0.04
0.24 0.09
0.05 0.01
0.43 0.03
1.1 0.2
0.8 0.2
0.7 0.1
2.0 0.5
0.25 0.1
0.28 0.02
0.27 0.4
1.2 0.8
1j
1k
1l
peak areas reveals a hydrate diastereomer distribution of ca. 7:3. In
the presence of Mpro, there is appearance of an additional peak due
to a hemithioacetal formed by binding of a single stereoisomer in
the active site of the enzyme. The hydrate diastereomer distribu-
tion remained the same as a result of interconversion between the
remaining diastereomeric unbound species, suggesting that this
equilibrium is dynamic (Fig. S5C). Analogous HSQC NMR experi-
ments with GC376 13C-labelled at the carbonyl carbon also
demonstrated a distribution of diastereomers, but only 3 distinct
signals were observed at a ratio of 36:46:18 (Fig. S5D). There are 4
different stereochemical permutations possible (Fig. S4), but the
range over which their chemical shift values in HSQC spectra may
be distributed is limited (Fig. S5E). Hence, it appears that the
middle signal is likely an aggregate resulting from spectral overlap
of 2 distinct stereochemical species. This study supports the pro-
posal that in aqueous solution, diastereomers of GC373, its hydrate,
and GC376, exist in a dynamic stereochemical equilibrium, with
only the correct aldehyde isomer binding as a single hemithioacetal
(Table 3), demonstrating that cation replacement is a viable strat-
egy to significantly increase aqueous solubility while retaining ac-
tivity. This approach represents an attractive general avenue for
obtaining more favorable solubility profiles in ion-paired drugs and
prodrugs.
It was observed that addition of H2O or D2O to powdered GC376
(sodium salt) initially formed clear, transparent solutions at a
concentration of ca. 440 mM (Fig. S6A). Further dilution of these
transparent solutions resulted in the formation of cloudy colloidal
suspensions (Fig. S6B) that eventually became transparent again at
sufficiently high dilutions (Fig S6C). While initially unexpected, this
led us to hypothesize that GC376 was forming micelles e a possi-
bility owing to the amphipathic nature of the compound. The
bisulfite moiety may function as a polar head group while the
largely non-polar peptide backbone and associated side chains may
function as a hydrophobic tail, akin in structure to that of a fatty
acid. To test this hypothesis, we conducted diffusion-ordered
spectroscopy (DOSY) experiments [33] in D2O at concentrations
of 440 mM and 5 mM (Fig. S7), as these concentrations both
resulted in the formation of clear solutions. Based on these exper-
in the active site of Mpro
.
iments,
a clear difference in diffusion coefficients could be
2.6. Increasing solubility by counter ion variation and investigating
aggregation phenomena
observed, corresponding to 1.2 ꢀ 10ꢁ10 m2/s at 440 mM and
4.0 ꢀ 10ꢁ10 m2/s at 5 mM. Accordingly, these diffusion order values
correspond to hydrodynamic radii of 20.6 Å and 6.2 Å (41.2 Å and
12.4 Å in diameter) respectively [34] e a distinct difference sug-
gesting the formation of micelles at 440 mM. For comparison, the
particle size of extensively studied dodecyl phosphocholine (DPC)
micelles in water are noted to be on the order of 64 Å to 72 Å in
diameter [35].
In order to help assess the potential of GC376 for administration
to humans, a number of solubility experiments were undertaken.
We sought to increase the aqueous solubility of GC376 and hy-
pothesized that the cation counterpart to the bisulfite adduct could
be replaced with alternative species that exhibited better proper-
ties. Potassium and choline were selected as possible candidates
owing to larger solvation radii and the generally regarded as safe
(GRAS) nature of choline [32]. The potassium and choline coun-
terparts to GC376 (designated as GC376-K (3a) and GC376-Cho
(3b)) were synthesized from GC373 using solutions of potassium
or choline bisulfite. Both derivatives demonstrated better solubility
in aqueous media, with increases in molar solubility of approxi-
mately 30% for GC376-K (3a) and over 90% for GC376-Cho (3b)
compared to GC376. Additionally, GC376-Cho did not appear to
form the same turbid solution as GC376 at 50 mM in water (see
below). Furthermore, for inhibitors 3a and 3b, cellular plaque
reduction assays with live virus revealed no significant difference in
inhibitory activity as compared to the parent compound GC376
Further support of this phenomenon is found when examining
the 1D 1H NMR spectra for these two solutions, with the 440 mM
sample producing a spectrum with a number of broad peaks, a
known phenomenon in samples exhibiting aggregation (Fig. S8A).
Contrasting this, much sharper peaks are observed in the 5 mM
sample (Fig. S8B), hinting at dispersed molecules in solution.
Expanding upon this, further DOSY experiments were conducted
with serially diluted samples of GC376 in D2O (Table S2). These
experiments showed that GC376 possessed a critical micelle con-
centration of approximately 96.7 mM (Fig. S9A), establishing a new
parameter of interest for the development of GC376 and related
bisulfite adduct prodrugs. This experiment was repeated with the
7

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
Table 3
Bisulfite adduct derivatives of GC373. GC376 and bisulfite adduct derivatives singly (2a, 2b) or doubly (2cd2f) modified at the P2 or P3 positions, or with variations in counter
ion (3a, 3b) are shown, along with SARS-CoV-2 Mpro IC50 and SARS CoV-2 antiviral EC50 values, both with and without co-administration of efflux inhibitor CP-100356 (þCP).
Cytotoxicity assays showed CC50 values of >200 mM for all compounds shown. ND indicates that value was not determined.
Entry
Structure
IC50
(
mM)
EC50
(
m
M)
EC50
(
m
M) þCP
GC376 [6]
0.19 0.04
0.9 0.2
0.25 0.02
2a
2b
0.08 0.03
0.07 0.02
0.9 0.3
1.7 0.3
0.27 0.2
0.32 0.04
2c
0.07 0.01
0.08 0.02
0.57 0.07
0.7 0.2
0.19 0.06
0.18 0.04
2d
2e
2f
0.04 0.01
0.05 0.03
0.20 0.04
1.8 0.4
1.2 0.2
0.8 0.3
0.29 0.03
0.5 0.1
ND
3a
3b
0.18 0.04
1.2 0.2
ND
Fig. 4. Key lead compounds identified as improved SARS-CoV-2 Mpro inhibitors. Both structures contain a cyclopropyl group at the P2 position. Differentiation occurs at the P3
position, where 2c contains a 3-fluorobenzyl group and 2d contains a 3-chlorophenylethyl group. Both inhibitors show substantially improved potency as demonstrated by IC50 and
EC50 values, both with and without CP-100356, as compared to the parent compound GC376.
choline version of GC376 (GC376-Cho, 3b), and a corresponding
critical micellar concentration of 91.1 mM was observed (Fig. S9B).
Previously reported effective dosages of GC376 for felines are
established at 5e10 mg/kg/day, twice daily [14]. In humans general
volume limits of subcutaneous injection are about 1.5 mL [36].
Administration of a micellar solution of GC376 is a possible method
for circumventing solubility issues and volume limits when
considering GC376 for use in human trials. Use of GC376-Cho, with
volume requirements as low as 0.11 mL per injection (calculated for
a 62 kg human, 5.66 M concentration, 360 mg per injection,
MW ¼ 588 g/mol), twice daily, could help to increase patient
acceptance. Administration of GC376 or related drugs as
a
8

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
concentrated micellar or colloid suspension along with the
increased solubility resulting from the use of an alternative coun-
terion may allow for significant improvements in the drug profile of
these types of peptide-aldehyde inhibitors.
and injection volume is an important issue. Our work has revealed
previously unreported self-aggregation and colloidal properties of
the compound. High concentrations (440 mM) of GC376 in water
form a clear, viscous solution. Upon dilution, the solution becomes
a cloudy colloidal dispersion and ultimately becomes fully homo-
geneous at sufficiently low concentrations (5 mM). Replacement of
the sodium counterion of GC376 with potassium or choline en-
hances solubility considerably. As the more potent analogs that
were synthesized for enzyme inhibition studies are structurally
related, they are likely to share many of these physical properties.
We believe that micellar administration of bisulfite adducts in
combination with cation replacement could enable the pharma-
ceutical development of a large number of inhibitors previously
limited by solubility issues. In conclusion, a number of alternative
compounds with more favorable drug-like properties were
designed and tested with desirable therapeutic indices of over 200.
The results of this study present insights and tools for drug
development based on cysteine protease inhibitors with implica-
tions not limited to just SARS-CoV-2.
3. Discussion
One of our objectives was to identify new drug candidates for
possible treatment of acute COVID-19 based on the antiviral pep-
tide aldehyde GC373 and its bisulfite prodrug GC376. A key goal was
to improve on inhibition of the essential viral main protease, SARS-
CoV-2 Mpro, and on antiviral effects in mammalian cells while
maintaining low toxicity [6]. The parent compounds are non-toxic
to mammals and effective in curing coronaviral infection in cats
[14] and more recently demonstrated, in mouse models [24,25]. A
number of new dipeptide derivatives were designed and synthe-
sized with improved IC50 enzyme inhibition and EC50 antiviral
values. Inhibitors 2c and 2d emerged as key compounds for Mpro
enzyme inhibition with better IC50 and cellular EC50 values
compared to the parent inhibitor GC376. Both 2c and 2d are also
easier to synthesize in high yield compared to related N-acyl indole
derivatives and also out-performs them in EC50 metrics. We note at
this point that emerging evidence indicates GC376 and related
compounds may exhibit a secondary mechanism of action resulting
from the inhibition of cathepsin L, a host-cell protease involved in
viral entry [37,38]. Taking this into account, and as a result of Vero
E6 cells lacking expression of TMPRSS2 (involved in an alternative
viral entry pathway), observed EC50 values may in part be the result
of inhibited viral entry, though this remains beyond the scope of
this study. X-ray crystallographic studies of four enzyme-inhibitor
complexes with SARS-CoV-2 Mpro suggest an unexplored avenue
for the design of inhibitors with bifurcations at the P3 position to
assist in capping the active site. Employment of this strategy may
facilitate further development of an additional family of new,
potent inhibitors.
4. Materials and methods
4.1. FRET peptide substrate synthesis
The FRET substrate Abz-SVTLQSG-Y(NO2)-R used was synthe-
sized according to the methods previously described in literature
[6]. Characterization data was in accordance as described.
4.2. Isolation and purification of SARS-CoV-2 Mpro
Pure SARS-CoV-2 Mpro was obtained as described previously and
confirmed according to established data [6].
4.3. Inhibition assay and determination of IC50 values
Another goal was to examine the properties of GC376 and GC373
in preparation for human clinical trials. Although the prodrug
GC376 has worked effectively to treat feline infectious peritonitis
(FIP) caused by a coronavirus in cats [14], there are issues that were
not reported in detail previously. These include limited aqueous
solubility of prodrug GC376 and its tendency to self-aggregate in
water, its occurrence as a mixture of stereoisomers, and the ten-
dency of the derived parent aldehyde inhibitor GC373 to isomerize
under aqueous conditions. Multidimensional NMR studies (HSQC)
of GC373 (aldehyde) and GC376 (bisulfite adduct) with a13C-label at
the C-terminal carbons indicate that they form a variety of species
in water, some of which are in a dynamic equilibrium with each
other. The stereochemically pure GC373 rapidly epimerizes in water
IC50 values for each inhibitor tested was determined via the
methods described previously in literature [6].
4.4. Crystallization of Mpro
The purified SARS-CoV-2 Mpro was dialyzed against buffer
containing 10 mM NaCl and 5 mM Tris HCl pH 8.0 overnight at 4 ꢂC.
Protein was concentrated with
a Millipore centrifugal filter
(10 kDa MW cutoff) to a concentration of 8 mg/mL. Protein was
incubated with 5 mM excess of inhibitor at 4 ꢂC for 1 h prior to
crystallization. The SARS-CoV-2 Mpro, the protein was subjected to
the JCSG plus and PACT crystallization screen (Molecular Di-
mensions), with hits identified in several conditions for all in-
hibitors. Best crystals were observed with hanging drop trays at
room temperature at a ratio of 1:1 with mother liquor 0.2 M So-
dium chloride, 0.1 M HEPES pH 7.0, 20% w/v PEG 6000. Prior to
freezing, crystals were incubated with 19% glycerol as a cryopro-
tectant. Data collection was take place at SSRL, beamline 12-2 with
Blu-Ice using the Web-Ice interface and Canadian Light Source
beamline 08B1-1 using MxDC.
due to the acidity of the
a-hydrogen adjacent to the aldehyde
carbonyl. This then forms a dynamic mixture of two diastereomeric
hydrates that predominate in water, but the stereochemically cor-
rect aldehyde form, which is one of the equilibrium structures, is
selectively captured as a hemithioacetal by the Mpro enzyme in the
inhibition process. This can be seen as a single species in the
enzyme active site both by NMR spectroscopy and protein X-ray
crystallography. Formation of bisulfite GC376, which is done under
aqueous conditions, can proceed from either the re or si faces of the
carbonyl for each of the aldehyde diastereomers. This nominally
results in formation of four diastereomeric bisulfite adducts, with at
least three being observed by HSQC NMR (chemical shifts for two
appear to overlap). However, in water the bisulfite adducts readily
revert to the aldehyde forms, which being readily epimerized,
provide the active inhibitory stereoisomer.
4.5. Diffraction data collection, phase determination, model
building, and refinement
X-ray diffraction data sets for SARS-CoV-2 Mpro and inhibitor
complex were collected at 100 K in a cold nitrogen stream using
beamlines at 12-2 at Stanford Synchrotron Radiation Lightsource
(SSRL) California, USA, of wavelength 0.97946, equipped with
Dectris PILATUS 6 M detector and Canadian light Source, CLSI
BEAMLINE 08B1-1 coupled with PILUUS 6 M. More than 80 data
In humans, weight-adjusted dosage for subcutaneous injections
of GC376 is higher than in cats, and therefore aqueous solubility
9

W. Vuong, C. Fischer, M.B. Khan et al.
European Journal of Medicinal Chemistry 222 (2021) 113584
sets were collected for all the five inhibitors, all of them processed
and the best ones selected based on data statistics. XDS and Scala
were used for processing of the data sets. The diffraction data set of
the Lig124 (XU4) SARS-CoV-2 Mpro was processed at a resolution of
1.93 Å, in an orthorhombic space group P21212 (Supplementary
Table 1). For the complex of SARS-CoV-2 Mpro with ligand 180
(XTJ), the data set collected, was processed at a resolution of 1.90 Å
in a space group C2, Ligand 182 (XV4) processed at resolution of
2.0 Å in a monoclinic space group P1. The data set collected for
Lig128 (XTP) at resolution 1.85 Å, processed in a space group C2.
The data set collected for Lig132 (XTM) in an orthorhombic space
group P212121 refined at resolution 1.95 Å. (Table S1). All the five
structures were determined by molecular replacement with the
crystal structure of the free enzyme of the SARS-CoV-2 Mpro (PDB
entry 6WTM) [6] as a search model, using the Phaser program from
Phenix, version v1.18.1e3855). Ligand was fit using Coot manually
for all the inhibitors in the density of pre-calculated map from
Phenix refinment. Refinement of all the structures was performed
with phenix.refine in Phenix software. The structure refinement
yielded final Rwork and Rfree of reasonable values. Statistics of
diffraction, data processing and model refinement are given in the
supplementary materials (Table S1). The model was inspected with
Ramachandran plots and all show good stereochemistry. Final
models displayed using PyMOL molecular graphics software
4.10. DOSY experiments
Diffusion Ordered Spectroscopy (DOSY) measurements were
performed on a 600 MHz four-channel Varian VNMRS spectrom-
eter equipped with a HCN Z-gradient probe using OpenVNMRJ 2.1A
as the acquisition and processing software. The Oneshot45 DOSY
pulse sequence [39,40]was used for all diffusion measurements. All
experiments were done at 27 ꢂC. The 90ꢂ pulse was optimized for
each sample using a simple pulse acquire NMR experiment with
presaturation of the residual HOD during the relaxation delay. The
diffusion delay was optimized for each sample using the Oneshot45
pulse sequence using 5 different pulsed field gradient strengths
from 2.4 G/cm to 59.5 G/cm. Based on the optimized diffusion delay,
for GC376 and 3b diffusion delays of 50 mse200 ms were used, the
final DOSY experiment was acquired using 15 different pulsed field
gradient strengths from 2 G/cm to 59.5 G/cm with 12 scans for each
value of gradient strength. The DOSY data were base line corrected
and then processed using a two component fit, were corrected for
non-uniform pulsed field gradient shapes and plotted using the
DOSY module in OpenVNMRJ 2.1A.
Funding
These investigations were supported by the Natural Sciences
and Engineering Research Council of Canada (NSERC) and the Ca-
nadian Institutes of Health Research (COVID-19 SOF-549297-2019
and VR3-172655).
€
(Version 2.0 Schrodinger, LLC).
4.6. Plaque reduction assay and EC50 determination
Author contributions
Plaque reduction assay for EC50 determination was carried out
with live virus in Vero cells using the methods that we have pre-
viously described [6].
W.V., C.F., J.A.N. and J.C.V. contributed to inhibitor design. W.V.
and C.F. contributed to inhibitor synthesis. T.L. and K.W. contributed
to FRET substrate synthesis. C.F. contributed to FRET assay studies.
W.V. and C.F. contributed to solubility studies. W.V. contributed to
NMR studies. C.F. and M.J.v.B. contributed to purified protein. M.B.K.
and M.J.L. contributed to protein crystallography. M.A.J., H.A.S, J.A.S,
and D.L.T. contributed to viral and toxicity studies. W.V. wrote the
initial draft. All authors read and approved the manuscript.
4.7. Evaluation of inhibitor cytotoxicity
Cytotoxicity of inhibitors was determined using the methods
that we have previously described [6].
4.8. Population distribution studies
Declaration of competing interest
The authors declare no competing interests.
Acknowledgments
13C-labelled substrate was prepared in accordance with pro-
cedures documented in the supplemental materials. NMR samples
of GC373 were prepared by dissolving the sample in CDCl3 or in the
case of D2O, as a solution in DMSO‑d6 that was subsequently diluted
to a final concentration of 1% DMSO‑d6 in D2O. Preparation of GC376
samples omitted the use of DMSO‑d6 as a co-solvent. Additional
information and protocols for samples containing Mpro enzyme can
be found previously documented in literature [6]. Experiments
were executed on a 700 MHz Agilent VNMRS spectrometer
equipped with a cryo-cooled HCN triple resonance Z-gradient
probe.
General: The authors thank Mark Miskolzie and Ryan McKay for
help with NMR analyses and assistance in DOSY experiments. We
are grateful to Randy Whittal, Jing Zheng, Angelina Morales-
ꢀ
Izquierdo, and Bela Reiz for assistance with mass spectrometry.
We also thank Wayne Moffatt and Jennifer Jones for assistance in
compound characterization. Helpful discussions with David Bruy-
ette (Anivive Lifesciences Inc., Long Beach CA) are gratefully
acknowledged.
4.9. Solubility studies
Solubility studies were carried out by depositing 10 mg of
bisulfite prodrug (differing in cation e Na, K, or choline) in a small
Appendix A. Supplementary data
conical vial and sequentially adding H2O in 0.5 mL increments at
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113584.
25 ꢂC. These mixtures were then alternately vortexed using a
desktop instrument (30 s) and sonicated in a water bath (30 s) 3x
each. The mixtures were then observed to see if and solids
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