Potent SARS-CoV-2 mRNA Cap Methyltransferase Inhibitors by Bioisosteric Replacement of Methionine in SAM Cosubstrate

Article abstract of DOI:10.1021/acsmedchemlett.1c00140

Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory activity of the synthesized compounds showed nanomolar to submicromolar IC50 values for five compounds. To evaluate selectivity, enzymatic inhibition of the human glycine N-methyltransferase involved in cellular SAM/SAH ratio regulation was also determined, which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16. No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.

Full text of DOI:10.1021/acsmedchemlett.1c00140

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pubs.acs.org/acsmedchemlett
Letter
Potent SARS-CoV‑2 mRNA Cap Methyltransferase Inhibitors by
Bioisosteric Replacement of Methionine in SAM Cosubstrate
̌
̌ ̌
̧s, Mihails Sisovs, Anna L. Bula,
Olga Bobile
Solveiga Grin
Kaspars Tars, and Kristaps Jaudzems*
̧
va, Raitis Bobrovs, Iveta Kan
̧
epe, Liene Patetko, Gints Kalnin
̌
̧is, Anna Ramata-Stunda, Nils Rostoks, Aigars Jirgensons,
̌
̅
berga, Martins Borodusk
̧
̅
̅
Cite This: ACS Med. Chem. Lett. 2021, 12, 1102−1107
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ABSTRACT: Viral mRNA cap methyltransferases (MTases) are emerging targets for the development of broad-spectrum antiviral
agents. In this work, we designed potential SARS-CoV-2 MTase Nsp14 and Nsp16 inhibitors by using bioisosteric substitution of the
sulfonium and amino acid substructures of the cosubstrate S-adenosylmethionine (SAM), which serves as the methyl donor in the
enzymatic reaction. The synthetically accessible target structures were prioritized using molecular docking. Testing of the inhibitory
activity of the synthesized compounds showed nanomolar to submicromolar IC50 values for five compounds. To evaluate selectivity,
enzymatic inhibition of the human glycine N-methyltransferase involved in cellular SAM/SAH ratio regulation was also determined,
which indicated that the discovered compounds are nonselective inhibitors of the studied MTases with slight selectivity for Nsp16.
No cytotoxic effects were observed; however, this is most likely a result of the poor cell permeability of all evaluated compounds.
KEYWORDS: SARS-CoV-2, MTase inhibitors, Nsp14, Nsp16, SAM analogues, antiviral drugs
Coronaviruses have evolved an mRNA capping apparatus to
protect their 5′-ends with a cap moiety that is indistinguishable
from eukaryotic mRNA cap structures.4 The capping is aided
by the MTases Nsp14 and Nsp16, which modify the N7 of the
guanosine cap and the 2′OH group of the two subsequent
nucleotides of viral mRNA, respectively.5 Nsp16 is activated
and stabilized by binding to Nsp10.6 The N7-methylguanosine
(m7G) cap is required for efficient translation of viral
proteins,7 whereas 2′-O-methylation of the first two nucleo-
tides is important for evasion of the host immune response.8
Studies on SARS-CoV-1 have shown that mutations in the
Nsp14 and Nsp16 genes lead to a significantly attenuated virus
that is recognized by the innate immune system.7,9,10 Further
he global COVID-19 pandemic has exposed a shortage of
Ttherapeutic treatment options against coronaviruses.
While repurposing studies of existing drugs have identified
numerous candidates, their usefulness in treating the
respiratory disease is limited due to poor efficacy.1 This has
urged the scientific community to embark on extensive drug
discovery research against the severe acute respiratory
syndrome coronavirus 2 (SARS-CoV-2).
The genome of SARS-CoV-2 codes for 29 proteins,2 several
of which have been explored as targets for development of new
antiviral drugs. The most notorious of these are the RNA-
dependent RNA polymerase (RdRp) Nsp12, the helicase
Nsp13, the main (Mpro) and papain-like proteases (PLpro)
Nsp5 and Nsp3d, the nucleocapsid protein N, as well as
mRNA cap methyltransferases (MTases) Nsp14 and Nsp16.3
Although future clinical development of the identified specific
SARS-CoV-2 inhibitors may take several years before approval,
these efforts are also crucial to avoid the breakout of another
coronavirus pandemic in the coming years.
Received: March 11, 2021
Accepted: June 8, 2021
Published: June 9, 2021
https://doi.org/10.1021/acsmedchemlett.1c00140
© 2021 American Chemical Society
ACS Med. Chem. Lett. 2021, 12, 1102−1107
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Figure 1. (A) Molecular structures of the MTase cosubstrate SAM, known pan-MTase inhibitor sinefungin, and general structures of the
compounds studied here. (B) Surface view of SAM binding pockets in the crystal structures of SARS-CoV-1 Nsp14−SAM complex (PDB ID:
5C8T), SARS-CoV-2 Nsp16−SAM complex (PDB ID: 6W4H), human glycine N-MTase−SAM complex, and human RNA guanine-N7-MTase−
SAH complex (PDB ID: 3BGV). The structure of SARS-CoV-2 Nsp14 is not available; however, both strains share 100% sequence identity within
10 Å distance of the SAM binding site. Amino acid side chains within 4 Å distance of SAM/SAH are shown as lines. Hydrogen bond and π-stacking
interactions are shown with yellow and cyan dashed lines. The figure was generated using PyMOL.22
data suggest that inhibition of Nsp16 can be used for
coronavirus treatment but requires the activation of interfer-
on-stimulated gene response for efficacy.11 Nsp14 and Nsp16
along with the RdRp Nsp12 and the helicase Nsp13 are among
the most conserved proteins of coronaviruses with sequence
identities above 50%. Therefore, drugs targeting the MTases of
SARS-CoV-2 could have broad-spectrum antiviral activity also
against other coronaviruses including SARS-CoV-1 and
MERS-CoV. Furthermore, crystallographic structures of
Nsp16 from SARS-CoV-212−14 and of Nsp14 from SARS-
CoV-115 are available facilitating the use of virtual screening
and structure-guided drug design approaches.
Most of the previous work to discover viral MTase inhibitors
has focused on S-adenosylmethionine (SAM) analogues with
minor (i.e., single/few atom) modifications in the amino acid
and adenine substituents.16−18 Thus, a few highly potent
inhibitors such as sinefungin (Figure 1A) have been identified
that are effective against MTases from various species.
However, all the discovered inhibitors lacked selectivity for
coronavirus MTases compared to human MTases, and many
showed poor cell permeability due to their zwitterionic nature.
For this reason, none of the early viral MTase inhibitors have
been advanced into clinical development. Aouadi et al.
performed high-throughput screening against SARS-CoV-1
Nsp14 and identified other new classes of active compounds,
mostly polyphenols.19 However, the most potent of these
compounds were similarly nonselective, although some of the
weaker inhibitors showed more selectivity toward one viral
MTase. Recently, two preprints appeared reporting new Nsp14
inhibitors discovered by screening 161 SAM competitive
MTase inhibitors and through structure-based design.20,21
The previous findings motivated us to compare the binding
pockets of SAM between viral and cellular MTases. Figure 1B
shows a side-by-side comparison of the SAM binding sites of
SARS-CoV-1 Nsp14, SARS-CoV-2 Nsp16, and human mRNA
(guanine-N7-) MTase and human glycine N-methyltransferase
(GNMT). Although the binding sites of the SARS-CoV Nsp14
and Nsp16 MTases look more secluded than those of the
human MTases, there are notable similarities in the shape of
the binding pockets. The adenine subpocket is very narrow in
all structures, whereas the amino acid binding subsite is wider
and potentially able to accommodate bulkier substituents.
Hence, any changes introduced in the adenine fragment have
in most cases led to reduced inhibitor potency against viral
MTases.16,17 In the Nsp16 structure, the amino acid binding
subsite is also tightly filled; however, the shape of the
methylation site would allow the introduction of bulkier or
branched substituents. Based on these considerations, we
decided to explore the structure−activity/selectivity relation-
ships of the methionine fragment with the aim to achieve
selectivity for the viral MTases. Bioisosteric replacements of
the SAM methionine substructure were proposed, and the
designed compounds were prioritized by docking against
SARS-CoV-1 Nsp14 and SARS-CoV-2 Nsp16. The docking
models suggested that aromatic groups replacing the aliphatic
side chain can engage in interactions with the Nsp14 residues
Phe401, Tyr420, Phe426, and Phe506 (Tyr47, Tyr132 in
Nsp16), which make up a narrow, hydrophobic surface groove
that accommodates the mRNA cap, and bioisosteres of the α-
amino acid group can interact with the charged/polar amino
acids Arg310, Asp331, Asn334, and Lys336 (Asn43, Lys46,
Lys170, Glu203 in Nsp16). As a result, a diverse set of the top
scoring compounds was prepared that could be grouped as S-
aryl-5′-thioadenosine and analogues (1), S-alkylaryl-
̀
(alkylhetaryl)-5-thioadenosines and analogues (2 and 3), and
other SAM analogues (4) (Table 1).
The synthesized compounds were tested for inhibition
against the MTase activities of recombinant SARS-CoV-2
Nsp14, Nsp16/Nsp10,23 and human GNMT using a
homogeneous time-resolved fluorescent energy transfer
(HTRF) assay,19 which is based on the quantification of
released S-adenosyl homocysteine (SAH) during the enzy-
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Table 1. Enzymatic Potency against Viral and Human MTases, Cytotoxicity and Cell Permeability of SAM Analogues
a
b
c
d
Limit of quantification, 0.2 μM or 1%. Limit of detection. Not determined. Literature IC50 values against SARS-CoV-1 Nsp14 and Nsp16 are
0.112,19 0.383,18 0.4965 μM, and 0.7365 μM, respectively.
matic reaction. GNMT was chosen as the off-target for SAM
analogues because of its high abundance24 and because it
competes with tRNA methyltransferases for SAM and thereby
regulates the relative levels of SAM and SAH in cells.25 The
pan-MTase inhibitor sinefungin was also tested for reference.
Of the 11 prepared compounds, one compound (2a) showed
nanomolar, four compounds (1a, 2b, 3a−b) submicromolar,
three compounds (1b, 4a−b) micromolar IC50 values, and
three compounds (2c, 3c, 4c) were inactive or had minimal
activity (Table 1). Notably, three compounds (1a, 2a−b) were
more potent than sinefungin. Although these compounds
showed no selectivity for the viral MTases over human
GNMT, they were more active against SARS-CoV-2 Nsp16
with selectivity factors of 2−4.
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The obtained data provide some insights into the structure−
activity relationships of amino acid-modified SAM analogues as
SARS-CoV-2 MTase inhibitors. Replacement of the sulfonium
by a thioether group leads to more potent inhibitors (1a, 2b,
3b) than by amide (1b) and amine (2c, 3c) functionalities,
indicating that conformational flexibility and bulkiness rather
than the positive charge of this part are required for efficient
binding of the inhibitors. Installation of an aromatic or
heteroaromatic group in place of the aliphatic amino acid side
chain results in improved activity (compounds 1a, 2a−b, 3a−
b) due to additional aromatic−aromatic interactions and
reduced entropic penalty of binding (Figure 2). The observed
donors/acceptors is not beneficial (1a, 2a compared to 2b,
3a−b). In contrast, the presence of a positively charged moiety
that replaces the amino group seems nonessential. This is
because the aromatic interactions direct the R group toward
Lys336/Arg310 and away from Asp331 (Asp130 in Nsp16,
Figure 2). Finally, replacement of the transferred methyl group
of SAM with larger substituents that could extend into the
mRNA cap binding pocket is not well tolerated as shown by
the poor activity of 4b.
Despite the high potency against human GNMT and
possibly other homologous cellular MTases, the compounds
exhibited virtually no cytotoxicity on mouse embryo fibroblast
(NIH 3T3), human liver cancer (HepG2), and adenocarci-
nomic human alveolar basal epithelial (A549) cell lines.
Therefore, we questioned whether the compounds are able to
permeate the cell membrane. Cell permeability was tested with
the A549 cell line, and compound concentrations were
determined in cell lysates and culture media by mass
spectrometry. The compound concentrations in the cell lysates
were indeed low and often below the detection limit (Table 1).
This indicates that the cell membrane crossing is a problem of
this class of compounds even though several of them are not
zwitterions as their parental compounds SAM and sinefungin.
However, the compounds retain high polarity, which could be
circumvented by utilizing derivatization or prodrug strategies
to improve lipophilicity or to promote the transport of the
nucleoside analogues.26
In summary, we have used SAM cosubstrate analogue design
to discover S-aryl-5′-thioadenosines and S-alkylaryl-
̀
(alkylhetaryl)-5-thioadenosines as novel classes of MTase
inhibitors. These compounds show nanomolar to submicro-
molar potency against SARS-CoV-2 mRNA cap MTases
Nsp14 and Nsp16, but they lack selectivity with respect to
human GNMT and have poor cell permeability. The relatively
facile synthesis and high potency of this class warranties further
studies with the aim to identify novel antiviral agents with
appropriate properties and selectivity toward coronavirus
MTases. The availability of the crystal structures of Nsp14,
Nsp16, and human GNMT should allow a structure-guided
approach to design SAM analogues with increased selectivity
for the coronavirus MTases.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acsmedchemlett.1c00140.
Description of synthesis of compounds 1−4, their NMR
spectra, description of molecular modeling, protein
expression and purification, description of enzymatic,
cytotoxicity, and cell permeability assays (PDF)
Figure 2. Docking models of compound 2a (colored salmon) in the
X-ray crystal structures of SARS-CoV-1 Nsp14−SAM complex (PDB
ID: 5C8T) and SARS-CoV-2 Nsp16−SAM complex (PDB ID:
6W4H) showing the bound SAM (green) and nearby side chains
located within 5 Å from it. Hydrogen bond and π-stacking
interactions are shown with yellow and cyan dashed lines. The figure
was generated using PyMOL.22
AUTHOR INFORMATION
Corresponding Author
■
Kristaps Jaudzems − Latvian Institute of Organic Synthesis,
Riga, LV 1006, Latvia; orcid.org/0000-0003-3922-
2447; Email: kristaps.jaudzems@osi.lv
improvements are larger for Nsp16 than for Nsp14, although
the number of formed interactions is equal. A possible
explanation of this is that the inhibitor’s adenine fragment
binds Nsp16 in a conformation that is closer to SAM than in
Nsp14 complexes. The presence of a carboxyl group 4−5
bonds apart from the S-adenosyl residue seems important for
high potency, and its substitution with other hydrogen bond
Authors
Olga Bobilȩ va − Latvian Institute of Organic Synthesis, Riga,
LV 1006, Latvia; orcid.org/0000-0001-9600-1603
Raitis Bobrovs − Latvian Institute of Organic Synthesis, Riga,
LV 1006, Latvia; orcid.org/0000-0002-0221-8658
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ACS Medicinal Chemistry Letters
Iveta Kan
pubs.acs.org/acsmedchemlett
Letter
Functional Analysis of the SARS-Coronavirus RNA Cap 2’-O-
Methyltransferase Nsp10/Nsp16 Complex. PLoS Pathog. 2011, 7
(5), No. e1002059.
(7) Chen, Y.; Cai, H.; Pan, J.; Xiang, N.; Tien, P.; Ahola, T.; Guo, D.
Functional Screen Reveals SARS Coronavirus Nonstructural Protein
Nsp14 as a Novel Cap N7Methyltransferase. Proc. Natl. Acad. Sci. U.
S. A. 2009, 106 (9), 3484−3489.
(8) Daffis, S.; Szretter, K. J.; Schriewer, J.; Li, J.; Youn, S.; Errett, J.;
Lin, T.-Y.; Schneller, S.; Zust, R.; Dong, H.; Thiel, V.; Sen, G. C.;
Fensterl, V.; Klimstra, W. B.; Pierson, T. C.; Buller, R. M.; Gale, M.;
Shi, P.-Y.; Diamond, M. S. 2’-O Methylation of the Viral MRNA Cap
Evades Host Restriction by IFIT Family Members. Nature 2010, 468
(7322), 452−456.
(9) Chen, Y.; Tao, J.; Sun, Y.; Wu, A.; Su, C.; Gao, G.; Cai, H.; Qiu,
S.; Wu, Y.; Ahola, T.; Guo, D. Structure-Function Analysis of Severe
Acute Respiratory Syndrome Coronavirus RNA Cap Guanine-N7-
Methyltransferase. J. Virol. 2013, 87 (11), 6296−6305.
(10) Menachery, V. D.; Yount, B. L.; Josset, L.; Gralinski, L. E.;
Scobey, T.; Agnihothram, S.; Katze, M. G.; Baric, R. S. Attenuation
and Restoration of Severe Acute Respiratory Syndrome Coronavirus
Mutant Lacking 2’-o-Methyltransferase Activity. J. Virol. 2014, 88 (8),
4251−4264.
(11) Menachery, V. D.; Debbink, K.; Baric, R. S. Coronavirus Non-
Structural Protein 16: Evasion, Attenuation, and Possible Treatments.
Virus Res. 2014, 194, 191−199.
̧
epe − Latvian Institute of Organic Synthesis, Riga,
LV 1006, Latvia
Liene Patetko − University of Latvia, Riga, LV 1004, Latvia
̌
Gints Kalniņs − Latvian Biomedical Research and Study
Centre, Riga, LV 1067, Latvia; orcid.org/0000-0002-
9593-6308
̌
̌
Mihails Sisovs − Latvian Biomedical Research and Study
Centre, Riga, LV 1067, Latvia
Anna L. Bula − Latvian Institute of Organic Synthesis, Riga,
LV 1006, Latvia
Solveiga Grinberga − Latvian Institute of Organic Synthesis,
̅
Riga, LV 1006, Latvia
̌
̌
̧is − University of Latvia, Riga, LV 1004,
Martin
Latvia
̧
s Borodusk
̅
Anna Ramata-Stunda − University of Latvia, Riga, LV 1004,
Latvia
Nils Rostoks − University of Latvia, Riga, LV 1004, Latvia
Aigars Jirgensons − Latvian Institute of Organic Synthesis,
Riga, LV 1006, Latvia; orcid.org/0000-0002-8937-8792
Kaspars Tars − Latvian Biomedical Research and Study
̅
Centre, Riga, LV 1067, Latvia; orcid.org/0000-0001-
8421-9023
Complete contact information is available at:
(12) Rosas-Lemus, M.; Minasov, G.; Shuvalova, L.; Inniss, N. L.;
Kiryukhina, O.; Brunzelle, J.; Satchell, K. J. F. High-Resolution
Structures of the SARS-CoV-2 2’-O-Methyltransferase Reveal
Strategies for Structure-Based Inhibitor Design. Sci. Signal. 2020, 13
(651), DOI: 10.1126/scisignal.abe1202.
(13) Viswanathan, T.; Arya, S.; Chan, S.-H.; Qi, S.; Dai, N.; Misra,
A.; Park, J.-G.; Oladunni, F.; Kovalskyy, D.; Hromas, R. A.; Martinez-
Sobrido, L.; Gupta, Y. K. Structural Basis of RNA Cap Modification
by SARS-CoV-2. Nat. Commun. 2020, 11 (1), 3718.
(14) Krafcikova, P.; Silhan, J.; Nencka, R.; Boura, E. Structural
Analysis of the SARS-CoV-2 Methyltransferase Complex Involved in
RNA Cap Creation Bound to Sinefungin. Nat. Commun. 2020, 11 (1),
3717.
https://pubs.acs.org/10.1021/acsmedchemlett.1c00140
Funding
This work was supported by the Latvian Council of Science
(grant No. VPP-COVID-2020/1−0014). R.B. acknowledges
European Regional Development Fund project No. 1.1.1.2/
VIAA/2/18/379 for financial support. O.B. acknowledges
European Regional Development Fund project No. 1.1.1.2/
VIAA/4/20/747 for financial support.
Notes
The authors declare no competing financial interest.
(15) Ma, Y.; Wu, L.; Shaw, N.; Gao, Y.; Wang, J.; Sun, Y.; Lou, Z.;
Yan, L.; Zhang, R.; Rao, Z. Structural Basis and Functional Analysis of
the SARS Coronavirus Nsp14-Nsp10 Complex. Proc. Natl. Acad. Sci.
U. S. A. 2015, 112 (30), 9436−9441.
(16) Pugh, C. S.; Borchardt, R. T.; Stone, H. O. Inhibition of
Newcastle Disease Virion Messenger RNA (Guanine-7-)-Methyl-
transferase by Analogues of S-Adenosylhomocysteine. Biochemistry
1977, 16 (17), 3928−3932.
(17) Pugh, C. S.; Borchardt, R. T. Effects of S-Adenosylhomocys-
teine Analogues on Vaccinia Viral Messenger Ribonucleic Acid
Synthesis and Methylation. Biochemistry 1982, 21 (7), 1535−1541.
(18) Sun, Y.; Wang, Z.; Tao, J.; Wang, Y.; Wu, A.; Yang, Z.; Wang,
K.; Shi, L.; Chen, Y.; Guo, D. Yeast-Based Assays for the High-
Throughput Screening of Inhibitors of Coronavirus RNA Cap
Guanine-N7-Methyltransferase. Antiviral Res. 2014, 104, 156−164.
(19) Aouadi, W.; Eydoux, C.; Coutard, B.; Martin, B.; Debart, F.;
Vasseur, J. J.; Contreras, J. M.; Morice, C.; Quérat, G.; Jung, M.-L.;
Canard, B.; Guillemot, J.-C.; Decroly, E. Toward the Identification of
Viral Cap-Methyltransferase Inhibitors by Fluorescence Screening
Assay. Antiviral Res. 2017, 144, 330−339.
ABBREVIATIONS
■
COVID-19, coronavirus disease 2019; SARS-CoV, severe
acute respiratory syndrome coronavirus; MERS-CoV, Middle
East respiratory syndrome coronavirus; Nsp, nonstructural
protein; MTase, methyltransferases; GNMT, human glycine
N-methyltransferase; SAM, S-adenosylmethionine; SAH, S-
adenosylhomocysteine; HTRF, homogeneous time-resolved
fluorescent energy transfer
REFERENCES
■
(1) Singh, T. U.; Parida, S.; Lingaraju, M. C.; Kesavan, M.; Kumar,
D.; Singh, R. K. Drug Repurposing Approach to Fight COVID-19.
Pharmacol. Rep. 2020, 72 (6), 1479−1508.
(2) Kim, D.; Lee, J.-Y.; Yang, J.-S.; Kim, J. W.; Kim, V. N.; Chang, H.
The Architecture of SARS-CoV-2 Transcriptome. Cell 2020, 181 (4),
914−921.e10.
(3) Rajarshi, K.; Khan, R.; Singh, M. K.; Ranjan, T.; Ray, S.; Ray, S.
Essential Functional Molecules Associated with SARS-CoV-2
Infection: Potential Therapeutic Targets for COVID-19. Gene 2021,
768, 145313.
(4) Decroly, E.; Ferron, F.; Lescar, J.; Canard, B. Conventional and
Unconventional Mechanisms for Capping Viral MRNA. Nat. Rev.
Microbiol. 2012, 10 (1), 51−65.
(5) Bouvet, M.; Debarnot, C.; Imbert, I.; Selisko, B.; Snijder, E. J.;
Canard, B.; Decroly, E. In Vitro Reconstitution of SARS-Coronavirus
MRNA Cap Methylation. PLoS Pathog. 2010, 6 (4), No. e1000863.
(6) Decroly, E.; Debarnot, C.; Ferron, F.; Bouvet, M.; Coutard, B.;
Imbert, I.; Gluais, L.; Papageorgiou, N.; Sharff, A.; Bricogne, G.;
Ortiz-Lombardia, M.; Lescar, J.; Canard, B. Crystal Structure and
(20) Devkota, K.; Schapira, M.; Perveen, S.; Yazdi, A. K.; Li, F.;
Chau, I.; Ghiabi, P.; Hajian, T.; Loppnau, P.; Bolotokova, A.; Satchell,
K. J. F.; Wang, K.; Li, D.; Liu, J.; Smil, D.; Luo, M.; Jin, J.; Fish, P. V.;
Brown, P. J.; Vedadi, M. Probing the SAM Binding Site of SARS-CoV-2
Nsp14 in Vitro Using SAM Competitive Inhibitors Guides Developing
Selective Bi-Substrate Inhibitors; preprint; bioRxiv, 2021,
DOI: 10.1101/2021.02.19.424337.
̌
(21) Otava, T.; Sála, M.; Li, F.; Fanfrlík, J.; Devkota, K.; Pakarian, P.;
Hobza, P.; Vedadi, M.; Boura, E.; Nencka, R. The Structure-Based
Design of SARS-CoV-2 Nsp14 Methyltransferase Ligands Yields
1106
https://doi.org/10.1021/acsmedchemlett.1c00140
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pubs.acs.org/acsmedchemlett
Letter
Nanomolar Inhibitors; preprint; 2021, DOI: 10.26434/chem-
rxiv.14075408.v1.
(22) The PyMOL Molecular Graphics System; Schrodinger, LLC.
̈
(23) Altincekic, N.; Korn, S. M.; Qureshi, N. S.; Dujardin, M.;
Ninot-Pedrosa, M.; Abele, R.; Abi Saad, M. J.; Alfano, C.; Almeida, F.
C. L.; Alshamleh, I.; de Amorim, G. C.; Anderson, T. K.; Anobom, C.
D.; Anorma, C.; Bains, J. K.; Bax, A.; Blackledge, M.; Blechar, J.;
Böckmann, A.; Brigandat, L.; Bula, A.; Butikofer, M.; Camacho-Zarco,
̈
A. R.; Carlomagno, T.; Caruso, I. P.; Ceylan, B.; Chaikuad, A.; Chu,
F.; Cole, L.; Crosby, M. G.; de Jesus, V.; Dhamotharan, K.; Felli, I. C.;
Ferner, J.; Fleischmann, Y.; Fogeron, M.-L.; Fourkiotis, N. K.; Fuks,
C.; Furtig, B.; Gallo, A.; Gande, S. L.; Gerez, J. A.; Ghosh, D.; Gomes-
̈
Neto, F.; Gorbatyuk, O.; Guseva, S.; Hacker, C.; Häfner, S.; Hao, B.;
Hargittay, B.; Henzler-Wildman, K.; Hoch, J. C.; Hohmann, K. F.;
́
̌
̧s, G.;
Hutchison, M. T.; Jaudzems, K.; Jovic, K.; Kaderli, J.; Kalnin
Kanepe, I.; Kirchdoerfer, R. N.; Kirkpatrick, J.; Knapp, S.;
̧
Krishnathas, R.; Kutz, F.; Zur Lage, S.; Lambertz, R.; Lang, A.;
Laurents, D.; Lecoq, L.; Linhard, V.; Löhr, F.; Malki, A.; Bessa, L. M.;
Martin, R. W.; Matzel, T.; Maurin, D.; McNutt, S. W.; Mebus-
Antunes, N. C.; Meier, B. H.; Meiser, N.; Mompeán, M.; Monaca, E.;
Montserret, R.; Marino Perez, L.; Moser, C.; Muhle-Goll, C.; Neves-
̃
Martins, T. C.; Ni, X.; Norton-Baker, B.; Pierattelli, R.; Pontoriero, L.;
Pustovalova, Y.; Ohlenschläger, O.; Orts, J.; Da Poian, A. T.; Pyper,
D. J.; Richter, C.; Riek, R.; Rienstra, C. M.; Robertson, A.; Pinheiro,
A. S.; Sabbatella, R.; Salvi, N.; Saxena, K.; Schulte, L.; Schiavina, M.;
Schwalbe, H.; Silber, M.; Almeida, M. da S.; Sprague-Piercy, M. A.;
Spyroulias, G. A.; Sreeramulu, S.; Tants, J.-N.; Tars, K.; Torres, F.;
̅
́
Töws, S.; Trevin
̃
o, M. A.; Trucks, S.; Tsika, A. C.; Varga, K.; Wang,
Y.; Weber, M. E.; Weigand, J. E.; Wiedemann, C.; Wirmer-
Bartoschek, J.; Wirtz Martin, M. A.; Zehnder, J.; Hengesbach, M.;
Schlundt, A. Large-Scale Recombinant Production of the SARS-CoV-
2 Proteome for High-Throughput and Structural Biology Applica-
tions. Front. Mol. Biosci. 2021, 8, 653148.
(24) Yeo, E. J.; Wagner, C. Tissue Distribution of Glycine N-
Methyltransferase, a Major Folate-Binding Protein of Liver. Proc. Natl.
Acad. Sci. U. S. A. 1994, 91 (1), 210−214.
(25) Luka, Z.; Mudd, S. H.; Wagner, C. Glycine N-Methyltransfer-
ase and Regulation of S-Adenosylmethionine Levels. J. Biol. Chem.
2009, 284 (34), 22507−22511.
(26) Li, F.; Maag, H.; Alfredson, T. Prodrugs of Nucleoside
Analogues for Improved Oral Absorption and Tissue Targeting. J.
Pharm. Sci. 2008, 97 (3), 1109−1134.
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