SAR Exploration of Tight-Binding Inhibitors of Influenza Virus PA Endonuclease

Article abstract of DOI:10.1021/acs.jmedchem.9b00747

Significant efforts have been reported on the development of influenza antivirals including inhibitors of the RNA-dependent RNA polymerase PA N-terminal (PA_N) endonuclease. Based on recently identified, highly active metal-binding pharmacophore

Full text of DOI:10.1021/acs.jmedchem.9b00747

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Article
SAR Exploration of Tight Binding Inhibitors
of Influenza Virus PA Endonuclease
Cy V. Credille, Christine N. Morrison, Ryjul Wynn Stokes, Benjamin
Dick, Yifan Feng, Jiaxing Sun, Yao Chen, and Seth M. Cohen
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00747 • Publication Date (Web): 19 Sep 2019
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Journal of Medicinal Chemistry
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SAR Exploration of Tight Binding Inhibitors of Influenza Virus PA
Endonuclease
Cy V. Credille,1† Christine N. Morrison,1† Ryjul W. Stokes,1† Benjamin Dick,1‡ Yifan Feng,2‡ Jiaxing Sun,2 and Yao Chen,2,* and Seth M.
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Cohen1,3,
*
1Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, CA 92093, United States
2State Key Laboratory of Medicinal Chemical biology, Nankai University, No.94 Weijin Road, Nankai District, Tianjin,
P.R.China 300071
3Lead corresponding author †Authors contributed equally ‡Authors contributed equally
Supporting Information Placeholder
ABSTRACT: Significant efforts have been reported on the development of influenza antivirals including inhibitors of the RNA-dependent
RNA polymerase PA N-terminal (PAN) endonuclease. Based on recently identified, highly active metal-binding pharmacophores (MBPs)
for PAN endonuclease inhibition, a fragment-based drug development (FBDD) campaign was pursued. Guided by coordination chemistry
and structure-based drug design (SBDD), MBP scaffolds were elaborated to improve activity and selectivity. Structure-activity relationships
(SARs) were established and used to generate inhibitors of influenza endonuclease with tight binding affinities. The activity of these
inhibitors was analyzed using a fluorescence quenching-based nuclease activity assay and binding was validated using differential scanning
fluorometry (DSF). Lead compounds were found to be highly selective for PAN endonuclease against several related dinuclear and
mononuclear metalloenzymes. Combining principles of bioinorganic and medicinal chemistry in this study has resulted in some of the most
active in vitro influenza PAN endonuclease inhibitors with high ligand efficiencies.
polymerase complex comprised of three distinct proteins (PA, PB1,
PB2).13,14 Following viral infection, the viral polymerase-RNA
complex is transported to the host cell nucleus where the
polymerase complex catalyzes both transcription and replication of
the viral genome.15 However, the polymerase complex is unable to
incorporate or synthesize the 5′-mRNA cap necessary for
eukaryotic translation.16,17 To overcome this limitation, the PB2
protein captures a capped mRNA or pre-mRNA by tightly binding
to the 5’-cap structure. The host mRNA is then cleaved 8-14
nucleotides downstream from the cap by the viral PA (or P3 in the
case of Influenza C) endonuclease domain.17-19 The sequestered,
capped RNA segment then serves as a primer for viral RNA
synthesis by the PB1 protein of the polymerase complex. This
‘cap-snatching’ mechanism enables formation of the viral mRNAs
which are translated to viral proteins by the host cell machinery.
The viral RNA polymerase is a validated target for the
development of new antiviral therapies. Of particular interest is the
PA endonuclease domain of influenza A and B viruses. The PA
endonuclease domain lacks a human homologue and is highly
conserved among all circulating influenza strains, including
pandemic and avian strains.3 The PA subunit can be divided into
two domains connected by a long flexible peptide chain: the C-
terminal domain, which is primarily structural in nature, and the N-
terminal domain which contains the endonuclease catalytic site.
The N-terminal domain of the PA (PAN) endonuclease is a
dinuclear metalloenzyme that can bind to either Mg2+ or Mn2+
cations, but shows a preference for Mn2+ in both metal ion affinity
and catalytic activity.17,20 The metal cations are coordinated by five
active site residues (His41, Ile120, Asp108, Glu80, Glu119) and
several water molecules, forming a dinuclear complex that is
bridged by Asp108 and a catalytically active water/hydroxide anion
in the native structure (Figure 1).17,21
INTRODUCTION
Annual influenza epidemics are responsible for substantial
morbidity and mortality, as well as significant financial burden
worldwide. Vaccinations against the influenza virus are generally
effective for healthy individuals, but they must be re-administered
annually and are less effective for the elderly,
immunocompromised, or other high-risk individuals. Vaccine
efficacy is also dependent on correctly predicting the major type
among circulating viral strains each season, and incorrect
predictions of the principal infectious strain have rendered previous
vaccines <30% effective (e.g., during the 2017-2018 flu season).1,2
Rapid mutation and antigenic variation, due to genetic
reassortment, drive both the emergence of novel pandemic strains
of the influenza virus as well as the appearance of antiviral
resistance to existing treatments.3,4 Although antiviral drugs have
been developed, the number of antivirals that can be used for
prophylaxis and therapeutic treatment of severe influenza infection
is limited.5 Three classes of small molecule treatments for
influenza have been approved by the U.S. Food and Drug
Administration (FDA). Two of these are M2 ion-channel blockers
and neuraminidase inhibitors. However, viral resistance to existing
inhibitors and the time-dependent effectiveness of neuraminidase
inhibitors persist as major challenges to current approved
therapies.6-10 Considering this, there is an urgent need for the
development of new drugs to prevent and treat influenza infection.
The N-terminal PA endonuclease component of the RNA-
dependent RNA polymerase (PAN) is an attractive target to
meet this need. The PAN endonuclease inhibitor baloxavir11
(Figure 1) was recently approved for use in the U.S.
All influenza viruses contain a segmented single-stranded
negative sense RNA genome.12 Each RNA segment is packaged in
complex with a single heterotrimeric RNA-dependent RNA
Previous studies have identified highly active metal-binding
pharmacophore (MBP) fragments for the PAN endonuclease metal
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site, including compound 1 which is reported to inhibit PAN
endonuclease activity with an IC50 value of 17 nM (Figure 1).22
Related fragments, such as 2-substituted-4,5-
Compound 1, which was previously identified as having
1
2
3
4
5
6
7
8
excellent metal coordination to the PAN endonuclease active site,22
was chosen for further hit-to-lead development. To help guide
initial structure-activity relationship (SAR) analysis, existing
inhibitors were examined that share similar metal-binding motifs
with compound 1 (Figure 1), including hydroxypyrimidinones,
hydroxypyridinones, β-keto acids, dihydroxybenzoic acids, and
catechol derivatives.24,25 Prior SAR studies on compound 1
suggested that the 6-position (methyl group) was preferred for
further derivatization.22 Elaboration of compound 1 at other ring
positions resulted in poorer activity.
dihydroxypyrimindines, have been identified from campaigns that
employed a novel fluorescence polarization assay.23 Herein, a
fragment-based drug discovery (FBDD) campaign was initiated
based on compound 1. Several of these leads were elaborated and
optimized, resulting in compounds that possess in vitro inhibitory
activity with IC50 values of <1 nM against PAN endonuclease in
fluorescence quenching-based nuclease activity assays. These are
among the most active in vitro inhibitors of this enzyme reported
to date and show high ligand efficiencies.
9
Comparing known inhibitors with compound 1, an aryl-halide
cross-coupling strategy for further elaboration was deemed most
viable. To generate the desired aryl-halide intermediate in
sufficient quantities, an efficient and scalable synthetic route was
developed beginning from commercially available furfural
(Scheme 1). Generation of the dibromo-hydroxypicolinonitrile
intermediate 2 is typically performed over five discrete steps but
was optimized into a one-pot reaction in >50% overall yield. This
advanced intermediate was then selectively functionalized at the 4-
position with an activated alcohol to generate compound 3. This
protected species was found to be compatible with Suzuki and
Sonogashira cross-coupling reactions; however, Buchwald-
Hartwig amination on 3 was unsuccessful. Acid-catalyzed
deprotection of the protected intermediates (4a-24a, Scheme 1)
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generated
the
desired
2-carboxy-3,4-hydroxypyridinone
derivatives (4-24, Scheme 1).
Using the aforementioned synthetic strategies, a small library of
functionalized derivatives of 1 were prepared (Table 1). The
compounds were evaluated using an established fluorescence
quenching-based nuclease assay.26,27 To calibrate our assay, four
known PAN endonuclease inhibitors with a wide range of reported
IC50 values were used as positive controls (Table S2). The four
control compounds were: epigallocatechin gallate (EGCG),28 L-
742,001,29 Baloxavir acid (the active form of FDA-approved
Baloxavir marboxil),30 and 2,4-dioxo-4-phenylbutanoic acid
(DPBA).29 The IC50 values for these control compounds, as
measured in our laboratory, tended to be ~5- to 10-fold more active
than reported values.28-30
The addition of a phenyl ring at the 6-position of the core MBP
scaffold (4) was well tolerated and resulted in a 3-fold increase in
activity versus 1 in fluorescence quenching-based nuclease assays
(see ESI for details), which is consistent with previously reported
SAR.26,27 Addition of a methyl group showed that derivatization
was tolerated at the 2′-, 3′-, and 4′-positions (5-7), with the 2′-
methyl substituent yielding a dramatic increase in inhibition
activity below the detection limit of the assay (IC50 values of <2
nM at 5 nM enzyme concentration). This SAR trend was validated
with bulkier substituents on the phenyl ring, as illustrated by
isopropyl (8-9) and phenyl ether derivatives (10-12) (Table 1).
Figure 1. Top: Structures of several reported metal-binding
inhibitors of PAN endonuclease.24,25 Bottom Left: Structure of the
dinuclear metal active site of PAN endonuclease with the active site
metal centers coordinated by compound 1 (IC50 = 17 nM),22
a
highly active MBP fragment (PDB 6E6V). The two Mn2+ cations
are coordinated by three acidic residues (Glu80, Asp108, Glu119),
the imidazole nitrogen of His41, and the carbonyl oxygen of Ile120,
as a bridged coordination complex. Coordinating waters are shown
as red spheres, and coordination bonds are shown as dashed yellow
lines. Bottom Right: Illustration of the active site metal centers
coordinated to compound 1. The three coordinating oxygen atoms
from the MBP replace the axial and bridging water/hydroxide
molecules that coordinate to the metal centers in the native protein.
To confirm the high binding affinity of these compounds,
differential scanning fluorimetry (DSF) assays were used to
identify the change in melting temperature (ΔTM) between
inhibitor-free and inhibitor-bound PAN endonuclease. A strong
linear correlation was observed between inhibitory activity (pIC50)
and ΔTM across multiple inhibitor chemotypes. Using this
relationship, the pIC50 values of compounds with common
chemical features were extrapolated to estimate values that were
otherwise below the detection limit of the fluorescence quenching-
based nuclease activity assay. X-ray crystallography studies of
these inhibitors bound to PAN endonuclease show key interactions
with active site residues, including Lys34, Arg124, and a small
pocket near Ile38. Several of these lead molecules were assayed
against H1N1 influenza A in cell-based viral neutralization studies.
The most active compound (23) displayed an IC50 value of 47 pM,
and several inhibitors were found to have EC50 values ranging from
~8-21 µM with no mammalian cellular toxicity up to 800 µM,
suggesting a wide therapeutic index for their use.
RESULTS AND DISCUSSION
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Scheme 1. Synthesis of PAN endonuclease inhibitors. Reagents
and conditions: (a) NH4OAc, KCN, EtOAc:water, r.t., 18 h; (b)
Br2, HBr, 5 ºC 30 min, r.t., 18 h; (c) BnOH, NaH, DMSO, 60 ºC,
18-24 h; (d) K2CO3, PdCl2(dppf), SPhos, boronic acid,
dioxanes:water, 95 ºC, 18 h; (e) HCl, HOAc, TFA, neat, 95 ºC, 18
h.
6
7
8
4.8 ± 1.3
<2.0
8.3
10.7
13.2
10.1
1
2
3
4
5
6
7
HN
O
O
O
O
OH OH
>8.7
8.2
HN
An extensive screen of 2′-substituents revealed that small alkyl
substituents are most active, with 2′-methyl, -ethyl, and -
trifluoromethyl derivatives (7, 13, 14) all resulting in IC50 values of
<2 nM, which is the detection limit of the in vitro nuclease assay
(at an enzyme concentration of 5 nM, Table 2). As the size or
hydrophilicity of the 2′-substituents increased, the compounds
became less active; for example, isopropyl and phenyl ether
derivatives 8 and 12 were found to be less active than smaller
methyl, ethyl, and methyl ether derivatives 7, 13, and 15. However,
many different functional groups were tolerated, indicating that 2′-
substituents were potentially suitable for late-stage chemical
modification for adsorption, distribution, metabolism, excretion,
and toxicity (ADMET) liabilities. Based on SAR found in other
PAN endonuclease inhibitors that show that polar and acidic 4′-
substituents can make favorable contacts with basic active site
residues, derivatives of 4 with carboxylic acid or tetrazole groups
at either the 3′- or 4′-positions were synthesized. All four
derivatives tested (17, 18, 19, 20, Table 2) show increased activity
relative to 4, with activity at or below the detection limit of the
assay (IC50 values of <2 nM at 5 nM enzyme concentration).
In an effort to maximize the activity of these inhibitors and based
on the assumption that the favorable 2′-alkyl and 4′-acidic
interactions were not mutually exclusive, a fragment merging
strategy was pursued. As shown in Table 3, derivatives were
synthesized combining a 2′-methyl group with a 4′-carboxylic acid
(21), 4′-tetrazole (22), or 4′-oxadiazolone (24) group. Gratifyingly,
these inhibitors displayed IC50 values of <2 nM, indicating that
merged inhibitors were as least as active as parent compounds 7,
17, and 19, and that the two functional groups were likely having
an additive effect on inhibitor activity. However, these derivatives
were too active to generate reliable inhibition data using the
nuclease activity assay, despite efforts to further optimize the assay
(i.e., the IC50 values were below the minimum usable concentration
of enzyme employed in the assay). This lower limit of detection is
also shared by other previously reported assays used to determine
PAN endonuclease inhibitor binding, which precluded their use as
orthogonal activity assays.31 Based on this, alternative biophysical
methods were sought to generate binding data for these tight-
binding compounds. Toward this goal, differential scanning
fluorometry (DSF) was employed as an orthogonal evaluation of
inhibitor binding.32-35 This method is more time- and cost-effective
than other techniques used to assess ligand binding and can be used
for both weak and strong binders.34
OH OH
6.3 ± 1.8
8
9
HN
O
O
O
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OH OH
9
3.2 ± 1.1
25 ± 6
8.5
7.6
11.6
8.1
HN
O
OH OH
10
O
HN
O
O
OH OH
11
12
14 ± 3
7.9
8.2
9.0
O
HN
O
O
OH OH
O
6.0 ± 1.6
10.4
HN
O
O
OH OH
Differential Scanning Fluorometry (DSF). Changes in
melting values (ΔTM) in DSF experiments are proportional to both
ligand concentration and ligand binding affinities.32,34 The thermal
stability of a protein generally increases with favorable protein-
ligand contacts made over the total interaction surface of the
protein, with tighter-binding interactions (lower KD) typically
resulting in larger increases to the observed melting temperature
compared to the native protein.32,34 DSF as an inhibitor screening
method has been successfully demonstrated with other
metalloenzymes such as human carbonic anhydrase I and II
(hCAI/hCAII) with sulfonamide-based inhibitors.34
We
determined the ΔTM values of L-742,001, Baloxavir acid, and
DPBA (Table S2); the values for Baloxavir acid and DPBA29,30
matched reported values well (no prior ΔTM value for L-742,001 is
available).
DSF analysis of compounds 4-20 (Table 1, Table 2) validates the
relative binding affinities of these inhibitors and gives insight into
compounds that are too active to properly rank-order using the
fluorescence quenching-based nuclease assay. Comparing ΔTM
(from DSF) and pIC50 values (from the nuclease assay) for
compounds 4-20 results in a linear correlation (r = 0.98, n = 12, p
< 2×10-8) for all compounds with activity above the detection limit
of the nuclease assay (Figure 2, 4-20, labeled as ‘series A’). To
evaluate the robustness of this correlation across chemotypes, the
ΔTM and pIC50 values of two additional, chemically distinct series
of inhibitors were compared (Figure S1). One series of compounds
(labeled ‘series B’ in Figure 2) is comprised of previously reported
PAN endonuclease inhibitors bearing a functionalized 3,4-
hydroxypyridinone MBP core (Figure S1).27 Compounds in series
B coordinate to the active site metal ions in PAN endonuclease and
make a variety of protein contacts, many of which are predicted to
be similar to those made by compounds 4-20. Series B also gave a
linear correlation (r = 0.99, n = 8, p < 8×10-7) between ΔTM and a
Table 1. Activity and binding affinities of derivatives of
compound 1.
Number Compound
IC50 (nM)
17 ± 3
pIC50 ΔTM (° C)
6
1
7.8
11.1
1 HN
5
2
4
O
O
O
O
3
OH OH
4
5
5.6 ± 1.2
7.2 ± 2.1
8.3
8.5
HN
OH OH
8.1
10.3
HN
O
O
OH OH
3
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wide range of pIC50 values. A third grouping of compounds,
1
2
3
4
5
6
7
8
(‘series C’, Figure S1) is comprised of various, non-elaborated
MBP fragments.22 These molecules coordinate to both active site
metal ions but make few other interactions with the remainder of
the protein active site. Interestingly, this series also shows a strong
linear correlation (r = 0.995, n = 9, p < 3×10-8) between ΔTM and
pIC50 values despite gaining most of their binding affinity from
metal coordination. Taken together, the DSF evaluation of
compounds in series A, B, and C (Figure S1) indicate that
qualitative inhibition data can be gained from extrapolation of
interrelated ΔTM and pIC50 data for PAN endonuclease inhibitors
with similar chemotypes.
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Using the observed linear relationship between DSF ΔTM
measurements and nuclease assay pIC50 values, we assigned
estimated IC50 values for tight binding compounds from series A,
as shown in Table 3 and Figure 2 (green diamonds). Based on DSF
extrapolation, 2′-methyl, -ethyl, and -trifluoromethyl derivatives
(7, 13, 14), were found to be nearly equipotent with IC50 values of
~1 nM. Compound 19, which contains a 4′-tetrazole moiety, was
found to be approximately twice as active as the equivalent
carboxylic acid-containing analogue 17, which is consistent with
literature reports.27,36 Merging 2′-methyl or trifluoromethyl and 4′-
acidic moieties (21-24) resulted in further improvements in
activity, with 22 and 23 displaying estimated IC50 values of ~110
pM and ~47 pM, respectively.
Figure 2. pIC50 values for three chemically distinct series of PAN
endonuclease inhibitors are plotted versus ΔTM measurements
obtained by DSF. The data show linear correlations within each
chemical series. In series A (4-20), inhibition values corresponding
to green diamonds are extrapolated from experimental ΔTM values
(shown as black triangles; see Tables 2, 3). This analysis allows
for estimation of inhibition for compounds with activity below the
detection limit of the nuclease assay. Chemical structures of
compounds in series B and C are provided in Figure S1, and a
version of this figure with all compounds labeled on the plot is
provided in Figure S2.
Table 3. Activity and binding affinities of PAN endonuclease
inhibitors derived from ΔTM values. IC50 values listed are
estimated from ΔTM values (see Figure 2).
Table 2. Activity and binding affinities of derivatives of
compound 4.
Number Compound ΔTM (° C) pIC50 Estimated
IC50 (pM)
Number
Compound
IC50 (nM) pIC50
ΔTM (° C)
13
<2.0
>8.7
>8.7
8.6
13.1
HN
O
7
13.2
13.3
9.0
9.1
920 ± 70
860 ± 70
HN
O
O
OH OH
O
OH OH
F3
C
F3
C
14
15
16
<2.0
13.3
11.9
11.8
HN
O
14
HN
O
O
O
O
OH OH
O
OH OH
O
OH
O
2.3 ± 0.6
3.2 ± 0.5
HN
O
O
1180
90
±
17
19
21
22
12.9
14.1
15.3
16.5
8.9
9.3
9.6
10.0
OH OH
HN
O
O
S
OH OH
N
N
8.5
HN
N
NH
OH OH
O
OH
510 ± 40
230 ± 20
110 ± 10
HN
O
O
17
18
<2.0
>8.7
8.6
11.9
11.7
OH OH
O
HN
OH
O
O
OH OH
O
HO
O
HN
O
2.3 ± 0.4
O
HN
OH OH
N
N
O
N
NH
OH OH
N
N
N
NH
HN
19
20
<2.0
<2.0
>8.7
>8.7
14.1
12.9
O
O
HN
OH OH
O
N
N
O
N
NH
OH OH
N
N
NH
N
23
17.8
10.3
47 ± 6
F3C
HN
HN
O
O
O
O
OH OH
OH OH
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O
The van der Waals contact with Ile38 has not been previously
O
N
NH
1
2
3
4
5
6
7
8
9
identified. The benefit of this contact is demonstrated by
comparing co-crystal structures of compounds 7 (2ʹ-methyl) and 13
(2ʹ-ethyl) as shown in Figure 4. Extending the methyl group to an
ethyl group causes compound 13 to tilt slightly outward so that the
terminal carbon of the 2ʹ-ethyl group makes the same van der Waals
contacts as the 2ʹ-methyl group in compound 7. This contact is
maintained despite sacrificing favorable, highly enthalpic metal
binding interactions; deviations from ideal coordination angle and
geometry have previously been shown to have pronounced
negative effects on the binding affinity of metal binding
inhibitors.37 Larger 2′-substituents (e.g., 9, 12) would require an
even greater outward tilt to maintain the same van der Waals
contact, and this likely contributes to the lower affinity of
compounds with larger 2′-substituents (Table 1).
24
16.3
9.9
120 ± 10
HN
O
O
OH OH
X-ray Crystallography. A subset of inhibitors was co-
crystallized with PAN endonuclease to elucidate the binding mode
for these particularly potent compounds. Tables of data collection
and refinement statistics for X-ray structures of PAN endonuclease
bound with compounds 1, 7, 13, 18, 20, 22, 24, and 40 (Tables S3-
S4) as well as figures displaying the electron density of each
inhibitor within the protein (Figure S3) are provided in the
Supporting Information. In the ligand-free structure, the Mn2+ ions
are coordinated by amino acid sidechains and water molecules to
achieve an octahedral coordination geometry for each metal ion.
Inhibitor molecules based on compound 1 were found to coordinate
to both Mn2+ metal centers by displacing the three coordinated
water molecules found in the ligand-free structure with a triad of
chelating oxygen atoms, including a carbonyl (coordinating to
Mn1), a hydroxyl (bridging Mn1 and Mn2), and a carboxylate
(coordinating to Mn2), as exemplified with compounds 1 (Figure 1)
and 22 (Figure 3). This binding motif was elaborated from the
bidentate chelation of compound 40 (previously reported as
compound Credille-63, Figure 3), the activity of which was
previously reported (Table 4),27 but no structure was available. The
protein-bound structure of 40 was solved (Figure 3), which shows
bidentate coordination of the ligand to Mn1, but only a
monodentate, bridging ligand to Mn2 (Figure 1).
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The inhibitors developed in this study, which bind both Mn2+
metal centers, are observed to make two specific types of contacts
with PAN endonuclease: van der Waals interactions with the side
wall of the active site near Ile38 via 2ʹ-alkyl substituents (i.e., 7, 13,
22, 24) and/or contacts with basic active site residues (i.e., 18, 20,
24). With respect to the latter, acidic 3′- and 4′-subsituents were
observed to make favorable hydrogen bonding interactions (or
solvent-mediated hydrogen bonding interactions) with Lys34,
Arg124, and/or Arg196 (compounds 18, 20, 24; Figure 4 and
Figure S3).
Figure 4. Left: Compounds 7 (green, PDB 6E3N) and 13 (yellow,
PDB 6E3O) bound in the PAN endonuclease active site. Compound
13 is tilted slightly outward relative to compound 7 to
accommodate the ethyl substituent. Right: Compound 24 (PDB
6E4C) bound in the active site of PAN endonuclease makes several
solvent-mediated or direct hydrogen bonding interactions with
Lys34, Arg124, and/or Arg196. Mn2+ ions and water molecules are
shown in purple and red spheres, respectively. Metal-coordination
and hydrogen bonds are represented with dashed yellow lines.
The protein-bound X-ray structure of compound 18 shows that
3ʹ-substitution forces the inhibitor to adopt an outward tilt (Figure
S4). This is accompanied by a decrease in activity, as demonstrated
by comparing the IC50 and ΔTM values of compounds 17 (4ʹ-acid)
and 18 (3ʹ-acid) (Table 2). In compound 18, the outward tilt of the
inhibitor is triggered by favorable hydrogen bonding interactions
between the 3ʹ-carboxylic acid moiety and Arg196 and solvent-
mediated interactions with Lys34. Compound 17 can make similar
interactions without tilting and hence without compromising a
more ideal metal binding geometry, which gives a structural basis
for its greater inhibitory activity.
Compound 20 occupies the active site in an unexpected way:
two inhibitor molecules are present, which form π-π stacking
interactions with each other (aryl-aryl distance ~3.2 Å). One
inhibitor molecule coordinates to the metal ions and tilts outward
from the active site pocket similar to compound 18 (Figure S4); the
second molecule forms polar contacts with Lys34 and Arg124
through its tetrazole ring. We reason that two molecules occupy
the active site because of increased available protein interactions as
a π-π stacked system. A similar phenomenon was observed in two
previous studies on PAN endonuclease inhibitors.31,38 Such stacked
interactions may occur because they mimic the binding
conformation of the native nucleic acid substrates of PAN
endonuclease.
Figure 3. Compounds 22 (left, PDB 6E6W) and 40 (right, PDB
6E6X) bound to the PAN endonuclease active site. In both panels,
the protein backbone is shown in gray cartoon. Mn2+ ions and
water molecules are shown as purple and red spheres, respectively.
Residues coordinating the Mn2+ ions are shown in sticks, and metal
coordination bonds are shown in dashed yellow lines. The electron
density of each inhibitor (green sticks) and the Mn2+ ions is
displayed as a blue mesh contoured to 1.5 σ.
Merging favorable 2ʹ- and 4ʹ- structural motifs was found to have
an additive effect on inhibitor binding, and the merged inhibitors
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22 and 24 were crystallographically observed to maintain the same
Based on an optimized MBP inhibitor core, a structure-guided
1
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8
interactions as parent inhibitors (7, 13, 40). For example,
compound 24 maintains the van der Waals contact near Ile38 and
solvent mediated or direct hydrogen bond interactions with Lys34,
Arg124, and Arg196 (Figure 4).
FBDD campaign was employed to generate highly active inhibitors
of PAN endonuclease. Structural elaborations of the optimized
MBP core identified key SAR trends, resulting in the development
of compounds with activity below the detection limit (IC50 <2 nM)
of the commonly used fluorescence quenching-based endonuclease
assay. DSF experiments were employed to further analyze
inhibitor binding, and a correlation between nuclease assay
inhibition values with ΔTM data was observed. X-ray co-crystal
structures of tight binding inhibitors highlight important protein
interactions necessary for tight binding. Key structural moieties
were merged to create inhibitors with subnanomolar inhibitory
activity against PAN endonuclease, while maintaining a high degree
of selectivity for PAN over similar metalloenzymes.
The lead compounds reported here are among the most active
inhibitors of PAN endonuclease reported in vitro. Even considering
that our in vitro nuclease assay tends to yield more potent IC50
values of reported inhibitors (vide supra), compounds 21-24 are
still among the most active PAN endonuclease inhibitors reported
to date.26,27,31,39 Importantly, compounds 21-24 are far more active
and ligand efficient then our previously reported inhibitors,27
showing that our FBDD approach, when combined with
biophysical and structural methods, can lead to substantial
improvements in in vitro potency.
Overall, the activity and LE of compounds 21-24 are very good.
However, relative to their very high in vitro activity, this class of
compounds has only modest performance against live virus in cell-
saving assays. This may be due to poor cell permeance and cell
uptake of these compounds (originating from the presence of three
ionizable functional groups), poor solubility, or other
physicochemical limitations. Nevertheless, the in vitro activity and
LE of these inhibitors indicates that they are good candidates for
pro-drugging and isostere strategies to improve cell permeance
with more lipophilic structural elaborations.
Inhibitor Selectivity. To evaluate the selectivity of these
inhibitors, compounds 22-24 were cross-screened against several
dinuclear and mononuclear human metalloenzyme targets,
including carbonic anhydrase II (hCAII), matrix metalloproteinase
12 (MMP-12), arginase I (ArgI), and methionine aminopeptidase
(MetAP; Mn2+ metalloform) (Table S5). Compounds 22-24 show
a high degree of selectivity for PAN endonuclease over these
metalloenzymes. To highlight the selectivity of 22-24 for PAN
endonuclease over other metalloenzymes bearing similar Mn2+
metal active sites, two dinuclear Mn2+ metalloenzymes (ArgI and
MetAP) were cross-screened at 200 µM inhibitor concentration. In
these assays, only compound 22 showed appreciable cross-
inhibition against MetAP (~44% inhibition) and none of these
compounds displayed any significant cross-inhibition against ArgI.
Little cross-inhibition was observed against the mononuclear Zn2+
metalloenzymes hCAII and MMP-12. Taken together, this data
indicates that these PAN endonuclease inhibitors have a high degree
of selectivity for their intended target over other competing
metalloenzymes, including those with similar dinuclear Mn2+
active sites.
Cytotoxicity and Antiviral Assays. Compounds 14, 19, 22, and
23 were selected for cytotoxicity and antiviral activity assays.
These assays were carried out in MDCK cells using influenza A
H1N1 virus obtained from the American Tissue Culture Collection
(A/California/04/2009 H1N1, ATCC VR-1737, see ESI for details).
Briefly, 80% confluent MDKC cells were incubated for a period of
48 h at 37 C 5% CO2 in the presence of varying concentrations of
inhibitor and influenza A virus at a theoretical multiplicity of
infection (MOI) of 0.05. After incubation, cells were analyzed for
CPE and viability using a CellTiter-Glo (Promega) luminescence
assay. Compounds 14 and 19 both showed modest antiviral activity
(EC50 = 10.4 ± 2.1 µM and 7.7 ± 3.3 µM, respectively), the positive
control L-742,001 exhibited anti-viral activity that is close to our
compound (EC50 = 7.4 ± 2.3 µM). The more active compounds 22
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EXPERIMENTAL
General Experimental Details
All solvents and reagents, unless otherwise noted, were obtained
from commercial sources and used without further purification. All
reactions, unless otherwise stated, were performed under a nitrogen
atmosphere. Silica chromatography was performed using a
CombiFlash Rf Teledyne ISCO system using hexane, ethyl acetate,
methylene chloride, or methanol as eluents. C18 reverse phase
chromatography was performed using the same instrument using
0.1% formic acid in methanol, acetonitrile, or water as eluent.
Separations were monitored by mass spectrometry via a Teledyne
ISCO RF+ PurIon ESI-MS detector with 1 Da resolution. 1H and
13C NMR spectra were obtained on a Varian (400 MHz)
spectrometer or Jeol (500 MHz) spectrometer or a VX (500 MHz)
equipped with XSens cold probe (Varian) spectrometer in the
Department of Chemistry and Biochemistry at U.C. San Diego.
The purity of all compounds used in assays was determined to be
≥95% by HPLC analysis. Standard resolution MS was performed
either at U.C. San Diego Molecular Mass Spectrometry Facility or
on the aforementioned Teledyne ISCO RF+ PurIon MS. HRMS
analysis was performed using an Agilent 6230 Accurate-Mass LC-
TOFMS located at the U.C. San Diego Molecular Mass
Spectrometry Facility.
and 23 showed similar antiviral activity (EC50 = 21.3 ± 4.8 µM and
11.5 ± 1.2 µM, respectively). While these antiviral activity values
are respectable, the large disparity between the in vitro PAN
endonuclease inhibitory activity and cell based antiviral activity
indicate that these compounds are likely limited by cellular uptake
and permeance, solubility, or other physiochemical attributes. The
other positive control Baloxavir acid showed antiviral activity
(EC50 = 2.2 ± 0.78 nM) that is significantly higher than L-742,001
and our compounds. However, Baloxavir acid also demonstrated
much higher cytotoxicity than the other compounds studied here
(~20 µM vs. >800 µM).
Cytotoxicity assays with MDCK cells (the host cell line for the
H1N1 virus) and 3T3 cells (a healthy cell line) were also performed.
All the tested compounds show nearly no influence on cell viability
at doses as high as 800 µM, indicating very low cytotoxicity (Table
S6). The selectivity index was determined as the ratio of the
concentration of the compound that reduced cell viability to 50%
(CC50) to the concentration of the compound needed to inhibit the
cytopathic effect to 50% of the control value (EC50). All the
compounds showed good selectivity indices (~35-75), with the
most potent inhibitor against H1N1 infected MDCK cells,
compound 19, giving a selectivity index of >100. The results reveal
excellent selectivity of these inhibitors towards virus over the host
cells. Efforts to improve cellular antiviral activity through pro-
drugging and isostere strategies are currently underway.
3-Hydroxy-6-methyl-4-oxo-1,4-dihydropyridine-2-carboxylic
acid (1). Synthesis of compound 1 was reported previously.22 1H
NMR (400 MHz, DMSO-d6): δ 6.92 (s, 1H), 2.48 – 2.38 (m, 3H).
13C NMR (126 MHz, DMSO-d6): δ 174.66 (s), 166.15 (s), 164.07
(s), 149.36 (s), 135.69 (s), 112.39 (s), 20.07 (d, J = 4.3 Hz). HR-
CONCLUSIONS
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Journal of Medicinal Chemistry
ESI-MS Experimental: 168.0302. Calculated for[C7H6NO4]-:
168.0302. Δ = 0.0 ppm.
min.
[1,1'-Bis(diphenylphosphino)ferrocene]palladium(II)
1
2
3
4
5
6
7
8
dichloride (30 mg, 5 mol%) was then added and the mixture was
heated to 95-100 C for 18-48 h under nitrogen. The reaction
progress was monitored by TLC. Upon completion, the reaction
mixture was filtered through a pad of celite and was rinsed once
with a 3:1 solution of dioxane:H2O. The filtrate was acidified to
pH<2 with 4M HCl and was concentrated under reduced pressure.
The isolated solids were then purified by silica chromatography.
However, in a majority of instances, the desired product compound
(identified by in-line MS detection and 1H NMR) was co-purified
with residual boronic acid as a significant contaminant, and
occasionally co-purified with significant quantities of other starting
materials. No further purification was attempted for these
compounds as experiments indicated that immediate deprotection
of contaminated materials provided a more reliable route for
purification and did not significantly impede the subsequent
deprotection reaction or purification. Intermediate yields, as
determined by 1H NMR spectroscopy varied from 30-77%.
4,6-Dibromo-3-hydroxypicolinonitrile (2). In a rapidly stirring
biphasic solution of EtOAc (50 mL) and H2O (12.5 mL) was
suspended ammonium acetate (14.0 g, 181 mmol) and KCN (4.2 g,
63 mmol). Furfural (5 mL, 60 mmol) was added slowly at room
temperature, and the mixture stirred over-night. A solution of
sodium thiosulfate should always be on hand when using KCN as
a means to quench cyanide in case of spill or splash, or to
decontaminate glassware, funnels, etc. after use. The reaction
mixture was then diluted with 25 mL of saturated sodium
bicarbonate solution, and the two layers were separated. After
phase separation, the organic layer was extracted once with 50 mL
of 5% aqueous HBr with agitation and frequent venting. The
organic layer was then further extracted with 2×20 mL of H2O. The
combined aqueous layers were combined and cooled to ~5 C and
Br2 (12.4 mL, 241 mmol) was added dropwise maintaining the
reaction in an ice bath. After addition, the mixture was allowed to
warm to room temperature and was stirred overnight and a yellow
precipitate formed. The reaction mixture was then cooled to 5-10
C and 25 mL of a 25% aqueous solution of NaHSO3 was added
slowly, maintaining the reaction mixture on an ice bath, to quench
excess Br2. The resulting suspension was stirred for 30 min then
filtered. The filter cake was washed twice with H2O and dried. The
desired product 2 was isolated as a tan solid (8.4 g, 30 mmol, 50%
yield; small quantities of a mono-brominated species may be
detected by NMR). 1H NMR (400 MHz, DMSO-d6): δ 8.25 (q, J
= 0.7 Hz, 1H). HR-ESI-MS Experimental: 274.8459. Calculated
for [C6HBr2N2O]-: 274.8461. Δ = -0.7 ppm.
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General procedure for the synthesis of 4-24
Purified or unpurified benzyl protected precursor compound (4a-
24a) were taken up in a 5:5:1 mixture of concentrated
HCl:HOAc:TFA (22 mL total volume) and was heated with stirring
to 95 C for 36-48 h. Reaction progress was monitored by TLC
and staining with a solution of FeCl3 (with the resulting deprotected
pyridinone compounds staining with FeCl3 as a pink-to-red spot on
TLC plates). After full deprotection solvents were removed under
high vacuum and the resulting solids were co-evaporated several
times with MeOH. The remaining solids were then purified by C18
chromatography eluting in a H2O/MeOH system to yield the target
compounds as off-white to lightly pink solids.
4-(Benzyloxy)-6-bromo-3-hydroxypicolinonitrile (3).
In a
flame-dried flask NaH (2.5 g, 63 mmol) was stirred in dry DMSO
(20 mL, dried on molecular sieves). Benzyl alcohol (9.4 mL, 90
mmol) was added dropwise to the mixture, pausing when excessive
foam formed. When the reaction was complete, as evidenced by
the cessation of hydrogen evolution, compound 2 (5 g, 18 mmol)
in dry DMSO (10 mL) was added dropwise to the stirring solution
and the mixture was heated to 60 C for 18 h, monitoring
conversion by TLC. When the reaction was complete, the mixture
was placed in an ice bath and 150 mL water was added. The
mixture continued to stir for 10 min, during which time the solution
clouded and then clarified. To remove residual benzyl alcohol, the
aqueous solution was extracted with 225 mL portions of diethyl
ether. Residual ether was then removed from the aqueous portion
under vacuum and the solution was cooled to 5 C on ice. The
addition of 4M HCl (until pH ~2) resulted in the precipitation of a
white solid. Note, it is imperative to maintain ice bath temperature
during acidification; otherwise, excess heat or residual ether
present in the solution will result in the formation of a gummy,
orange oil in lieu of off-white crystallites. If an oil forms, it can be
slowly dissolved in basic (pH >10) water with sonication to repeat
the precipitation. The acidified mixture was stirred for 30 min on
ice and the resultant solids were filtered, rinsed with H2O, and
3-Hydroxy-4-oxo-6-phenyl-1,4-dihydropyridine-2-carboxylic
acid (4). Compound 4 was prepared according to the general
procedure and was isolated as a pink solid in 68% yield. 1H NMR
(400 MHz, DMSO-d6): δ 7.81-7.73 (m, 2H), 7.48 (dd, J = 4.1, 2.3
Hz, 3H), 7.31 (s, 1H). 13C NMR (126 MHz, DMSO-d6): δ 166.8,
151.5, 145.2, 134.0, 130.4, 129.0, 128.4, 127.6, 112.0. HR-ESI-
MS Experimental: 230.0458. Calculated for [C12H8NO4]-:
230.0459. Δ = -1.4 ppm.
3-Hydroxy-4-oxo-6-(p-tolyl)-1,4-dihydropyridine-2-carboxylic
acid (5). Compound 5 was prepared according to the general
procedure and was isolated as a white solid in 62% yield. H NMR
(400 MHz, DMSO-d6): δ 7.67 (d, J = 8.1 Hz, 2H), 7.34 – 7.26 (m,
3H), 2.36 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 166.5,
161.4, 151.6, 144.9, 140.3, 130.9, 129.6, 128.3, 127.3,
111.7, 21.4. HR-ESI-MS Experimental: 244.0615. Calculated
for [C13H10NO4]-: 244.0615. Δ = 0.0 ppm.
1
3-Hydroxy-4-oxo-6-(m-tolyl)-1,4-dihydropyridine-2-
carboxylic acid (6). Compound 6 was prepared according to the
general procedure and was isolated as a light-pink solid in 68%
yield. 1H NMR (400 MHz, DMSO-d6): δ 7.60 (s, 1H), 7.54 (d, J
= 7.5 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.30 (d, J = 4.4 Hz, 2H),
2.37 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 166.5, 161.2,
151.7, 145.0, 138.3, 133.65, 131.1, 129.0, 127.4, 125.5, 111.9,
21.4. HR-ESI-MS Experimental: 244.0615. Calculated for
[C13H10NO4]-: 244.0615. Δ = 0.0 ppm.
dried.
Purification via silica gel chromatography in an
EtOAc/hexanes system afforded the desired product 3 as an off-
white solid (4.2 g, 13.7 mmol, 76% yield). 1H NMR (400 MHz,
acetone-d6): δ 10.38 (s, 1H), 7.51 (d, J = 6.5 Hz, 2H), 7.47 – 7.35
(m, 4H), 5.30 (s, 2H). HR-ESI-MS Experimental: 302.9771.
Calculated for [C13H8BrN2O2]-: 302.9775. Δ = -1.3 ppm.
3-Hydroxy-4-oxo-6-(o-tolyl)-1,4-dihydropyridine-2-carboxylic
acid (7). Compound 7 was prepared according to the general
procedure and was isolated as a white solid in 57% yield. H NMR
(400 MHz, DMSO-d6): δ 7.39 (t, J = 7.9 Hz, 1H), 7.35 – 7.24 (m,
3H), 6.98 (s, 1H), 2.18 (s, 3H). 13C NMR (126 MHz, DMSO-d6):
δ 165.1, 162.4, 152.2, 144.5, 136.7, 130.6, 130.2, 130.1, 126.8,
General procedure for the synthesis of 4a-24a
1
Compound 3 (250 mg, 0.8 mmol), SPhos (35 mg, 0.08 mmol),
K2CO3 (340 mg, 2.4 mmol), and desired boronic acid (1.6 mmol)
were dissolved in a 3:1 solution of dioxane:H2O (20 mL). This
solution was de gassed under reduced pressure with stirring for 15
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126.1, 113.94, 20.0. HR-ESI-MS Experimental: 244.0614.
prepared according to the general procedure and was isolated as a
1
2
3
4
5
6
7
8
Calculated for [C13H10NO4]-: 244.0615. Δ = -0.4 ppm.
dark pink solid in 55% yield. 1H NMR (500 MHz, DMSO-d6): δ
7.83 (d, J = 7.5 Hz, 1H), 7.72 (dd, J = 12.2, 7.5 Hz, 2H), 7.58 (d, J
= 7.2 Hz, 1H), 7.01 (s, 1H). 13C NMR (126 MHz, DMSO-d6): δ
165.8, 161.6, 152.4, 142.4, 132.9, 132.6, 132.4, 130.6, 127.8 (q, J
= 30.1 Hz, 1C), 127.1, 126.4 (q, J = 4.8 Hz, 1C), 124.2 (q, J=272.7
Hz, 1C), 114.1. HR-ESI-MS Experimental: 298.0329. Calculated
for [C13H7F3NO4]-: 298.0333. Δ = -1.3 ppm.
3-Hydroxy-6-(3-isopropylphenyl)-4-oxo-1,4-dihydropyridine-
2-carboxylic acid (8). Compound 8 was prepared according to the
general procedure and was isolated as a white solid in 51% yield.
1H NMR (400 MHz, DMSO-d6): δ 7.64 (s, 1H), 7.58 – 7.51 (m,
1H), 7.45 – 7.34 (m, 2H), 7.31 (s, 1H), 2.97 (s, 1H), 1.24 (dd, J =
6.9, 1.6 Hz, 6H). 13C NMR (126 MHz, DMSO-d6): δ 166.4, 161.4,
151.7, 149.2, 145.2, 133.6, 129.1, 128.5, 127.4, 126.6, 125.9,
112.04, 33.9, 24.2. HR-ESI-MS Experimental: 272.0927.
Calculated for [C15H14NO4]-: 272.0928. Δ = -0.4 ppm.
3-Hydroxy-6-(2-methoxyphenyl)-4-oxo-1,4-dihydropyridine-2-
carboxylic acid (15). Compound 15 was prepared according to the
general procedure and was isolated as a white solid in 39% yield.
1H NMR (400 MHz, DMSO-d6): δ 7.58 (d, J = 7.7 Hz, 1H), 7.51
(t, J = 8.4 Hz, 1H), 7.27 (s, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.09 (t, J
= 7.6 Hz, 1H), 3.87 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ
164.9, 162.7, 157.3, 151.9, 141.0, 132.7, 130.6, 125.9, 121.4,
120.43, 112.8, 112.6, 56.6. HR-ESI-MS Experimental: 260.0563.
Calculated for [C13H10NO5]-: 260.0564. Δ = -0.4 ppm.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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3-Hydroxy-6-(2-isopropylphenyl)-4-oxo-1,4-dihydropyridine-
2-carboxylic acid (9). Compound 9 was prepared according to the
general procedure and was isolated as a white solid in 56% yield.
1H NMR (400 MHz, CDCl3): δ 7.36 (m, 1H), 7.18 (dd, J = 6.4, 1.5
Hz, 2H), 6.98 (s, 1H), 2.97 (dq, J = 13.3, 6.7 Hz, 1H), 1.19 – 1.04
(m, 6H). 13C NMR (126 MHz, DMSO-d6): δ 165.2, 162.4, 152.2,
147.1, 144.6, 132.8, 130.5, 130.1, 126.7, 125.9, 125.8, 114.1, 30.2,
24.2. HR-ESI-MS Experimental: 272.0926. Calculated for
[C15H14NO4]-: 272.0928. Δ = -0.7 ppm.
3-Hydroxy-6-(2-(methylthio)phenyl)-4-oxo-1,4-
dihydropyridine-2-carboxylic acid (16). Compound 16 was
prepared according to the general procedure and was isolated as a
1
white solid in 56% yield. H NMR (400 MHz, DMSO-d6): δ 7.48
3-Hydroxy-4-oxo-6-(4-phenoxyphenyl)-1,4-dihydropyridine-2-
(dd, J = 11.3, 3.7 Hz, 1H), 7.41 (d, J = 7.9 Hz, 1H), 7.36 – 7.32 (m,
1H), 7.26 (t, J = 7.4 Hz, 1H), 7.02 (s, 1H), 2.41 (s, 3H). 13C NMR
(126 MHz, DMSO-d6): δ 165.3, 161.8, 152.5, 143.3, 138.2, 132.9,
130.9, 130.6, 127.0, 126.6, 125.2, 114.2, 16.0. HR-ESI-MS
carboxylic acid (10). Compound 10 was prepared according to the
1
general procedure and was isolated as a pink solid in 61% yield. H
NMR (400 MHz, DMSO-d6): δ 7.79 (d, J = 8.7 Hz, 2H), 7.42 (t, J
= 7.9 Hz, 2H), 7.27 (s, 1H), 7.19 (t, J = 7.4 Hz, 1H), 7.07 (dd, J =
8.1, 3.8 Hz, 4H). 13C NMR (126 MHz, DMSO-d6): δ 166.9, 160.9,
158.9, 156.3, 151.3, 144.7, 130.7, 130.3, 129.0, 127.5, 124.6,
119.8, 118.4, 111.6. HR-ESI-MS Experimental: 322.0715.
Calculated for [C18H12NO5]-: 322.0721. Δ = -1.9 ppm.
Experimental:
276.0333.
Calculated for [C13H10NO4S]-:
276.0336. Δ = -1.1 ppm.
6-(4-Carboxyphenyl)-3-hydroxy-4-oxo-1,4-dihydropyridine-2-
carboxylic acid (17). Compound 17 was prepared according to the
1
general procedure and was isolated as a pink solid in 22% yield. H
3-Hydroxy-4-oxo-6-(3-phenoxyphenyl)-1,4-dihydropyridine-2-
carboxylic acid (11). Compound 11 was prepared according to the
general procedure and was isolated as a white solid in 67% yield.
1H NMR (400 MHz, DMSO-d6): δ 7.84 (s, 1H), 7.61 – 7.35 (m,
5H), 7.18 – 6.93 (m, 4H). 13C NMR (126 MHz, DMSO-d6): δ
167.9, 157.0, 157.0, 151.2, 144.6, 137.2, 130.8, 130.6,
124.1, 123.9, 123.0, 120.1, 119.1, 119.0, 118.4, 111.9. HR-
ESI-MS Experimental: 322.0720. Calculated for [C18H12NO5]-:
322.0721. Δ = -0.3 ppm.
NMR (400 MHz, DMSO-d6): δ 7.98 (q, J = 8.4 Hz, 4H), 7.32 (s,
1H). 13C NMR (126 MHz, DMSO-d6): δ 168.7, 167.4, 159.0,
151.0, 144.7, 139.9, 131.5, 129.9, 129.3, 127.8, 112.0. HR-ESI-
MS Experimental: 274.0356. Calculated for [C13H8NO6]-:
274.0357. Δ = -0.4 ppm.
6-(3-Carboxyphenyl)-3-hydroxy-4-oxo-1,4-dihydropyridine-2-
carboxylic acid (18). Compound 18 was prepared according to the
1
general procedure and was isolated as a pink solid in 43% yield. H
NMR (400 MHz, DMSO-d6): δ 8.33 (s, 1H), 8.03 (dd, J = 16.0, 7.8
Hz, 2H), 7.60 (t, J = 7.8 Hz, 1H), 7.37 (s, 1H). 13C NMR (126
MHz, DMSO-d6): δ 167.8, 167.4, 159.7, 151.1, 144.8, 135.4,
132.3, 131.4, 130.6, 129.4, 129.0, 128.6, 112.0. HR-ESI-MS
Experimental: 274.0359. Calculated for [C13H8NO6]-: 274.0357.
Δ = 0.8 ppm.
3-Hydroxy-4-oxo-6-(2-phenoxyphenyl)-1,4-dihydropyridine-2-
carboxylic acid (12). Compound 12 was prepared according to the
general procedure and was isolated as a white solid in 54% yield.
1H NMR (400 MHz, DMSO-d6): δ 7.64 (d, J = 7.7 Hz, 1H), 7.50
(t, J = 7.8 Hz, 1H), 7.34 (t, J = 7.8 Hz, 2H), 7.27 (t, J = 7.5 Hz, 1H),
7.21 (s, 1H), 7.10 (t, J = 7.4 Hz, 1H), 7.01 – 6.95 (m, 3H). 13C
NMR (126 MHz, DMSO-d6): δ 165.3, 161.9, 156.4, 154.4, 152.0,
140.8, 132.4, 131.8, 130.5, 126.8, 124.8, 124.3, 124.2, 119.3,
119.0, 113.8. HR-ESI-MS Experimental: 322.0717. Calculated
for [C18H12NO5]-: 322.0721. Δ = -1.2 ppm.
6-(4-(1H-tetrazol-5-yl)phenyl)-3-hydroxy-4-oxo-1,4-
dihydropyridine-2-carboxylic acid (19). Compound 19 was
prepared according to the general procedure and was isolated as a
1
white solid in 31% yield. H NMR (400 MHz, DMSO-d6): δ 8.11
(q, J = 8.2 Hz, 4H), 7.45 (s, 1H). 13C NMR (126 MHz, DMSO-d6):
δ 168.6, 158.9, 151.0, 144.7, 138.2, 129.0, 128.6, 127.5, 125.2,
112.0, 40.9. HR-ESI-MS Experimental: 320.0398. Calculated for
[C13H7N5O4Na]-: 320.0401. Δ = -0.9 ppm.
6-(2-Ethylphenyl)-3-hydroxy-4-oxo-1,4-dihydropyridine-2-
carboxylic acid (13). Compound 13 was prepared according to the
general procedure and was isolated as a light pink solid in 52%
1
yield. H NMR (400 MHz, DMSO-d6): δ 7.46 – 7.39 (m, 1H), 7.34
6-(3-(1H-tetrazol-5-yl)phenyl)-3-hydroxy-4-oxo-1,4-
(d, J = 7.5 Hz, 1H), 7.30 – 7.26 (m, 2H), 6.98 (s, 1H), 2.52 – 2.46
(m, 2H), 1.01 (t, J = 7.6 Hz, 3H). 13C NMR (126 MHz, DMSO-
d6): δ 165.2, 162.2, 152.2, 144.6, 142.6, 133.2, 130.4, 130.2, 128.8,
126.8, 126.0, 114.0, 26.0, 15.5. HR-ESI-MS Experimental:
258.0771. Calculated for [C14H12NO4]-: 258.0772. Δ = -0.4 ppm.
dihydropyridine-2-carboxylic acid (20). Compound 20 was
prepared according to the general procedure and was isolated as a
light pink solid in 38% yield. 1H NMR (500 MHz, DMSO-d6): δ
8.51 (d, J = 25.9 Hz, 1H), 8.23 – 7.99 (m, 2H), 7.78 (d, J = 28.4
Hz, 1H), 7.46 (s, 1H). HR-ESI-MS Experimental: 320.0397.
Calculated for [C13H7N5O4Na]-: 320.0401. Δ = -1.3 ppm.
3-Hydroxy-4-oxo-6-(2-(trifluoromethyl)phenyl)-1,4-
dihydropyridine-2-carboxylic acid (14). Compound 14 was
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Journal of Medicinal Chemistry
6-(4-Carboxy-2-methylphenyl)-3-hydroxy-4-oxo-1,4-
dihydropyridine-2-carboxylic acid (21). Compound 21 was
(10 mL). The reaction mixture was heated to 125 C for 20 min in
1
2
3
4
5
6
7
8
a microwave reactor. After heating, the solvent was removed under
vacuum and the residue was taken up in 2M HCl and EtOAc. The
layers were separated and the aqueous phase was extracted once
with EtOAc. The combined organic fractions were dried and
concentrated to yield a brown oil, which was purified by silica
chromatography (EtOAc:hexanes) to produce 26 as a white solid in
89% yield. (1.1 g, 4.5 mmol). 1H NMR (500 MHz, CD3OD): δ
7.83 (d, J = 7.7 Hz, 1H), 7.52 (s, 1H), 7.49 (d, J = 7.7 Hz, 1H), 2.56
(s, 3H), 1.37 (s, 12H). HR-ESI-MS Experimental: 266.1326.
Calculated for [C14H18BNO2Na]+: 266.1325. Δ = 0.4 ppm.
prepared according to the general procedure and was isolated as a
1
white solid in 21% yield. H NMR (400 MHz, DMSO-d6): δ 7.85
(s, 1H), 7.81 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 7.7 Hz, 1H), 6.98 (s,
1H), 2.24 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 167.4,
165.5, 162.2, 152.3, 149.0, 143.7, 138.1, 137.3, 132.0, 131.2,
130.5, 126.8, 113.8, 19.9. HR-ESI-MS Experimental: 288.0512.
Calculated for [C14H10NO6]-: 288.0514. Δ = -0.7 ppm.
3-Hydroxy-6-(2-methyl-4-(1H-tetrazol-5-yl)phenyl)-4-oxo-1,4-
dihydropyridine-2-carboxylic acid (22). Compound 22 was
prepared according to the general procedure and was isolated as a
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
5-(3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)-1H-tetrazole (27). Compound 26 (2.0 g, 8.2 mmol),
NH4Cl (1.32 g, 24.6 mmol), and NaN3 (0.80 g, 12.3 mmol) were
combined in DMF (15 mL) and stirred. The reaction mixture was
heated to 110 C for 3 h. Upon cooling, the mixture was poured
into 150 mL of cool water (note: maintain the concentration of
azide in solution <5%) and was acidified to pH 2 with 4M HCl. A
white precipitate formed and the solution was stirred in an ice bath
for 30 min, after which time the solids were collected by filtration
and dried to produce 27 as a white solid in 93% yield (2.2 g, 7.6
mmol). 1H NMR (400 MHz, DMSO-d6): δ 7.85 (s, 1H), 7.80 (t, J
= 6.6 Hz, 2H), 2.54 (s, 3H), 1.30 (s, 12H). HR-ESI-MS
1
white solid in 24% yield. H NMR (400 MHz, DMSO-d6): δ 7.98
(s, 1H), 7.92 (d, J = 8.0 Hz, 1H), 7.55 (d, J = 7.9 Hz, 1H), 6.97 (s,
1H), 2.30 (s, 3H). 13C NMR (126 MHz, DMSO-d6): δ 165.7,
163.0, 155.6, 152.3, 143.6, 138.2, 136.6, 131.3, 128.9,
126.9, 125.7, 124.6, 114.0, 20.1. HR-ESI-MS Experimental:
312.0735. Calculated for [C14H10N5O4]-: 312.0738. Δ = -1.0 ppm.
3-Hydroxy-6-(2-(trifluoromethyl)-4-(1H-tetrazol-5-yl)phenyl)-
4-oxo-1,4-dihydropyridine-2-carboxylic acid (23). Compound
23 was prepared according to the general procedure and was
1
isolated as a white solid in 29% yield. H NMR (400 MHz, DMSO-
Experimental:
287.1673.
Calculated for [C14H20BN4O2]+:
d6): δ 8.45 (s, 1H), 8.39 (d, J = 8.0 Hz, 1H), 7.85 (d, J = 8.0 Hz,
1H), 7.10 (s, 1H). 13C NMR (126 MHz, DMSO-d6): δ 166.8,
162.7, 156.5, 152.2, 141.9, 135.4, 133.5, 130.2, 128.6 (q, J = 30.4
Hz, 1C), 124.2 (q, J = 5.0 Hz, 1C), 123.9 (q, J = 274.3 Hz, 1C),
114.1, 66.8. HR-ESI-MS Experimental: 366.0450. Calculated for
[C14H7F3N5O4]-: 366.0456. Δ = -1.6 ppm.
287.1676. Δ = -1.0 ppm.
4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-3-
(trifluoromethyl)benzonitrile (28). In a microwave vessel were
combined 4-bromo-3-(trifluoromethyl)benzonitrile (2.6 g, 10.4
mmol), bis(pinacolato)diboron (3.96 g, 15.6 mmol), potassium
acetate (2.55 g, 26.0 mmol), and Pd(dppf)Cl2 (0.85 g, 1.0 mmol) in
dry DMF (15 mL). The reaction mixture was heated to 125 C in
a microwave reactor for 25 min. After heating, the solvent was
removed under vacuum and the residue was taken up in 2M HCl
and EtOAc. The layers were separated and the aqueous phase was
extracted once with EtOAc. Combined organic fractions were
dried and concentrated to yield a brown oil, which was purified by
silica chromatography (EtOAc:hexanes) to produce 28 as a white
3-Hydroxy-6-(2-methyl-4-(5-oxo-4,5-dihydro-1,2,4-oxadiazol-
3-yl)phenyl)-4-oxo-1,4-dihydropyridine-2-carboxylic acid (24).
Compound 24 was prepared according to the general procedure and
was isolated as a white solid in 36% yield. 1H NMR (400 MHz,
DMSO-d6): δ 8.31 (s, 1H), 8.19 (s, 1H), 7.74 (s, 1H), 7.69 (d, J =
7.8 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 7.09 (s, 1H), 2.40 (s, 3H).
13C NMR (126 MHz, DMSO-d6): δ 172.5, 160.4, 160.4, 157.6,
154.2, 148.8, 147.7, 143.3, 137.4, 130.7, 130.6, 128.5, 124.0,
123.0, 115.0, 20.8. HR-ESI-MS Experimental: 328.0571.
Calculated for [C15H10N3O6]-: 328.0575. Δ = -1.2 ppm.
1
solid in 66% yield (2.1 g, 6.8 mmol). H NMR (400 MHz, CDCl3):
δ 7.93 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H), 7.79 (d, J = 7.7 Hz, 1H),
1.37 (s, 12H).
3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzoic acid (25). In a 30 mL microwave vessel were combined
5-(4-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-3-
4-bromo-3-methylbenzoic
acid
(1.0
g,
4.6
mmol),
(trifluoromethyl)phenyl)-1H-tetrazole (29). Compound 28 (2.0
g, 6.7 mmol), NH4Cl (1.08 g, 20.2 mmol), and NaN3 (0.66 g, 10.1
mmol) were combined in DMF (15 mL) and stirred. The reaction
mixture was heated to 110 C for 3 h. Upon cooling, the mixture
was poured into 150 mL cool water (note: maintain the
concentration of azide in solution <5%) and was acidified to pH 2
with 4M HCl. A white precipitate formed and the solution was
stirred in an ice bath for 30 min, after which time the solids were
collected by filtration and dried to produce 29 as a white solid in
bis(pinacolato)diboron (1.77 g, 6.9 mmol), potassium acetate (1.14
g, 11.6 mmol), and Pd(dppf)Cl2 (0.38 g, 0.46 mmol) in dry DMF
(10 mL). The reaction mixture was heated to 125 C for 20 min in
a microwave reactor. After heating, the solvent was removed under
vacuum and the residue was taken up in 2M HCl and EtOAc. The
layers were separated and the aqueous phase was extracted once
with EtOAc. Combined organic fractions were dried and
concentrated to yield a brown oil, which was purified by silica
chromatography (EtOAc:hexanes) to produce 25 as a white solid in
74% yield (900 mg, 3.4 mmol). Evidence of hydrolysis products
of the boronic ester were observed in the NMR spectrum but were
found to still be useful in the subsequent coupling reaction. 1H
NMR (500 MHz, CD3OD): δ 7.80 (s, 1H), 7.77 (d, J = 0.96 Hz,
2H), 2.55 (s, 3H), 1.36 (s, 12H). HR-ESI-MS Experimental:
261.1308. Calculated for [C14H18BO4]-: 261.1306. Δ = 0.8 ppm.
1
94% yield (2.15 g, 6.3 mmol). H NMR (400 MHz, DMSO-d6): δ
8.37 (s, 1H), 8.32 (d, J = 7.7 Hz, 1H), 7.95 (d, J = 7.7 Hz, 1H), 1.32
(d, J = 3.0 Hz, 12H). HR-ESI-MS Experimental: 341.1398.
Calculated for [C14H17BF3N4O2]+: 341.1394. Δ = 1.2 ppm.
4-Bromo-N'-hydroxy-3-methylbenzimidamide (30).
To a
solution of 4-bromo-3-methylbenzonitrile (2.0 g, 10.2 mmol) in
EtOH (20 mL) was added hydroxylamine hydrochloride (1.42 g,
20.4 mmol), and TEA (2.8 mL, 20.4 mmol) with stirring. The
reaction mixture was heated to reflux for 3 h, after which time
solvent was removed under vacuum and the residue was taken up
in 1M HCl. The acidic solution was washed with 40 mL CH2Cl2
3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzonitrile (26). In a microwave vessel were combined 4-
bromo-3-methylbenzonitrile
(1.0
g,
5.1
mmol),
bis(pinacolato)diboron (1.94 g, 7.6 mmol), potassium acetate (1.25
g, 12.7 mmol), and Pd(dppf)Cl2 (0.41 g, 0.51 mmol) in dry DMF
9
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Journal of Medicinal Chemistry
Page 10 of 13
and cooled in an ice bath with stirring. The pH of the cooled
fluorescent signal, but had an IC50 detection limit of ~15 nM. Using
1
2
3
4
5
6
7
aqueous solution was then adjusted to >10 with 6M NaOH and a
white precipitate formed. The basic solution stirred on ice for 30
min and the resultant solids were isolated by filtration to give 30 as
an enzyme concentration of 5 nM, the detection limit was observed
to be ~2 nM; however, the change in fluorescent signal was
significantly weaker than that from an enzyme concentration of 25
nM. These detection limits prompted the use of thermal shift assay
to distinguish between the highly potent inhibitors reported herein.
1
a white solid in 77% yield (1.8 g, 7.9 mmol). H NMR (400 MHz,
DMSO-d6): δ 9.65 (d, J = 6.1 Hz, 1H), 7.61 (s, 1H), 7.55 – 7.50
(m, 1H), 7.38 (dd, J = 5.5, 2.8 Hz, 1H), 5.80 (s, 2H), 2.32 (d, J =
5.5 Hz, 3H). HR-ESI-MS Experimental: 228.9971. Calculated for
[C8H10BrN2O]+: 228.9971. Δ = 0.0 ppm.
Thermal Shift Assay (DSF)
To each well of
a 96-well 0.2 mL optical MicroAmp
(ThermoFisher) thermocycler plate was added 9.5 µL of buffer
(150 mM NaCl, 2 mM MnCl2, 20 mM HEPES pH 7.5), 4 µL of
PAN endonuclease in buffer (250 µg/mL), 4 µL of inhibitor in
buffer (1 mM; 2% DMSO), and 2.5 µL of 8  SYPRO orange
Thermal Shift® dye (ThermoFisher) in buffer. This results in a final
well volume of 20 µL containing final concentrations of 1 µg PAN
endonuclease, 200 µM inhibitor, and 1 dye in buffer with 0.4%
DMSO. The presence of this small concentration of DMSO was
found to have a negligible effect on ΔTM values of native PAN
endonuclease. Thermocycler plate wells were sealed prior to
analysis, and the plate was then heated in a thermocycler from 25
C to 99 C at a ramp rate of 0.05 C/sec. Fluorescence was read
using the ROX filter channel (x = 580 nm; em = 623 nm) and
fluorescence signal was fitted to a first derivative curve to identify
TM. Native PAN endonuclease was generally observed to melt with
a TM = 58-60 C. All assayed compounds were observed to cause
either no significant ΔTM or a positive ΔTM.
8
9
3-(4-Bromo-3-methylphenyl)-1,2,4-oxadiazol-5(4H)-one (31).
To a stirring solution of compound 30 (1.0 g, 4.4 mmol) and TEA
(0.91 mL, 6.5 mmol) in DMF (10 mL) was added ethyl
chloroformate (0.46 mL, 4.8 mmol) dropwise at room temperature.
The reaction mixture stirred for 30 min, after which time the
mixture was heated to 130 C for 1.5 h. After heating, the DMF
solution was poured into 125 mL of cool water and 25 mL 1M
NaOH. The aqueous solution was washed once with CH2Cl2 and
was cooled in an ice bath with stirring. The basic solution was then
acidified with 4M HCl to pH <2. A white solid formed and the
solution stirred for 15 min. The solid was then isolated by filtration
to produce 31 as a white solid in 63% yield (700 mg, 2.7 mmol).
1H NMR (400 MHz, DMSO-d6): δ 7.69 (s, 1H), 7.53 (d, J = 8.3
Hz, 1H), 7.46 (d, J = 8.2 Hz, 1H), 2.32 (s, 3H). HR-ESI-MS
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Experimental:
252.9618.
Calculated for [C9H6BrN2O2]-:
252.9618. Δ = 0.0 ppm.
3-(3-Methyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)phenyl)-1,2,4-oxadiazol-5(4H)-one (32). In 30 mL
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website at DOI:
a
microwave vessel were combined compound 31 (500 mg, 1.96
mmol), bis(pinacolato)diboron (750 mg, 2.95 mmol), potassium
acetate (480 mg, 8.1 mmol), and Pd(dppf)Cl2 (200 mg, 0.25 mmol)
in dry DMF (10 mL). The reaction mixture was heated to 125 C
for 20 min in a microwave reactor. The solvent was then
evaporated under vacuum and the residue was taken up in 2M HCl
and EtOAc. The layers were separated and the aqueous phase was
extracted once with EtOAc. The combined organic fractions were
dried and concentrated to yield a brown oil, which was purified by
silica chromatography (EtOAc:hexanes) to produce 32 as a white
solid in 59% yield (350 mg, 1.16 mmol). Evidence of hydrolysis
products of the boronic ester were observed in the NMR spectrum
but were found to still be useful in the subsequent coupling
reaction. 1H NMR (400 MHz, CD3OD): δ 7.81 (d, J = 7.7 Hz,
1H)), 7.59 – 7.47 (m, 2H)), 2.54 (s, 3H), 1.35 (s, 12H). HR-ESI-
MS Experimental: 301.1366. Calculated for [C15H18BN2O4]-:
301.1368. Δ = -0.7 ppm.
Synthesis of boronic esters; protein expression and
purification; assay data of PAN endonuclease control
inhibitors; protein crystallography; cross-inhibition
assays; cell-based assays; NMR spectra of select lead
inhibitors.
SMILES strings for all compounds (PDF)
SMILES strings formula (CSV)
PDB file of 1 modeled in PAN (PDB)
PDB file of 7 modeled in PAN (PDB)
PDB file of 13 modeled in PAN (PDB)
PDB file of 18 modeled in PAN (PDB)
PDB file of 20 modeled in PAN (PDB)
PDB file of 22 modeled in PAN (PDB)
PDB file of 24 modeled in PAN (PDB)
PDB file of 40 modeled in PAN (PDB)
PAN Enzymatic Nuclease Activity Assay
PAN endonuclease activity assays were carried out as previously
reported.27 Briefly, assay were performed using Black Costar 96-
well plates. Each well contained a total volume of 100 μL
comprised of: buffer (20 mM Tris, 150 mM NaCl, 2 mM MnCl2,
10 mM β-mercaptoethanol, 0.2% Triton-X100, pH 8.0), influenza
PAN endonuclease (5 or 25 nM), inhibitor (various concentrations)
Corresponding Author
*scohen@ucsd.edu, chenyao@nankai.edu.cn
Notes
S.M.C. is a co-founder, has an equity interest, and receives
income as member of the Scientific Advisory Board for Cleave
Biosciences and is a co-founder, has an equity interest, and a
member of the Scientific Advisory Board for Forge Therapeutics.
Both companies may potentially benefit from the research results
of certain projects in the laboratory of S.M.C. The terms of this
arrangement have been reviewed and approved by the University
of California, San Diego in accordance with its conflict of interest
policies.
in buffer, and fluorescent ssDNA-oligo substrate (200 nM).
single-stranded, 17-mer DNA oligonucleotide labeled with a 5′-
FAM fluorophore and 3′-TAMRA quencher ([6-
A
a
FAM]AATCGCAGGCAGCACTC[TAM], Sigma-Aldrich) was
employed as the substrate. Upon addition of the substrate, the
change in fluorescence was measured over 45 min at 37 C (ex
=
485 nm; em = 528 nm). The gain was set to 100 and the first 15
min of data were excluded from the activity calculations. Dose-
response curves were generated, fitted, and analyzed using
Origin16 graphing software. For most experiments, the enzyme
concentration was 25 nM, which afforded a strong change in
ACCESSION CODES
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Journal of Medicinal Chemistry
PDB ID code for protein structures of PAN endonuclease
J. Crystal Structures of Oseltamivir-Resistant Influenza Virus
1
2
3
4
5
6
7
8
(compound co-crystallized with protein given in parentheses):
6E6V (compound 1), 6E3N (compound 7), 6E3O (compound 13),
6E3M (compound 18), 6E3P (compound 20), 6E6W (compound
22), 6E4C (compound 24), 6E6X (compound 40). Authors will
release the atomic coordinates and experimental data upon article
publication.
Neuraminidase Mutants. Nature 2008, 453, 1258-1261.
(10) Moscona, A. Global Transmission of Oseltamivir-Resistant
Influenza. N. Engl. J. Med. 2009, 360, 953-956.
(11) Heo, Y. A. Baloxavir: First Global Approval. Drugs 2018, 78,
693-697.
(12) Huang, T. S.; Palese, P.; Krystal, M. Determination of
Influenza-Virus Proteins Required for Genome Replication. J.
Virol. 1990, 64, 5669-5673.
(13) Fodor, E. The RNA Polymerase of Influenza A Virus:
Mechanisms of Viral Transcription and Replication. Acta Virol.
2013, 57, 113-122.
(14) Resa-Infante, P.; Jorba, N.; Coloma, R.; Ortin, J. The
Influenza Virus RNA Synthesis Machine: Advances in its Structure
and Function. RNA Biol. 2011, 8, 207-215.
(15) Hutchinson, E. C.; Fodor, E. Transport of the Influenza Virus
Genome From Nucleus to Nucleus. Viruses 2013, 5, 2424-2446.
(16) Plotch, S. J.; Bouloy, M.; Ulmanen, I.; Krug, R. M. A Unique
Cap(m7GpppXm)-Dependent Influenza Virion Endonuclease
Cleaves Capped RNAs to Generate the Primers that Initiate Viral
RNA Transcription. Cell 1981, 23, 847-858.
(17) Dias, A.; Bouvier, D.; Crepin, T.; McCarthy, A. A.; Hart, D.
J.; Baudin, F.; Cusack, S.; Ruigrok, R. W. The Cap-Snatching
Endonuclease of Influenza Virus Polymerase Resides in the PA
Subunit. Nature 2009, 458, 914-918.
(18) Yuan, P. W.; Bartlam, M.; Lou, Z. Y.; Chen, S. D.; Zhou, J.;
He, X. J.; Lv, Z. Y.; Ge, R. W.; Li, X. M.; Deng, T.; Fodor, E.; Rao,
Z. H.; Liu, Y. F. Crystal Structure of an Avian Influenza
Polymerase PA(N) Reveals an Endonuclease Active Site. Nature
2009, 458, 909-913.
(19) Guilligay, D.; Tarendeau, F.; Resa-Infante, P.; Coloma, R.;
Crepin, T.; Sehr, P.; Lewis, J.; Ruigrok, R. W. H.; Ortin, J.; Hart,
D. J.; Cusack, S. The Structural Basis for Cap Binding by Influenza
Virus Polymerase Subunit PB2. Nat. Struct. Mol. Biol. 2008, 15,
500-506.
(20) Crepin, T.; Dias, A.; Palencia, A.; Swale, C.; Cusack, S.;
Ruigrok, R. W. H. Mutational and Metal Binding Analysis of the
Endonuclease Domain of the Influenza Virus Polymerase PA
Subunit. J. Virol. 2010, 84, 9096-9104.
(21) Rao, P.; Yuan, W. M.; Krug, R. M. Crucial Role of CA
Cleavage Sites in the Cap-Snatching Mechanism for Initiating
Viral mRNA Synthesis. Embo J. 2003, 22, 1188-1198.
(22) Credille, C. V.; Dick, B. L.; Morrison, C. N.; Stokes, R. W.;
Adamek, R. N.; Wu, N. C.; Wilson, I. A.; Cohen, S. M. Structure-
Activity Relationships in Metal-Binding Pharmacophores for
Influenza Endonuclease. J. Med. Chem. 2018, 61, 10206-10217.
(23) Bauman, B. M.; Slavish, P. J.; DuBois, R. M.; Boyd, V. A.;
White, S. W.; Webb, T. R. Identification of Influenza Endonuclease
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Chem Biol 2012, 7, 526-534.
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X. Y.; Zhan, P. Inhibitors of Influenza Virus Polymerase Acidic
(PA) Endonuclease: Contemporary Developments and
Perspectives. J. Med. Chem. 2017, 60, 3533-3551.
(25) Rogolino, D.; Carcelli, M.; Sechi, M.; Neamati, N. Viral
Enzymes Containing Magnesium: Metal Binding as a Successful
Strategy in Drug Design. Coordin. Chem. Rev. 2012, 256, 3063-
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Xiang, A.; Parhi, A. K.; Martinez-Sobrido, L.; LaVoie, E. J.; Das,
K.; Arnold, E. Crystallographic Fragment Screening and Structure-
Based Optimization Yields a New Class of Influenza Endonuclease
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ABBREVIATIONS
ArgI, arginase I; DPBA, 2,4-dioxo-4-phenylbutanoic acid; DSF,
differential scanning fluorimetry; EGCG, epigallocatechin gallate;
FBDD, fragment-based drug discovery; FDA, U.S. Food and Drug
Administration; hCA, human carbonic anhydrase; LE, ligand
efficiency; MBP, metal-binding pharmacophore; MetAP,
methionine aminopeptidase; MMP, matrix metalloproteinase; PAN,
N-terminal domain of the polymerase acidic (PA) subunit of the
RNA-dependent RNA polymerase complex from the influenza A
virus; PSA, polar surface area; SAR, structure-activity relationship;
SBDD, structure-based drug design;
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ACKNOWLEDGMENT
The authors acknowledge Dr. Yongxuan Su (U.C. San Diego,
Molecular Mass Spectrometry Facility) for aid with HR-MS and
HPLC compound purity analysis; Prof. Carlo Ballatore (U.C. San
Diego) for access to a preparative HPLC system; and Dr. Curtis
Moore and Dr. Milan Gembicky (UC San Diego X-ray Facility),
the Prof. J. P. Noel (Salk Institute), and the staff at beam lines 5.0.1
and 5.0.2 at the Advanced Light Source for assistance with X-ray
crystallography studies. This work was supported by grants from
the National Institutes of Health (R01 GM098435 to S.M.C.; F32
GM125233 to C.N.M.), and by the University of California
President’s Postdoctoral Fellowship (to C.N.M.). B.L.D. was
supported, in part, by the National Institute of Health Molecular
Biophysics Training Grant (T32GM008326-26). R.W.S. was
supported, in part, by the Graduate Research Fellowship Program
(GRFP) from the National Science Foundation.
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