Discovery, X-ray Crystallography and Antiviral Activity of Allosteric Inhibitors of Flavivirus NS2B-NS3 Protease

Article abstract of DOI:10.1021/jacs.9b02505

Flaviviruses, including dengue, West Nile and recently emerged Zika virus, are important human pathogens, but there are no drugs to prevent or treat these viral infections. The highly conserved Flavivirus NS2B-NS3 protease is essential for viral replication and therefore a drug target. Compound screening followed by medicinal chemistry yielded a series of drug-like, broadly active inhibitors of Flavivirus proteases with IC₅₀ as low as 120 nM. The inhibitor exhibited significant antiviral activities in cells (EC₆₈: 300-600 nM) and in a mouse model of Zika virus infection. X-ray studies reveal that the inhibitors bind to an allosteric, mostly hydrophobic pocket of dengue NS3 and hold the protease in an open, catalytically inactive conformation. The inhibitors and their binding structures would be useful for rational drug development targeting Zika, dengue and other Flaviviruses.

Full text of DOI:10.1021/jacs.9b02505

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Cite This: J. Am. Chem. Soc. XXXX, XXX, XXX−XXX
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Discovery, X‑ray Crystallography and Antiviral Activity of Allosteric
Inhibitors of Flavivirus NS2B-NS3 Protease
†
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†,⊥
†,⊥
†,⊥
†
,†
†
Yuan Yao, Tong Huo, Yi-Lun Lin, Shenyou Nie, Fangrui Wu, Yuanda Hua, Jingyu Wu,
‡
‡,§
‡
Alexander R. Kneubehl, Megan B. Vogt, Rebecca Rico-Hesse, and Yongcheng Song*
†
‡
§
Department of Pharmacology and Chemical Biology, Department of Molecular Virology and Microbiology, Integrative Molecular
and Biomedical Sciences Graduate Program, Baylor College of Medicine, 1 Baylor Plaza, Houston, Texas 77030, United States
*
S Supporting Information
ZIKV/DENV contain a single-stranded, positive-sense
RNA with ∼10,800 nucleotides, encoding a viral polyprotein.
The polyprotein is site-specifically cleaved by the viral NS2B-
NS3 protease and several host proteases to produce
ABSTRACT: Flaviviruses, including dengue, West Nile
and recently emerged Zika virus, are important human
pathogens, but there are no drugs to prevent or treat these
viral infections. The highly conserved Flavivirus NS2B-
NS3 protease is essential for viral replication and therefore
a drug target. Compound screening followed by medicinal
chemistry yielded a series of drug-like, broadly active
inhibitors of Flavivirus proteases with IC50 as low as 120
nM. The inhibitor exhibited significant antiviral activities
1
,3
functional proteins (Supporting Information Figure S1).
The NS2B-NS3 protease is essential for viral replication and,
1
,9,10
therefore, a promising drug target.
A number of peptide-
based covalent inhibitors of Flavivirus proteases have been
1
,11,12
reported,
but they did not demonstrate significant
antiviral activities in cells or animal models due to low cell
permeability and metabolic stability. Nonpeptidic inhibitors
have also been reported, but their inhibitory activities are
relatively weak and how these compounds bind to the
in cells (EC : 300−600 nM) and in a mouse model of
6
8
Zika virus infection. X-ray studies reveal that the inhibitors
bind to an allosteric, mostly hydrophobic pocket of
dengue NS3 and hold the protease in an open,
catalytically inactive conformation. The inhibitors and
their binding structures would be useful for rational drug
development targeting Zika, dengue and other Flavivi-
ruses.
1
,13,14
protease is unknown.
Homology analysis showed Flavivirus proteases are evolu-
tionally conserved (Figure S2) and highly stable (Figure S3).
NS3 contains an N-terminal serine protease domain, but
complexation with NS2B is required to become an active
11,15−17
18−20
enzyme. Previous X-ray
and NMR
studies show
the protease can adopt a “closed” or “open” conformation. In
the closed state that is catalytically active, NS2B is fully tied
around NS3 (Figure S4), and becomes part of the active site.
In the open and inactive conformation, NS2B is partially
bound to NS3 and far from the active site.
engue (DENV), West Nile and recently emerged Zika
D
(ZIKV) viruses belong to the genus Flavivirus in the
Flaviviridae family of RNA viruses. These viruses are
transmitted primarily by Aedes mosquitos. Four serotypes of
DENV infect ∼400 million people each year with 100 million
developing dengue fever. ∼500,000 cases develop serious
dengue hemorrhagic fever, causing ∼22,000 deaths each
11
21
We produced a Gly -Ser-Gly linked and binary form of
4
4
recombinant ZIKV protease (ZVpro), containing NS2B (47−
95) and NS3 (1−170). ∼1,200 compounds in our laboratory
that were synthesized targeting histone modifying enzymes
1
year. Moreover, patients recovered from one serotype are
2
2
still susceptible to other serotypes with an increased
likelihood of a more severe disease due to existing
including lysine specific demethylase 1 (LSD1) were
screened against the linked-ZVpro. Compounds 1 and 2
were identified to be novel inhibitors with IC50 of 21.7 and
3.1 μM (Table 1).
2
antibodies. ZIKV has caused three major outbreaks in
Pacific Ocean islands (2007 and 2013), Brazil and other
American countries (2015−2016), in which >1 million
infections were reported and a large number of patients
Scheme 1 shows the general synthesis for medicinal
chemistry studies. 6-Chloro-2-aminopyrazine (10) was
selectively iodized using N-iodosuccinimide, and the 2-
amino group was converted to a hydroxyl, which was
alkylated using a Mitsunobu reaction to give 12 with a
protected piperidin-4-yl-methoxy group. Two selective Suzuki
reactions were performed to introduce different aryl groups
3
sought medical treatment. More seriously, ZIKV infection
has been correlated with a 20-fold increased incidence of
serious neurological disorders, including Guillain-Barre
syndrome and >4,000 cases of microcephaly in newborns.
5,6́
4
Since 2015, ZIKV has quickly spread to 48 pan-American
countries. Recently, ZIKV was found to be transmitted
5
6
R and R to produce, after deprotection, compounds 3−5, 7
7
and 9. Monosubstitution of 1,6-dibromo-pyridine or -pyrazine
through sex or body fluids. Despite these serious outcomes
(
15) with (N-Boc-piperidin-4-yl)methylamine, followed by
as well as possible future outbreaks, there have been no
antiviral drugs to prevent or treat ZIKV and DENV
infections. A licensed dengue vaccine, Dengvaxia, has raised
concerns about efficacy and increased risk of severe disease
iodination produced the intermediate 16. Selective replace-
Received: March 6, 2019
8
for seronegative people during clinical trials.
©
XXXX American Chemical Society
A
DOI: 10.1021/jacs.9b02505
J. Am. Chem. Soc. XXXX, XXX, XXX−XXX

Journal of the American Chemical Society
Communication
Table 1. Structures and Activity of Compounds 1−9
Cpd
R5
4-Br-Ph
R6
4-Br-Ph
4-t-Bu-Ph
Indol-5-yl
4-(furan-3-yl)-Ph
4-(pyrazol-4-yl)-Ph
4-(furan-3-yl)-Ph
4-(furan-3-yl)-Ph
4-(furan-3-yl)-Ph
4-(furan-3-yl)-Ph
linked-ZVpro IC50 (μM)
ZIKV-FLR EC68 (μM)
a
1
2
3
4
5
6
7
8
9
21.7 ± 1.5
3.14 ± 0.09
1.12 ± 0.07
1.05 ± 0.07
0.71 ± 0.07
0.79 ± 0.03
0.53 ± 0.06
0.40 ± 0.05
0.20 ± 0.01
>10
4-t-Bu-Ph
5.0
2.50
2.50
2.50
1.20
1.20
1.20
0.30−0.60
4-(NH CH )-Ph
2
2
4-(NH CO)-Ph
2
4-(NH CH )-Ph
2
2
4-(NH CH )-Ph
2
2
4-(MeNHCH )-Ph
2
4-(NH CH )-Ph
2
2
4-(NH CH )-Ph
2
2
a
EC68 cannot be determined because 1 showed cytotoxicity at >10 μM. All other compounds had no significant cytotoxicity.
a
Scheme 1. General Synthesis for Compounds 2−9
Table 2. IC50 (μM) of 3-9 against Flavivirus Proteases
linked-ZVpro
DV2pro
DV3pro
WVpro
3
4
5
6
7
8
9
1.12 ± 0.07
1.05 ± 0.07
0.71 ± 0.07
0.79 ± 0.03
0.53 ± 0.06
0.40 ± 0.05
0.20 ± 0.01
0.64 ± 0.03
0.98 ± 0.02
0.21 ± 0.04
0.86 ± 0.01
0.73 ± 0.02
0.29 ± 0.06
0.59 ± 0.02
0.54 ± 0.01
0.93 ± 0.02
0.20 ± 0.07
0.86 ± 0.02
0.53 ± 0.01
0.21 ± 0.07
0.52 ± 0.06
0.93 ± 0.03
1.34 ± 0.01
0.12 ± 0.02
1.27 ± 0.03
0.87 ± 0.02
0.51 ± 0.04
0.78 ± 0.02
results show 9 is a broadly active inhibitor of Flavivirus
proteases with a high selectivity.
We determined X-ray structures of DV2pro in complex
with compounds 5, 8 and 9 at 2.7−3.0 Å. Statistics for
diffraction data and structure refinement are shown in Table
S3. The three structures are very similar to each other, with
each asymmetric unit containing two inhibitor-bound
proteins (Figure 1a−c, Figure S6 and S7). Similar to the
a
Reagents and conditions: (i) N-Iodosuccinimide, DMSO; (ii)
NaNO , H SO (Conc.); (iii) N-Boc-piperidin-4-ylmethanol, PPh ,
diisopropyl azodicarboxylate, THF; (iv) R -boronic acid, Pd(PPh ) ,
16
2
2
4
3
5
apoprotein, the DV2pro-inhibitor complexes adopt an open
3
4
6
conformation, with NS2B binding partially to NS3 (Figure
S8a). The inhibitor-bound NS3 does not deviate significantly
from the apo- or substrate-bound protein, except that the
residues 152−164 are disordered (with no observed electron
density) upon inhibitor binding. In contrast, the U-shaped
peptide segment is well organized in both the apo- and
substrate-bound NS3 (Figure S8b,c). In the latter case,
residues 152−164 constitute part of the S1 and S2 pockets of
Na CO , 1,4-dioxane-H O, 80 °C; (v) R -boronic acid, Pd(PPh ) ,
2
3
2
3 4
Na CO , 1,4-dioxane-H O, 110 °C; (vi) 4 M HCl, CH Cl , 0 °C;
2
3
2
2
2
(vii) (N-Boc-piperidin-4-yl)methylamine, K CO , DMF, 100 °C.
2 3
ment of the 5-iodo and 6-bromo substituent using a Suzuki
reaction gave compound 6 or 8.
Compound 9 was found to be a potent inhibitor of the
linked- and binary-ZVpro with IC50 of 200 and 220 nM,
respectively (Figure S5). Tables 1 and S1 summarize the
inhibitory activities of selected analogs 3-8. Changing the
15
the active site and have interactions with the substrate.
Inhibitor binding pushes the loops 71−75 and 117−122
outward by ∼1.3 and 3 Å (Figure S8d). All of these
movements remodel the surface of NS3 and create a deep, L-
shaped pocket (Figures 1d and S8e) that accommodates the
inhibitors. The compounds are allosteric inhibitors, which do
not occupy the substrate binding site (Figure 1e).
Mechanistically, these inhibitors bind to and stabilize
DV2pro in the open conformation, which prevents NS2B
from folding into the active site as well as the binding of the
substrate.
−
O− linkage at 2-position to an −NH− in 8 (IC : 400 nM)
5
0
resulted in a 2-fold activity reduction. Changing the central
pyrazine ring in 8 to a pyridine in 6 (IC : 790 nM) further
reduced the potency. As compared with 7 (IC : 530 nM)
with a N-methyl secondary amine or 4 (IC : 1.1 μM) with
an amide at the 5-position, the primary amine in 9 is more
favored. Changing the furan-3-yl group in 9 to a pyrazol-4-yl
in 5 (IC : 710 nM) or a fused pyrrole ring in 3 (IC : 1.1
50
50
50
5
0
50
μM) also decrease the inhibitory activity.
Compounds 3−9 also inhibited DENV serotype-2, -3, and
The inhibitor-protein interactions are illustrated in Figures
1d,f and S9. The central pyrazine ring of 9 is located at the
junction of the L-shaped pocket. The furanylphenyl group is
deeply inserted into the pocket with favorable hydrophobic
interactions. The 2- and 5-substituents occupy a deep surface
groove, having mostly hydrophobic interactions. The
15,16
West Nile protease (DV2pro, DV3pro and WVpro)
with
IC50 values of 120−1340 nM (Table 2). However, 9
exhibited negligible activities against several human serine-,
cysteine-, aspartic- and metallo-proteases (Table S2). These
B
DOI: 10.1021/jacs.9b02505
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Journal of the American Chemical Society
Communication
Figure 1. X-ray structure of the DV2pro-9 complex. (A) The overall structure of DV2pro in complex with compound 9 (ball and stick model
with C atoms in brown). NS3 is shown in green and NS2B in purple; (B) The 2F -F electron density map of DV2pro-9 at the inhibitor
o
c
binding site (contoured at 1σ) and (C) the Fo-Fc omit map (at 3σ); (D) Compound 9 is located in an L-shaped, deep pocket of NS3 (shown
as an electrostatic surface) that is mostly hydrophobic; (E) Aligned structures of DV2pro-9 with the substrate-bound DV3pro (PDB: 3U1I, NS3
in cyan and NS2B in yellow), showing 9 occupies a different binding site from the substrate (tube model with C atoms in black); (F)
Interactions between compound 9 and DV2pro.
Figure 2. Cellular and in vivo antiviral activities of compound 9. (A−C) Treatment of U87 cells with 9 caused dose-dependent reduction of
(A) RNA copies of ZIKV (FLR strain), (B) infectious ZIKV (FLR strain) and (C) infectious ZIKV (HN16 strain). (D) Treatment with
compound 9 significantly reduced ZIKV RNA in plasma (left) and brains (right) of ZIKV-infected mice. (E) Treatment with compound 9
significantly prolonged the survival of ZIKV-infected mice.
positively charged −NH2 of 9 has hydrogen-bond and
electrostatic interactions with Asp75, one of the protease
catalytic triad.
High structural and sequence similarities between DV2pro
and ZVpro, particularly for the inhibitor-interacting residues
(Figure S10a,b), suggest compound 9 binds to ZVpro
C
DOI: 10.1021/jacs.9b02505
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Journal of the American Chemical Society
Communication
similarly. Enzyme kinetics studies showed that 9 is a
noncompetitive inhibitor of DV2pro (Figure S10c), con-
sistent with its X-ray structure. Similar enzyme kinetics results
support 9 is also an allosteric inhibitor of ZVpro (Figure
S10d).
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jacs.9b02505.
■
*
S
Experimental details (PDF)
Anti-ZIKV activity of 9 was evaluated in U87 glioma
2
3,24
25
cells.
The passage-3 stock of ZIKV FLR strain was used
AUTHOR INFORMATION
Corresponding Author
ysong@bcm.edu
ORCID
Yongcheng Song: 0000-0003-2611-2476
Author Contributions
These authors contributed equally.
for clinical relevance. Upon infection with ZIKV-FLR, U87
cells were incubated with 9 for 48 h. Newly generated ZIKV
viruses in the media were determined quantitatively.
Compound 9 significantly reduced ZIKV RNA in a dose-
■
*
4
dependent manner (Figure 2a). Because ∼1/10 of RNAs
25
represent infectious viruses, an end-point dilution assay was
used to determine viral titers more accurately. Half-log
⊥
(
0.32×) serial dilutions of the media were added to Vero
Notes
cells in quadruplicate. Upon incubation for 7 days, ZIKV
infection in each sample was determined with cytopathic
effects. TCID50 (tissue culture infective dose) was calculated
based on the highest dilution in which ≥50% of the
quadruplicate samples were infected with ZIKV. As shown
in Figure 2b for a representative experiment, compound 9
reduced infectious ZIKV viruses by 68% at 300 nM, 90% at
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the staff at the beamline 19-ID/-BM, Structural
Biology Center at the Advanced Photon Source for assistance
in X-ray data collection. This work was supported by a grant
(
W81XWH-18-1-0368) from USAMRAA of the U.S. Depart-
6
00 nM, 97% at 1.2 μM, 99% at 2.5 μM, and 99.7% at 5 μM.
ment of Defense and grants (RP150129 and RP180177) from
Cancer Prevention and Research Institute of Texas to Y.S.,
NIH grants R01AI099483 and R01AI098715 to R.R.-H.
Multiple experiments showed that EC68 of 9 was 300 or 600
nM. Compound 9 exhibited similar antiviral activities against
26
ZIKV HN16 strain (Figure 2c). 9 also showed significant
27
activity against DENV-2 (strain K0049), inhibiting viral
replication in Vero cells by 97% at 5 μM. These results
demonstrate compound 9 has potent cellular antiviral activity
against Zika and dengue viruses. Moreover, cellular antiviral
activities of compounds 1−9 are generally correlated with
their biochemical activities against ZVpro (Table 1).
Compound treatment also dose-dependently inhibited the
viral proteins capsid, NS3 and NS5 in infected Vero cells
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DOI: 10.1021/jacs.9b02505
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