Quinoxaline-Based Linear HCV NS3/4A Protease Inhibitors Exhibit Potent Activity against Drug Resistant Variants

Article abstract of DOI:10.1021/acsmedchemlett.8b00150

A series of linear HCV NS3/4A protease inhibitors was designed by eliminating the P2-P4 macrocyclic linker in grazoprevir, which, in addition to conferring conformational flexibility, allowed structure-activity relationship (SAR) exploration of diverse quinoxalines at the P2 position. Biochemical and replicon data indicated preference for small hydrophobic groups at the 3-position of P2 quinoxaline for maintaining potency against resistant variants R155K, A156T, and D168A/V. The linear inhibitors, though generally less potent than the corresponding macrocyclic analogues, were relatively easier to synthesize and less susceptible to drug resistance. Three inhibitor cocrystal structures bound to wild-type NS3/4A protease revealed a conformation with subtle changes in the binding of P2 quinoxaline, depending on the 3-position substituent, likely impacting both inhibitor potency and resistance profile. The SAR and structural analysis highlight inhibitor features that strengthen interactions of the P2 moiety with the catalytic triad residues, providing valuable insights to improve potency against resistant variants.

Full text of DOI:10.1021/acsmedchemlett.8b00150

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Letter
Quinoxaline-Based Linear HCV NS3/4A Protease Inhibitors
Exhibit Potent Activity Against Drug Resistant Variants
Linah N Rusere, Ashley N. Matthew, Gordon J Lockbaum, Muhammad Jahangir, Alicia Newton,
Christos J. Petropoulos, Wei Huang, Nese Kurt Yilmaz, Celia A. Schiffer, and Akbar Ali
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00150 • Publication Date (Web): 17 May 2018
Downloaded from http://pubs.acs.org on May 18, 2018
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ACS Medicinal Chemistry Letters
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Quinoxaline-Based Linear HCV NS3/4A Protease Inhibitors Exhibit
Potent Activity Against Drug Resistant Variants
†
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Linah N. Rusere, Ashley N. Matthew, Gordon J. Lockbaum, Muhammad Jahangir, Alicia Newton,
‡
‡
†
†,
†,
Christos J. Petropoulos, Wei Huang, Nese Kurt Yilmaz, Celia A. Schiffer *, and Akbar Ali *
†
Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Mas-
‡
sachusetts 01605, United States Monogram Biosciences, South San Francisco, California 94080, United States
Keywords: HCV; NS3/4A protease; protease inhibitor; quinoxaline; crystal structure; drug resistance
ABSTRACT: A series of linear HCV NS3/4A protease inhibitors was designed by eliminating the P2–P4 macrocyclic linker in
grazoprevir, which, in addition to conferring conformational flexibility, allowed SAR exploration of diverse quinoxalines at the P2
position. Biochemical and replicon data indicated preference for small hydrophobic groups at the 3-position of P2 quinoxaline for
maintaining potency against resistant variants R155K, A156T and D168A/V. The linear inhibitors, though generally less potent
than the corresponding macrocyclic analogues, were relatively easier to synthesize and less susceptible to drug resistance. Three
inhibitor co-crystal structures bound to wild-type NS3/4A protease revealed a conformation with subtle changes in the binding of
P2 quinoxaline, depending on the 3-position substituent, likely impacting both inhibitor potency and resistance profile. The SAR
and structural analysis highlight inhibitor features that strengthen interactions of the P2 moiety with the catalytic triad residues,
providing valuable insights to improve potency against resistant variants.
Hepatitis C virus (HCV) is the leading cause of liver diseas-
es such as cirrhosis, fibrosis and liver cancer. The most recent
estimates indicate that at least 70 million people worldwide
have chronic HCV infection, and about 1.75 million new cases
1
are recorded each year. The treatment of HCV infection has
been challenging due to high genetic diversity of the virus and
a lack of antiviral drugs that effectively target all HCV geno-
2
types. Fortunately, recent advances in direct-acting antivirals
(DAAs) targeting the HCV NS3/4A, NS5A and NS5B pro-
teins have significantly improved sustained virological re-
3
sponse (SVR) in patients across genotypes. However, the
high genetic diversity of HCV and the rapid emergence of
drug resistance necessitate the use of combination therapies
with two or three drugs from different classes to effectively
treat infected patients.
HCV NS3/4A protease inhibitors (PIs) are a key component
3
of new all-oral combination therapies. The five FDA ap-
proved PIs are all macrocyclic acylsulfonamides with different
4
Figure 1. Structures of the HCV NS3/4A protease inhibitors gra-
zoprevir (1), P1–P3 macrocyclic analogue 5172-mcP1P3 (2),
linear analogue 5172-linear (3), and SAR compounds.
heterocyclic moieties at the P2 position. Of these, paritaprevir
5
,6
and grazoprevir are active against most HCV genotypes,
while the recently approved glecaprevir and voxilaprevir ex-
7
,8
hibit pan-genotypic activity. These new PIs are also signifi-
cantly more active against most polymorphic and drug re-
pending on the heterocyclic moiety at the P2 position and the
12,13
location of the macrocycle.
Grazoprevir (1) (Figure 1) was
9
sistant HCV variants. The development of NS3/4A PIs with
the first NS3/4A PI to show activity against most HCV geno-
pan-genotypic activity and improved resistance profiles marks
a major milestone in anti-HCV drug discovery.
types and resistant variants due to its unique binding confor-
12,14
mation.
The P2 quinoxaline moiety in 1 largely avoids
Despite remarkable improvements in potency and resistance
profiles, the current NS3/4A PIs are still susceptible to drug
direct interactions with residues Arg155 and Asp168, but in-
1
2
stead interacts with the catalytic His57 and Asp81. As a re-
sult, 1 maintains potency against substitutions at Arg155 and
is only moderately susceptible to substitutions at Asp168.
However, the P2–P4 macrocycle in 1 causes steric clashes
with larger substitutions at Ala156, resulting in drastically
resistance due to substitutions at one or more amino acid posi-
10,11
tions in the target protein.
The most common resistance-
associated substitutions (RASs) in the NS3/4A protease occur
at residues Arg155, Ala156, and Asp168. Inhibitors show var-
ying susceptibility to RASs at these and other positions de-
1
2
reduced potency. Both glecaprevir and voxilaprevir are also
ACS Paragon Plus Environment

ACS Medicinal Chemistry Letters
P2–P4 macrocyclic compounds structurally similar to 1 and
Page 2 of 7
resistant variants R155K and D168A (Table 1). For a subset
of compounds, cellular antiviral potencies (EC50) were deter-
mined using replicon-based antiviral assays against WT HCV
and resistant variants R155K, A156T, D168A, and D168V
(Table 2). Grazoprevir (1) was used as a control in all assays.
1
2
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5
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are expected to bind in similar conformations, as indicated by
their particularly high susceptibility to substitutions at
9,15
Ala156.
These three PIs currently in clinical use indeed
exhibit similar resistance profiles and thus are prone to cross-
resistance. Therefore, more robust NS3/4A PIs need to be
developed, preferably incorporating diverse structural features.
Scheme 1. Synthesis of HCV NS3/4A protease inhibitorsa
We have shown that the unique binding conformation of 1
does not depend on the P2–P4 macrocycle, as the P1−P3 mac-
rocyclic and linear analogues, 5172-mcP1P3 (2) and 5172-
linear (3) (Figure 1), still bind in a similar conformation as the
0
1
2
3
4
5
6
7
8
9
0
1
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1
6
parent compound. In the absence of the P2–P4 macrocyclic
constraint, the conformationally flexible P2 quinoxaline moie-
ty can allow the inhibitor to better adapt to structural changes
in the protease active site due to RASs, particularly at Ala156.
Accordingly, compared to 1, the P1–P3 macrocyclic analogue
2 exhibits similar potency against RASs at Arg155 and
Asp168 but is significantly less susceptible to substitutions at
1
3
Ala156, resulting in an overall improved resistance profile.
In addition to conferring conformational flexibility, elimina-
tion of the P2–P4 macrocycle in 1 provides opportunities to
further improve potency and resistance profile by exploring
structure-activity relationship (SAR) at the 3-position of P2
quinoxaline in both the P1–P3 macrocyclic (2) and linear (3)
analogues. Toward this end, we recently developed a substrate
envelope guided design strategy that aims to exploit interac-
tions with the invariant catalytic triad, minimize interactions
with the S2 subsite residues and incorporate conformational
flexibility at the P2 moiety, which led to the identification of
P1–P3 macrocyclic analogues of 2 with exceptional potency
aReagents and conditions: (a) 4 N HCl in dioxane, CH
3 h; (b) Boc-Tle-OH, HATU, DIEA, DMF, RT, 4 h; (c) Li-
OH.H O, THF, H O, RT, 24 h; (d) HATU, DIEA, DMF, RT, 2 h;
e) 4 N HCl in dioxane, RT, 3 h; (f) N-(cyclopentyloxy-
carbonyloxy)-succinimide, DIEA, CH CN, RT, 36 h.
2
2
Cl , RT,
1
7
and resistance profiles. These exciting findings encouraged
us to explore SAR of linear compound 3 to identify analogues
with improved potency against drug resistant HCV variants. In
this letter, we report the structure-guided design, synthesis,
and SAR studies of a series of quinoxaline-based linear
NS3/4A PIs. In addition, we report co-crystal structures of
three linear inhibitors with different P2 quinoxaline moieties
bound to NS3/4A protease, providing valuable structural in-
sights to further optimize potency and resistance profiles.
2
2
(
3
Compared to the macrocyclic PIs 1 and 2, the linear ana-
logue 3 exhibited significantly lower potency against WT pro-
tease (K = 19 nM) and experienced an even larger reduction in
i
1
3
antiviral potency (EC50 = 24 nM), as reported previously.
Compound 3 was also less potent than 1 and 2 against the re-
sistant variants R155K, D168A and D168V in both enzyme
inhibition and replicon assays. The significant potency losses
for the linear inhibitor 3 are likely due to the increase in con-
formational flexibility and associated entropic penalty of bind-
ing to the protease. However, close examination of the overall
resistance profile revealed that fold losses in potency were
generally lower for compound 3 than 1 in both enzyme inhibi-
tion and replicon assays (Tables S1 and 2). Moreover, while 1
was highly susceptible to the A156T variant (EC50 = 200 nM),
with >1600-fold loss in potency compared to WT, compound
The synthesis of linear NS3/4A PIs with diverse P2 quinox-
aline moieties is outlined in Scheme 1. The key Boc-protected
P2 intermediates 4a-e were prepared from the corresponding
3
-substituted 7-methoxy-quinoxalin-2-ones by an S
N
2 dis-
placement reaction with the activated cis-hydroxyproline de-
1
7
rivative as described previously. Deprotection of the Boc
group and reaction with N-Boc-L-tert-leucine under HATU
coupling conditions gave the P2–P3 intermediates 5a-e. After
ester hydrolysis, the resulting acids 6a-e were coupled with the
P1–P1' acylsulfonamide intermediates 7 and 8 to afford the
target inhibitors 3, 9b-e and 10a-e. The tert-butyl-capped
compounds were converted to the corresponding cyclopentyl
derivatives in two steps, involving Boc deprotection and reac-
tion with N-(cyclopentyloxycarbonyloxy)-succinimide, to
provide the desired compounds 11a-e and 12a-e.
3
showed better antiviral potency against this variant (EC50
=
7
3 nM). The reduced susceptibility to RASs, particularly at
Ala156, observed for 3 demonstrates that removal of the mac-
rocyclic linker and the resulting conformational flexibility
allows the inhibitor to adapt to substitutions in the S2 subsite.
To improve potency, analogues of inhibitor 3 with modifi-
cations at the P1' and P4 capping groups were prepared and
tested. Replacement of the P1' cyclopropylsulfonamide with a
more hydrophobic 1-methylcyclopropyl-sulfonamide moiety
generally improved potency of the resulting analogues. Thus,
compared to 3, analogue 10a afforded a slight increase in en-
zyme potency against WT protease and resistant variants
R155K and D168A. Similarly, replacing the tert-butyl P4 cap-
The SAR exploration of 3 was focused on optimizing inter-
actions of the P2 quinoxaline moiety and minimizing direct
interactions with S2 subsite residues. In addition, modifica-
2
3
tions at the P1' (R ) and the N-terminal capping groups (R )
were also investigated (Figure 1). The potency and resistance
profiles of the resulting linear PIs were evaluated using bio-
chemical and replicon assays. The enzyme inhibition constants
i
(K ) were determined against WT GT1a NS3/4A protease and
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ACS Medicinal Chemistry Letters
ping group with a bulkier cyclopentyl moiety in 11a provided
P1' position did not show much improvement in enzyme po-
tency compared to 9b. Compounds 11b and 12b with the cy-
clopentyl P4 capping group were slightly more potent than the
corresponding tert-butyl analogues 9b and 10b against WT
and D168A. Furthermore, compounds 11b and 12b showed
nanomolar potency in replicon assays against WT HCV, and
despite losing about 5–20-fold potency, 12b maintained signif-
icant potency against all variants tested. Although both the 3-
ethyl and 3-methyl-quinoxaline compounds showed similar
potencies against WT and R155K protease variants, PIs with
the smaller methyl substituent were generally more potent
against the D168A variant. Together, the enzyme inhibition
and replicon data indicate a preference for smaller substituents
at the 3-position of the P2 quinoxaline to maintain potency
against resistant variants.
1
2
3
4
5
6
7
8
9
compounds with improved potency. Analogue 12a with the 1-
methyl-cyclopropylsulfonamide moiety at P1' and cyclopentyl
P4 capping group was 2- and 4-fold more potent than 3 against
the WT protease (Ki = 6.9 nM) and D168A variant (Ki = 145
nM), respectively. Thus, minor modifications at the P1' and P4
moieties of inhibitor 3 provided analogues with improved po-
tency against WT protease and the D168A variant.
Table 1. Inhibitory activity against wild-type HCV NS3/4A
protease and drug resistant variants
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Next, a larger isopropyl group was incorporated in com-
pounds 9c and 10c to further explore the optimal size of the
substituent at the 3-position of P2 quinoxaline that can be ac-
commodated in the S2 subsite without causing unfavorable
interactions. These compounds displayed considerably lower
potency compared to the 3-methyl- and 3-ethyl-quinoxaline
compounds across all variants in enzyme inhibition assays.
Moreover, compounds with a larger isopropyl group at this
position were highly susceptible to RASs at Arg155 and
Ki (nM)
Compd.
R1
R2
WT
R155K
17 ± 2.3
14 ± 1.1
15 ± 2.2
13 ± 2.7
8.5 ± 2.1
14 ± 1.7
9.6 ± 0.9
10 ± 1.3
49 ± 11
D168A
642 ± 101
385 ± 31
350 ± 30
145 ± 14
290 ± 24
265 ± 26
144 ± 23
140 ± 13
1086 ± 137
1179 ± 170
985 ± 106
1000 ± 80
128 ± 16
119 ± 16
99 ± 10
3
Et
Et
Et
H
Me
H
Me
H
Me
H
Me
H
Me
H
Me
H
Me
H
Me
H
Me
H
19 ± 2.7
16 ± 1.3
9.8 ± 2.0
6.9 ± 0.5
18 ± 1.6
14 ± 2.1
9.2 ± 0.9
7.1 ± 1.1
32 ± 5.1
29 ± 9.4
17 ± 3.2
21 ± 2.6
7.8 ± 1.1
6.1 ± 1.1
3.8 ± 0.6
3.9 ± 0.7
87 ± 18
46 ± 9.6
34 ± 8.3
22 ± 3.4
2.0 ± 0.1
1
1
1
0a
1a
2a
i
Asp168, with K values in the millimolar range against the
D168A protease variant. Analogues 11c and 12c, with a cy-
clopentyl P4 capping group, showed similar trends to the cor-
responding tert-butyl analogues across all protease variants
tested. These findings further support our hypothesis that large
substituents at the 3-position of the P2 quinoxaline are detri-
mental to potency against resistant variants.
Et
9
b
Me
Me
Me
Me
i-Pr
i-Pr
i-Pr
i-Pr
Cl
1
1
0b
1b
12b
9
c
1
1
1
0c
1c
2c
27 ± 5.6
55 ± 11
43 ± 11
After determining optimal size of the substituent at the 3-
position of the P2 quinoxaline, isosteric replacements of the
alkyl group with different electronic properties were explored.
Thus, a set of compounds bearing a 3-chloroquinoxaline P2
moiety, with comparable size to the 3-methylquinoxaline, was
analyzed. In general, compounds with the 3-chloroquinoxaline
were significantly more potent than the corresponding 3-ethyl-
and 3-methyl-quinoxaline analogues. Compounds 9d and 10d,
with a tert-butyl P4 capping group, showed about 2-fold better
potency than the corresponding 3-methylquinoxaline ana-
logues 9b and 10b against WT, R155K and D168A proteases.
Similarly, the cyclopentyl-capped compounds 11d and 12d
9
d
2.2 ± 0.4
3.8 ± 0.6
4.1 ± 0.5
5.2 ± 0.8
24 ± 3.3
12 ± 1.7
26 ± 7.7
22 ± 6.9
3.1 ± 0.34
1
0d
Cl
Cl
Cl
11d
12d
51 ± 6.0
9
e
CF
CF
CF
CF
3
3
3
3
723 ± 80
513 ± 50
703 ± 63
516 ± 61
91 ± 10
1
1
1
0e
1e
2e
Me
2
1
0.20 ± 0.1 0.80 ± 0.3
40 ± 5.0
i
(WT K = 3.8 and 3.9 nM, respectively) were more potent than
the corresponding 11b and 12b, showing excellent potency
against WT protease and resistant variants. In fact, both 11d
We have shown that minimizing inhibitor interactions in the
S2 subsite resulted in an overall improvement in potency and
resistance profiles.1 Although the P2 quinoxaline in 3 largely
avoids direct interactions with residues in the S2 subsite, the
ethyl group at the 3-position of this moiety makes hydropho-
bic interactions with the hydrocarbon portion of the Arg155
side chain as well as with Ala156. Thus, in an effort to opti-
mize interactions with these residues, changes at the 3-position
of P2 quinoxaline moiety were explored. Based on the co-
crystal structures and SAR results from the P1–P3 macrocyclic
and 12d exhibited K
variants in the same range as the macrocyclic inhibitor 2 (WT
= 2.0 nM), indicating that potency of the quinoxaline-based
i
values against WT protease and resistant
7
K
i
linear PIs could be improved significantly by SAR explora-
tion. In replicon assays, the 3-chloroquinoxaline compounds
exhibited the best overall potency profile among the linear
compounds, with PIs 9d, 10d and 12d showing significant
improvement in replicon potency against the multidrug re-
sistant HCV variants D168A/V (EC50 = 12–38 nM). However,
these compounds were more susceptible to the A156T substi-
17
series, replacing the ethyl group with a smaller methyl group
at this position, while reducing overall inhibitor interactions in
the S2 subsite, was expected to maintain key hydrophobic
interactions with side chains of Arg155 and Ala156. As antici-
pated, compound 9b incorporating the 3-methylquinoxaline
was 2-fold more potent than 3 against the R155K and D168A
protease variants in enzyme inhibition assays. However, ana-
logue 10b with the 1-methylcyclopropylsulfonamide moiety at
17
tution than the corresponding macrocyclic analogues. The
improved potency profiles of the 3-chloroquinoxaline com-
pounds compared to the corresponding 3-methylquinoxaline
analogues indicate that the chloro group likely renders more
favorable electronic properties to the quinoxaline moiety,
which improves the critical π-π stacking interactions with cata-
lytic residue His57.
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Page 4 of 7
D81
D81
D81
(a)
(b)
(c)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
H57
H57
H57
R155
R155
D168
R155
D168
D168
S139
S139
S139
A156
A156
A156
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 2. Co-crystal structures of WT1a HCV NS3/4A protease in complex with linear inhibitors (a) 12b, (b) 12c, and (c) 12d. The prote-
ase active site is presented as a light grey surface with bound inhibitors depicted as orange sticks. The catalytic triad is highlighted in yel-
low, and drug resistance residues Arg155, Ala156, and Asp168 are shown as sticks.
Table 2. Antiviral activity against wild-type HCV and drug
resistant variants
with the catalytic triad residues (Figure 2). These structures
confirm that the quinoxaline moiety maintains this unique
binding conformation irrespective of macrocyclization and the
substituent at the 3-position. Inhibitors 12b-d span the S1'–S4
pockets in the active site, with a conserved hydrogen bond
network present in all WT NS3/4A protease structures. The
hydrogen bonds between the P1 amide and the backbone car-
bonyl of Arg155 as well as the P3 amide and Ala157 back-
bone are maintained. The P1' acylsulfonamide moiety is posi-
tioned in the oxyanion hole, stabilized by hydrogen bonds
with His57, Gly137, Ser138 and Ser139. Although the overall
binding mode of linear analogues is similar to that of com-
pound 1, there are subtle changes in the binding of P2 quinox-
aline that may impact inhibitor potency.
Replicon EC50 (nM) (fold change)
Compd.
WT
24
R155K
50 (2.1)
A156T
D168A
D168V
3
73 (3.0) >500 (>21) >500 (>21)
48 (4.8) 164 (16)
28 (6.2) 87 (19)
10 (1.5) 107 (16)
10 (1.6) 100 (16)
40 (5.4) 292 (40)
27 (8.7) 163 (53)
1
1
1b
2b
10
101 (10)
45 (10)
38 (5.8)
30 (4.8)
50 (6.8)
25 (8.1)
6.3 (19)
150 (15)
59 (13)
12 (1.8)
12 (1.9)
54 (7.3)
23 (7.4)
9.1 (28)
5.3 (44)
4.5
6.6
6.3
7.4
3.1
9
d
1
1
0d
1d
12d
2
0.33 1.8 (5.5) 9.7 (29)
0.12
1
1.9 (16) 200 (1667) 11 (92)
The differences between the WT co-crystal structures of 1
and 3-methylquinoxaline inhibitor 12b occur predominantly in
the S2 subsite. Relative to WT-1, the Asp168 side chain in the
WT-12b structure is shifted to allow additional hydrogen
bonding with the side chain of Arg155 (Figures 2A and 3A).
This conformation of Asp168, which allows the P4 cyclopen-
tyl capping group to occupy the S4 pocket, is observed in most
To further investigate the effect of electron-withdrawing
groups on the activity of the inhibitors, derivatives with a
more electronegative, although relatively larger, 3-
trifluoromethylquinoxaline were examined. In contrast to the
3
-chloroquinoxaline inhibitors, compounds 9e and 10e showed
considerable loss in potency against WT protease. However,
despite relatively lower potency against WT, the 3-
trifluoromethylquinoxaline analogues were slightly more po-
tent than the corresponding 3-isopropylquinoxaline PIs against
the resistant variants R155K and D168A. Similar trends were
observed for inhibitors 11e and 12e with the cyclopentyl P4
capping groups. While it is difficult to separate the effects of
electronic properties of the chloro- and trifluoromethyl-
quinoxaline moieties, it is likely that size played a more im-
portant role in determining the overall potency profile of these
inhibitors.
1
2,17
WT
protease-inhibitor
complexes.
The
3-
methylquinoxaline moiety is shifted away from the catalytic
residues toward the S2 subsite relative to the conformation of
P2 quinoxaline in WT-1 structure. This shift was also ob-
served in the parent compound 3, though to a lesser extent,
likely to accommodate the larger ethyl group at the 3-position
of quinoxaline (Figure S1). However, despite larger shift of
the 3-methylquinoxaline moiety, inhibitor 12b has an im-
proved potency profile against resistant variants compared to
3
, likely due to weaker contacts of the smaller methyl group
In an effort to explain the observed potency and resistance
profiles, crystal structures of three linear PIs incorporating
different P2 quinoxaline moieties were determined in complex
with WT NS3/4A protease (Table S2). The crystal structures
of co-complexes WT-12b, -12c and -12d were compared with
with residues in the S2 subsite that mutate to confer resistance.
Thus, while a slight shift of the P2 quinoxaline toward the S2
subsite does not appear to affect the overall potency profile,
the substituent at the 3-position of this moiety significantly
impacts inhibitor potency against resistant variants.
our previously determined structures of 1 and 3 (PDB IDs
12,16
The shift of the quinoxaline moiety toward the S2 subsite
residues was also observed in the WT-12c and WT-12d com-
plexes (Figure 3B–C), as well as in WT-2 and other structures
of the P1–P3 macrocyclic analogues. However, the crystal
structure of inhibitor 12c with the 3-isopropylquinoxaline re-
vealed an additional rearrangement of the P2 moiety. Com-
pared to 1, the P2 quinoxaline in 12c, with a larger isopropyl
3
(
SUD and 5EQQ, respectively).
These high-resolution
1.78–1.80 Å) structures provided details of protein-inhibitor
interactions to elucidate the structural differences that underlie
varied potency and susceptibility to resistant variants.
1
7
The overall binding mode of linear inhibitors 12b-d is simi-
lar to that of macrocyclic inhibitors 1–2 and the parent com-
pound 3, where the P2 quinoxaline predominately interacts
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(
(aa ))
(b)
(c)
1
2
3
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9
D81
D81
D81
R155
R155
R155
H57
H57
H57
D168
D168
D168
A156
S/A139
A156
S/A139
A156
S/A139
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0
H57
H57
H57
D81
D81
D81
R155
R155
R155
A156
A156
A156
S/A139
S/A139
S/A139
D168
D168
D168
Figure 3. Superposition of WT-1 and (a) WT-12b, (b) WT-12c, and (c) WT-12d complexes, focusing on the differences at the P2 quinoxa-
line. The protease is in ribbon representation (light grey), with bound inhibitors 1 (blue) and 12b-d (orange) depicted as sticks. The side
chains of catalytic triad and drug resistance residues Arg155, Ala156, and Asp168 are shown as ball and sticks.
substituent, packs less against the catalytic His57 and moves
toward the solvent exposed surface of the binding pocket
(Figure 3B). This movement of the quinoxaline away from
the catalytic His57 is not observed in the inhibitor complexes
with smaller substituents at the 3-position (Figure 3A and
C). Interestingly, this conformation is reminiscent of the
conformation of 1 when bound to the A156T protease variant
PDB ID 3SUG) (Figure S2), where the larger threonine resi-
due causes steric clash with the P2–P4 macrocycle.
accommodate the larger side chain in the A156T protease,
inhibitor 1 undergoes a rearrangement resulting in the shift of
the P2 quinoxaline moiety toward the solvent exposed binding
surface, weakening the critical π–π interactions with the cata-
lytic His57 (Figure S2A). This altered conformation of 1 re-
shift of the P2 quinoxaline moiety toward the S2 subsite. In-
terestingly, comparison of the WT-12b and WT-12d structures
(Figure 3C) did not reveal any noticeable difference in bind-
ing poses for the 3-methyl- and 3-chloro-quinoxaline ana-
logues that could explain the disparity in inhibitory activity.
The electronic effects of the chloro group appear to improve
stacking interactions of the quinoxaline moiety with the cata-
lytic residue His57, which are crucial for the binding of inhibi-
tors with a P2 quinoxaline moiety. Thus, enhancing interac-
tions with the catalytic triad residues by modifying the inhibi-
tor P2 quinoxaline moiety is likely to improve overall binding
energy and potency profiles.
3
(
1
2,16
To
In summary, we have investigated the SAR of quinoxaline-
based linear HCV NS3/4A PIs using a structure-guided design
strategy to improve potency against resistant variants. Co-
crystal structures of three inhibitors with different P2 moieties
bound to WT protease revealed the structural basis for the
observed potency and resistance profiles. Inhibitors with small
substituents at the 3-position of the P2 quinoxaline were pre-
ferred for maintaining potency against resistant variants due to
decreased interactions with the S2 subsite residues. Com-
pounds with larger groups at this position cause the P2
quinoxaline moiety to shift out of the active site, weakening
critical stacking interactions with the catalytic His57. These
findings further support our hypothesis that reducing inhibitor
interactions with the S2 subsite residues in the protease active
site results in improved resistance profiles. Moreover, in the
absence of a macrocycle, the quinoxaline-based linear PIs
could be optimized by SAR exploration to provide compounds
with potent activity against resistant variants.
1
2
sults in dramatic potency losses against RASs at Ala156.
Similar to the A156T-1 complex, the altered conformation
of the 3-isopropylquinoxaline results in significantly reduced
interactions with the catalytic His57. Any perturbation to the
protease active site due to RASs may further reduce interac-
tions with His57. Indeed, the 3-isopropylquinoxaline com-
pounds exhibit reduced potency against WT relative to the
parent compound 3, and are the most susceptible to resistant
variants. These results suggest that modifications of the inhibi-
tor scaffold that cause movement of the quinoxaline away
from His57 toward the solvent are highly detrimental to po-
tency. Whereas, movement of the quinoxaline away from
His57 toward the S2 subsite residues has less of an effect on
inhibitor potency when a smaller substituent is present at the
3-position of P2 quinoxaline.
The 3-chloroquinoxaline series exhibited an excellent po-
tency profile against WT and resistant variants even with a
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Di Filippo, M.; Gates, A. T.; Graham, D. J.; Harper, S.; Hazuda, D. J.;
ASSOCIATED CONTENT
Supporting Information
1
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McHale, C.; Monteagudo, E.; Pucci, V.; Rowley, M.; Rudd, M. T.;
Soriano, A.; Stahlhut, M. W.; Vacca, J. P.; Olsen, D. B.; Liverton, N.
J.; Carroll, S. S. MK-5172, a selective inhibitor of hepatitis C virus
NS3/4A protease with broad activity across genotypes and resistant
variants. Antimicrob. Agents Chemother. 2012, 56, 4161–4167.
7. Kwo, P. Y.; Poordad, F.; Asatryan, A.; Wang, S.; Wyles, D. L.;
Hassanein, T.; Felizarta, F.; Sulkowski, M. S.; Gane, E.; Maliakkal,
B.; Overcash, J. S.; Gordon, S. C.; Muir, A. J.; Aguilar, H.; Agarwal,
K.; Dore, G. J.; Lin, C. W.; Liu, R.; Lovell, S. S.; Ng, T. I.; Kort, J.;
Mensa, F. J. Glecaprevir and pibrentasvir yield high response rates in
patients with HCV genotype 1-6 without cirrhosis. J. Hepatol. 2017,
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Tables S1–S2, Figures S1–S2, experimental procedures and char-
acterization data for intermediates and final compounds, and de-
tails of protein expression, purification, x-ray crystallography,
enzyme inhibition, and antiviral assays (PDF)
Accessions Codes
The PDB accession codes for X-ray crystal structures of WT
NS3/4A protease in complex with inhibitors 12b, 12c and 12d are
6
7, 263–271.
0
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CVW, 6CVX and 6CVY. Authors will release the atomic coor-
8. Bourliere, M.; Gordon, S. C.; Flamm, S. L.; Cooper, C. L.;
dinates and experimental data upon article publication.
Ramji, A.; Tong, M.; Ravendhran, N.; Vierling, J. M.; Tran, T. T.;
Pianko, S.; Bansal, M. B.; de Ledinghen, V.; Hyland, R. H.; Stamm,
L. M.; Dvory-Sobol, H.; Svarovskaia, E.; Zhang, J.; Huang, K. C.;
Subramanian, G. M.; Brainard, D. M.; McHutchison, J. G.; Verna, E.
C.; Buggisch, P.; Landis, C. S.; Younes, Z. H.; Curry, M. P.; Strasser,
S. I.; Schiff, E. R.; Reddy, K. R.; Manns, M. P.; Kowdley, K. V.;
Zeuzem, S.; Polaris; Investigators, P.-. Sofosbuvir, velpatasvir, and
voxilaprevir for previously treated HCV infection. N. Engl. J. Med.
2017, 376, 2134–2146.
AUTHOR INFORMATION
Corresponding Author
Akbar Ali: Phone: +1 508 856 8873; Fax: +1 508 856 6464; E-
mail: Akbar.Ali@umassmed.edu
Celia A. Schiffer: Phone: +1 508 856 8008; Fax: +1 508 856
6
464; E-mail: Celia.Schiffer@umassmed.edu
9
. Ng, T. I.; Tripathi, R.; Reisch, T.; Lu, L.; Middleton, T.;
ORCID
Hopkins, T. A.; Pithawalla, R.; Irvin, M.; Dekhtyar, T.; Krishnan, P.;
Schnell, G.; Beyer, J.; McDaniel, K. F.; Ma, J.; Wang, G.; Jiang, L. J.;
Or, Y. S.; Kempf, D.; Pilot-Matias, T.; Collins, C. In vitro antiviral
activity and resistance profile of the next-generation hepatitis C virus
NS3/4A protease inhibitor glecaprevir. Antimicrob. Agents
Chemother. 2017.
Akbar Ali: 0000-0003-3491-791X
Celia A. Schiffer: 0000-0003-2270-6613
Nese Kurt Yilmaz: 0000-0002-5036-676X
Present Addresses
§Department of Chemistry, GC University Lahore, Katchery
Road, Lahore 54000, Pakistan
Notes
The authors declare no competing financial interest. AN, CJP and
WH are employees of Monogram Biosciences.
10. Pawlotsky, J. M. Hepatitis C virus resistance to direct-acting
antiviral drugs in interferon-free regimens. Gastroenterology 2016,
1
51, 70–86.
11. Sarrazin, C. The importance of resistance to direct antiviral
drugs in HCV infection in clinical practice. J. Hepatol. 2016, 64,
86–504.
2. Romano, K. P.; Ali, A.; Aydin, C.; Soumana, D.; Özen, A.;
4
1
ACKNOWLEDGMENT
Deveau, L. M.; Silver, C.; Cao, H.; Newton, A.; Petropoulos, C. J.;
Huang, W.; Schiffer, C. A. The molecular basis of drug resistance
against hepatitis C virus NS3/4A protease inhibitors. PLoS Pathog.
2012, 8, e1002832.
This work was supported by a grant from the National Institute of
Allergy and Infectious Diseases of the NIH (R01 AI085051).
ANM was also supported by the National Institute of General
Medical Sciences of the NIH (F31 GM119345). MJ was support-
ed by a Postdoctoral Fellowship from the Higher Education
Commission (HEC) of Pakistan.
1
3. Ali, A.; Aydin, C.; Gildemeister, R.; Romano, K. P.; Cao, H.;
Özen, A.; Soumana, D.; Newton, A.; Petropoulos, C. J.; Huang, W.;
Schiffer, C. A. Evaluating the role of macrocycles in the susceptibility
of hepatitis C virus NS3/4A protease inhibitors to drug resistance.
ACS Chem. Biol. 2013, 8, 1469–1478.
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TOC Graphic
D81
OMe
N
H57
R155
D168
N
R1
1
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O
O
S139
A156
O
O
O
N
H
HN
S
R3
R2
O
N
N
O
H
O
1
R = Me, Et, i-Pr, Cl, CF3
Teal (Me), cyan (Et), magenta (i-Pr), orange (Cl)
7
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