Target-guided screening of fragments (TGSOF) in the discovery of inhibitors against EV-A71 3C protease

Article abstract of DOI:10.1039/c8cc00469b

Target-guided screening of fragments (TGSOF) was developed and employed in the identification of EV-A71 3C protease (3C^pro) inhibitors. We identified 4-acetylpyridine and 3-acetylpyridine as effective P3 fragments of an inhibitor and obtained the corresponding irreversible inhibitors 12c and 12fvia this method. Furthermore, based on 12c and 12f, we have obtained reversible inhibitors 17c and 17f. These results demonstrated that TGSOF is a useful strategy for identifying suitable fragments in developing leads in drug discovery.

Full text of DOI:10.1039/c8cc00469b

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc
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Target-guided Screening of Fragments (TGSOF) in the Discovery of
Inhibitors against EV-A71 3C Protease
Received 00th January 20xx,
Accepted 00th January 20xx
a
a
a
a
a
a
*,a
Shengjun Fu, Quandeng Nie, Yuying Ma, Ping Song, Xuejiao Ren, Cheng Luo, Luqing Shang,
*,a
and Zheng Yin
DOI: 10.1039/x0xx00000x
www.rsc.org/
Target-guided screening of fragments (TGSOF) was developed and the last decade, “target-guided synthesis (TGS)”, “tethering in
pro
employed in the identification of EV-A71 3C protease (3C
)
situ click chemistry (TISCC)” and “templated assembly of
inhibitors. We identified 4-acetylpyridine and 3-acetylpyridine as protein-binding fragments” have demonstrated great promise
effective P3 fragments of an inhibitor and obtained the in streamlining drug discovery processes by combining
corresponding irreversible inhibitors 12c and 12f via this method. screening and synthesis into a single step, where the biological
Furthermore, based on 12c and 12f, we have obtained reversible target molecule is used as a template for the construction of
inhibitors 17c and 17f. These results demonstrated that TGSOF is a its own inhibitor through self-assembly within the active site of
4
-5
useful strategy for identifying suitable fragments in developing the target. These strategies are based on the notion that
leads in drug discovery.
when the reaction product binds tightly with the target
molecule, the reaction will be driven by the 3D environment of
6
the target molecule. Inspired by these methods, we present a
Human enterovirus A71 (EV-A71) is a major etiologic agent of
1
hand, foot and mouth disease (HFMD). Several large
related approach, i.e., target-guided screening of fragments
outbreaks of HFMD have occurred in children since 1997,
2
mostly in the Asia-Pacific region. Several approaches have
(
TGSOF) (Figure 1). Similar to those previously described
methods, if a reactive substrate binds tightly to the active
pocket of the biomolecule, the corresponding screening
reaction in the pocket could be promoted by the 3D
environment of the biomolecule. Therefore, TGSOF is
proposed for screening drug fragments and obtaining lead
compounds. In TGSOF, according to the screening of different
fragments of the inhibitor, the corresponding ligation building
block (LBB) is designed and synthesized. A functional group can
be introduced at a specific site of the target biomolecule using
a LBB. Therefore, compared to TGS, TISCC, and “templated
assembly of protein-binding fragments”, it is ensured that the
screening reaction in the TGSOF can occur in a designated
location. Furthermore, the screening efficiency was proved
and a certain fragment or region of an inhibitor was precisely
found. TGSOF can rapidly provide effective fragments for drug
discovery and highly active inhibitors are also more likely to be
obtained by assembling the corresponding fragment and LBB.
Herein, TGSOF was employed to screen the P3 fragment of a
been attempted to prevent enterovirus infections, but no
effective antiviral agents are available for the clinical
1
treatment of EV-A71. EV-A71, a member of the genus
Enterovirus in the family Picornaviridae, contains a single-
stranded, positive-sense RNA genome that encodes a large
2
poly-protein precursor. The poly-protein is cleaved into four
structural proteins (VP1-VP4) and seven non-structural
2
pro
proteins (2A-2C and 3A-3D). 3C is required for processing
all the cleavage sites on the polyprotein, except VP1/2A and
3
C/3D. The critical roles that it plays for the viral replication
pro
make 3C an attractive drug target for EV-A71 infection.
pro
Therefore, the critical role of the 3C makes it an attractive
1
target for anti-EV-A71 agent discovery.
With the aim of developing antivirals for the treatment of
EV-A71, several efforts have been made to discover inhibitors
pro
1, 3
of 3C using traditional medicinal chemistry methods. In a
typical and traditional process, biologically active compounds
4
need to be synthesized prior to being assessed for activity.
This is a burdensome process as organic synthesis often is the
most time-consuming part of a medicinal chemistry project. In
a.
College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and
Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin
3
00071, China. E-mail: shanglq@nankai.edu.cn, zheng_yin@nankai.edu.cn
Figure 1.The TGSOF strategy. First, a reactive functional group is site-specifically
introduced to the active pocket of the target protein using the ligation building
block (LBB). Second, fragment molecules are screened by the modified protein
with the LBB. Finally, potential inhibitors are assembled.
†
Electronic Supplementary Informaꢀon (ESI) available: Experimental procedures,
characterization data, and additional spectra. See DOI: 10.1039/x0xx00000x
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pro
pro
Figure 4. UV/vis (left) and CD (right) spectra of native 3C and 3C modified
with LBB 8.
conversion rate of 100.0% was quantitatively determined by
MS analysis (Figure 3 and Figure S25-26, the detailed
8
procedure is provided in Part D and Part F of the SI). Site-
pro
（8） interacting with EV-A71 3C . The
Figure 2. Schematic diagram of the LBB
(
specific modification was further confirmed by trypsin
digestion. LC−MS/MS analysis of the resulꢀng pepꢀde
fragments revealed that the Michael addition occurred site-
specifically with Cys147 at the catalytic site (see Figure S2-S5 in
the SI). The comparison of circular dichroism (CD) and UV/vis
spectra of the native protease and the protease modified by
S)-δ-lactam ring and the benzene group were required to ensure specificity for
p
r
o
p
r
o
the 3C . The α,β-unsaturated ester was used as the linker to 3C . The α-
ketoaldehyde was used for the reaction screening.
pro
3C
inhibitor. Suitable P3 fragments have been identified,
leading to several highly active inhibitors. The positive results
obtained demonstrate the feasibility of TGSOF.
Michael addition with
8 showed few changes, which provided
With the aim of selecting the P3 fragment of the EV-A71
pro
evidence that the structure/conformation of the protein was
not changed after the modification (Figure 4).
3C
inhibitor, a LBB was designed based on the following
requirements (Figure 2). First, as Cys147 plays a critical role in
pro
the catalytic site of EV-A71 3C , a covalent-irreversible group,
To verify the feasibility of the method, 28 commercially
available ketones (Figure 5) were screened to investigate
pro
α,β-unsaturated ester, was introduced to obtain strong
pro
whether the aldol reaction occurred in the S3 pocket of 3C
binding of the LBB with the active site of 3C . Second, the P1
pro
modified with LBB (8). At the beginning, the mixture of several
and P2 substituents of EV-A71 3C
inhibitors should
ketones was applied as a group for screening. In such
experiments, a small amount of protein deposition was,
presumably because the overall concentration of ketones was
too high. After the screening of mixed ketones, we screened
the 28 ketones indvidually, in order to avoid affecting the
protein structure and to enhance the fidelity of the screening
results.
preferentially feature a (S)-δ-lactam ring and a benzyl group,
respectively, to efficiently occupy the catalytic pockets of the
pro 1
EV-A71 3C . Therefore, they were introduced to the LBB for
pro
the identification of 3C . More importantly, a desired
functional group used for the screening reaction should be
introduced into the LBB. To avoid the formation of non-
biocatalyzed adducts, an ideal screening reaction applied in
the method should have a low yield and slow kinetics in the
absence of a template protein. Our recent report provided
evidence that under biochemical conditions, direct aldol
The modified protease solution was subjected to different
ketones in 1.5 mL plastic tubes with a final protein
concentration of 40 μM and a ketone concentration of 80 mM
in PB buffer (25 mM, pH= 7.0). The solution was then
incubated at room temperature for 12 h. Subsequently, the
small molecules and precipitate were removed using three
ultra-filtration steps to prepare the protease solution for
7
condensation reaction barely proceeds in aqueous buffers.
Therefore, aldol condensation reaction was selected as the
screening reaction. As mentioned above, LBB (8) was designed
and synthesized with a terminal aldehyde group as the
pro
functional group to screen the P3 fragment of the EV-A71 3C
pro
analysis. Interestingly, the desired signal of modified 3C
reacted with 4-acetylpyridine was detected. A conversion rate
of 40.5% was achieved as quantitatively analyzed by LC-ESI-
inhibitor. (The detailed procedure is given in Part A of the SI).
pro
3C
was subjected to LBB (8) in 1.5 mL plastic tubes with a
Orbitrap MS analysis
，according to the published protocol
final protein concentration of 40 μM and an LBB (8)
(
Figure 6 and Figure S27, the detailed procedure is given in
concentration of 2.4 mM in PB buffer (25 mM, pH = 7.0). The
solution was incubated at room temperature for 12 hours.
Subsequently, the small molecules were removed by three
ultra-filtration steps to prepare the protease solution for
8
Part D and Part F in the SI). In addition, the desired signal of
pro
modified 3C reacted with 3-acetylpyridine was also detected
and the conversion rate was 29.8% (Figure 6 and Figure S28,
8
the detailed procedure is given in Part D and Part F in the SI).
analysis using LC-ESI-Orbitrap MS analysis. As expected, the
pro
desired signal of 3C modified with 8 was observed, and a
Except for 4-acetylpyridine and 3-acetylpyridine, the aldol
pro pro
Figure 3. LC-ESI-Orbitrap analysis of native and modified 3C : native 3C (top)
pro
and 3C modified with aldehyde
8
(bottom)
.
Figure 5. The collection of commercial ketones used for screening.
2
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reaction did not proceed with other tested ketones under the Table1. Enzyme-inhibition activity of 8，12a-12g and 17a-17g against EV-
pro
pro
same conditions. Native 3C was employed as a control, and
A71 3C
no product was observed (see Figure S20 and Figure S21 in the
SI).
The site-specific modification of the protease product was
further confirmed by trypsin digestion. LC−MS/MS analysis of
the resulting peptide fragments showed that the aldol reaction
occurred site-specifically between the terminal aldehyde of
LBB and 4-acetylpyridine or 3-acetylpyridine (see Figure S6-9 in
the SI).
α, b
-1 -1 c
NO
IC50 (µM)
kobs/[I](M s )
8
16.38 ± 0.12
6.23 ± 0.20
5347 ± 121
1
2a
10320 ± 372
pro
12b
12c
6.54 ± 0.21
1.43 ± 0.10
1.41 ± 0.05
1.44 ± 0.07
6.20 ± 0.13
6.39 ± 0.13
12743 ± 425
216347± 5752
236307 ± 7230
182500 ± 5607
21586 ± 794
14680 ± 472
12549 ± 289
In the absence of EV-A71 3C , the reaction of 4-
acetylpyridine or 3-acetylpyridine with LBB (
8) was also
performed. LBB (8) and 4-acetylpyridine or 3-acetylpyridine
pro
12c-1
12c-2
were mixed and shaken in the absence of EV-A71 3C . The
reaction conditions, including the pH, the concentrations and
the temperature, were identical with those used for the
1
1
2d
2e
1
screening reaction. The aldol reaction, monitored by H-NMR
and HPLC, did not proceed and no new compound was
detected. These data confirmed that the aldol reaction
(7S,2'S,10S,
3R)-12e
7S,2'S,10S,
3S)-12e
2f
6
.42 ± 0.15
1
(
between LLB (8) and 4-acetylpyridine or 3-acetylpyridine was
pro
driven by EV-A71 3C in the desired active site (see Figure
S10-19 in SI).
6.34 ± 0.13
1.61 ± 0.07
6.72 ± 0.12
16388 ± 476
103887 ± 3346
21327 ± 689
1
1
Because the aldol reaction of 4-acetylpyridine or 3-
acetylpyridine and LLB (
pro
8
) occurred only in the presence of the
12g
7a
17b
7c
3
C , 4-acetylpyridine and 3-acetylpyridine were postulated to
1
1.52 ± 0.02
1.41 ± 0.01
0.15 ± 0.01
1.45 ± 0.03
have higher binding affinity to the protease than other ketones.
To verify our assumption, 12a-g, corresponding to pinacolone,
2'-acetonaphthone, 4-acetylpyridine, 2-acetylthiophene, acet-
1
ophenone, 3-acetylpyridine, and 1-(2-nitrophenyl)-ethanone
were synthesized and evaluated for their enzyme-inhibition
activity (Part A of the SI and Table 1).
1
1
7d
7e
1.37 ± 0.02
For the evaluation of inhibition activities of 12a-g, rupintrivir,
with the α,β-unsaturated ester, was used as the positive
control. Compounds 12a-g showed significantly better active-
(2S,2'S,5S,8
R)-17e
1
.36 ± 0.02
(
2S,2'S,5S,8
S)-17e
7f
1.47 ± 0.01
0.17 ± 0.01
1.55 ± 0.03
ties than
fragment is important for the inhibitors. As shown in Table 1,
2c (IC50 = 1.43 ± 0.10 µM) presented approximately 4- to 5-
fold better activity than 12a (IC50 = 6.23 ± 0.20 µM), 12b (IC50
8 (IC50 = 16.38 ± 0.12 µM), which suggests that the P3
1
1
1
7g
=
d
6
0
.54 ± 0.21 µM), 12d (IC50 = 6.20 ± 0.13 µM), 12e (IC50 = 6.39 ±
.13 µM), and 12g (IC50 = 6.72 ± 0.12 µM). In addition,
12f (IC50 = 1.61 ± 0.07 µM) showed lower
Rupintrivir
-----
-----
1.13 ± 0.02
0.32 ± 0.02
615418 ± 22517
e
1.8k
a
b
compared with 12c
,
The pH of the biochemical assay for the inhibitors was 7.0. All values are the
9
c
mean of three independent experiments. Analytical methods were carried out
d
activity,
than 12a
but it presented approximately 4-fold better activity as reported in the literature. Rupintrivir was used as the positive
e
control. Compound 1.8k, as reported in the reference, was used as the positive
control.
1
, 12b, 12d, 12e and 12g. Furthermore, the first-order
rate constant (kobs) was introduced to verify the comparison of
the obtained enzyme-inhibition activity (IC50). As listed in Table
the idea that 4-acetylpyridine or 3-acetylpyridine are better fit
with the S3 pocket than the other commercial ketones. Such
results also support the feasibility of TGSOF in screening for a
fragment of the desired inhibitor. To elucidate the binding
1, the kobs/[I] (12a-g) showed good correlation with IC . These
results were consistent with the screening results, supporting
50
pro
mode of 12c and 12f with EV-A71 3C , a docking study was
conducted using MOE (see Figure S22 in the SI). The striking
difference to other ketones was that the 4-acetylpyridine of
12c or 3-acetylpyridine of 12f interacted with Asn165. Because
the nitrogen atom of 4-acetyl pyridine or 3-acetylpyridine is
electron-rich, it formed a hydrogen bond with Asn165. For 12a
1
,
2b, 12d, 12e and 12g such an interaction was not observed.
This specific binding element might be critical for the activity
difference, resulting in 12c or 12f exhibiting better activity.
Further, compared with 12c, the hydrogen bond distance
Figure 6. LC-ESI-Orbitrap analysis of the aldol reaction of modified protease with
4-acetylpyridine (top); LC-ESI-Orbitrap analysis of the aldol reaction of modified
protease with 3-acetylpyridine (bottom).
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between 12f with Asn165 was a little farther away (2.2 Å for
In summary, target-guided screening of fragments (TGSOF)
pro
has been employed in the study of EV-A71 3C inhibitors. We
12f vs. 1.9 Å for 12c), so 12f exhibited slightly lower activity.
To define the absolute configuration of the two isomers of identified 4-acetylpyridine and 3-acetylpyridine as effective P3
the aldol reaction product, the epimeric mixture 12e was fragments and obtained the corresponding irreversible
separated to yield (7S,2'S,10S,13R)-12e and (7S,2'S,10S,13S)- inhibitor 12c (IC50 = 1.43 ± 0.10 µM) and 12f (IC50 = 1.61 ± 0.07
1
2e. The absolute configuration of the chiral carbon of the µM) using the method. In view of our previous study of EV-A71
pro
hydroxyl group was determined by the modified Mosher’s 3C inhibitors and based on 12c and 12f, the reversible
10
1
method. The result was further supported by H-NMR (see inhibitor 17c and 17f were obtained, and they exhibited
Part B in SI). The epimeric mixture 12e (IC50 = 6.39 ± 0.13 µM) excellent inhibitory activity (IC50 = 0.15 ± 0.01 µM and IC50
and the isolated isomers (7S,2'S,10S,13R)-12e (IC50 = 6.42 ± 0.17 ± 0.01 µM). These findings strongly support the use of
.15 µM) and (7S,2'S,10S,13S)-12e (IC50 = 6.34 ± 0.13 µM) TGSOF as an efficient way to screen for a specific inhibitor
=
0
pro
exhibited equipotent inhibitory activity against EV-A71 3C
.
fragment and as a novel strategy to discover lead compounds.
In TGSOF, the functional group used to screen fragment can
Similar results were observed for the epimeric mixture 12c
(IC50 = 1.43 ± 0.10 µM) and both isomers 12c-1 (IC50 = 1.41 ± be introduced at a specific site of the target biomolecule using
0.05 µM) and 12c-2 (IC50 = 1.44 ± 0.07 µM).
LBB. Furthermore, the screening reaction of the modified
The interactions of epimeric mixture 12c docked to EV-A71 protein and fragments is to happen in a specific location of
pro
3C
could explain the unsubstantial difference in the IC50 interest. TGSOF can be employed to screen other fragments of
pro
values. As shown in Figure S24, the two isomers had the same EV-A71 3C inhibitors, and then novel inhibitors could be
interaction distances between the 4-acetylpyridine nitrogen obtained by the assembly of those screened fragments.
atom and Asn165 (1.9 Å). Moreover, the OH oxygen of the two Therefore, TGSOF offer a way to rapidly identify effective
isomers could form a similar weak hydrogen bond with Gly164 fragments for drug discovery. For future studies, TGSOF might
(2.8 Å vs. 2.9 Å).
be applied to study other cysteine proteases, such as
To obtain more potent inhibitors, we then optimized the cathepsins. Moreover, instead of aldol condensation reaction,
structure of 12c and 12f. Based on the characterization of the other reactions can also be applied in TGSOF (i.e. the
binding modes, the inhibitors can be divided into reversible substitution reaction of C–N bond formation, the substitution
4
inhibitors (e.g., aldehydes) and irreversible inhibitors (e.g., α,β- reaction of C–S bond formation).
pro
1
unsaturated ester). The previous study of EV-A71 3C
1
This work was supported by the National Basic Research
inhibitors provided evidence that the aldehyde group fits Program of China (973 program, Grant Nos. 2013CB911104,
pro
readily into the S1'pocket of EV-A71 3C and the interaction 2013CB911100), the National Natural Science Foundation of
is more favorable than the unsaturated ester. Therefore, the China (Grant Nos. 21672115), and the "111" Project of the
warhead α,β-unsaturated ester of 12c and 12f were replaced Ministry of Education of China (Project No. B06005).
by an aldehyde group to obtain 17c and 17f. In addition, 17a
,
17b, 17d, 17e and 17g corresponding to pinacolone, 2'-
Notes and references
acetonaphthone, 2-acetylthiophene, acetophenone and 1-(2-
nitrophenyl)-ethanone were also synthesized and evaluated
for their enzyme-inhibition activity (Table 1 and Part A of the
SI). Similar to examination of the absolute configuration of the
two isomers of 12e, the epimeric mixture 17e was separated
to yield (2S,2'S,5S,8R)-17e and (2S,2'S,5S,8S)-17e (Part B in SI).
1
2
3
Y. Y. Zhai, Y. Y. Ma, F. Ma, Q. D. Nie, X. J. Ren, Y. X. Wang, L. Q.
Shang and Z. Yin, Eur. J. Med. Chem., 2016, 124, 559-573.
L. Q. Shang, M. Y. Xu and Z. Yin, Antiviral Res., 2013, 97, 183-
194.
Y. Y. Zhai, X. S. Zhao, Z. J. Cui, M. Wang, Y. X. Wang, L. F. Li, Q.
Sun, X. Yang, D. B. Zeng, Y. Liu, Y. N. Sun, Z. Y. Lou, L. Q. Shang
and Z. Yin, J. Med. Chem., 2015, 58, 9414-9420.
For reversible inhibitors 17a-g, 1,8k with the aldehyde group
reported in previous literature, was applied as the positive
control. The enzyme-inhibition activity indicated that aldehyde
4
5
X. D. Hu and R. Manetsch, Chem. Soc. Rev., 2010, 39, 1316-1324.
(a) Y. Wang, J. J. Zhu and L. W. Zhang, J. Med. Chem., 2017, 60,
17c (IC50 = 0.15 ± 0.01 µM) exhibited approximately 10-fold
2
63-272; (b) E. Oueis, C. Sabot and P. Y. Renard, Chem.
more potent activity than 12c, and 17f (IC50 = 0.17 ± 0.01 µM)
exhibited similar result in the protease assay. Compared to the
α,β-unsaturated ester, the aldehyde group formed a reversible
covalent bond with Cys147, which fit in the catalytic site of EV-
Commun., 2015, 51, 12158-12169; (c) D. Becker, Z. Kaczmarska,
C. Arkona, R. Schulz, C. Tauber, G. Wolber, R. Hilgenfeld, M. Coll
and J. Rademann, Nat Commun, 2016, 7.
A. Herrmann, Chem. Soc. Rev., 2014, 43, 1899-1933.
P. Wang, S. M. Zhang, Q. Y. Meng, Y. Liu, L. Q. Shang and Z. Yin,
Org. Lett., 2015, 17, 1361-1364.
6
7
pro
A71 3C . In addition, as shown in Table 1, 17c and 17f
presented approximately 9-fold better activity than 17a (IC50
=
8
9
Q. Ruan, Q. C. Ji, M. E. Arnold, W. G. Humphreys and M. S. Zhu,
Anal. Chem., 2011, 83, 8937-8944.
J. Z. Tan, S. George, Y. Kusov, M. Perbandt, S. Anemuller, J. R.
Mesters, H. Norder, B. Coutard, C. Lacroix, P. Leyssen, J. Neyts
and R. Hilgenfeld, J. Virol., 2013, 87, 4339-4351.
1
0
0
.52 ± 0.02 µM), 17b (IC50 = 1.41 ± 0.01 µM), 17d (IC50 = 1.45 ±
.03 µM), 17e (IC50 = 1.37 ± 0.02 µM), and 17g (IC50 = 1.55 ±
.03 µM). These findings verified that 4-acetylpyridine and 3-
acetylpyridine were still the more potent P3 fragments for the
inhibitors with aldehyde warhead. Therefore, based on 12c
and 12f, we successfully obtained 17c and 17f as reversible
1
1
0 F. Freire, J. M. Seco, E. Quinoa and R. Riguera, J. Org. Chem.,
2
005, 70, 3778-3790.
inhibitors with excellent enzyme-inhibition activity against EV-
pro
A71 3C
1 H.-H. Otto and T. Schirmeister, Chem. Rev., 1997, 97, 133-172.
.
4
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Graphical Abstract
Target-guided screening of fragments (TGSOF) was developed and employed in the identification of
pro
EV-A71 3C protease (3C ) inhibitors.