Michael Acceptor-Based Peptidomimetic Inhibitor of Main Protease from Porcine Epidemic Diarrhea Virus

Article abstract of DOI:10.1021/acs.jmedchem.7b00103

Porcine epidemic diarrhea virus (PEDV) causes high mortality in pigs. PEDV main protease (M^pro) plays an essential role in viral replication. We solved the structure of PEDV M^pro complexed with peptidomimetic inhibitor N3 carrying a Michael acceptor warhead, revealing atomic level interactions. We further designed a series of 17 inhibitors with altered side groups. Inhibitors M2 and M17 demonstrated enhanced specificity against PEDV M^pro. These compounds have potential as future therapeutics to combat PEDV infection.

Full text of DOI:10.1021/acs.jmedchem.7b00103

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Brief Article
Michael Acceptor-Based Peptidomimetic Inhibitor of
Main Protease from Porcine Epidemic Diarrhea Virus
Fenghua Wang, Cheng Chen, Kailin Yang, Yang Xu, Xiaomei Liu, Fan
Gao, He Liu, Xia Chen, Qi Zhao, Xiang Liu, Yan Cai, and Haitao Yang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b00103 • Publication Date (Web): 13 Mar 2017
Downloaded from http://pubs.acs.org on March 14, 2017
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Michael Acceptor-Based Peptidomimetic Inhibitor of Main Protease from Porcine Epidemic Diarrhea Virus
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Fenghua Wang,†,+ Cheng Chen,†,‡,+ Kailin Yang,#,+ Yang Xu,‡ Xiaomei Liu,‡ Fan Gao,‡ He Liu,‡ Xia Chen,† Qi
Zhao,§ Xiang Liu,‡ Yan Cai,‡,* and Haitao Yang†,‡,*
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†School of Life Sciences, Tianjin University, Tianjin 300072, China
‡Tianjin International Joint Academy of Biotechnology and Medicine, Tianjin 300457, China
#Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44195, United
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States
§Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520, United
States
ABSTRACT: Porcine epidemic diarrhea virus (PEDV) causes high mortality in pigs. PEDV main protease (Mpro)
plays an essential role in viral replication. We solved the structure of PEDV Mpro complexed with peptidomimetic
inhibitor N3 carrying a Michael acceptor warhead, revealing atomic level interactions. We further designed a series
of 17 inhibitors with altered side groups. Inhibitors M2 and M17 demonstrated enhanced specificity against PEDV
Mpro. These compounds have potential as future therapeutics to combat PEDV infection.
passive immunity through maternal antibody transfer
via milk. However, the low to moderate efficacy of
PEDV vaccines on the market, possibly caused by
antigenic and genetic variation, underscores the need
for effective antiviral therapeutics. Broadꢀspectrum
antiviral agents such as ribavirin have been tested in
vitro.12, 13 The genome of PEDV is approximately 28
kb in length and contains a large 5’ open reading frame
INTRODUCTION
Porcine epidemic diarrhea virus (PEDV) is an
enveloped, single strand, positiveꢀsense RNA virus
belonging to the genus Alphacoronavirus in the
subfamily Coronavirinae.1,
Unlike
human
2
coronaviruses, which cause respiratory diseases,
including severe acute respiratory syndrome (SARS)
and Middle East respiratory syndrome (MERS), PEDV
infects the gastrointestinal tract in feeder pigs and
fattening swine and causes porcine epidemic diarrhea
(PED). PED symptoms include severe diarrhea,
vomiting, and high rates of morbidity and mortality
(approaching 100% in young piglets). Although the
symptoms of PED are similar to the gastroenteritis
caused by another coronavirus porcine transmissible
gastroenteritis virus (TGEV), PEDV causes more
severe disease and a higher death rate among nursing
piglets. PEDV was initially identified in the early
(ORF1) and at least 5 smaller ORFs (ORF2−6) in the 3’
end.1, 2 ORF1 encodes two large polypeptides of the
replicase machinery: pp1a, and through ribosomal
frameshift, pp1ab. Both pp1a and pp1ab are coꢀ
translationally cleaved into 16 nonstructural proteins
(Nsps) that are mediated by two proteases: the main
protease (Mpro, also called Nsp5) and the papainꢀlike
protease (PLP or Nsp3).14 Given its essential role in
proteolytic processing in viral replication, Mpro is
highly conserved in different coronavirus species at the
levels of amino acid sequence and three dimensional
structure.15ꢀ18 Furthermore, the lack of a human
homolog makes Mpro an ideal drug target, which has
been explored in the pharmaceutical development
against human coronaviruses such as SARS and
MERS.15, 19
1970s and had been mainly limited to Europe.3,
4
However, since the 1990s, additional severe PEDV
outbreaks have been reported in Asian countries,
including South Korea and China, causing significant
economic losses in these countries that heavily rely on
pork as a food source.5, 6 The first epidemic of PEDV
infection in the United States was reported in 2013,7
and a total of 3 etiological PEDV strains have been
identified to date.8 The epidemic quickly spread across
the entire continent into Mexico and Canada and
resulted in the deaths of over 8 million newborn piglets
during a one year period1. Therefore, the global spread
of PEDV has caused severe and dramatic negative
economic impacts on the food industry.9
RESULTS AND DISCUSSION
Peptidomimetic inhibitors carrying a Michael acceptor
warhead achieve optimal pharmaceutical activity
against viral protease through mechanismꢀbased
irreversible inhibition.15 In a previous study, we
designed and evaluated a series of wideꢀspectrum
inhibitors targeting the main proteases of
coronaviruses;19 however, their therapeutic efficacy for
PEDV had not been explored. In the current study
focusing on PEDV Mpro, we first used a potent
Vaccines against epidemic PEDV strains have
been used in several countries with limited success.10, 11
Through active vaccination of sows, piglets gain
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inhibitor, compound 1 (N3), designed in our previous
determined based on the plot. (C) Reaction scheme
work19 as an initial structural scaffold for SARS
studies. The α,βꢀunsaturated ester group in compound
1 is commonly used to irreversibly inactivate cysteine
proteases. As shown in Figure 1, the Michael acceptor
was positioned between the P1 and P1’ sites of the
peptidomimetic backbone, thus maximizing its
electrophilic potential with the cysteine residue of the
CysꢀHis catalytic dyad. Compound 1 carries a
comparatively bulky lactam ring at the P1 site to
accommodate the absolute requirement for a glutamine
residue at the P1 position of almost all coronavirus
Mpro substrates. Beyond the preservation of critical
hydrogen bonds required for S1 recognition, the lactam
ring also participates in multiple van der Waals
interactions. An alkyl leucine side chain was included
at the P2 site to accommodate the hydrophobic S2
pocket. The valine side chain at the P3 site is solventꢀ
exposed, and the Ala side chain at the P4 position
favors compound 1 to readily enter into the
comparatively small S4 pocket. To enhance the
binding affinity, an isoxazole group was incorporated
at the P5 position to increase the van der Waals
contacts with flanking residues at either side.
showing the twoꢀstep mechanismꢀbased irreversible
inactivation of PEDV Mpro by compound 1.
To assess the efficacy of compound 1 on
PEDV Mpro, timeꢀdependent kinetic analyses were
performed using a fluorescently labeled substrate,
MCAꢀAVLQ↓SGFRꢀLys(Dnp)ꢀLysꢀNH2. Interestingly,
the progress curves of compound 1 indicated timeꢀ
dependent irreversible inhibition of PEDV Mpro, as the
observed rate of inactivation (kobs) increased with
increasing concentration of the inhibitor (Figure 2A).
Further, the hyperbolic shape of this curve (Figure 2B)
supports the mechanism of twoꢀstep irreversible
inactivation (Figure 2C). The inhibitor first forms a
reversible complex with Mpro at the active site (EI)
with a dissociation constant Ki; then, conjugate
addition of the thiol group of Cys144 onto the Michael
acceptor of compound 1 induces the formation of a
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covalent bond between Mpro and compound 1 (E
−I).
The Ki of compound 1 toward PEDV Mpro was
determined to be 19.4 ± 0.7 µM. The rate constant, k3,
of Michael acceptor addition was determined to be
71.7 ± 4.2 10ꢀ3 sꢀ1, higher than 18.0 ± 1.1 10ꢀ3 sꢀ1 of
HCoVꢀ229E and 3.1 ± 0.5 10ꢀ3 sꢀ1 of SARSꢀCoV,19
thus indicating a favorable conformation created in EI
for covalent bond formation.
Figure 1. Scheme of Michael acceptor mediated
irreversible inhibition of PEDV Mpro.
Figure 3. The 2.44 Å complex structure of compound
1 (colored green) bound to PEDV Mpro (PDB ID
5GWZ), indicating that 1 occupies the substrateꢀ
binding site.
The structure of PEDV Mpro dimer bound to
Figure 2. Inhibition of PEDV Mpro by compound 1
carrying a Michael acceptor warhead. (A) Reaction
progress curves in the presence of increasing inhibitor
concentration, indicating timeꢀdependent inactivation
of PEDV Mpro. (B) Plot of observed rate of inactivation
constant (kobs) over the concentration of compound 1.
compound
1
confirmed that the hypothesized
mechanismꢀbased irreversible inhibition occurred at
the active site of the enzyme (Figure 3). Considering
monomer A, for example, compound 1 is located at
the cleft between domain I and II of PEDV Mpro, with a
buried surface area of 706.4 Å2 (approximately 72.3%
of the total surface area) (Figure 4A and S3,
A
secondꢀorder inactivation rate constant was
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Supporting Information). A 1.83ꢀÅ C
−
S covalent bond
3 and 15 (Table S3, Supporting Information), achieved
was observed between the Cβ atom of vinyl group of
compound 1 and the Sγ atom of Cys144, evidencing
Michael acceptor addition (Figure 4B and S4,
Supporting Information). Furthermore, compound 1 is
stabilized by a network of 8 hydrogen bonds involving
the Nε2 atom of His162; the carbonyl group of Gln163,
Glu165, and Thr189; the amide nitrogen of Cys144
and Glu165; and the Oε2 atom of Glu165 (Figure 4B
and Table S2, Supporting Information). In addition, as
shown in Figure 3, the P1 and P2 subsites of
compound 1 insert favorably into S1 (consisting of
much better inhibitory activity than compound 1, with
percentage inactivation of PEDV Mpro as 96% and 93%,
respectively. The Ki parameters of compounds 3 and
15 toward PEDV Mpro were determined to be 7.1 ± 0.2
µM and 8.3 ± 0.3 µM, respectively (Figure S6,
Supporting
Information),
which
demonstrated
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improved activity compared with that of compound 1.
The rate constant k3 of Michael acceptor addition for
compounds 3 and 15 was determined to be 59.2 ± 2.8
10ꢀ3 sꢀ1 and 66.7 ± 2.7 10ꢀ3 sꢀ1, respectively (Figure S6,
Supporting Information), both of which remained at
the same level with compound 1, but much higher than
the k3 rate constant for other coronaviruses. Therefore,
the two newly designed compounds, 3 and 15,
demonstrated improved inhibitory specificity towards
PEDV Mpro compared to the lead compound 1.
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residues Phe139
and His171) and S2 (consisting of His41, Thr47, Tyr53,
Gln163 Leu164, Met180, and Glu185 Pro188)
−Asn141, Cys144, His162−Glu165,
−
−
substrateꢀbinding pockets, respectively. The P3 site of
1 is solvent facing, while the P4 and P5 sites are
accommodated by one loop formed by residues
188
165
−
−
191 and another loop/strand spanning residues
167. Structural comparison of the compound 1ꢀ
complex structure with similar inhibitorꢀprotease
structures from other coronaviruses including SARSꢀ
CoV,19 HCoVꢀNL63,18 HCoVꢀHKU1,17 and TGEV19 is
shown in Figure S5, Supporting Information. Clearly,
compound 1 adopts a similar binding mode in the
substrateꢀbinding pocket of PEDV Mpro compared with
other CoV Mproꢀcompound 1 complex structures. The
differences lie in the orientations of P5 and P1´. One
possibility is that subsites S5 and S1´ in CoV Mpros are
very shallow, making P5 and P1´ partially solvent
exposed. Thus, strong interactions are not readily
achieved at these two subsites.
The above structural data based on lead
compound 1 permitted further rational optimization of
a Michael acceptorꢀbased peptidomimetic inhibitor to
target the substrateꢀbinding pocket of PEDV Mpro. We
then designed a series of 17 new compounds derived
from the backbone structure of compound 1 with
altered side chains (Table S3, Supporting Information).
Briefly, in the molecular design, we kept the lactam
ring at P1 and the side chain of Leu at P2, but replaced
P3, P4, P5 and P1´ with various functional groups to
obtain lead compounds with better inhibition.
Specifically, in the newly synthesized compound 3
(M2), the P3 site is a cyclopentane; in compound 15
(M17), the P1´ site is a fluorideꢀsubstituted benzyl
ester.20
Figure 4. Graphical representation demonstrating the
key interactions between compound 1 and PEDV Mpro
(PDB ID 5GWZ). (A) Interaction between compound 1
and PEDV Mpro. (B) Detailed view of key covalent and
hydrogen bonds between compound 1 and PEDV Mpro.
To screen for improved inhibitory activity of
these 17 newly designed compounds, we determined
the percentage inactivation of the initial enzymatic
activity of PEDV Mpro (defined as the initial slope of
the reaction curve). As shown in Table 1, compound 1
was used as the reference, which achieved 89%
inhibition of PEDV Mpro. Two of the new compounds,
CONCLUSION
In summary, we have presented the application of
Michael
acceptorꢀbased peptidomimetic
small
molecule inhibitor to target PEDV Mpro, an essential
and conserved enzyme in the viral replication. We
obtained the coꢀcrystal structure of wideꢀspectrum
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compound 1 bound to PEDV Mpro, revealing the
refinement using PHENIX23 led to a final model with a
detailed irreversible inhibition mechanism at the active
site of the enzyme. Based on this structure, we further
designed and synthesized another series of 17
inhibitors with altered side groups. Two compounds, 3
and 15, demonstrated improved inhibitory activity
against PEDV Mpro, particularly with improved affinity
for the active site as demonstrated by the much lower
Ki. Therefore, the class of peptidomimetic inhibitor
carrying a Michael acceptor has the potential to enter
crystallographic R factor (Rcryst) of 18.5% and a free R
factor (Rfree) of 21.9% at 2.44ꢀÅ resolution. All
parameters of Xꢀray data processing and refinement
are detailed in Table S1, Supporting Information. A
summary of the covalent bond and all hydrogen bonds
between PEDV Mpro and compound 1 are listed in
Table S2, Supporting Information.
Enzymatic Activity and Inhibition Assays
Enzymatic assays were performed using a fluorogenic
substrate with a consensus sequence of CoVMpro,
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pharmaceutical development as
a
promising
therapeutic to treat acute gastroenteritis in pigs and
especially nursing piglets infected by PEDV.
MCAꢀAVLQ↓SGFRꢀLys(Dnp)ꢀLysꢀNH2
(>95%
purity, GL Biochem Shanghai Ltd., Shanghai, China),
as previously reported.16, 17, 19 Fluorescence intensity
was monitored using a Fluoroskan Ascent instrument
(Thermo Scientific, USA) with excitation and emission
wavelengths of 320 and 405 nm, respectively. The
assay was performed in a buffer solution consisted of
50 mM TrisꢀHCl (pH 7.3) and 1 mM EDTA at 30 °C.
Kinetic parameters, including Km and kcat of PEDV
Mpro (Figure S1 and S2, Supporting Information) and
Ki and k3 of the inhibitors,17, 19 were determined using
methods described in detail in the Supporting
Information.
Table 1. Evaluation of the inhibitor activity
targeting PEDV Mpro for compound 1 and the 17
newly synthesized compounds. Inhibition ratio (Ir)
is defined as the percentage inactivation of the
initial enzymatic activity of PEDV Mpro.
Inhibition ratio
Inhibitory
activitya
+ + + +
compound
(Ir)
89%
1
2
87%
96%
80%
ꢀ
+ + + +
+ + + + +
+ + +
N/Ab
+
3
Chemical Synthesis of Compounds 3 and 15
4
The synthetic pathway of compound 3 was detailed in
Supporting Information, which was similar to that of
compound 1.19 Compound 15 was synthesized from 1:
7 mg LiOH was added to a solution of compound 1
(0.1 g) in 3 mL THF, and water was added until the
solution became clear. Then it was stirred for 1 hour.
After completion of the reaction, 1 M HCl (aq) was
add to adjust the pH to 3; then, the solvent was
evaporated and purification followed to yield a white
solid (0.06 g), which was then dissolved in 1 mL DMF,
with 1ꢀ(1ꢀbromoethyl)ꢀ2ꢀfluorobenzene (41 mg) and
34 µL DIEA. The solution was stirred overnight, and
after completion of the reaction, DMF was removed,
and the residue was purified by silica gel
chromatography to yield compound 15. Both
compounds 3 and 15 were further purified using
preparative HPLC.
5
12%
72%
10%
62%
75%
20%
ꢀ
6
7
+ + +
+
8
9
+ +
10
11
12
13
14
15
16
17
18
+ + +
+
N/A
ꢀ
N/A
80%
93%
90%
89%
90%
+ + +
+ + + + +
+ + ++
+ + ++
+ + ++
aPercentage inhibitory activity: + + + + +, >90%; + + + +,
80%−90%; + + +, 70%−80%; + +, 20%−70%; +, <20%. bNo
inhibition was observed.
Characterization of Compounds.
All compounds used in this study (Table 1) were
characterized with 1H and 13C NMR spectroscopy, ESIꢀ
MS, and HPLC. The purity of all compounds was > 95%
according to analytical HPLC using UV detection at
215 nm. All data for compound characterization are
provided in Supporting Information.
EXPERIMENTAL SECTION
Structure Determination and Analysis
The structure of the PEDV Mproꢀcompound 1 complex
was determined using molecular replacement from that
of HCoVꢀ229E Mpro (PDB entry 2ZU2). All crossꢀ
rotation and translation searches for molecular
replacement were performed with Phaser.21 Cycles of
manual adjustment using Coot22 and subsequent
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge
on the ACS Publications website at DOI:
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Enzymatic activity of PEDV Mpro, structural
2.
virus:
Song, D.; Park, B. Porcine epidemic diarrhoea
comprehensive review of molecular
overview and comparison of PEDV Mproꢀ
compound 1 interaction, inhibition kinetics of
compounds 3 and 15, Xꢀray data collection and
refinement statistics, details and characterization
data including purity for all 17 compounds,
additional methods (PDF)
a
epidemiology, diagnosis, and vaccines. Virus Genes
2012, 44, 167ꢀ175.
3.
particles associated with porcine epidemic diarrhoea.
Res. Vet. Sci. 1978, 25, 255ꢀ256.
Chasey, D.; Cartwright, S. F. Virusꢀlike
9
PDB coordinates for complex structure of PEDV
4.
Wood, E. N. An apparently new syndrome of
Mpro with compound 1 (PDB)
porcine epidemic diarrhoea. Vet. Rec. 1977, 100, 243ꢀ
244.
5.
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Molecular formula strings and some data (CSV)
Li, W.; Li, H.; Liu, Y.; Pan, Y.; Deng, F.; Song,
Accession Codes
13
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Atomic coordinates for the crystal structure of PEDV
Mpro in complex with compound 1 can be accessed
using PDB ID 5GWZ. Authors will release the atomic
coordinates and experimental data upon article
publication.
Y.; Tang, X.; He, Q. New variants of porcine epidemic
diarrhea virus, China, 2011. Emerging Infect. Dis.
2012, 18, 1350ꢀ1353.
6.
Sun, R. Q.; Cai, R. J.; Chen, Y. Q.; Liang, P.
S.; Chen, D. K.; Song, C. X. Outbreak of porcine
epidemic diarrhea in suckling piglets, China. Emerging
Infect. Dis. 2012, 18, 161ꢀ163.
AUTHOR INFORMATION
Corresponding Authors
7.
Stevenson, G. W.; Hoang, H.; Schwartz, K. J.;
*H.Y.: phone, +86 22 27403906; fax, +86 22
27403902; eꢀmail, yanght@tju.edu.cn.
*C.Y.: phone, +86 22 65378009; fax, +86 22
65378036; eꢀmail, caiyan_86@163.com.
Author Contributions
Burrough, E. R.; Sun, D.; Madson, D.; Cooper, V. L.;
Pillatzki, A.; Gauger, P.; Schmitt, B. J.; Koster, L. G.;
Killian, M. L.; Yoon, K. J. Emergence of porcine
epidemic diarrhea virus in the United States: clinical
signs, lesions, and viral genomic sequences. J. Vet.
Diagn. Invest. 2013, 25, 649ꢀ654.
+F.W., C.C., and K.Y. contributed equally and are
considered as coꢀfirst authors. All authors have given
approval to the final version of this manuscript.
Notes
8.
Marthaler, D.; Bruner, L.; Collins, J.; Rossow,
K. Third strain of porcine epidemic diarrhea virus,
United States. Emerging Infect. Dis. 2014, 20, 2162ꢀ
2163.
The authors declare no competing financial interest.
9.
Schulz, L. L.; Tonsor, G. T. Assessment of the
economic impacts of porcine epidemic diarrhea virus
in the United States. J. Anim. Sci. 2015, 93, 5111ꢀ5118.
ACKNOWLEDGEMENTS
We thank Dunquan Jiang for data collection at
beamline BLꢀ5A (the Photon Factory, KEK, Japan)
and Steve C. Ding for helpful discussion. This work
was supported by the National Key Basic Research
10.
Kweon, C. H.; Kwon, B. J.; Lee, J. G.; Kwon,
G. O.; Kang, Y. B. Derivation of attenuated porcine
epidemic diarrhea virus (PEDV) as vaccine candidate.
Vaccine 1999, 17, 2546ꢀ2553.
Program
of
China
(973
program,
grant
2015CB859800), the Binhai New Area Special Fund
for Innovations in Science and Technology, the
National Natural Science Foundation of China (grant
31528006), the National Key Research Program of
China (No. 2016YFD0500300), and the Tianjin Marine
Science and Technology Program (grant KJXH2014ꢀ
16).
11.
Song, D. S.; Oh, J. S.; Kang, B. K.; Yang, J. S.;
Moon, H. J.; Yoo, H. S.; Jang, Y. S.; Park, B. K. Oral
efficacy of Vero cell attenuated porcine epidemic
diarrhea virus DR13 strain. Res. Vet. Sci. 2007, 82,
134ꢀ140.
12.
Kim, S. H.; Lee, J. M.; Jung, J.; Kim, I. J.;
Hyun, B. H.; Kim, H. I.; Park, C. K.; Oem, J. K.; Kim,
Y. H.; Lee, M. H.; Lee, K. K. Genetic characterization
of porcine epidemic diarrhea virus in Korea from 1998
to 2013. Arch. Virol. 2015, 160, 1055ꢀ1064.
ABBREVIATIONS USED
PED, porcine epidemic diarrhea; PEDV, Porcine
Epidemic Diarrhea Virus; Mpro, main protease; SARS,
severe acute respiratory syndrome; MERS, Middle
East respiratory syndrome.
13.
Lee, D. K.; Park, C. K.; Kim, S. H.; Lee, C.
Heterogeneity in spike protein genes of porcine
epidemic diarrhea viruses isolated in Korea. Virus Res.
2010, 149, 175ꢀ182.
14.
Brian, D. A.; Baric, R. S. Coronavirus genome
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