Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element

Article abstract of DOI:10.1016/j.ejmech.2018.03.004

There are currently no approved vaccines or small molecule therapeutics available for the prophylaxis or treatment of Middle East Respiratory Syndrome coronavirus (MERS-CoV) infections. MERS-CoV 3CL protease is essential for viral replication; consequently, it is an attractive target that provides a potentially effective means of developing small molecule therapeutics for combatting MERS-CoV. We describe herein the structure-guided design and evaluation of a novel class of inhibitors of MERS-CoV 3CL protease that embody a piperidine moiety as a design element that is well-suited to exploiting favorable subsite binding interactions to attain optimal pharmacological activity and PK properties. The mechanism of action of the compounds and the structural determinants associated with binding were illuminated using X-ray crystallography.

Full text of DOI:10.1016/j.ejmech.2018.03.004

Accepted Manuscript
Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL
protease that utilize a piperidine moiety as a novel design element
Anushka C. Galasiti Kankanamalage, Yunjeong Kim, Vishnu C. Damalanka, Athri D.
Rathnayake, Anthony R. Fehr, Nurjahan Mehzabeen, Kevin P. Battaile, Scott Lovell,
Gerald H. Lushington, Stanley Perlman, Kyeong-Ok Chang, William C. Groutas
PII:
S0223-5234(18)30239-3
10.1016/j.ejmech.2018.03.004
EJMECH 10269
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2017
Revised Date: 28 February 2018
Accepted Date: 1 March 2018
Please cite this article as: A.C. Galasiti Kankanamalage, Y. Kim, V.C. Damalanka, A.D. Rathnayake,
A.R. Fehr, N. Mehzabeen, K.P. Battaile, S. Lovell, G.H. Lushington, S. Perlman, K.-O. Chang, W.C.
Groutas, Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease
that utilize a piperidine moiety as a novel design element, European Journal of Medicinal Chemistry
(2018), doi: 10.1016/j.ejmech.2018.03.004.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Structure-Guided Design of Potent and Permeable Inhibitors
of MERS Coronavirus 3CL Protease that Utilize a Piperidine
Moiety as a Novel Design Element
+
^
Anushka C. Galasiti Kankanamalage,^ Yunjeong Kim, Vishnu C. Damalanka, Athri D.
#
@
&
Rathnayake,^ Anthony R. Fehr, Nurjahan Mehzabeen, Kevin P. Battaile, Scott
@
%
#
*+
Lovell, Gerald H. Lushington, Stanley Perlman, Kyeong-Ok Chang, William C.
Groutas,*^
^Department of Chemistry, Wichita State University, Wichita, Kansas 67260
+
Department of Diagnostic Medicine & Pathobiology, College of Veterinary Medicine,
Kansas State University, Manhattan, Kansas 66506
@
Protein Structure Laboratory, The University of Kansas, Lawrence, Kansas 66047,
USA
&
IMCA-CAT, Hauptman-Woodward Medical Research Institute, APS Argonne National
Laboratory, Argonne, IL 60439, USA
%
LiS Consulting, Lawrence, KS 66046, USA
#
Department of Microbiology and Immunology, Carver College of Medicine, University
of Iowa, Iowa City, IA 52242
*
authors to whom correspondence should be addressed.
Department of Chemistry, Wichita State University, Wichita, KS 67260
Tel. (316) 978 7374; Fax: (316) 978 3431
e-mail: bill.groutas@wichita.edu
Department of Diagnostic Medicine & Pathobiology, Manhattan, KS 66506
Tel. (785) 532 3849; Fax: (785) 532 4039
e-mail: kchang@vet.ksu.edu
1

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Abstract
There are currently no approved vaccines or small molecule therapeutics available for
the prophylaxis or treatment of Middle East Respiratory Syndrome coronavirus (MERS-
CoV) infections. MERS-CoV 3CL protease is essential for viral replication;
consequently, it is an attractive target that provides a potentially effective means of
developing small molecule therapeutics for combatting MERS-CoV. We describe herein
the structure-guided design and evaluation of a novel class of inhibitors of MERS-CoV
3CL protease that embody a piperidine moiety as a design element that is well-suited to
exploiting favorable subsite binding interactions to attain optimal pharmacological
activity and PK properties. The mechanism of action of the compounds and the
structural determinants associated with binding were illuminated using X-ray
crystallography.
2

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1. Introduction
Coronaviruses are enveloped, positive-sense, single-stranded RNA viruses of the family
Coronaviridae [1]. Human and animal coronaviruses are classified into at least 25
species in four genera, α to δ coronaviruses [1]. Mouse hepatitis virus (MHV) is the
most prominently studied coronavirus both in vitro and in vivo, serving as the prototype
coronavirus. Human coronaviruses, including 229E, NL63, OC43, and HKU1 strains,
have been implicated in respiratory infections, otitis media, exacerbations of asthma,
diarrhea, myocarditis, and neurological disease [1-4]. In contrast, severe acute
respiratory distress syndrome coronavirus (SARS-CoV) and Middle East respiratory
syndrome coronavirus (MERS-CoV) pose a significant threat to public health worldwide
due to their ability to cause serious human disease with high mortality rates [2-4].
MERS-CoV has become a global threat due to continuous outbreaks in countries on the
Arabian peninsula and the potential of spread to other countries with a high mortality
rate [5-8]. Coronaviruses are also important pathogens in animals and are associated
with respiratory and enteric illnesses, including transmissible gastroenteritis virus
(
TGEV), porcine epidemic diarrhea virus (PEDV), feline infectious peritonitis virus
FIPV), and bovine coronavirus [1]. There are currently no effective vaccines or small
(
molecule therapeutics for the treatment of MERS (or SARS) coronavirus infection [9-
1].
1
The highly conserved genome of coronaviruses contains two open reading frames,
ORF1a and ORF1b, on the 5’ end that encode nonstructural proteins. Genes encoding
3

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the coronavirus structural proteins, S (spike glycoprotein), E (envelope protein), M
(membrane glycoprotein), and N (nucleocapsid protein), which play a critical role in
virion-cell receptor binding, replication and virion assembly, are located at the 3’ end of
the genome [1,12]. Coronavirus entry is initiated by the binding of the spike protein (S)
to cell receptors, specifically, dipeptidyl peptidase 4 (DDP4) and angiotensin converting
enzyme 2 (ACE2) for MERS-CoV and SARS-CoV, respectively [1-5]. Entry into cells
requires host proteases for cleavage at two sites in the S protein, in the case of most
CoV [13-14] Translation of the genomic mRNA of ORF1a yields polyprotein pp1a, while
a second polyprotein (pp1b) is the product of a ribosomal frame shift that joins ORF1a
together with ORF1b. ORF1a encodes a papain-like cysteine protease (PLpro) and a
3
C-like cysteine protease (3CLpro). Polyproteins pp1a and pp1b are processed by
CLpro (11 cleavage sites) and PLpro (3 cleavage sites) resulting in sixteen mature
3
nonstructural proteins, including RNA-dependent RNA polymerase (RdRp) and
helicase, which play important roles in the transcription and replication of coronaviruses
(Figure 1). Both proteases are essential for viral replication, making them attractive
targets for drug development [9-10, 15-17].
MERS-CoV 3CLpro is a chymotrypsin-like cysteine protease having a catalytic Cys148-
His41 dyad and an extended binding site [18-21]. The protease displays a stringent
primary substrate specificity for a P Gln residue [22] and has a strong preference for a
1
P
2
Leu residue. The P
3
residue side chain is oriented toward the solvent while the S
4
subsite is shallow, preferring a small hydrophobic P
4
residue (Ala). Functional and
structural studies have delineated the similarities between the 3CLpro of coronaviruses
that can be exploited in the design of broad-spectrum inhibitors [23].
4

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We have recently reported the first demonstration of clinical efficacy of a coronavirus
protease inhibitor (a dipeptidyl aldehyde bisulfite adduct inhibitor designated GC376)
[
24-25]. Specifically, administration of GC376 to cats infected with FIPV, a coronavirus
that is 100% fatal in cats, reversed the progression of fatal FIP and resulted in clinical
remission in a majority of animals (>90%). Since FIP disease progression is quite rapid
and its pathogenesis primarily immune-mediated, features shared by MERS-CoV, we
hypothesized that a viral protease inhibitor could reverse the pathogenesis of MERS-
CoV in affected hosts. Interrogation of this hypothesis entailed, as a first step, the
design of a new and versatile class of peptidomimetic inhibitors of MERS-CoV 3CL
protease. We describe herein the structure-guided design of inhibitors of MERS-CoV
3CLpro that embody a piperidine moiety as a novel design element, as well as pertinent
structural and biochemical studies. These inhibitors were also examined against other
coronaviruses, including SARS-CoV, FIPV and MHV to evaluate the spectrum of activity
against multiple coronaviruses.
2. Results and Discussion.
2.1. Inhibitor design rationale.
The structure-guided design of inhibitor (I) encompassed the following steps: (a) we first
determined a high resolution X-ray crystal structure of MERS-CoV 3CLpro in complex
with GC376 (Figure 2/Panel A). Examination of the active site of the complex revealed
that the aldehyde bisulfite adduct had reverted to the precursor peptidyl aldehyde, which
subsequently formed a tetrahedral hemi-thioacetal upon reaction with the active site
Cys148. Notably, the electron density at this stereocenter was consistent with the
formation of both R and S enantiomers at the covalent binding site (also observed for
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the other structures described in the following sections). The structure reveals a network
of backbone hydrogen bonds which ensure correct positioning of the inhibitor to the
active site, as well as two critical hydrogen bonds with the P Gln surrogate [26] side
1
chain. The inhibitor P Leu side chain is ensconced in the hydrophobic S subsite of the
2
2
enzyme. Importantly, the structure shows a hydrophobic-driven interaction between the
benzyl group of the inhibitor and the γ-lactam ring of the Gln surrogate side chain; (b)
based on the forgoing, we reasoned that extending the “cap” would allow the inhibitor to
assume an extended conformation and orient the phenyl ring toward the hydrophobic S
4
pocket of the enzyme. Validation of this idea was obtained by synthesizing extended
inhibitor GC813 and determining a high resolution X-ray crystal structure of the MERS-
CoV 3CLpro:GC813 complex (Figure 2/Panel B). The m-Cl phenethyl side chain is
clearly shown to occupy the hydrophobic S subsite. In addition to an array of H-bonds
4
with Gln192, Glu169, and Gln167 and the backbone of the inhibitor, which serve to
correctly position the inhibitor at the active site, the inhibitor interacts with the S
subsites, but not the S subsite; (c) we hypothesized that the attachment of a
piperidine ring to the peptidyl component would yield a structurally novel peptidomimetic
I) capable of (1) orienting recognition elements R and R in a correct vector
1 2
, S and
S
4
3
(
3
4
relationship for optimal interactions with the S and S subsites, (2) rendering a
3
4
dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor with potentially diminished PK
liabilities and, (3) providing a flexible means for the structure-guided parallel
optimization of ADMET/PK and physicochemical properties using diversity sites R and
3
4
R in inhibitor (I) (Figure 3). In summary, the piperidine-based design strategy is a
hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a
6

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1
tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four S -
S subsites but which, however, is anticipated to display diminished PK liabilities due to
4
its reduced peptidyl character. Furthermore, the aforementioned piperidine-based
design strategy has wide applicability and can be extended to any protease with an
extended binding site. Preliminary evidence in support of this approach is provided by
the results of enzyme and cell-based screening of derivatives of (I) (Tables 1 and 2), as
well as the results of structural studies (vide infra).
2.2. Chemistry.
The synthesis of final compounds 9(a-f) and 10(a-f) is outlined in Scheme 1. 1-Boc-4-
piperidinone was reacted with different Grignard reagents to yield the corresponding 1-
Boc-4-piperidinol derivatives (1c and 1e). Refluxing (L) leucine methyl ester
hydrochloride with trichloromethyl chloroformate yielded the isocyanate which was
reacted with (1c and 1e, or commercially-available N-substituted 4-piperidinol 1a) to
form the corresponding carbamate adducts (4a, 4c and 4e) that were hydrolyzed to the
corresponding acids (5a, 5c and 5e) with lithium hydroxide in aqueous THF.
Subsequent coupling with glutamine surrogate methyl ester hydrochloride 11 afforded
the desired dipeptidyl esters (6a, 6c and 6e) which were either treated with lithium
borohydride directly or were first treated with dry HCl in dioxane followed by reaction
with an alkyl sulfonyl chloride or alkyl chloroformate, to yield esters (7b, 7d and 7f) prior
to reduction with lithium borohydride, to yield alcohols 8 (a-f). Dess-Martin oxidation,
followed by flash chromatography, yielded pure aldehydes 9(a-f). The enantiomeric
purity of the aldehydes was consistently high, with the amount of epimerized aldehyde
ranging between 0-10%. The corresponding bisulfite adducts 10(a-f) were readily
7

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obtained as white solids by stirring the aldehydes with sodium bisulfite in an ethyl
acetate/water mixture. The synthesized compounds are listed in Table 1.
2.3. Biochemical Studies.
The inhibitory activity of the synthesized compounds against 3CLpro of MERS-CoV,
SARS-CoV or FIPV, and the antiviral activity of two representative compounds
(compounds 10a and 10c) in a cell-based system including MERS-CoV, FIPV and MHV
were evaluated as described in the experimental section. The IC50, EC50, and CC50
values, are listed in Tables 1 and 2. These are the average of at least two
determinations.
It is evident that derivatives of (I) function as highly potent inhibitors of all tested
coronaviruses in enzyme (Table 1) and cell based assays (Table 2 and Figure 4). More
importantly, representative aldehyde bisulfite adduct compounds 10a and 10c display
potent inhibition toward MERS-CoV in both enzyme and cell-based systems, with low
cytotoxicity (CC > 100 µM) (Table 2 and Figure 4). For example, compound 10a has a
50
selectivity index (SI = CC /EC ) of > 250. With the exception of compounds 9e-10e,
50
50
the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro
potency toward MERS-CoV 3CLpro. Furthermore, pharmacological activity was found to
3
be dependent on the nature of the R group (compounds 9e-10e are 10-fold less active
toward MERS-CoV 3CLpro than compounds 9a-d, 10a-d and 9f-10f).
In order to establish the mechanism of action of (I), as well as obtain structural
information that can be used to guide the optimization of pharmacological activity, the
high resolution X-ray crystal structures of several derivatives of (I) bound to MERS-CoV
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3CLpro were determined, including the cocrystal structure of the MERS-CoV
3CLpro:inhibitor 10c complex (Figure 5A). The formation of a tetrahedral adduct via the
reaction of the aldehyde, generated from aldehyde bisulfite adduct 10c under the
crystallization conditions used [27-28], with the active site cysteine (Cys148) is clearly
evident, confirming the mechanism of action of (I). Inspection of the structure reveals
the presence of prominent electron density consistent with the structure of inhibitor 10c;
however, the N-Boc-piperidinyl moiety was disordered. The position and orientation of
the benzyl group suggest that the piperidine ring is likely projecting toward the S
4
subsite. Inhibitor 10c is bound to the active site of the enzyme via a network of
backbone H-bonds with Gln192, Gln167, and Glu169 (Figure 5B). Additionally, a H-
bond with His41 serves to stabilize the hemi-thioacetal tetrahedral adduct. Also clearly
evident are three critical H-bonds involving the P Gln surrogate ring oxygen and
1
nitrogen with Glu169, His166 and Phe143. The H-bonding interactions are near
identical to those of inhibitor GC813 (Figure 2/Panel B). The structural complementarity
of inhibitors 10c and GC813 is also evident in the electrostatic surface representation of
the enzyme with the two inhibitors nestled in the active site (Figure 6).
The cocrystal structure of the MERS-CoV 3CLpro:aldehyde bisulfite adduct 10e
complex also showed that, under the crystallization conditions used, the aldehyde
bisulfite adduct reverted to the precursor aldehyde, which subsequently formed a
tetrahedral adduct with the active site cysteine (Cys148) (Figure 7A). The piperidinyl
moiety was disordered and consequently its precise location could not be discerned.
However, inhibitor 10e is engaged in the same H-bonding interactions as inhibitor 10c
(Figure 7B).
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3. Conclusion
MERS-CoV constitutes a global public health concern. There are currently no licensed
vaccines or antiviral drugs for the prevention and treatment of coronavirus infections.
We disclose herein for the first time the design and utilization of a general class of
piperidine-based peptidomimetic inhibitors of coronavirus 3CL proteases. Attachment of
the piperidine moiety to a dipeptidyl component permits the resultant hybrid inhibitor to
engage in favorable binding interactions with the S and S subsites of the enzyme.
3
4
More importantly, the approach disclosed herein can be extended to other proteases of
medical relevance. Finally, the disclosed compounds potently inhibit MERS-CoV, and
their mechanism of action and mode of binding to MERS-CoV 3CL protease have been
illuminated using X-ray crystallography.
1
0

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4. Experimental section
4.1. General
Reagents and dry solvents were purchased from various chemical suppliers (Aldrich,
Acros Organics, Chem-Impex, TCI America, and Bachem) and were used as obtained.
Silica gel (230-450 mesh) used for flash chromatography was purchased from Sorbent
Technologies (Atlanta, GA). Thin layer chromatography was performed using Analtech
silica gel plates. Visualization was accomplished using UV light and/or iodine. NMR
3 6
spectra were recorded in CDCl or DMSO-d using a Varian XL-400 spectrometer.
Melting points were recorded on a Mel-Temp apparatus and are uncorrected. High
resolution mass spectrometry (HRMS) was performed at the University of Kansas Mass
Spectrometry lab using an LCT Premier mass spectrometer (Waters, Milford, MA)
equipped with a time of flight mass analyzer and an electrospray ion source. The purity
of the compounds was determined by high-performance liquid chromatography (HPLC)
using a Waters Alliance HPLC system with a reverse phase column (Symmetry® C18
3
.5 µm, 4.6 x 75 mm) and a Varian Pro-star HPLC system with a normal phase column
Kinetex 2.6u HILIC 100A, 75 x 4.6mm) at 254 nm. Analysis was conducted using two
different methods. Method A: Revered phase system with gradient elution. Gradient was
(
1
1

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started with water 98%: methanol 2% to water 60%: methanol 40% over a period of 15
minutes, mobile phase flow rate 1.0 mL/min. Method B: Normal phase system with
isocratic with 20% acetonitrile and 80% dichloromethane, mobile phase flow rate 1.0
mL/min. All final compounds had a purity of ≥95% by both methods.
4.1.1. Synthesis of Boc-piperidinol derivatives 1c and 1e. General procedure.
An appropriate Grignard reagent (11 mmol/1.1 eq) was added dropwise under a
N
2
atmosphere to a solution of 1-Boc-4-piperidinone (10 mmol) in dry THF (15 mL) in an
o
ice bath kept at 0 C. The reaction mixture was stirred for 3 h at room temperature under
a N atmosphere while monitoring completion of the reaction by TLC. The reaction
2
mixture was diluted with water (25 mL) and the solution was acidified to pH ~3 using 5%
hydrochloric acid. The solvent was removed on the rotary evaporator and the residue
was extracted with ethyl acetate (75 mL) and the layers separated. The organic layer
then washed with brine (40 mL) and dried over anhydrous sodium sulfate, filtered and
concentrated to yield a colorless oily product which was purified by flash
chromatography to yield 1c and 1e.
4.1.2. Synthesis of (L) leucine methyl ester isocyanate 3.
(L) Leucine methyl ester hydrochloride 2 (100 mmol) was placed in a dry 500-mL
RB flask and then dried overnight on the vacuum pump. The flask was flushed with
nitrogen and dry dioxane (200 mL) was added followed by trichloromethyl chloroformate
(29.67 g, 150 mmol), and the reaction mixture was refluxed for 10 h. The solvent was
1
2

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removed on the rotary evaporator and the residue was vacuum distilled to yield pure
isocyanate 3 as a colorless oil [27-28].
4
.1.3. Synthesis of substituted piperidine-derived carbamates 4a, 4c and 4e. General
procedure.
A solution of substituted or unsubstituted 1-Boc-4-piperidinol (1c, 1e or 1a) (20
mmol) in dry acetonitrile (15 mL) was treated with triethylamine (4.05 g, 40 mmol)
followed by the amino acid methyl ester isocyanate 3 (20 mmol). The resulting solution
was refluxed for 2 h and then allowed to cool to room temperature. The solution was
concentrated and the residue was taken up in ethyl acetate (75 mL). The organic layer
was washed with 5% HCl (2 x 20 mL) and brine (20 mL). The organic layer was dried
over anhydrous sodium sulfate, filtered and concentrated, leaving compounds 4a, 4c
and 4e as colorless oils.
4.1.4. Synthesis of acids 5a, 5c and 5e. General procedure.
A solution of ester (4a, 4c or 4e) (20 mmol) in tetrahydrofuran (30 mL) was
treated with 1M LiOH (40 mL). The reaction mixture was stirred for 3 h at room
temperature and the disappearance of the ester was monitored by TLC. Most of the
solvent was evaporated off and the residue was diluted with water (25 mL). The solution
was acidified to pH ~3 using 5% hydrochloride acid (20 mL) and the aqueous layer was
extracted with ethyl acetate (3 x 100 mL). The combined organic layers were dried over
anhydrous sodium sulfate, filtered, and concentrated to yield the corresponding
compounds 5a, 5c and 5e as colorless oils.
1
3

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4.1.5. Synthesis of compounds 6a, 6c and 6e. General Procedure.
EDCI (2.40 g, 12.5 mmol, 1.25 eq) and HOBt (1.92 g, 12.5 mmol, 1.25 eq) were
added to a solution of compound (5a, 5c or 5e) (10 mmol) in dry DMF (20 mL) and the
mixture was stirred for 30 minutes at room temperature. In a separate flask, a solution
of deprotected glutamine surrogate 11 (2.23 g, 10 mmol) in DMF (15 mL) cooled to 0-5
o
C was treated with diisopropylethylamine (DIEA) (9.5 g, 40 mmol, 4 eq), stirred for 30
minutes, and then added to the reaction mixture containing the acid. The reaction
mixture was stirred for 12 h while monitoring the reaction by TLC. The solvent was
removed and the residue was partitioned between ethyl acetate (100 mL) and 10% citric
acid (2 x 40 mL). The layers were separated and the ethyl acetate layer was further
washed with saturated aqueous NaHCO (40 mL), followed by saturated NaCl (50 mL).
3
The organic layer was dried over anhydrous sodium sulfate, filtered and concentrated to
yield a yellow-colored oily product. Purification by flash chromatography yielded esters
6a, 6c and 6e as white solids.
4.1.6. Synthesis of compounds 7b, 7d and 7f. General Procedure.
4M HCl in dioxane (8 mL) was added to a solution of compound (6a, 6c and 6e)
(10 mmol) in dry DCM (5 mL) and the mixture was stirred for 1 h at room temperature.
The solvent was removed and the residue was dried under high vacuum for 2 h before
the product was dissolved in dry THF (20 mL). An appropriate alkyl sulfonyl chloride or
alkyl chloroformate derivative (11 mmol/ 1.1 eq) was added to the solution with stirring.
The reaction mixture was stirred for 12 h at room temperature and the residue was
1
4

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dissolved in ethyl acetate (50 mL) and washed with 5% HCl (2 x 20 mL). The ethyl
acetate layer was further washed with saturated NaCl (20 mL). The organic layer was
dried over anhydrous sodium sulfate, filtered and concentrated to yield a crude product.
Purification by flash chromatography yielded the corresponding esters 7b, 7d and 7f as
white solids.
4.1.7. Synthesis of alcohols 8 (a-f). General procedure.
Lithium borohydride (2M in THF, 7.5 mL, 15 mmol) was added dropwise to a solution
of ester (6 or 7) (5 mmol) in anhydrous THF (30 mL), followed by absolute ethyl alcohol
15 mL) and the reaction mixture was stirred at room temperature overnight. The
(
reaction mixture was then acidified by adding 5% HCl and the pH adjusted to ~2.
Removal of the solvent left a residue which was taken up in ethyl acetate (100 mL). The
organic layer was washed with brine (25 mL), dried over anhydrous sodium sulfate,
filtered, and concentrated to yield compounds 8 (a-f) as white solids.
4.1.8. Synthesis of aldehydes 9a-f. General procedure.
Compound 8 (a-f) (5 mmol) was dissolved in anhydrous dichloromethane (50
o
mL) under a nitrogen atmosphere and cooled to 0 C. Dess-Martin periodinane reagent
(
3.18 g, 7.5 mmol, 1.5 eq) was added to the reaction mixture with stirring. The ice bath
was removed and the reaction mixture was stirred at room temperature for 3 h
monitoring by TLC indicated complete disappearance of the starting material). A
(
solution of 10% aqueous sodium thiosulfate (20 mL) was added and the solution was
stirred for another 15 minutes. The aqueous layer was removed and the organic layer
1
5

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was washed with 10% aqueous sodium thiosulfate (20 mL), followed by saturated
aqueous sodium bicarbonate (2 x 20 mL), water (2 x 20 mL) and brine (20 mL). The
organic layer was dried over anhydrous sodium sulfate, filtered and concentrated. The
yellow residue was purified by flash chromatography (silica gel/methylene chloride/ethyl
acetate/methanol) to yield a white solid 9 (a-f).
4.1.8.1.
tert-Butyl
4-({[(S)-4-methyl-1-oxo-1-({(S)-1-oxo-3-[(S)-2-oxopyrrolidin-3-
yl]propan-2-yl}amino)pentan-2-yl]carbamoyl}oxy)piperidine-1-carboxylate (9a).
0
1
White solid (yield 73%), M.p 62-64 C. H NMR (400 MHz, CDCl -d): δ ppm 0.88 - 1.03
3
(m, 6 H), 1.46 (s, 9 H), 1.54 - 1.74 (m, 3 H), 1.76 - 1.90 (m, 2 H), 1.92 - 2.04 (m, 1 H),
2.43 (d, J=8.89 Hz, 2 H), 3.14 - 3.26 (m, 2 H), 3.32 - 3.43 (m, 2 H), 3.60 (br. s., 1 H),
3.65 - 3.77 (m, 3 H), 3.94 - 4.02 (m, 1 H), 4.17 - 4.26 (m, 1 H), 4.28 - 4.37 (m, 1 H), 4.75
- 4.84 (m, 1 H), 5.22 - 5.29 (m, 1 H), 6.12 (s, 1 H), 7.81 - 7.86 (m, 1 H), 8.37 - 8.42 (m, 1
13
H), 9.49 (s, 1 H). C NMR (400 MHz, DMSO-d6): δ ppm 200.84, 178.90, 172.33,
1
2
4
55.39, 153.90, 78.71, 69.23, 59.74, 53.29, 52.92, 40.78, 40.14, 37.49, 31.90, 30.68,
9.29, 28.04, 24.23, 22.95. HRMS (ESI) calcd for C H N O : [M+H]: 497.2975. Found:
24
40
4
7
97.2865. HPLC 96.2% (Method A).
.1.8.2. 1-(Methylsulfonyl)piperidin-4-yl {(S)-4-methyl-1-oxo-1-[{(S)-1-oxo-3-[(S)-2-
4
oxopyr rolidin-3-yl]propan-2-yl}amino]pentan-2-yl}carbamate (9b).
0
1
White solid (yield 70%), M.p 53-55 C. H NMR (400 MHz, CDCl -d): δ ppm 0.92 - 1.02
3
(m, 6 H), 1.50 - 1.74 (m, 5 H), 1.78 - 1.89 (m, 2 H), 1.91 - 2.06 (m, 4 H), 2.26 - 2.53 (m,
1
1
H), 2.80 (s, 3 H), 3.14 - 3.25 (m, 2 H), 3.38 (dd, J=7.62, 4.74 Hz, 4 H), 4.24 - 4.34 (m,
H), 4.84 (d, J=3.47 Hz, 1 H), 5.20 - 5.26 (m, 1 H), 5.77 - 5.83 (m, 1 H), 7.32 - 7.34 (m,
1
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1
34 4 7
H), 8.51 - 8.56 (m, 1 H), 9.48 (s, 1 H). HRMS (ESI) calcd for C20H N O S: [M+H]:
475.2226. Found: 475.2230. HPLC 98.8% (Method A).
4.1.8.3.
tert-Butyl
4-benzyl-4-([{(S)-4-methyl-1-oxo-1-[{(S)-1-oxo-3-[(S)-2-
oxopyrrolidin-3-yl]propan-2-yl}amino]pentan-2-yl}carbamoyl]oxy)piperidine-1-
carboxylate (9c).
0
1
White solid (yield 76%), M.p 57-59 C. H NMR (400 MHz, CDCl -d): δ ppm 0.78 - 0.91
3
(m, 6 H), 1.33 (s, 9 H), 1.35 - 1.47 (m, 2 H), 1.51 - 1.72 (m, 6 H), 1.79 - 1.88 (m, 2 H),
2.14 - 2.40 (m, 3 H), 2.74 - 2.96 (m, 1 H), 3.12 - 3.27 (m, 2 H), 3.42 - 3.60 (m, 1 H), 3.78
-
3.94 (m, 1 H), 4.07 - 4.18 (m, 1 H), 4.22 (d, J=4.78 Hz, 1 H), 4.93 - 5.03 (m, 2 H), 5.94
(d, J=5.52 Hz, 1 H), 6.99 - 7.08 (m, 1 H), 7.19 - 7.29 (m, 4 H), 7.64 - 7.70 (m, 1 H), 8.21
-
8.26 (m, 1 H), 9.37 (s, 1 H). HRMS (ESI) calcd for C H N O : [M+H]: 587.3445.
31
46
4
7
Found: 587.3452. HPLC 100% (Method A).
4.1.8.4.
4-Benzyl-1-(methylsulfonyl)piperidin-4-yl
{(S)-4-methyl-1-oxo-1-[{(S)-1-
oxo-3-[(S)-2-oxopyrrolidin-3-yl]propan-2-yl}amino]pentan-2-yl}carbamate (9d).
0
1
White solid (yield 69%), M.p 56-58 C. H NMR (400 MHz, CDCl -d): δ ppm 0.89 - 1.04
3
(m, 6 H), 1.52 (td, J=8.69, 4.52 Hz, 2 H), 1.60 - 1.76 (m, 5 H), 1.80 - 1.89 (m, 1 H), 1.95
(dd, J=6.53, 3.53 Hz, 2 H), 2.26 - 2.50 (m, 2 H), 2.80 (s, 3 H), 3.25 - 3.37 (m, 2 H), 3.44
- 3.52 (m, 1 H), 3.54 - 3.64 (m, 2 H), 3.91 - 4.00 (m, 1 H), 4.19 - 4.28 (m, 1 H), 4.29 -
4.37 (m, 1 H), 5.10 (d, J=2.10 Hz, 2 H), 5.90 (br. s., 1 H), 7.13 (d, J=7.08 Hz, 1 H), 7.30
13
-
7.38 (m, 4 H), 7.77 - 7.82 (m, 1 H), 8.33 - 8.37 (m, 1 H), 9.49 (s, 1 H). C NMR (400
MHz, DMSO-d6): δ ppm 200.84, 178.94, 172. 68, 155.90, 137.16, 130.44, 128.27,
27.86, 77.93, 65.20, 53.38, 48.57, 42. 38, 41.25, 40.83, 37.49, 37.24, 32.85, 32.44,
1
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7

ACCEPTED MANUSCRIPT
40 4 7
27.67, 24.20, 22.96. HRMS (ESI) calcd for C27H N O S: [M+H]: 565.2696. Found:
565.2678. HPLC 100% (Method A).
4.1.8.5.
tert-Butyl
4-ethyl-4-({[(S)-4-methyl-1-oxo-1-{[(S)-1-oxo-3-{(S)-2-
oxopyrrolidin-3-yl}propan-2-yl]amino}pentan-2-yl]carbamoyl}oxy)piperidine-1-
carboxylate (9e).
0
1
White solid (yield 75%), M.p 58-60 C. H NMR (400 MHz, DMSO-d ): δ ppm 0.77 (t,
6
J=7.30 Hz, 3 H), 0.86 (dd, J=6.80, 6.70 Hz, 6 H), 1.39 (s, 9 H), 1.50 (q, J=5.80 Hz, 2 H),
1
.57 - 1.67 (m, 2 H), 1.72 - 1.87 (m, 4 H), 1.96 - 2.06 (m, 1 H), 2.07 - 2.18 (m, 2 H), 2.19
2.30 (m, 1 H), 2.97 - 3.20 (m, 2 H), 3.67 - 3.80 (m, 2 H), 3.85 - 3.94 (m, 1 H), 3.96 -
.08 (m, 1 H), 4.16 - 4.25 (m, 1 H), 4.35 - 4.44 (m, 1 H), 4.47 - 4.55 (m, 1 H), 4.60 - 4.74
-
4
(
m, 1 H), 7.60 - 7.73 (m, 1 H), 8.41 - 8.43 (m, 1 H), 8.56 - 8.59 (m, 1 H), 9.41 (s, 1 H).
HRMS (ESI) calcd for C H N O : [M+H]: 525.3288. Found: 525.3276. HPLC 100%
26
44
4
7
(
Method A).
.1.8.6.
yl]propan-2-yl}amino]pentan-2-yl}carbamoyl]oxy)piperidine-1-carboxylate (9f).
4
Ethyl
4-([{(S)-4-methyl-1-oxo-1-[{(S)-1-oxo-3-[(S)-2-oxopyrrolidin-3-
0
1
White solid (yield 82%), mp 60-61 C. H NMR (400 MHz, CDCl -d): δ ppm 0.96 (dd,
3
J=9.07, 5.87 Hz, 6 H), 1.26 (t, J=7.10 Hz, 3 H), 1.48 - 1.73 (m, 4 H), 1.80 - 1.90 (m, 2
H), 1.94 - 2.03 (m, 1 H), 2.35 - 2.52 (m, 2 H), 3.19 - 3.30 (m, 2 H), 3.33 - 3.46 (m, 2 H),
3.67 - 3.80 (m, 2 H), 4.08 - 4.20 (m, 5 H), 4.33 (d, J=8.71 Hz, 1 H), 4.78 - 4.87 (m, 2 H),
5.30 (s, 1 H), 6.14 - 6.25 (m, 1 H), 8.38 (s, 1 H), 9.49 (s, 1 H). HRMS (ESI) calcd for
22 36 4 7
C H N O : [M+H]: 469.2662. Found: 469.2643. HPLC 100% (Method A).
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8

ACCEPTED MANUSCRIPT
4.1.9. Synthesis of bisulfite adducts 10a-f. General procedure.
Absolute ethanol (12 mL) was added to a solution of aldehyde 9 (a-f) (5 mmol) in
dry ethyl acetate (20 mL) with stirring, followed by a solution of sodium bisulfite (540
0
mg; 5 mmol) in water (5 mL) and the reaction mixture was stirred for 3 h at 50 C. The
reaction mixture was allowed to cool to room temperature and then vacuum filtered. The
solid was thoroughly washed with absolute ethanol and the filtrate was dried over
anhydrous sodium sulfate, filtered, and concentrated to yield a yellowish oil. The oily
product was treated with ethyl ether (2 x 50 mL) to form a white solid. The white solid
was stirred with ethyl ether (30 mL) and ethyl acetate (15 mL) for 5 minutes. Careful
removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts
10 (a-f) as white solids.
4.1.9.1.
Sodium
(2S)-2-{(S)-2-[({[1-(tert-butoxycarbonyl)piperidin-4-yl]oxy}
carbonyl)amino]-4-methylpentanamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl]propane-1-
sulfonate (10a).
0
1
White solid (yield 72%), M.p 87-89 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0
.80 - 0.90 (m, 6 H), 1.04 - 1.13 (m, 4 H), 1.20 (d, J=6.35 Hz, 1 H), 1.39 (s, 9 H), 1.51 -
.67 (m, 2 H), 1.69 - 1.84 (m, 4 H), 1.99 - 2.31 (m, 2 H), 3.10 (dt, J=15.85, 7.87 Hz, 2
1
H), 3.55 - 3.66 (m, 2 H), 3.71 - 3.80 (m, 1 H), 3.83 - 3.90 (m, 1 H), 3.93 - 4.04 (m, 2 H),
.59 - 4.74 (m, 2 H), 7.20 (s, 1 H), 7.54 (s, 1 H), 8.44 - 8.48 (m, 1 H). HRMS (ESI) calcd
10SNa: [M+H]: 601.2519. Found: 601.25. HPLC 97.6 % (Method B).
4
41 4
for C24H N O
1
9

ACCEPTED MANUSCRIPT
4.1.9.2.
Sodium (2S)-1-hydroxy-2-{(S)-4-methyl-2-[({[1-(methylsulfonyl)piperidin-4-
yl]oxy}carbonyl)amino]pentanamido}-3-[(S)-2-oxopyrrolidin-3-yl]propane-1-sulfonate
10b).
(
0
1
White solid (yield 76%), M.p 77-80 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0.79 - 0.95 (m, 6 H), 1.14 - 1.23 (m, 2 H), 1.31 - 1.47 (m, 2 H), 1.61 (br. s., 4 H), 1.90 (d,
J=6.54 Hz, 2 H), 2.03 - 2.22 (m, 2 H), 2.26 - 2.36 (m, 1 H), 2.87 (s, 3 H), 3.02 - 3.20 (m,
4
H), 3.83 - 3.97 (m, 2 H), 4.20 (d, J=7.66 Hz, 1 H), 4.33 (dd, J=14.72, 4.56 Hz, 1 H),
.64 (d, J=3.61 Hz, 2 H), 7.27 (d, J=8.20 Hz, 1 H), 7.63 - 7.69 (m, 1 H), 8.38 - 8.47 (m, 1
4
H). HRMS (ESI) calcd for C H N O S Na: [M+H]: 579.1771. Found: 579.18. HPLC
20
35
4
10 2
98.7 % (Method B).
4.1.9.3.
Sodium (2S)-2-{(S)-2-[({[4-benzyl-1-(tert-butoxycarbonyl)piperidin-4-yl]oxy}
carbonyl)amino]-4-methylpentanamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-yl] propane-
1-sulfonate (10c).
0
1
White solid (yield 71%), M.p 65-67 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0.87 (d, J=7.03 Hz, 6 H), 1.03 - 1.15 (m, 2 H), 1.38 (s, 9 H), 1.58 - 1.69 (m, 4 H), 1.79
(
d, J=10.91 Hz, 2 H), 2.01 - 2.32 (m, 4 H), 2.96 - 3.17 (m, 4 H), 3.70 - 3.81 (m, 3 H),
.93 - 4.12 (m, 2 H), 4.62 - 4.76 (m, 1 H), 4.95 - 5.08 (m, 2 H), 7.12 - 7.26 (m, 1 H), 7.34
br. s., 4 H), 7.52 (s, 1 H), 7.62 - 7.72 (m, 1 H), 8.49 (d, J=6.86 Hz, 1 H). HRMS (ESI)
calcd for C H N O SNa: [M+H]: 691.2989. Found: 691.2954. HPLC 100% (Method
3
(
31
47
4
10
B).
2
0

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4.1.9.4.
Sodium
(2S)-2-{(S)-2-[({[4-benzyl-1-(methylsulfonyl)piperidin-4-
yl]oxy}carbonyl)amino]-4-methylpentanamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-
yl]propane-1-sulfonate (10d).
0
1
White solid (yield 71%), M.p 68-70 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0
.80 - 0.97 (m, 6 H), 1.04 - 1.14 (m, 2 H), 1.32 - 1.53 (m, 4 H), 1.57 - 1.69 (m, 2 H), 1.74
1.82 (m, 1 H), 2.06 - 2.34 (m, 2 H), 2.85 (s, 3 H), 3.00 - 3.19 (m, 2 H), 3.31 - 3.46 (m, 2
-
H), 3.50 - 3.64 (m, 2 H), 3.72 - 3.86 (m, 3 H), 3.93 - 4.10 (m, 2 H), 4.95 - 5.09 (m, 2 H),
.16 (d, J=6.25 Hz, 1 H), 7.34 (d, J=2.29 Hz, 4 H), 7.47 - 7.56 (m, 1 H), 7.62 - 7.70 (m, 1
7
13
H), 8.49 (d, J=7.23 Hz, 1 H). C NMR (400 MHz, DMSO-d6): δ ppm 179.40, 172. 68,
1
56.33, 137.56, 130.86, 128.70, 127.96, 126.78, 65.63, 64.31, 61.62, 53. 81, 49.00,
2.38, 41.25, 37.92, 34.73, 32.85, 32.16, 28.08, 24.63, 22.30. HRMS (ESI) calcd for
4
27 41 4 10 2
C H N O S Na: [M+H]: 669.2240. Found: 669.22. HPLC 100% (Method B).
4.1.9.5.
Sodium
(2S)-2-{(S)-2-[({[1-(tert-butoxycarbonyl)-4-ethylpiperidin-4-
yl]oxy}carbonyl)amino]-4-methylpentanamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-
yl]propane-1-sulfonate (10e).
0
1
White solid (yield 71%), M.p 61-63 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0.81 - 0.97 (m, 6 H), 1.08 (dq, J=13.44, 6.83 Hz, 3 H), 1.39 (s, 9 H), 1.47 - 1.65 (m, 4
H), 1.79 (d, J=11.18 Hz, 4 H), 1.99 - 2.33 (m, 4 H), 3.00 - 3.20 (m, 2 H), 3.62 (d, J=4.98
Hz, 2 H), 3.74 (dd, J=14.18, 7.05 Hz, 2 H), 3.94 - 4.09 (m, 2 H), 4.16 - 4.24 (m, 1 H),
4
1
6
.42 (d, J=10.74 Hz, 2 H), 4.60 - 4.75 (m, 1 H), 7.53 (d, J=6.83 Hz, 1 H), 7.63 - 7.72 (m,
H), 8.37 (d, J=7.96 Hz, 1 H). HRMS (ESI) calcd for C H N O SNa: [M+H]:
26
45
4
10
29.2832. Found: 629.28. HPLC 95.9% (Method B).
2
1

ACCEPTED MANUSCRIPT
4.1.9.6.
Sodium
(2S)-2-{(S)-2-[({[1-(ethoxycarbonyl)piperidin-4-
yl]oxy}carbonyl)amino]-4-methylpentanamido}-1-hydroxy-3-[(S)-2-oxopyrrolidin-3-
yl]propane-1-sulfonate (10f).
0
1
White solid (yield 76%), M.p 86-88 C. H NMR (400 MHz, DMSO-d ): δ ppm
6
0.80 - 0.90 (m, 6 H), 1.03 - 1.12 (m, 1 H), 1.18 (t, J=7.05 Hz, 3 H), 1.38 - 1.50 (m, 4 H),
1.60 (d, J=6.88 Hz, 2 H), 1.75 - 1.85 (m, 2 H), 2.04 - 2.23 (m, 2 H), 3.06 (dd, J=12.40,
7.96 Hz, 2 H), 3.16 (d, J=6.93 Hz, 2 H), 3.61 - 3.75 (m, 2 H), 3.80 - 3.88 (m, 1 H), 3.95
(dd, J=10.42, 5.98 Hz, 1 H), 4.00 - 4.07 (m, 2 H), 4.18 - 4.25 (m, 1 H), 4.33 - 4.41 (m, 1
H), 4.62 - 4.74 (m, 2 H), 7.22 (s, 1 H), 7.45 (s, 1 H), 7.60 (d, J=6.10 Hz, 1 H). HRMS
37 4
(ESI) calcd for C22H N O10SNa: [M+H]: 573.2206. Found: 573.22. HPLC 94.8%
(Method B).
4.2. Enzyme Assays and Inhibition Studies. FRET protease assays.
The FRET protease assay was performed by preparing stock solutions of the
substrate (Dabcyl-KTSAVLQ/SGFRKME-Edans derived from the cleavage sites on the
viral polyproteins of SARS-CoV) and inhibitor in DMSO and diluting into assay buffer
which was comprised of 20 mM HEPES buffer, pH 8, containing NaCl (200 mM), EDTA
(0.4 mM), glycerol (60%), and 6 mM dithiothreitol (DTT). The expression and
purification of the 3CLpro of MERS-CoV, SARS-CoV or FIPV was performed by a
standard method described previously by our lab [24, 29]. The protease (3CLpro of
MERS-CoV, SARS-CoV or FIPV) was mixed with serial dilutions of each compound or
o
with DMSO in 25 µL of assay buffer and incubated at 37 C for 30 min, followed by the
2
2

ACCEPTED MANUSCRIPT
addition of 25 µL of assay buffer containing substrate. Fluorescence readings were
obtained using an excitation wavelength of 360 nm and an emission wavelength of 460
nm on a fluorescence microplate reader (FLx800; Biotec, Winoosk, VT) 1 h following the
addition of substrate. Relative fluorescence units (RFU) were determined by subtracting
background values (substrate-containing well without protease) from the raw
fluorescence values, as described previously [29]. The dose-dependent FRET inhibition
curves were fitted with a variable slope by using GraphPad Prism software (GraphPad,
La Jolla, CA) in order to determine the IC50 values of the inhibitors.
4.3. Antiviral Assays/Cell-based inhibition assays.
The effects of compounds 10a and 10c on the replication of MERS-CoV, FIPV or
MHV-A59 were examined in Vero81, CRFK or CCL9.1 cells, respectively [30]. Briefly,
confluent and semi-confluent cells were infected at an MOI of 0.01 PFU/cell. Following
adsorption, cells were incubated with medium containing DMSO (<0.1%) or each
compound (up to 100 µM) for 48 h. After incubation, viral titers were determined with a
TCID50 (FIPV or MHV) or plaque assay (MERS-CoV). EC50 values were determined
using GraphPadPrism software [31].
4.4. Nonspecific cytotoxic effects.
The cytotoxic dose for 50% cell death (CC ) for compounds 10a and 10c was
5
0
determined in Vero81, CRFK or CCL9.1 cells. Confluent cells grown in 96-well plates
were treated with various concentrations (1 to 100 µM) of each compound for 72 h. Cell
cytotoxicity was measured by a CytoTox 96 nonradioactive cytotoxicity assay kit
2
3

ACCEPTED MANUSCRIPT
(Promega, Madison, WI). The in vitro therapeutic index was calculated by dividing the
CC by the EC
50.
50
4.5. X-ray Crystallographic studies. Crystallization and Data Collection.
Purified MERS-CoV 3CLpro, in 100 mM NaCl, 20 mM Tris pH 8.0, was
concentrated to 8 mg/mL (0.5 mM). Stock solutions of 100 mM GC376, GC813,
compound 10c or compound 10e were prepared in DMSO and the complex with MERS
3CLpro was prepared by mixing the concentrated protein supplemented with 3 mM
o
compound and incubating overnight at 4 C. All crystallization experiments were
conducted using Compact 300 (Rigaku Reagents) sitting drop vapor diffusion plates at
o
2
0 C using equal volumes of protein and crystallization solution equilibrated against 75
µL of the latter. Crystals of MERS 3CLpro in complex with GC813, compound 10c and
compound 10e that displayed a prismatic morphology were obtained from the Index HT
screen (Hampton Research) condition G10 (25% (w/v) PEG 3350, 100 mM Bis-Tris pH
5
.5, 200 mM MgCl
Index HT screen (Hampton Research) condition E6 (30% (v/v) PEG 550 MME, 100 mM
Bis-Tris pH 6.5, 50 mM CaCl ). Samples were transferred to a fresh drop containing
0% crystallant and 20% (v/v) PEG 200 before storing in liquid nitrogen. X-ray
2
) in 1-2 days. Crystals of the GC376 complex were obtained from the
2
8
diffraction data were collected at the Advanced Photon Source beamline 17-ID using a
Dectris Pilatus 6M pixel array detector.
4.6. Structure Solution and Refinement.
Intensities were integrated using XDS [32-33] using Autoproc [34] and the Laue
class analysis and data scaling were performed with Aimless [35], which suggested that
2
4

ACCEPTED MANUSCRIPT
the highest probability Laue class was 2/m and space group C2. Structure solution was
conducted by molecular replacement with Phaser [36] using a previously determined
isomorphous structure of MERS 3CLpro (PDB: 4RSP [37]) as the search model.
Structure refinement and manual model building were conducted with Phenix [38] and
Coot [39], respectively. Disordered side chains were truncated to the point for which
electron density could be observed. Structure validation was conducted with MolProbity
[
40] and figures were prepared using the CCP4MG package [41].
4.7. Accession Codes
Coordinates and structure factors for the MERS 3CLpro inhibitor complexes were
deposited to the Worldwide Protein Data Bank (wwPDB) with the accession codes:
WKJ (GC376), 5WKK (GC813), 5WKL (inhibitor 10c) and 5WKM (inhibitor 10e).
5
Acknowledgments
Use of the University of Kansas Protein Structure Laboratory was supported by a grant
from the National Institute of General Medical Sciences (P30GM110761) of the National
Institutes of Health. Use of the IMCA-CAT beamline 17-ID at the Advanced Photon
Source was supported by the companies of the Industrial Macromolecular
Crystallography Association through a contract with Hauptman-Woodward Medical
Research Institute. Use of the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy Sciences under
contract no. DE-AC02-06CH11357. We are grateful to Dr. Terry L. Bowlin, Microbiotix,
Inc. for assistance in determining the cell-based values of compounds GC376 and
GC813. The research was supported in part by grants from the National Institutes of
Health (R01 AI109039 to K.O.C. and P01 AI060699 and R01 AI129269 to S. P.) and a
2
5

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faculty development KU Endowment Dolph Simons Award in Biomedical Sciences
(W.C.G.)
2
6

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