key: cord-252600-bvh1o64r
authors: Galasiti Kankanamalage, Anushka C.; Kim, Yunjeong; Damalanka, Vishnu C.; Rathnayake, Athri D.; Fehr, Anthony R.; Mehzabeen, Nurjahan; Battaile, Kevin P.; Lovell, Scott; Lushington, Gerald H.; Perlman, Stanley; Chang, Kyeong-Ok; Groutas, William C.
title: Structure-guided design of potent and permeable inhibitors of MERS coronavirus 3CL protease that utilize a piperidine moiety as a novel design element
date: 2018-04-25
journal: European Journal of Medicinal Chemistry
DOI: 10.1016/j.ejmech.2018.03.004
sha: 
doc_id: 252600
cord_uid: bvh1o64r

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.

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 0 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 [1e5] . 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 3C-like cysteine protease (3CLpro). Polyproteins pp1a and pp1b are processed by 3CLpro (11 cleavage sites) and PLpro (3 cleavage sites) resulting in sixteen mature nonstructural proteins, including RNA-dependent RNA polymerase (RdRp) and helicase, which play important roles in the transcription and replication of coronaviruses (Fig. 1 ). Both proteases are essential for viral replication, making them attractive targets for drug development [9,10,15e17] .

MERS-CoV 3CLpro is a chymotrypsin-like cysteine protease having a catalytic Cys148-His41 dyad and an extended binding site [18e21] . The protease displays a stringent primary substrate specificity for a P 1 Gln residue [22] and has a strong preference for a 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] .

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.

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 ( Fig. 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 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 1 Gln surrogate [26] side chain. The inhibitor P 2 Leu side chain is ensconced in the hydrophobic S 2 subsite of the enzyme. Importantly, the structure shows a hydrophobic-driven interaction between the benzyl group of the inhibitor and the g-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 ( Fig. 2/Panel B) . The m-Cl phenethyl side chain is clearly shown to occupy the hydrophobic S 4 subsite. In addition to an array of H-bonds 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 1 , S 2 and S 4 subsites, but not the S 3 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 3 and R 4 in a correct vector relationship for optimal interactions with the S 3 and S 4 subsites, (2) rendering a 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 3 and R 4 in inhibitor (I) (Fig. 3) . In summary, the piperidine-based design strategy is a hitherto unrecognized effective means of rendering a dipeptidyl inhibitor equivalent to a tetrapeptidyl inhibitor capable of engaging in optimal binding interactions with all four S 1 -S 4 subsites but which, however, is anticipated to display diminished PK liabilities due to 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) (see Table 3 ).

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 Nsubstituted 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 and 10%. The corresponding bisulfite adducts 10(a-f) were readily 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 .

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 cellbased system including MERS-CoV, FIPV and MHV were evaluated as described in the experimental section. The IC 50, EC 50, and CC 50 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 Fig. 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 50 > 100 mM) ( Table 2 and Fig. 4 ). For example, compound 10a has a selectivity index (SI ¼ CC 50 /EC 50 ) of >250. With the exception of compounds 9e-10e, the aldehyde and aldehyde bisulfite adducts were found to have comparable in vitro potency toward MERS-CoV 3CLpro. Furthermore, pharmacological activity was found to be dependent on the nature of the R 3 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 3CLpro were determined, including the cocrystal structure of the MERS-CoV 3CLpro:inhibitor 10c complex (Fig. 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-Bocpiperidinyl 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 (Fig. 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 1 Gln surrogate ring oxygen and nitrogen with Glu169, His166 and Phe143. The H-bonding interactions are near identical to those of inhibitor GC813 (Fig. 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 (Fig. 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) (Fig. 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 (Fig. 7B ).

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 piperidinebased 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 3 and S 4 subsites of the enzyme. 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.

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 (230e450 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 dropwise under a N 2 atmosphere to a solution of 1-Boc-4piperidinone (10 mmol) in dry THF (15 mL) in an ice bath kept at 0 C. The reaction mixture was stirred for 3 h at room temperature under a N 2 atmosphere while monitoring completion of the reaction by TLC. The reaction 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 removed on the rotary evaporator and the residue was vacuum distilled to yield pure isocyanate 3 as a colorless oil [27, 28] . Table 3 Crystallographic data for MERS-CoV 3CLpro in complex with compounds GC376, GC813, 10c and 10e.

MERS-CoV 3CLpro: GC376

MERS-CoV 3CLpro: Compound 10c MERS-CoV 3CLpro: Compound 10e hkl. c R factor ¼ Ʃ hkl jjF obs (hkl) j -jF calc (hkl) jj/Ʃ hkl jF obs (hkl)j; Rfree is calculated in an identical manner using 5% of randomly selected reflections that were not included in the refinement. d R meas ¼ redundancy-independent (multiplicity-weighted) R merge. [42, 43] R pim ¼ precision-indicating (multiplicity-weighted) R merge. [44, 45] . e CC 1/2 is the correlation coefficient of the mean intensities between two random half-sets of data [46, 47] .

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 Â 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 1 M 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 Â 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.

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 min 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 0e5 C was treated with diisopropylethylamine (DIEA) (9.5 g, 40 mmol, 4 eq), stirred for 30 min, 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 Â 40 mL). The layers were separated and the ethyl acetate layer was further washed with saturated aqueous NaHCO 3 (40 mL), followed by saturated NaCl (50 mL). 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 4 M 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 dissolved in ethyl acetate (50 mL) and washed with 5% HCl (2 Â 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.

Lithium borohydride (2 M 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 tõ 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.

Compound 8 (a-f) (5 mmol) was dissolved in anhydrous dichloromethane (50 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 min. The aqueous layer was removed and the organic layer was washed with 10% aqueous sodium thiosulfate (20 mL), followed by saturated aqueous sodium bicarbonate (2 Â 20 mL), water (2 Â 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). 

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 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 Â 50 mL) to form a white solid. The white solid was stirred with ethyl ether (30 mL) and ethyl acetate (15 mL) for 5 min. Careful removal of the solvent using a pipette left the corresponding aldehyde bisulfite adducts 10 (a-f) as white solids. 

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 with DMSO in 25 mL of assay buffer and incubated at 37 C for 30 min, followed by the addition of 25 mL 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 dosedependent FRET inhibition curves were fitted with a variable slope by using GraphPad Prism software (GraphPad, La Jolla, CA) in order to determine the IC 50 values of the inhibitors.

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 mM) for 48 h. After incubation, viral titers were determined with a TCID 50 (FIPV or MHV) or plaque assay (MERS-CoV). EC 50 values were determined using GraphPadPrism software [31].

The cytotoxic dose for 50% cell death (CC 50 ) for compounds 10a and 10c was determined in Vero81, CRFK or CCL9.1 cells. Confluent cells grown in 96-well plates were treated with various concentrations (1e100 mM) of each compound for 72 h. Cell cytotoxicity was measured by a CytoTox 96 nonradioactive cytotoxicity assay kit (Promega, Madison, WI). The in vitro therapeutic index was calculated by dividing the CC 50 by the EC 50.

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 compound and incubating overnight at 4 C. All crystallization experiments were conducted using Compact 300 (Rigaku Reagents) sitting drop vapor diffusion plates at 20 C using equal volumes of protein and crystallization solution equilibrated against 75 mL 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 2 ) in 1e2 days. Crystals of the GC376 complex were obtained from the Index HT screen (Hampton Research) condition E6 (30% (v/v) PEG 550 MME, 100 mM Bis-Tris pH 6.5, 50 mM CaCl 2 ). Samples were transferred to a fresh drop containing 80% crystallant and 20% (v/v) PEG 200 before storing in liquid nitrogen. X-ray diffraction data were collected at the Advanced Photon Source beamline 17-ID using a Dectris Pilatus 6 M pixel array detector.

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 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] .

Coordinates and structure factors for the MERS 3CLpro inhibitor complexes were deposited to the Worldwide Protein Data Bank (wwPDB) with the accession codes: 5WKJ (GC376), 5WKK (GC813), 5WKL (inhibitor 10c) and 5WKM (inhibitor 10e).

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Supplementary data related to this article can be found at https://doi.org/10.1016/j.ejmech.2018.03.004.