We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC 50 value of 20 M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARSCoV viral replication in Vero E6 cells with an EC50 of 15 M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.ubiquitin-specific protease ͉ noncovalent cysteine protease inhibitor ͉ severe acute respiratory syndrome antiviral ͉ X-ray structure

We describe here the design, synthesis, molecular modeling, and biological evaluation of a series of small molecule, nonpeptide inhibitors of SARS-CoV PLpro. Our initial lead compound was identified via high-throughput screening of a diverse chemical library. We subsequently carried out structure-activity relationship studies, and optimized the lead structure to potent inhibitors that have shown antiviral activity against SARS-CoV infected Vero E6 cells. Based upon the X-ray crystal structure of one of the potent inhibitors 24-bound to SARS-CoV PLpro, a drug-design template was created. Our structure-based modification led to the design of a more potent inhibitor, 2 (enzyme IC50 = 0.46 μM; antiviral EC50 = 12.5 μM). Interestingly, its methylamine derivative 49 displayed good enzyme inhibitory potency (IC50 = 1.3 μM) and most potent SARS antiviral activity (EC50 = 2.5 μM) in the series. We have carried out computational docking studies and generated a predictive 3D-QSAR model for SARS-CoV PLpro inhibitors.

Design, synthesis and biological evaluation of a series of 5-chloropyridine ester-derived severe acute respiratory syndrome-coronavirus chymotrypsin-like protease inhibitors is described. Position of the carboxylate functionality is critical to potency. Inhibitor 10 with a 5-chloropyridinyl ester at position 4 of the indole ring is the most potent inhibitor with a SARS 3Clpro IC 50 value of 30 nM and antiviral EC 50 value of 6.9 μM. Molecular Docking studies have provided possible binding modes of these inhibitors.© 2008 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. 4 While SARS is contained in the world and no more cases have been reported since April 2004, there is expectation that this epidemic will strike again in an even more severe form. Furthermore, the nature of its unpredictable outbreak is a potential threat to the global economy and public heath. To date, no effective therapy exists for this viral illness. NIH Public AccessThe SARS coronavirus is a positive-strand RNA virus. The 5′ two-thirds of the genome encodes two overlapping polyproteins, pp1a and pp1ab, which are processed to generate the viral replication complex. During viral replication, the replicase polyprotein undergoes extensive processing by two viral proteases namely, chymotrypsin-like protease (3CLpro) and papainlike protease (PLpro). 5,6 Because of their essential roles in viral replication, both proteases are recognized as attractive targets for development of anti-SARS therapeutics. 7 The structure and activity of active sites of both SARS-CoV 3CLpro and SARS-CoV PLpro have been elucidated. Thus far, inhibitor design efforts are mostly limited to SARS-CoV 3CLpro and numerous covalent and noncovalent inhibitors have been reported. 7 In our continuing interest in the design and development of SARS-CoV 3CLpro inhibitors, we recently reported structurebased design of a number of potent peptidomimetic SARS-CoV 3CLpro inhibitors (1 and 2). The SARS-CoV 3CLpro active site contains a catalytic dyad where a cysteine residue acts as a nucleophile and a histidine residue acts as the general acid base. 9 The inhibitors bind to SARS-CoV-3CLpro through covalent bonding with the active site cysteine 145 residue. These inhibitors contain peptidomimetic scaffolds and lacked adequate potency, particularly antiviral activity suitable for drug-development. Recently, Wong and co-workers reported a new class of potent small molecule benzotriazole ester-based 3CLpro inhibitors. Compound 3 is the most potent inhibitor among the benzotriazole esters. 10 The mode of acti...