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<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds</title><author><name sortKey="Baez Santos, Yahira M" sort="Baez Santos, Yahira M" uniqKey="Baez Santos Y" first="Yahira M." last="Báez-Santos">Yahira M. Báez-Santos</name></author><author><name sortKey="St John, Sarah E" sort="St John, Sarah E" uniqKey="St John S" first="Sarah E." last="St. John">Sarah E. St. John</name></author><author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25554382</idno><idno type="pmc">5896749</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5896749</idno><idno type="RBID">PMC:5896749</idno><idno type="doi">10.1016/j.antiviral.2014.12.015</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">001280</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">001280</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds</title><author><name sortKey="Baez Santos, Yahira M" sort="Baez Santos, Yahira M" uniqKey="Baez Santos Y" first="Yahira M." last="Báez-Santos">Yahira M. Báez-Santos</name></author><author><name sortKey="St John, Sarah E" sort="St John, Sarah E" uniqKey="St John S" first="Sarah E." last="St. John">Sarah E. St. John</name></author><author><name sortKey="Mesecar, Andrew D" sort="Mesecar, Andrew D" uniqKey="Mesecar A" first="Andrew D." last="Mesecar">Andrew D. Mesecar</name></author></analytic><series><title level="j">Antiviral Research</title><idno type="ISSN">0166-3542</idno><idno type="eISSN">1872-9096</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><title>Highlights</title><p><list list-type="simple" id="l0005"><list-item id="o0005"><label>•</label><p id="p0235">HTS and structure-based design produced naphthalene-based lead compounds with inhibition of SARS-CoV PLpro in the nM range.</p></list-item><list-item id="o0010"><label>•</label><p id="p0240">These naphthalene-based lead compounds have antiviral potency against SARS-CoV in cell culture.</p></list-item><list-item id="o0015"><label>•</label><p id="p0245">SARS-CoV PLpro naphthalene-based inhibitors are non-toxic and highly selective for SARS-CoV PLpro.</p></list-item><list-item id="o0020"><label>•</label><p id="p0250">Designed SARS-CoV PLpro inhibitors act through a non-covalent, competitive mechanism of inhibition.</p></list-item><list-item id="o0025"><label>•</label><p id="p0255">Lessons from design of SARS-CoV PLpro inhibitors have profound implications for other USPs implicated in disease processes.</p></list-item></list></p></div></front><back><div1 type="bibliography"><listBibl><biblStruct><analytic><author><name sortKey="Angelini, M M" uniqKey="Angelini M">M.M. 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<front><journal-meta><journal-id journal-id-type="nlm-ta">Antiviral Res</journal-id><journal-id journal-id-type="iso-abbrev">Antiviral Res</journal-id><journal-title-group><journal-title>Antiviral Research</journal-title></journal-title-group><issn pub-type="ppub">0166-3542</issn><issn pub-type="epub">1872-9096</issn><publisher><publisher-name>Elsevier B.V.</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25554382</article-id><article-id pub-id-type="pmc">5896749</article-id><article-id pub-id-type="publisher-id">S0166-3542(14)00366-0</article-id><article-id pub-id-type="doi">10.1016/j.antiviral.2014.12.015</article-id><article-categories><subj-group subj-group-type="heading"><subject>Article</subject></subj-group></article-categories><title-group><article-title>The SARS-coronavirus papain-like protease: Structure, function and inhibition by designed antiviral compounds</article-title></title-group><contrib-group><contrib contrib-type="author" id="au005"><name><surname>Báez-Santos</surname><given-names>Yahira M.</given-names></name><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au010"><name><surname>St. John</surname><given-names>Sarah E.</given-names></name><xref rid="fn1" ref-type="fn">1</xref></contrib><contrib contrib-type="author" id="au015"><name><surname>Mesecar</surname><given-names>Andrew D.</given-names></name><email>amesecar@purdue.edu</email><xref rid="cor1" ref-type="corresp">⁎</xref></contrib></contrib-group><aff id="af005">Department of Biological Sciences, Purdue University, West Lafayette, IN, USA</aff><aff id="af010">Department of Chemistry, Purdue University, West Lafayette, IN, USA</aff><aff id="af015">Center for Drug Discovery, Purdue University, West Lafayette, IN, USA</aff><aff id="af020">Center for Cancer Research, Purdue University, West Lafayette, IN, USA</aff><author-notes><corresp id="cor1"><label>⁎</label>Corresponding author at: Department of Biological Sciences, Purdue University, West Lafayette, IN, USA. Tel.: +1 765 494 1924. <email>amesecar@purdue.edu</email></corresp><fn id="fn1"><label>1</label><p id="np005">These authors contributed equally to this work.</p></fn></author-notes><pub-date pub-type="pmc-release"><day>29</day><month>12</month><year>2014</year></pub-date><pmc-comment> PMC Release delay is 0 months and 0 days and was based on .</pmc-comment>
<pub-date pub-type="ppub"><month>3</month><year>2015</year></pub-date><pub-date pub-type="epub"><day>29</day><month>12</month><year>2014</year></pub-date><volume>115</volume><fpage>21</fpage><lpage>38</lpage><history><date date-type="received"><day>29</day><month>8</month><year>2014</year></date><date date-type="rev-recd"><day>17</day><month>12</month><year>2014</year></date><date date-type="accepted"><day>19</day><month>12</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 Elsevier B.V. All rights reserved.</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>Elsevier B.V.</copyright-holder><license><license-p>Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.</license-p></license></permissions><abstract abstract-type="author-highlights" id="ab005"><title>Highlights</title><p><list list-type="simple" id="l0005"><list-item id="o0005"><label>•</label><p id="p0235">HTS and structure-based design produced naphthalene-based lead compounds with inhibition of SARS-CoV PLpro in the nM range.</p></list-item><list-item id="o0010"><label>•</label><p id="p0240">These naphthalene-based lead compounds have antiviral potency against SARS-CoV in cell culture.</p></list-item><list-item id="o0015"><label>•</label><p id="p0245">SARS-CoV PLpro naphthalene-based inhibitors are non-toxic and highly selective for SARS-CoV PLpro.</p></list-item><list-item id="o0020"><label>•</label><p id="p0250">Designed SARS-CoV PLpro inhibitors act through a non-covalent, competitive mechanism of inhibition.</p></list-item><list-item id="o0025"><label>•</label><p id="p0255">Lessons from design of SARS-CoV PLpro inhibitors have profound implications for other USPs implicated in disease processes.</p></list-item></list></p></abstract><abstract id="ab010"><p>Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of invited articles in <italic>Antiviral Research</italic> on “From SARS to MERS: 10 years of research on highly pathogenic human coronaviruses.”</p></abstract><kwd-group id="kg005"><title>Abbreviations</title><kwd>PLpro, papain-like protease</kwd><kwd>CoV, coronavirus</kwd><kwd>nsp, nonstructural protein</kwd><kwd>USP, ubiquitin-specific protease</kwd><kwd>DUB, de-ubiquitinating enzyme</kwd></kwd-group><kwd-group id="kg010"><title>Keywords</title><kwd>Papain-like protease</kwd><kwd>3C-like protease</kwd><kwd>Nsp3</kwd><kwd>SARS-CoV</kwd><kwd>MERS-CoV</kwd><kwd>Ubiquitin</kwd></kwd-group></article-meta></front><body><sec id="s0005"><label>1</label><title>Introduction</title><sec id="s0010"><label>1.1</label><title>SARS-CoV genome and replication</title><p id="p0005">Coronavirus (CoV) replication is a highly orchestrated process involving complex replication machineries to protect the virus genome, the largest known RNA genome, and the viral proteins from the host’s antiviral defense mechanisms (<xref rid="b0055" ref-type="bibr">Brian and Baric, 2005</xref>). Before the zoonotic emergence of the first human coronavirus that caused the 2002/2003 epidemic of severe acute respiratory syndrome (SARS-CoV), there was a paucity of information pertaining to CoV genomes and mechanisms of replication. Since then, the numbers of CoVs sequenced and studied have increased dramatically and several potential drug targets have emerged. Many of the SARS-CoV drug targets are encoded in the 5′-terminal two thirds of the genome, within the two open reading frames (ORF) that encode for the replicase or non-structural proteins (nsps) (see <xref rid="f0005" ref-type="fig">Fig. 1</xref>) (<xref rid="b0330" ref-type="bibr">Tong, 2009</xref>).<fig id="f0005"><label>Fig. 1</label><caption><p>Genome and proteome organization of SARS-CoV non-structural proteins highlighting nsp3 domain organization and PLpro cleavage sites. The ∼30 kb genome of SARS-CoV and its associated replicase, structural and accessory proteins are indicated and the sizes of boxes representing each protein are to scale (top of figure). The replicase genes encoded by ORF1a and ORF1b are shaded in grey. The nsp3 multi-domain protein is shown with the amino acids defining the approximate boundaries of each domain indicated underneath. Downstream of the SARS-CoV PLpro cleavage site between nsp2/3 (818/819aa) is a ubiquitin-like domain (Ubl-1), PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2GRI" id="ir0005">2GRI</ext-link>, a N-terminal Glu-rich acidic-domain (AC), ADP-ribose-1″-phosphatase (ADRP) domain (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2FAV" id="ir0010">2FAV</ext-link>) the SARS unique domain SUD (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2WCT" id="ir0015">2WCT</ext-link>, <ext-link ext-link-type="uri" xlink:href="pdb:2JZE" id="ir0020">2JZE</ext-link>, <ext-link ext-link-type="uri" xlink:href="pdb:2KAF" id="ir0025">2KAF</ext-link>,), a papain-like protease (PLpro) containing a second UBl-2 domain at the N-terminus (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2FE8" id="ir0030">2FE8</ext-link>) followed by the nucleic acid binding domain (NAB), PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2K87" id="ir0035">2K87</ext-link>, the marker domain G2M and four predicted transmembrane domains (TM1–TM4) forming an additional domain containing a metal-binding region (ZF). Finally, the remainder of nsp3 is composed of so-called Y domains (Y1–3), which precede the C-terminal PLpro cleavage sequence at nsp3/4 (2740/2741aa). An alignment of the SARS-CoV PLpro cleavage sequences (right bottom corner) shows a comparison of the P-sites and P′-sites from the nsps to the C-terminal sequences of the cellular proteins ubiquitin (Ub) and ISG15 shown with an isopeptide bond at the P′-sites.</p></caption><graphic xlink:href="gr1"></graphic></fig></p><p id="p0010">Production of the replicase proteins is initiated by the translation of ORF1a and ORF1ab via a –1 ribosomal frame-shifting mechanism (<xref rid="b0050" ref-type="bibr">Bredenbeek et al., 1990</xref>). This mechanism produces two large viral polyproteins, pp1a and pp1ab, that are further processed by two virally encoded cysteine proteases (<xref rid="f0005" ref-type="fig">Fig. 1</xref>), the papain-like protease (PLpro) and a 3C-like protease (3CLpro), which is sometimes referred to as main protease (Mpro) (<xref rid="b0325" ref-type="bibr">Thiel et al., 2003</xref>, <xref rid="b0360" ref-type="bibr">Ziebuhr, 2004</xref>, <xref rid="b0365" ref-type="bibr">Ziebuhr et al., 2000</xref>, <xref rid="b0370" ref-type="bibr">Ziebuhr et al., 2001</xref>). Processing of the viral polyprotein is required for the release and maturation of 16 viral proteins (non-structural proteins or nsps) involved in the formation of a membrane-associated, cytoplasmic enzyme complex, the replicase complex, which is responsible for directing the replication and transcription of the viral genome. It is thought that the establishment of viral replication sites is initiated by the recruitment of replicase proteins to host membranes, a process mediated by several viral transmembrane domain-containing proteins such as the nsp3, nsp4 and nsp6 (<xref rid="b0190" ref-type="bibr">Kanjanahaluethai et al., 2007</xref>, <xref rid="b0255" ref-type="bibr">Oostra et al., 2008</xref>, <xref rid="b0260" ref-type="bibr">Oostra et al., 2007</xref>, <xref rid="b0335" ref-type="bibr">van Hemert et al., 2008</xref>).</p><p id="p0015">Electron tomography and three-dimensional reconstruction imaging studies have revealed that the replicase complex utilizes a reticulovesicular network of double-membrane vesicles (DMVs) with interconnected outer membranes originating from the endoplasmic reticulum (ER) (<xref rid="b0005" ref-type="bibr">Angelini et al., 2013</xref>, <xref rid="b0145" ref-type="bibr">Gosert et al., 2002</xref>, <xref rid="b0195" ref-type="bibr">Knoops et al., 2008</xref>). As a result, the viral replication mechanism is localized, increasing the concentration and cooperation of viral macromolecules and forming a framework of RNA synthesis. Most importantly, this mechanism provides a microenvironment for the protection of viral RNA from host nucleases, and of double-stranded RNA intermediates from the host cell’s innate immune surveillance. Among the components of the replicase complex, nsp3 is of special interest since it is believed to be part of the central scaffolding protein of the replicase complex due to the large number of interactions with other nsp’s (<xref rid="b0005" ref-type="bibr">Angelini et al., 2013</xref>, <xref rid="b0170" ref-type="bibr">Imbert et al., 2008</xref>, <xref rid="b0305" ref-type="bibr">Snijder et al., 2003</xref>).</p></sec><sec id="s0015"><label>1.2</label><title>The multi-domain protein nsp3</title><p id="p0020">The SARS-CoV nsp3 multi-domain protein is the largest replicase subunit at 1,922 amino acids (<xref rid="b0305" ref-type="bibr">Snijder et al., 2003</xref>, <xref rid="b0325" ref-type="bibr">Thiel et al., 2003</xref>). Nsp3 is thought to play an essential role during the formation of virus replication complexes via its insertion into host membranes and its numerous interactions with other nsps (<xref rid="b0170" ref-type="bibr">Imbert et al., 2008</xref>) especially nsp4 and nsp6 (<xref rid="b0005" ref-type="bibr">Angelini et al., 2013</xref>). Numerous domains have now been identified in nsp3 (<xref rid="f0005" ref-type="fig">Fig. 1</xref>) and many are predicted to be conserved in all CoV (<xref rid="b0245" ref-type="bibr">Neuman et al., 2008</xref>). Due to the large size of the nsp3 multi-domain protein, <italic>in vitro</italic> and <italic>in cellular</italic> studies have mainly utilized truncated nsp3 constructs that represent the predicted domains boundaries (<xref rid="f0005" ref-type="fig">Fig. 1</xref>). Using this approach, the three dimensional structures of most of the domains from the nsp3 of SARS-CoV have been determined by X-ray crystallography or NMR spectroscopy (<xref rid="b0065" ref-type="bibr">Chatterjee et al., 2009</xref>, <xref rid="b0105" ref-type="bibr">Egloff et al., 2006</xref>, <xref rid="b0185" ref-type="bibr">Johnson et al., 2010</xref>, <xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>, <xref rid="b0290" ref-type="bibr">Saikatendu et al., 2005</xref>, <xref rid="b0295" ref-type="bibr">Serrano et al., 2007</xref>, <xref rid="b0300" ref-type="bibr">Serrano et al., 2009</xref>, <xref rid="b0320" ref-type="bibr">Tan et al., 2009</xref>).</p><p id="p0025">The N-terminal region of the nsp3 (181–1000) is highly conserved among CoVs, containing a ubiquitin-like (Ubl) globular fold followed by a flexible, extended acidic-domain (AC domain) rich in glutamic acid (38%) (<xref rid="b0295" ref-type="bibr">Serrano et al., 2007</xref>). Next to the AC domain is a catalytically active ADP-ribose-1″-phosphatase (ADRP, <italic>app-1″-pase</italic>) domain (also called <italic>macro</italic> domain or X domain) thought to play a role during synthesis of viral subgenomic RNAs (<xref rid="b0105" ref-type="bibr">Egloff et al., 2006</xref>, <xref rid="b0290" ref-type="bibr">Saikatendu et al., 2005</xref>). SARS Unique Domain (SUD), a domain not yet identified in other coronaviruses from alphacoronavirus and betacoronavirus, follows next (<xref rid="b0315" ref-type="bibr">Tan et al., 2007</xref>). The SUD domain binds oligonucleotides known to form G-quadruplexes (<xref rid="b0320" ref-type="bibr">Tan et al., 2009</xref>). Downstream of the SUD domain is a second Ubl domain and the catalytically active PLpro domain (<xref rid="b0035" ref-type="bibr">Barretto et al., 2006</xref>) that proteolytically processes the nsp1/2, nsp2/3 and nsp3/4 cleavage sites (<xref rid="b0160" ref-type="bibr">Harcourt et al., 2004</xref>). Downstream of PLpro are found a nucleic acid-binding domain (NAB) with a nucleic acid chaperon function (<xref rid="b0245" ref-type="bibr">Neuman et al., 2008</xref>), which is conserved in betacoronavirus and gammacoronavirus, and one uncharacterized domain termed the marker domain (G2M) (<xref rid="b0245" ref-type="bibr">Neuman et al., 2008</xref>). Following the G2M are two predicted double-pass transmembrane domains (TM1–2 and TM3–4) (<xref rid="b0245" ref-type="bibr">Neuman et al., 2008</xref>, <xref rid="b0305" ref-type="bibr">Snijder et al., 2003</xref>), a putative metal binding region (ZN) and the Y domain of unknown function (subdomains Y1–3) (<xref rid="b0305" ref-type="bibr">Snijder et al., 2003</xref>, <xref rid="b0325" ref-type="bibr">Thiel et al., 2003</xref>, <xref rid="b0370" ref-type="bibr">Ziebuhr et al., 2001</xref>). Interestingly, comparative genome and proteome analyses of two bovine CoV (BCoV) isolates showed a predominant clustering of mutations within the nsp3 multi-domain (<xref rid="b0090" ref-type="bibr">Chouljenko et al., 2001</xref>). Consequently, the multi-functionality of the nsp3, the frequency of point mutations observed in nsp3 domains, and the involvement of nsp3 in structural arrangements of the replicase complex and double-membrane vesicles may engender pleiotropic effects, not only in SARS-CoV pathogenicity, but also on future emerging coronaviruses (<xref rid="b0305" ref-type="bibr">Snijder et al., 2003</xref>).</p></sec><sec id="s0020"><label>1.3</label><title>The SARS-CoV PLpro domain within the nsp3</title><p id="p0030">The SARS-CoV PLpro catalytic domain is flanked by numerous catalytically active enzymes, transmembrane domains, and domains of unknown function, and the entire nsp3 is localized to the ER membranes where the majority of the domains reside in the cytosol of the cell (<xref rid="f0005" ref-type="fig">Fig. 1</xref>) (<xref rid="b0150" ref-type="bibr">Hagemeijer et al., 2010</xref>, <xref rid="b0255" ref-type="bibr">Oostra et al., 2008</xref>). In the cytosol, the membrane associated PLpro domain recognizes the P4–P1 consensus cleavage sequence LXGG, found in the boundaries of nsp1/2, nsp2/3 and nsp3/4 where membrane association is required for cleavage of the nsp3/4 (<xref rid="b0155" ref-type="bibr">Han et al., 2005</xref>, <xref rid="b0160" ref-type="bibr">Harcourt et al., 2004</xref>). Proteolytic cleavage of the peptide bond after the glycine at position P1 results in the release of nsp1, nsp2 and nsp3 from the viral polyprotein (<xref rid="f0005" ref-type="fig">Fig. 1</xref>, left bottom panel), a process that is essential for viral replication. Therefore, SARS-CoV PLpro is proposed to be an excellent candidate as a drug target for the development of anti-CoV therapeutics.</p></sec><sec id="s0025"><label>1.4</label><title>SARS-CoV PLpro is a protease, a deubiquitinating (DUB) and deISGylating enzyme</title><p id="p0035">Reminiscent of the overall architecture of human deubiquitinating enzymes (DUBs) in the ubiquitin specific protease family (USP), the molecular structure of the PLpro catalytic domain consists of a canonical, right-handed thumb–palm–fingers architecture which is flanked at the N-terminus by an additional ubiquitin-like (UBL) domain of unknown function (<xref rid="f0010" ref-type="fig">Fig. 2</xref>a and b) (<xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>). The <italic>in vitro</italic> characterization of PLpro enzymatic activities reveal that PLpro can recognize and hydrolyze the cellular proteins ubiquitin (Ub) (<xref rid="b0030" ref-type="bibr">Barretto et al., 2005</xref>, <xref rid="b0035" ref-type="bibr">Barretto et al., 2006</xref>, <xref rid="b0205" ref-type="bibr">Lindner et al., 2005</xref>, <xref rid="b0210" ref-type="bibr">Lindner et al., 2007</xref>) and the UBL protein ISG15 (interferon-induced gene 15) (<xref rid="b0210" ref-type="bibr">Lindner et al., 2007</xref>, <xref rid="b0250" ref-type="bibr">Nicholson et al., 2008</xref>, <xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>), both bearing the LXGG recognition motif at their C-terminus (<xref rid="f0005" ref-type="fig">Fig. 1</xref>, right bottom panel). Ubiquitin and ISG15 are important cellular modifiers that are covalently attached to target proteins via the formation of an isopeptide bond (<xref rid="f0005" ref-type="fig">Fig. 1</xref>, right bottom panel) between their C-terminus and the ε-amino group of a lysine side chain on a target protein. These isopeptide bonds can be hydrolyzed by the isopeptidase activities of DUB and deISGlating enzymes to remove Ub and ISG15 from host cell proteins.<fig id="f0010"><label>Fig. 2</label><caption><p>Multi-domain architecture of PLpro and its two ubiquitin binding subsites. (a) The SARS-CoV PLpro monomer (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2FE8" id="ir0040">2FE8</ext-link>) consists of four domains: starting from N- to the C-terminus, the extended UBL, the thumb, the palm and the fingers domain. (b) An overlay of SARS-CoV PLpro (blue) with USP7 (yellow, PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4M5W" id="ir0045">4M5W</ext-link>) displaying the USP fold. (c) A solvent-accessible surface representation of SARS-CoV PLpro is shown in blue. A K48-linked di-Ub molecule (orange) superimposed onto an ISG15 (yellow) molecule is shown with molecules represented as ribbons. The ISG15 structure consists of two tandem UBL folds (Ub1 and Ub2). The two Ub and UBL binding subsites of SARS-CoV PLpro (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>) are shown in the solvent accessible surface representation with SUb1 shaded white and SUb2 shaded yellow.</p></caption><graphic xlink:href="gr2"></graphic></fig></p><p id="p0040">Kinetic studies on the catalytic efficiency of SARS-CoV PLpro toward different substrates have shown that ISGylated and ubiquitinated substrates are more readily hydrolyzed than small peptide substrates such as RLRGG-AMC, which represents the C-terminus sequence of Ub and ISG15, suggesting a more complex mechanism for substrate recognition that extends well beyond the S4 to S1 enzyme recognition subsites for the LXGG peptide (<xref rid="b0020" ref-type="bibr">Baez-Santos et al., 2014b</xref>, <xref rid="b0205" ref-type="bibr">Lindner et al., 2005</xref>, <xref rid="b0210" ref-type="bibr">Lindner et al., 2007</xref>, <xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>). More recently, a comprehensive analysis of PLpro substrate specificity by X-ray crystallography and mutational analyses demonstrated that two distinct Ub binding subsites (SUb1 and SUb2) exist distant from the catalytic site, providing SARS-CoV PLpro a unique bivalent mechanism of interaction with Ub-like modifier substrates (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>). For most USPs and SARS-CoV PLpro, the primary Ub/UBL binding subsite (SUb1) is distal to the isopeptide bond and located at the boundaries of palm domain and fingers regions (<xref rid="f0010" ref-type="fig">Fig. 2</xref>c). For SARS-CoV PLpro, a second, distal Ub binding subsite (SUb2) exists which is located in a ridge region of the thumb domain (<xref rid="f0010" ref-type="fig">Fig. 2</xref>c). This region provides interactions for a second Ub molecule on a K48-linked di-Ub chain, and for ISG15, which has structural resemblance to K48-linked di-Ub molecules (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>).</p></sec><sec id="s0030"><label>1.5</label><title>SARS-CoV PLpro innate immune functions</title><p id="p0045">The DUB and deISGylating activities of SARS-CoV PLpro have significant functional implications in the innate immune response during SARS-CoV infection. Both Ub and ISG15 are important signaling elements of the host innate immune response against viral infection, which can be negatively regulated by viral DUB and deISGylating enzymes (<xref rid="b0060" ref-type="bibr">Calistri et al., 2014</xref>). SARS-CoV PLpro has been shown to act as a strong antagonist of many Ub-dependent cellular responses to viral infection (<xref rid="b0230" ref-type="bibr">Mielech et al., 2014a</xref>). Although the mechanism of PLpro-mediated antagonism of cellular pathways is not well understood, the evidence strongly suggests that catalytic activity is important for antagonism and, therefore, DUB and deISGylating activities have been proposed as a mechanism.</p><p id="p0050">Because the multiple cellular roles of CoV PLpro DUB/deISGylating activities have been reviewed elsewhere (<xref rid="b0230" ref-type="bibr">Mielech et al., 2014a</xref>), only a brief overview is presented here and the reader should visit other sources for a complete assessment on this topic. SARS-CoV PLpro antagonistic activities have been shown to block the production of important cytokines involved in the activation of the host’s innate immune response against viral infection, including the Type I interferon β (IFNβ) and chemokines such as CXCL10 and CCL5. Based on the <italic>in vitro</italic> and cell culture data available on SARS-CoV PLpro and other homologous coronaviral PLPs, a summary of the possible mechanisms for how PLpro can interfere with the expression of proinflamatory cytokines, such as interferon β (IFN-β), and block the establishment of the antiviral state is illustrated in <xref rid="f0015" ref-type="fig">Fig. 3</xref>. Initially, SARS-CoV PLpro was shown to interfere with the activation of the transcription factors IRF3 (<xref rid="b0100" ref-type="bibr">Devaraj et al., 2007</xref>) and NF-κB (<xref rid="b0095" ref-type="bibr">Clementz et al., 2010</xref>, <xref rid="b0120" ref-type="bibr">Frieman et al., 2009</xref>). Devaraj et al. showed that SARS-CoV PLpro-mediated antagonism of Type I interferon (IFN) production functions upstream of IRF3 activation by blocking IRF3 phosphorylation, homodimerization, and consequently nuclear translocation (<xref rid="b0100" ref-type="bibr">Devaraj et al., 2007</xref>, <xref rid="b0310" ref-type="bibr">Sun et al., 2012</xref>). On the other hand, Frieman et al. demonstrated that PLpro stabilizes the NF-κB inhibitor, IκBα, and thereby blocks the activation of the NF-κB signaling pathway (<xref rid="b0120" ref-type="bibr">Frieman et al., 2009</xref>). More recently, Mielech et al. demonstrated SARS-CoV PLpro ability to decrease endogenous levels of proinflamatory cytokines and chemokines in activated cells (<xref rid="b0235" ref-type="bibr">Mielech et al., 2014b</xref>).<fig id="f0015"><label>Fig. 3</label><caption><p>Currently proposed sites of action for SARS-CoV PLpro-mediated antagonism of the innate immune response. Viral infection is sensed by RIG-I (RIG-I-like helicase) and MDA-5 (melanoma differentiation-associated protein 5) recognition of viral RNA. Recruitment of adaptor proteins MAVS transduces signals to the downstream kinase complex, which activates the transcription factor, IRF-3 and NF-κB, which coordinates the expression of type I interferons (IFN-β and -α). Type I IFN induces the activation of STAT transcription factors resulting in the expression of ISGs (IFN-stimulated genes) and the establishment of an antiviral state in surrounding cells. PLpro can act on different branches of these pathways by interacting with or recognizing and deISGylating and/or deubiquitinating proteins within these pathways. The net effect of these different functions is to help SARS-CoV evade the host antiviral response via antagonizing the establishment of an antiviral state.</p></caption><graphic xlink:href="gr3"></graphic></fig></p><p id="p0055">Interestingly, in some instances, mutation of the active site catalytic cysteine residue of SARS-CoV PLpro only had a small decrease in antagonism of certain pathways, suggesting that the protease activity is not the only requirement for antagonism (<xref rid="b0100" ref-type="bibr">Devaraj et al., 2007</xref>). However, Clementz et al. demonstrated that the SARS-CoV PLpro-mediated block of NF-κB activation can be reversed by the addition of a PLpro competitive inhibitor, emphasizing the importance for catalytic activity during antagonism of this cellular pathway (<xref rid="b0095" ref-type="bibr">Clementz et al., 2010</xref>). Nevertheless, it is important to consider that a mutation of the catalytic machinery of PLpro may not affect its ability to interact with Ub and ISG15 molecules as the competitive inhibitor does. Therefore, a protein-to-protein interaction mediated by PLpro’s strong affinity for Ub/ISG15 molecules on ubiquitinated/ISGylated proteins could still lead to the interference of the IRF3/NF-κB signaling pathways.</p></sec></sec><sec id="s0035"><label>2</label><title>SARS-CoV PLpro structure and function</title><sec id="s0040"><label>2.1</label><title>Active site structure and catalytic mechanism of SARS-CoV PLpro</title><sec id="s0045"><label>2.1.1</label><title>Active site structure</title><p id="p0060">SARS-CoV PLpro belongs to the peptidase clan CA (family C16). The active site contains a classic catalytic triad, composed of Cys112–His273–Asp287, that is well-aligned, functional, and located at the interface of the thumb and palm sub-domains (<xref rid="f0020" ref-type="fig">Fig. 4</xref>) (<xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>). The catalytic cysteine (Cys112) is situated at the foot (N-terminus) of α-helix α4 in the thumb domain. The side chain sulfur atom of Cys112 is positioned 3.7 Å from the <italic>pros</italic>(π)-nitrogen atom of the catalytic histidine (His273) (<xref rid="b0220" ref-type="bibr">McNaught and Wilkinson, 1997</xref>), which is located at the foot of the palm domain and adjacent to the flexible loop BL2 (also called the G267–G272 loop (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>) or β-turn (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>)). One of the oxygen atoms of the side chain of catalytic aspartic acid (Asp287) is located 2.7 Å from the <italic>tele</italic>(τ)-nitrogen of the catalytic histidine at the foot of the palm domain (<xref rid="f0020" ref-type="fig">Fig. 4</xref>). The side chain of Trp107 is located within the oxyanion hole and the indole-ring nitrogen is proposed to participate in the stabilization of negatively-charged tetrahedral intermediates produced throughout catalysis (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>, <xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). The overall tertiary structure of PLpro is remarkably similar to the cellular ubiquitin specific proteases (USP) including USP14 and USP7 (or HAUSP) (<xref rid="f0010" ref-type="fig">Fig. 2</xref>b). However, in their unbound states, the catalytic triad of PLpro aligns well structurally only with the catalytic triad of USP14 and not USP7. In order for the catalytic triad of USP7 to adopt the proper orientation for catalysis, a substrate-induced alignment must occur upon association with ubiquitin or other regulatory domains (<xref rid="b0165" ref-type="bibr">Hu et al., 2002</xref>, <xref rid="b0240" ref-type="bibr">Molland et al., 2014</xref>, <xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>). The fingers domain of PLpro, which contains a zinc ion that is tetrahedrally coordinated by four cysteines, is essential for catalysis because it maintains the structural integrity of PLpro (<xref rid="b0030" ref-type="bibr">Barretto et al., 2005</xref>, <xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>).<fig id="f0020"><label>Fig. 4</label><caption><p>Catalytic center of the active site of SARS-CoV PLpro. Unliganded X-ray crystal structure of SARS-CoV PLpro (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2FE8" id="ir0050">2FE8</ext-link>). The catalytic triad residues, Cys112, His273 and Asp287 are shown along with the oxyanion hole-stabilizing residue Trp107 (right panel). The flexible BL2 loop that interacts with the substrates is indicated as are the zinc ion (purple sphere) and α-helix α4 in the thumb domain. Distances between atoms are given in units of Ångströms (Å) and are indicated by dashed lines.</p></caption><graphic xlink:href="gr4"></graphic></fig></p></sec><sec id="s0050"><label>2.1.2</label><title>Catalytic mechanism</title><p id="p0065">SARS-CoV PLpro functions through a proposed cysteine protease catalytic cycle where Cys112 acts as nucleophile, His273 functions as a general acid-base, and Asp287 is paired with histidine helping to align it and promote deprotonation of Cys112 (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). Currently, there are no experimental studies defining the protonation state of Cys112 prior to nucleophilic attack on the substrate, or whether the reactive nucleophilic species is the thiolate ion of the thiolate–histidine pair, or the neutral thiol, that acts via a mechanism analogous to that of serine peptidases where a thionium ion must be produced after reaction with the ligand (<xref rid="b0025" ref-type="bibr">Barrett et al., 2012</xref>). Because of the extremely low pKa of the thionium intermediate that must be produced in this case, we have chosen to show the classically accepted nucleophilic thiolate form in our general mechanism.<fig id="f0025"><label>Fig. 5</label><caption><p>Catalytic cycle and proposed chemical mechanism of the SARS-CoV PLpro catalyzed reaction. Active site residues of the catalytic triad (Cys112, His273, Asp287) and oxyanion hole residue Trp107 are shown in black. The peptide substrate is shown in green and a catalytic water molecule is shown in blue.</p></caption><graphic xlink:href="gr5"></graphic></fig></p><p id="p0070">In its unliganded form, “<bold>E</bold>”, the catalytic residues of SARS-CoV PLpro are within hydrogen-bonding distance of one another, indicating that the protonation state of Cys112 may exist in equilibrium with that of His273 and that the event of substrate binding drives this equilibrium toward the reactive thiolate (<xref rid="f0020" ref-type="fig">Fig. 4</xref>, <xref rid="f0025" ref-type="fig">Fig. 5</xref>, step a). Alternatively, deprotonation of Cys112 may occur during substrate binding to form the “<bold>ES</bold>” complex (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step a). An addition–elimination sequence follows, where attack of the thiolate of Cys112 on the carbonyl carbon of the peptide bond forms the first negatively-charged tetrahedral intermediate “<bold><italic>TI-1</italic></bold>” or “<bold>FP</bold>,” (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step b). The oxyanion of the tetrahedral intermediate is stabilized via the presence of an oxyanion hole adjacent to the PLpro enzymatic active site (<xref rid="b0085" ref-type="bibr">Chou et al., 2014</xref>, <xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>, <xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>). This oxyanion hole contains a tryptophan residue (Trp107), which our group has confirmed is crucial for the enzymatic function of PLpro through site-directed mutagenesis (<xref rid="b0010" ref-type="bibr">Baez-Santos, 2012</xref>). In addition, we recently showed via the X-ray structure of SARS-CoV PLpro covalently modified with ubiquitin-aldehyde that indole-ring nitrogen of Trp107 acts as a hydrogen-bond donor to the hemithioacetal intermediate (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>), confirming its function in the oxyanion hole. Furthermore, an asparagine residue (Asn110, not depicted) has been found to be highly conserved among coronaviral PLP2s and may contribute to stabilization in the oxyanion hole, as it is positioned above the catalytic cysteine. Elimination of the C-terminal amine of the substrate cleaves the peptide bond, subsequently forming the thioester intermediate “<bold>F</bold>” (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step c). A second addition–elimination sequence follows, where the nucleophilic addition of water to the carbonyl carbon of the thioester forms a second, negatively-charged tetrahedral intermediate “<bold><italic>TI-2</italic></bold>” or “<bold>FQ</bold>” (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step d). The oxyanion of this intermediate is again stabilized via the oxyanion hole of the PLpro active site. Elimination of cysteine from the tetrahedral intermediate results in the formation of the N-terminal carboxylic acid “<bold>EQ</bold>”, which may be transiently stabilized in the PLpro active site via a hydrogen-bond between the carbonyl carbon of the newly formed acid and the indole ring nitrogen of Trp107 (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step e) (<xref rid="b0085" ref-type="bibr">Chou et al., 2014</xref>). Elimination of the cleaved N-terminus of the peptide substrate then completes the catalytic cycle where the final product “<bold>Q</bold>” is released from the active site and the free enzyme “<bold>E</bold>” is regenerated (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, step f).</p></sec></sec></sec><sec id="s0055"><label>3</label><title>SARS-CoV PLpro inhibitor reactions with Cys112</title><p id="p0075">Cysteine proteases are known to react with a variety of electrophilic or “warhead” functionalities within covalent inhibitor molecules. These warhead inhibitors typically function by first forming a noncovalent interaction complex within the cysteine protease active site, where the warhead group of the inhibitor is positioned in the proximity of the reactive cysteine nucleophile. The reaction proceeds via a nucleophilic attack of the thiolate on the electrophilic carbon of the warhead group, forming a covalently modified enzyme–inhibitor complex, which subsequently inactivates the enzyme. Examples of such reactive warhead groups that are known to inhibit cysteine proteases include aldehydes, epoxy-ketones, Michael acceptors, activated ketones, activated esters, vinyl sulfones, acrylamides, alkynes, alkylhalides, and nitriles (<xref rid="f0030" ref-type="fig">Fig. 6</xref>) (<xref rid="b0135" ref-type="bibr">Ghosh et al., 2006</xref>, <xref rid="b0140" ref-type="bibr">Ghosh et al., 2005</xref>, <xref rid="b0175" ref-type="bibr">Jacobs et al., 2013</xref>, <xref rid="b0180" ref-type="bibr">Jain et al., 2004</xref>, <xref rid="b0345" ref-type="bibr">Xue et al., 2008</xref>, <xref rid="b0350" ref-type="bibr">Yang et al., 2006</xref>, <xref rid="b0355" ref-type="bibr">Zhang et al., 2007</xref>).<fig id="f0030"><label>Fig. 6</label><caption><p>Examples of cysteine protease inhibitors with covalent “warheads.” The chemical “warhead” groups are shown in boxes and the reactive, electrophilic portions are colored in red, with the corresponding electrophilic “warhead” carbon indicated with an asterisk. The resulting covalent inhibitor–cysteine complexes are shown outside the boxes and the cysteine portions of the covalent adducts are colored in blue.</p></caption><graphic xlink:href="gr6"></graphic></fig></p><p id="p0080">Our lab successfully solved the first X-ray crystal structure of SARS-CoV PLpro covalently modified with an electrophilic “warhead.” (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>). The SARS-CoV PLpro enzyme was co-crystallized with a semisynthetic version of ubiquitin, where the C-terminus of ubiquitin was modified with an aldehyde functional group (Ubal). This intentional modification of ubiquitin was introduced as a way to modify the catalytic cysteine with a covalent-reversible group thereby trapping the PLpro–Ubal complex and mimicing the reaction intermediate <bold><italic>TI-2</italic></bold> in <xref rid="f0025" ref-type="fig">Fig. 5</xref>. The SARS-CoV PLpro–Ubal structure was determined to a resolution of 2.5 Å and is the first X-ray crystal structure of the hemithioacetal, tetrahedral intermediate in SARS-CoV PLpro (<xref rid="f0035" ref-type="fig">Fig. 7</xref>a and b). The catalytic cysteine is positioned 1.6 Å from the carbon of the tetrahedral intermediate, a distance irrefutably indicating a covalent C–S bond. The tetrahedral, hemithioacetal intermediate is stabilized via the oxyanion hole of SARS-CoV PLpro by a hydrogen bond to the indole nitrogen of Trp107, and is also within hydrogen-bonding distance to the <italic>pros</italic>(π)-nitrogen atom of His273.<fig id="f0035"><label>Fig. 7</label><caption><p>X-ray structure of SARS-CoV PLpro in complex with ubiquitin-aldehyde (Ubal). (a) Pictorial representation of the tetrahedral hemithioaminal intermediate generated upon PLpro–Ubal complex formation, where the Ubal C-terminal residues are shown in orange and the SARS-CoV PLpro residues are shown in black. (b) X-ray crystal structure of PLpro–Ubal complex (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4MM3" id="ir0055">4MM3</ext-link> (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>)) where the PLpro is shown in blue and the residues His273, Cys112, Asp287, and Trp107 are shown in ball-and-stick and colored according to atom type, and Ubal is shown in orange and colored according to atom type. Distances between atoms are given in units of ångströms (Å) and are indicated by dashed lines.</p></caption><graphic xlink:href="gr7"></graphic></fig></p></sec><sec id="s0060"><label>4</label><title>SARS-CoV PLpro inhibitor classes</title><sec id="s0065"><label>4.1</label><title>Inhibitors identified via a designed yeast-based screen</title><p id="p0085">In 2011, two new PLpro inhibitors were reported by Frieman and coworkers (<xref rid="b0115" ref-type="bibr">Frieman et al., 2011</xref>). These inhibitors were identified via a yeast-based screening assay that was developed to discover inhibitors of SARS-CoV PLpro. The assay involved the expression of SARS-CoV PLpro in <italic>S</italic><italic>accharomyces</italic><italic>cerevisiae</italic> that results in a slow-growth phenotype. A 2000 compound NIH Diversity Set library was screened for molecules capable of reversing the PLpro-induced slow-growth phenotype. Compounds identified as hits in this primary assay were then tested for efficacy against SARS-CoV replication and inhibition of PLpro function in cell culture models. This screening method resulted in the identification of <bold>NSC158362</bold>, a compound capable of specifically inhibiting SARS-CoV replication in cell culture without cytotoxic effects. However, <bold>NSC158362</bold> was found incapable of inhibiting the protease, deubiquitinase and anti-interferon functions of PLpro – pointing to a potential new mechanism for inhibition of SARS-CoV replication. A second compound, <bold>NSC158011</bold> was found capable of inhibiting the protease activity of PLpro in cell-based assays; however, it was unsuccessful at blocking SARS-CoV viral replication. The structures of <bold>NSC15832</bold> and <bold>NSC158011</bold> are shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>a (<xref rid="b0115" ref-type="bibr">Frieman et al., 2011</xref>).<fig id="f0040"><label>Fig. 8</label><caption><p>SARS-CoV PLpro inhibitors identified through small-scale screening efforts. Positions within each molecule that are susceptible to covalent modification through nucleophilic attack (“warhead” positions) are indicated in red where the electrophilic carbons are indicated with an asterisk. (a) Inhibitors identified via yeast-based screen (<xref rid="b0115" ref-type="bibr">Frieman et al., 2011</xref>), (b) thiopurine inhibitors (<xref rid="b0070" ref-type="bibr">Chen et al., 2009</xref>, <xref rid="b0080" ref-type="bibr">Chou et al., 2008</xref>), (c) natural product inhibitors (<xref rid="b0075" ref-type="bibr">Cho et al., 2013</xref>, <xref rid="b0265" ref-type="bibr">Park et al., 2012a</xref>, <xref rid="b0270" ref-type="bibr">Park et al., 2012b</xref>), where the most potent natural product of each class is shown and the IC<sub>50</sub> detailed.</p></caption><graphic xlink:href="gr8"></graphic></fig></p></sec><sec id="s0070"><label>4.2</label><title>Thiopurine compounds</title><p id="p0090">Chou and coworkers screened a small library of 960 compounds for the inhibition of SARS-CoV PLpro using a FRET-based assay and purified SARS-CoV PLpro enzyme (<xref rid="b0070" ref-type="bibr">Chen et al., 2009</xref>, <xref rid="b0080" ref-type="bibr">Chou et al., 2008</xref>). Two thiocarbonyl-containing compounds, <bold>6MP</bold> and <bold>6TG</bold>, were found to be effective inhibitors of PLpro with IC<sub>50</sub> values of 21.6 and 5 μM, respectively. In their assay, zinc ion (Zn<sup>2+</sup>) was also found to be an effective inhibitor of PLpro (<italic>vide infra</italic>). Both <bold>6MP</bold> and <bold>6TG</bold> were found to be slow-binding, competitive, reversible, and selective inhibitors of SARS-CoV PLpro. Through comparison to molecular analogs of <bold>6MP</bold> and <bold>6TG</bold> that lacked the thiocarbonyl functionality, the thiocarbonyl was found to be the crucial moiety for these compound’s inhibition of SARS-CoV PLpro and it is postulated to react covalently with the active site cysteine. Currently, both <bold>6MP</bold> and <bold>6TG</bold> are used clinically in the treatment of leukemia, and the anti-cancer action of <bold>6TG</bold> is dependent upon its conversion to <bold>6TG</bold> nucleotides and incorporation into DNA, preventing replication. This, and the acute toxicities associated with both <bold>6MP</bold> and <bold>6TG</bold>, makes them feasible as leads for future PLpro inhibitors, but not as SARS-CoV drugs themselves (<xref rid="f0040" ref-type="fig">Fig. 8</xref>b) (<xref rid="b0070" ref-type="bibr">Chen et al., 2009</xref>, <xref rid="b0080" ref-type="bibr">Chou et al., 2008</xref>).</p></sec><sec id="s0075"><label>4.3</label><title>Natural product inhibitors</title><p id="p0095">A variety of natural products have been found to inhibit SARS-CoV PLpro activity including tanshinones derived from <italic>Salvia miltiorrhiza</italic> (<xref rid="b0270" ref-type="bibr">Park et al., 2012b</xref>), diarylheptanoids derived from <italic>Alnus japonica</italic> (<xref rid="b0265" ref-type="bibr">Park et al., 2012a</xref>), and geranylated flavonoids isolated from the fruits of the <italic>Paulownia tomentosa</italic> tree (<xref rid="b0075" ref-type="bibr">Cho et al., 2013</xref>).</p><sec id="s0080"><label>4.3.1</label><title>Tanshinones</title><p id="p0100">Park and coworkers assayed seven different tanshinones for SARS-CoV PLpro inhibition using a continuous fluorometric assay (<xref rid="b0270" ref-type="bibr">Park et al., 2012b</xref>). The tanshinones were found to inhibit SARS-CoV PLpro activity with IC<sub>50</sub> values ranging from 0.8 to 30.0 μM. Interestingly, the authors found SARS-CoV PLpro inhibition increased as the pre-incubation time of PLpro with tanshinone increased, suggesting a slow-binding mechanism where inhibition of PLpro occurs through the rapid formation of an enzyme–inhibitor complex that slowly isomerizes to form a modified enzyme complex and possible covalent inhibition. The most potent tanshinone inhibitor of SARS-CoV PLpro enzymatic activity is shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>c, left panel (<xref rid="b0270" ref-type="bibr">Park et al., 2012b</xref>).</p></sec><sec id="s0085"><label>4.3.2</label><title>Diarylheptanoids</title><p id="p0105">The diarylheptanoid inhibitors were identified via activity-guided fractionation of the ethanolic extracts from the air-dried bark of <italic>A.</italic><italic>japonica</italic> and assayed for inhibition of SARS-CoV PLpro using a continuous fluorometric assay (<xref rid="b0265" ref-type="bibr">Park et al., 2012a</xref>). The most active diarylheptanoid inhibitor was found to contain an α,β-unsaturated carbonyl group – suggesting that inhibition of SARS-CoV PLpro may occur through formation of a covalent bond with the active site cysteine (<italic>vide supra</italic>). The most potent diarylheptanoid inhibitor of SARS-CoV PLpro enzymatic activity is shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>c, middle panel (<xref rid="b0265" ref-type="bibr">Park et al., 2012a</xref>).</p></sec><sec id="s0090"><label>4.3.3</label><title>Geranylated flavonoids</title><p id="p0110">Using activity-guided fractionation of the methanolic extracts of <italic>P. tomentosa</italic> fruits, Cho and coworkers identified five new geranylated flavonoid derivatives and seven known flavonoids that exhibit micromolar inhibition of SARS-CoV PLpro (<xref rid="b0075" ref-type="bibr">Cho et al., 2013</xref>). Using a fluorogenic assay, the 12 flavonoids were found to inhibit SARS-CoV PLpro with IC<sub>50</sub> values between 5.0 and 14.4 μM. The most active inhibitor was found to be a novel geranylated flavonoid. In general, it was shown that compounds containing the dihydro-2<italic>H</italic>-pyran moiety displayed better PLpro inhibition than their parent compounds. The most potent geranylated flavonoid inhibitor of SARS-CoV PLpro enzymatic activity is shown in <xref rid="f0040" ref-type="fig">Fig. 8</xref>c, right panel (<xref rid="b0075" ref-type="bibr">Cho et al., 2013</xref>).</p></sec></sec><sec id="s0095"><label>4.4</label><title>Zinc Ion (Zn<sup>2+</sup>) and zinc conjugate inhibitors</title><p id="p0115">Using a fluorogenic inhibitor-screening platform that utilized nanomolar concentrations of SARS-CoV PLpro, Han and coworkers established that zinc ion (Zn<sup>2+</sup>) was capable of inhibiting PLpro protease activity with an IC<sub>50</sub> value of 1.3 μM (<xref rid="b0155" ref-type="bibr">Han et al., 2005</xref>). Additionally, they found the zinc conjugates <italic>N</italic>-ethyl-<italic>N</italic>-phenyldithiocarbamic acid–Zn<sup>(II)</sup> and hydroxypyridine-2-thione–Zn<sup>(II)</sup> to inhibit PLpro protease activity with IC<sub>50</sub> values of 3.3 and 3.7 μM, respectively. In this study, inhibition of PLpro activity by Zn<sup>(II)</sup> appeared to be specific as the other divalent metals tested (Mg<sup>(II)</sup>, Mn<sup>(II)</sup>, Ca<sup>(II)</sup>, Ni<sup>(II)</sup>, and Co<sup>(II)</sup>) showed no inhibitory effect on the activity of SARS-CoV PLpro when tested at a concentration of 10 μM; though Cu<sup>(II)</sup> was found to inhibit SARS-CoV PLpro protease activity to 70% at a concentration of 10 μM (<xref rid="b0205" ref-type="bibr">Lindner et al., 2005</xref>).</p></sec><sec id="s0100"><label>4.5</label><title>Naphthalene inhibitors</title><sec id="s0105"><label>4.5.1</label><title>Compound <bold>24</bold></title><p id="p0120">The class of naphthalene inhibitors was first reported in 2008, and was discovered by our group through the implementation of a high-throughput screen (HTS) of 50,080 compounds for inhibitors of SARS-CoV PLpro (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). Two promising SARS-CoV PLpro inhibitors were identified, <bold>7724772</bold> and <bold>6577871</bold>, having IC<sub>50</sub> values of 20.1 μM and 59 μM, respectively (<xref rid="f0045" ref-type="fig">Fig. 9</xref>a1 and a2). Interestingly, both of these hits from the primary HTS contained a naphthylmethylamine moiety. The more potent naphthyl inhibitor identified, compound <bold>7724772</bold>, was identified as a racemate, and upon further investigation the (<italic>R</italic>)-enantiomer was found to be a more potent inhibitor, having and IC<sub>50</sub> value of 8.7 ± 0.7 μM (<xref rid="f0045" ref-type="fig">Fig. 9</xref>a1). Analogs of (<italic>R</italic>)-<bold>7724772</bold> were synthesized to explore the effects of adding different substituents at the <italic>ortho</italic>- and <italic>meta</italic>-positions of the benzene ring and the effects of changing the linkage to either the 1- or 2-naphthyl position. Preliminary structure–activity relationship (SAR) analysis indicated that substitutions at the <italic>ortho</italic>-benzene position other than a methyl group were unfavorable and decreased binding affinity, while changing the linkage to the 1-napthyl position increased inhibitor potency by 4-fold. Addition of a nitro-substituent at the <italic>meta-</italic>position to the amide on the benzene ring decreased inhibitory potency, while addition of an amine at that same position increased inhibition of PLpro by 4-fold (<xref rid="f0045" ref-type="fig">Fig. 9</xref>b1). These SAR studies guided the construction of inhibitor <bold>24</bold>, which has a significantly increased inhibitory potency against SARS-CoV PLpro relative to <bold>7724772</bold> (IC<sub>50</sub> = 0.6 ± 0.1 μM vs. 20.1 ± 1.1 μM). Compound <bold>24</bold> was also found to inhibit SARS-CoV viral replication in Vero cells with an EC<sub>50</sub> of 14.5 ± 0.8 μM (<xref rid="f0045" ref-type="fig">Fig. 9</xref>c1) (<xref rid="b0125" ref-type="bibr">Ghosh et al., 2009</xref>, <xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). More in depth kinetic and biochemical studies demonstrated <bold>24</bold> to be a potent, noncovalent, competitive inhibitor of SARS-CoV PLpro (<italic>K</italic><sub>i</sub> = 0.49 ± 0.08 μM).<fig id="f0045"><label>Fig. 9</label><caption><p>Discovery and design pipeline for SARS-CoV PLpro inhibitors <bold>24</bold> and <bold>15g</bold>. Results of the primary high-throughput screen identified hit compounds <bold>7724772</bold> (a1) and <bold>6577871</bold> (a2). SAR diagrams for analogs of <bold>7724772</bold> (b1) and <bold>6577871</bold> (b2) (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>, <xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). Structures of potent, first-generation leads <bold>24</bold> (c1) and <bold>15g</bold> (c2). X-ray crystal structures of <bold>24</bold> (d1) (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:3E9S" id="ir0060">3E9S</ext-link>) and <bold>15g</bold> (d2) (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:3MJ5" id="ir0065">3MJ5</ext-link>) bound to SARS-CoV PLpro highlighting the residues involved in binding each inhibitor. Hydrogen bonds are shown as dashed lines.</p></caption><graphic xlink:href="gr9"></graphic></fig></p><p id="p0125">The X-ray structure of <bold>24</bold> in complex with SARS-CoV PLpro was determined to a resolution of 2.5 Å, where <bold>24</bold> was found to bind to SARS-CoV PLpro within the S3 and S4 subsites in a cleft leading to the active site (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). The amide group of <bold>24</bold> forms two hydrogen bonds with the Asp165 and Gln270 residues of SARS-CoV PLpro, while the rest of the interactions between the inhibitor and SARS-CoV PLpro were hydrophobic. The naphthyl group of the inhibitor forms hydrophobic interactions with the aromatic rings of the Tyr265 and Tyr269 residues and with the side chains of Pro248 and Pro249. The (<italic>R</italic>)-methyl group of <bold>24</bold> is positioned directly into a polar cavity, between residues Tyr265 and Thr302. The <italic>ortho</italic>-methyl substituent on the benzene ring is directed toward the floor of this cavity, and surrounded by residues Tyr265, Tyr274, and Leu163. The amino-substituent of the benzene ring is surrounded by several polar groups that may serve as hydrogen-bond acceptors (<xref rid="f0045" ref-type="fig">Fig. 9</xref>d1).</p></sec><sec id="s0110"><label>4.5.2</label><title>Compound <bold>15g</bold></title><p id="p0130">The second, less potent naphthylmethylamine hit identified from our primary HTS campaign (<bold>6577871</bold>, <xref rid="f0045" ref-type="fig">Fig. 9</xref>a2) was also found to be a competitive, noncovalent inhibitor of SARS-CoV PLpro (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). Interestingly, <bold>6577871</bold> had a 1-substituted naphthalene ring, which was found to be the more active linkage in the <bold>7724772</bold> series (<italic>vide supra</italic>). A small library of <bold>6577871</bold> analogs was synthesized to investigate the effect of substitution at the 1- or 2-naphthyl positions, at the benzylic-naphthyl position, and at positions on the benzene ring (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>). Similar to the SAR trends found for <bold>7724772</bold>, a 1-napthyl linkage was found to be most favorable for increased PLpro inhibition. Again, substitution of a methyl group at the benzylic-naphthyl position was found to increase inhibition; however, in the case of the <bold>6577871</bold> series, the stereochemistry of this chiral center had a much less dramatic effect, where the preference for an (<italic>R</italic>)- or (<italic>S</italic>)-stereochemistry changed depending upon the substituents on the benzene ring. A 2-methoxy substituent on the benzene ring was found to have a 3.5-fold decrease in inhibition, while positioning the methoxy substituent at the 3- or 4-positions on the benzene ring did not measurably change the IC<sub>50</sub> values. A series of benzodioxole analogs was also investigated and found to have minimally increased inhibitory potency relative to the methoxy-substituted benzene analogs (<xref rid="f0045" ref-type="fig">Fig. 9</xref>b2). These studies and SAR analyses ultimately led to the generation of inhibitor <bold>15g</bold>, which has a significantly increased inhibitory potency against SARS-CoV PLpro relative to <bold>6577871</bold> (IC<sub>50</sub> = 0.32 ± 0.01 μM vs. 59 ± 7.8 μM). Compound <bold>15g</bold> was found to inhibit SARS-CoV viral replication in Vero cells with an EC<sub>50</sub> = 9.1 ± 0.5 μM. Interestingly, the (<italic>S</italic>)-enantiomer of <bold>15g</bold>, compound <bold>15h</bold>, was found to have only slightly decreased inhibition of SARS-CoV PLpro, having an IC<sub>50</sub> value of 0.56 ± 0.03 μM. Moreover, <bold>15g</bold> showed better inhibition of SARS-CoV PLpro and better inhibition of SARS-CoV replication than the previously identified compound, <bold>24</bold> (<xref rid="f0045" ref-type="fig">Fig. 9</xref>c2). Additional kinetic and biochemical studies demonstrated that <bold>15g</bold> is a potent, noncovalent, competitive inhibitor of PLpro (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>).</p><p id="p0135">The X-ray structure of <bold>15g</bold> in complex with SARS-CoV PLpro was determined to a resolution of 2.63 Å and <bold>15g</bold> was found to bind in the same cleft leading to the active site as <bold>24</bold> (<xref rid="f0045" ref-type="fig">Fig. 9</xref>d1 and d2). Both compounds, <bold>15g</bold> and <bold>24</bold>, bind with their napthylmethylamine moieties positioned in the same orientation and location within the enzyme active site. The naphthyl moieties sit in a hydrophobic pocket surrounded by PLpro residues Tyr269, Tyr265, Pro248, Pro249, and Thr302. The (<italic>R</italic>)-configured methyl group of <bold>15g</bold> extends into a small pocket that is lined with both hydrophobic and polar groups that are bound to water molecules. The piperidine nitrogen of <bold>15g</bold> is located in an analogous position to the carboxamide nitrogen of <bold>24</bold>, and at physiological pH the piperidine nitrogen acts as a nitrogen-centered hydrogen-bond donor, forming a hydrogen-bond with the side chain carboxylate of Asp165. The remaining portion of the <bold>15g</bold> molecule, containing the amide and benzodioxole, extends to occupy space within the binding cleft not utilized by <bold>24</bold>, where the amide nitrogen of <bold>15g</bold> makes a hydrogen bond with the backbone carbonyl oxygen of Tyr269, further anchoring the inhibitor in the binding site. The presence of these additional interactions within the PLpro binding cleft is likely responsible for the increased inhibitory ability of <bold>15g</bold> relative to <bold>24</bold> (<xref rid="f0045" ref-type="fig">Fig. 9</xref>d2).</p></sec></sec><sec id="s0115"><label>4.6</label><title>Second generation naphthalene inhibitors</title><sec id="s0120"><label>4.6.1</label><title>Compounds <bold>3k</bold> and <bold>3j</bold></title><p id="p0140">Though compounds <bold>24</bold> and <bold>15g</bold> had low micromolar inhibitory potency and minimal cytotoxicity in SARS-CoV infected Vero E6 cells, further optimization of their antiviral potencies and physiochemical properties was still required to make them therapeutically viable. Using the SARs previously described, and the structural information obtained from the X-ray crystal structures of <bold>24</bold> and <bold>15g</bold>, a second generation of 30 inhibitors was synthesized and evaluated for SARS-CoV PLpro inhibition. This second generation library was designed to probe the steric and electronic demands of the benzylic-naphthyl position, benzylic position, substituent and bioisoster tolerance of the benzyl and naphthyl rings, and the effect of distance between the aromatic rings. The common features of <bold>15g</bold> and <bold>24</bold> required for good PLpro were held constant, namely the 1-naphthyl substitution and position of the hydrogen-donating nitrogen (<xref rid="f0050" ref-type="fig">Fig. 10</xref>a).<fig id="f0050"><label>Fig. 10</label><caption><p>Discovery and design pipeline of compounds <bold>3k</bold>, <bold>3j</bold>, <bold>3e</bold>, and <bold>5c</bold>. (a) Initial SARS-CoV PLpro inhibitor leads, <bold>15g</bold> and <bold>24</bold>, (b) SARs derived from small <bold>15g</bold> analog library, (c) top SARS-CoV PLpro inhibitor candidates, <bold>3k</bold>, <bold>3j</bold>, <bold>3e</bold>, and <bold>5c</bold> (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>).</p></caption><graphic xlink:href="gr10"></graphic></fig></p><p id="p0145">Increasing the size of the benzylic-naphthyl substituent from a methyl group to anything with greater steric demands decreased SARS-CoV PLpro inhibition in a trend proportional to substituent size, indicating that the pocket accessed by the (<italic>R</italic>)-methyl group was less accessible than initially indicated by the crystal structures of <bold>24</bold> and <bold>15g</bold> (<xref rid="f0050" ref-type="fig">Fig. 10</xref>b, position R<sub>1</sub>). Adding a variety of substituents to the benzylic position did not increase inhibitory potency beyond that of <bold>15g</bold>, indicating that additional functionalization at this position does not further engage the residues of the PLpro binding cavity (<xref rid="f0050" ref-type="fig">Fig. 10</xref>b, position R<sub>2</sub>). The effect of substitution on the benzene ring was found to be completely substituent and position dependent, with no clear SAR trend; however two analogs with <italic>meta</italic>- and <italic>para</italic>-fluoro substituents were found to have the greatest PLpro inhibition, <bold>3k</bold> and <bold>3j</bold> (<xref rid="f0050" ref-type="fig">Fig. 10</xref>b, position R<sub>3</sub>). The effect of extending the linkage between the amide and benzene ring was examined and lengthening the chain by even one carbon atom was found to weaken PLpro inhibition. A variety of bioisosteres were also investigated where the W, X, Y, and Z positions (<xref rid="f0050" ref-type="fig">Fig. 10</xref>b) were substituted with nitrogen. In general, these bioisosteres did not increase the inhibitory potency of the compounds, with the exception of <bold>5c</bold>, which had a similar IC<sub>50</sub> and EC<sub>50</sub> to <bold>15g</bold> (<xref rid="f0050" ref-type="fig">Fig. 10</xref>b and c).</p><p id="p0150">The X-ray structures of SARS-CoV PLpro in complex with lead inhibitors <bold>3k</bold> and <bold>3j</bold> were determined to resolutions of 2.1 Å and 2.5 Å, respectively (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). Similar to the X-ray structures of SARS-CoV PLpro in complex with lead inhibitors <bold>24</bold> and <bold>15g</bold>, <bold>3k</bold> and <bold>3j</bold> were found to bind adjacent to the active site, within the S3 and S4 sub-sites of the SARS-CoV PLpro enzyme and in the same orientation. The inhibitors bind to SARS-CoV PLpro through an induced-fit mechanism, where the BL2 loop, which contains Tyr269, adopts a closed conformation in order to interact strongly with the inhibitors. The interactions between the BL2 loop and the inhibitor occur through different bonds including a hydrogen-bond between the backbone carbonyl of Tyr269 and the amide nitrogen of the inhibitors. The motions associated with the BL2 loop and its different conformations are further discussed below in Section <xref rid="s0135" ref-type="sec">5</xref>.</p><p id="p0155">The naphthyl-rings of <bold>3k</bold> and <bold>3j</bold> are located in identical chemical space to the inhibitors <bold>15g</bold> and <bold>24</bold> within the hydrophobic pocket hedged by Pro248 and Pro249 and lined by Tyr265, Tyr269, and Thr302 (<xref rid="f0055" ref-type="fig">Fig. 11</xref>a). The higher resolution X-ray structures of <bold>3k</bold> and <bold>3j</bold> allowed for a more definitive placement of three conserved water molecules present in the small cavity occupied by the (<italic>R</italic>)-methyl substituent. These water molecules decrease the effective size of the cavity and support our previously described SAR for substituents at the benzylic-naphthyl position. The superposition of compounds <bold>3k</bold>, <bold>3j</bold>, and <bold>15g</bold> shows a 1.0 Å difference in the position of the amide nitrogen of <bold>15g</bold> compared to <bold>3k</bold> and <bold>3j</bold>, allowing a slight tilt in the orientation of the benzene ring to accommodate the different substituents (<xref rid="f0055" ref-type="fig">Fig. 11</xref>b). This tilt allows <bold>3k</bold> to form stronger interactions with Tyr269 and Gln270 and is postulated to explain the increased inhibitory potency of <bold>3k</bold>.<fig id="f0055"><label>Fig. 11</label><caption><p>X-ray crystal structures of SARS-CoV PLpro in complex with inhibitors <bold>3k</bold> and <bold>3j</bold> and comparison to <bold>15g</bold>. (a) An overlay of SARS-CoV PLpro (blue cartoon, gray surface) in complex with <bold>3k</bold> (orange stick, PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4OW0" id="ir0070">4OW0</ext-link>) and PLpro (cyan cartoon, gray surface) in complex with 3j (pink sticks, PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4OVZ" id="ir0075">4OVZ</ext-link>) depicting amino acids important for inhibitor binding (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). (b) An overlay of PLpro–<bold>3k</bold> (PLpro in blue cartoon, <bold>3k</bold> in orange sticks), PLpro–<bold>3j</bold> (PLpro in cyan cartoon, <bold>3j</bold> in pink sticks) and PLpro–<bold>15g</bold> (green cartoon and sticks, (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:3MJ5" id="ir0080">3MJ5</ext-link>, (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>)) complexes.</p></caption><graphic xlink:href="gr11"></graphic></fig></p></sec><sec id="s0125"><label>4.6.2</label><title>Metabolically stable naphthalene-based SARS-CoV PLpro inhibitors</title><p id="p0160">The most potent identified inhibitor of PLpro, compound <bold>3k</bold>, was expected to have greater metabolic stability than the previously identified lead compound, <bold>15g</bold>, as the metabolically labile dioxolane of <bold>15g</bold> had been replaced with a fluoro-substituent (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). Additionally, as <bold>3k</bold> has a lower topological polar surface area relative to <bold>15g</bold>, it was expected to have increased cell permeability and subsequently, greater antiviral activity in comparison to <bold>15g</bold>. The most potent analogs of <bold>15g</bold> were subjected to anti-SARS-CoV assays in cell culture to determine EC<sub>50</sub> values, which resulted in the identification of four top compounds with good antiviral activity: <bold>3k</bold>, <bold>3j</bold>, <bold>3e</bold>, and <bold>5c</bold> (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). To assess the potential stability of these compounds <italic>in vivo</italic>, their stability to Phase I metabolism by mouse liver microsomes was evaluated. Surprisingly, <bold>3k</bold> was found to be rapidly metabolized; however, the compounds <bold>3e</bold> and <bold>5c</bold> showed significantly improved metabolic stability relative to the best first generation inhibitor, <bold>15g</bold> (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). Because of their improved metabolic stability, inhibitors <bold>3e</bold> and <bold>5c</bold> are promising candidates for advancing to animal efficacy models.</p></sec><sec id="s0130"><label>4.6.3</label><title>Selectivity of naphthalene-based SARS-CoV PLpro inhibitors</title><p id="p0165">There are at least 98 functional deubiquitinating enzymes in the human genome which fall into one of 6 different classes, where the number of members within each class are indicated in parenthesis; USP (56), OTU (15), JAMM (12), MCPIP (7), MJD (4) and UCH (4) (<xref rid="b0110" ref-type="bibr">Fraile et al., 2012</xref>). The USP class, to which coronaviral PLpro’s belong, represent the largest class with 56 members. As the catalytic domains among the USP class are highly conserved, the question immediately arises as to how selective the designed inhibitors are for SARS-CoV PLpro and against human USPs? At least two approaches have been used so far in an attempt to answer this question. The first was a chemical biology approach pioneered by Hidde Ploeugh and colleagues (<xref rid="b0040" ref-type="bibr">Borodovsky et al., 2001</xref>, <xref rid="b0045" ref-type="bibr">Borodovsky et al., 2002</xref>, <xref rid="b0215" ref-type="bibr">Love et al., 2007</xref>). In this approach, the active-site-directed probe HA–Ub–vinyl sulfone (VS) was used to treat cellular lysates of untransfected cells and cells transfected with SARS-CoV PLpro both in the absence and presence of inhibitors (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). Since HA–Ub–VS potently inhibits a number of human USPs, the ability of compounds to block modification of USPs or PLpro from cellular lysates can be determined via Western blot analysis by using an anti-HA antibody. Using this approach, we found that the naphthalene-based class of inhibitors do not compete against HA–Ub–VS modification of cellular DUBs but does compete against modification of SARS-CoV PLpro.</p><p id="p0170">A second approach for testing selectivity against USPs, other DUBs and cysteine proteases is to test for compound inhibition against purified enzymes, as many of these enzymes are now commercially available. We used this approach to test the naphthalene-based inhibitors against purified USP2, USP7, USP8, USP18, USP20, USP21, USP28, UCH-L1, UCH-L3, DEN1, caspase 3 and cathepsin K in biochemical-based assays (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>, <xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>). We found that none of these human enzymes was significantly inhibited by the naphthalene-based PLpro inhibitors up to concentrations as high as 100 μM. Importantly, the selective inhibition of SARS-CoV PLpro by designed, non-covalent inhibitors is the first example demonstrating that it is possible to design selective inhibitors against the USP class of enzymes which is of universal importance for selectively targeting DUBs involved in promoting different diseases.</p></sec></sec></sec><sec id="s0135"><label>5</label><title>X-ray crystal structures of SARS-CoV PLpro</title><sec id="s0140"><label>5.1</label><title>Structure of SARS-CoV PLpro in an unbound state</title><p id="p0175">The X-ray structure of the 35-kDa catalytic domain of PLpro (residues 1541–1855 of the SARS-CoV nsp3 polyprotein domain) was originally determined to a resolution of 1.85 Å in its unbound state, providing the first structure of a coronaviral PLpro (<xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>). Although gel filtration analyses indicate that PLpro is predominately monomeric in solution (<xref rid="b0155" ref-type="bibr">Han et al., 2005</xref>), the unliganded structure of PLpro crystallized in space group <italic>C2</italic> as three closely packed monomers in the crystallographic asymmetric unit (<xref rid="f0060" ref-type="fig">Fig. 12</xref>a, left panel). Structural alignment of the three monomers shows no significant conformational differences in the central body (palm and thumb domains) of the enzyme or within the active site. However, significant conformational differences between all three monomers were detected at the fingers domain and the BL2 loop of the palm domain at the entrance of the active site, suggesting high plasticity at these regions (<xref rid="f0060" ref-type="fig">Fig. 12</xref>a, right panel).<fig id="f0060"><label>Fig. 12</label><caption><p>X-ray crystal structures of SARS-CoV PLpro in different asymmetric units. PLpro is shown as a ribbon representation with each monomer within an asymmetric unit colored in a different shade of blue. The BL2 loop is highlighted in red and the zinc ion is shown as a pink sphere. (a) The crystallographic trimer of unliganded PLpro (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:2FE8" id="ir0085">2FE8</ext-link>, (<xref rid="b0285" ref-type="bibr">Ratia et al., 2006</xref>)) is shown in the left panel and a structural superposition of the three monomers is shown in the right panel. (b) The crystallographic dimer of the SARS-CoV PLpro–<bold>15g</bold> inhibitor complex (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:3MJ5" id="ir0090">3MJ5</ext-link>, (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>)) is shown in the left panel and a structural superposition of the two monomers is shown in the right panel. Compound <bold>15g</bold> is shown in orange. (c) The crystallographic monomer of the SARS-CoV PLpro–<bold>24</bold> inhibitor complex (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:3E9S" id="ir0095">3E9S</ext-link>, (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>)) is shown in the left panel. Compound <bold>24</bold> is shown in green. The right panel depicts the structural superposition of the SARS-CoV PLpro–<bold>15g</bold> and SARS-CoV PLpro–<bold>24</bold> inhibitor complexes. (D) The crystallographic dimer of the SARS-CoV PLpro–<bold>3k</bold> inhibitor complex (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4OW0" id="ir0100">4OW0</ext-link>, (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>)) is shown in the left panel and a structural superposition of the two monomers in the asymmetric unit is shown in the right panel.</p></caption><graphic xlink:href="gr12"></graphic></fig></p></sec><sec id="s0145"><label>5.2</label><title>Structure of SARS-CoV PLpro in complex with inhibitors</title><p id="p0180">Generally, SARS-CoV PLpro has been highly challenging to crystallize. The crystallization conditions that yielded the SARS-CoV PLpro crystals in its unliganded state were often irreproducible or did not to produce diffraction quality crystals, preventing the crystallization of SARS-CoV PLpro in complex with inhibitors via soaking experiments. Attempts to co-crystallize SARS-CoV PLpro with inhibitors using the crystallization conditions identified for the unliganded SARS-CoV PLpro enzyme were also unsuccessful, presumably due to the tight packing interactions of SARS-CoV PLpro monomers and the positioning of the active site on the interior of the crystallographic trimer (<xref rid="f0060" ref-type="fig">Fig. 12</xref>a). Eventually, co-crystals of SARS-CoV PLpro–inhibitor complexes were achieved by the screening of thousands of crystallization conditions using a crystallization robot. Ultimately, the SARS-CoV PLpro–inhibitor complexes were found to crystallize in different crystallization conditions (<xref rid="t0005" ref-type="table">Table 1</xref>). The co-crystal of SARS-CoV PLpro–<bold>15g</bold> complex diffracted to a slightly lower resolution of 2.63 Å, and similarly to unliganded SARS-CoV PLpro, it belongs to the space group <italic>C2</italic> but with two monomers per asymmetric unit (<xref rid="b0130" ref-type="bibr">Ghosh et al., 2010</xref>) (<xref rid="f0060" ref-type="fig">Fig. 12</xref>b). Crystals of the SARS-CoV PLpro–inhibitor <bold>24</bold> complex (also known as compound <bold>GRL0617</bold>) grew in space group I222 with one monomer per asymmetric unit and diffracted to a resolution of 2.5 Å (<xref rid="b0125" ref-type="bibr">Ghosh et al., 2009</xref>, <xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>) (<xref rid="f0060" ref-type="fig">Fig. 12</xref>c). Unfortunately, as observed before for the unliganded SARS-CoV PLpro crystals, the crystallization conditions that yielded the inhibitor-bound SARS-CoV PLpro crystals were irreproducible and not suitable for the crystallization of SARS-CoV PLpro in complex with newly developed inhibitors.<table-wrap position="float" id="t0005"><label>Table 1</label><caption><p>Summary of SARS-CoV PLpro crystallization conditions and crystal properties.</p></caption><table frame="hsides" rules="groups"><thead><tr><th>Protein form</th><th>PDB code</th><th>Resolution range (Å)</th><th>Crystallization conditions</th><th>Crystallization method</th><th>Space group</th><th>Molecules per asymmetric unit</th><th>Reference</th></tr></thead><tbody><tr><td>Apo</td><td>2FE8</td><td>1.85</td><td>1.1–20 mg/mL PLpro (in 20 mM Tris pH 7.5, 10 mM DTT), 100 mM sodium citrate, pH 5.2, 3 M ammonium sulfate</td><td>Vapor diffusion, sitting drop</td><td><italic>C2</italic></td><td>3</td><td><xref rid="b0275" ref-type="bibr">Ratia et al. (2014)</xref></td></tr><tr><td>Inhibitor <bold>15g</bold> complex</td><td>3MJ5</td><td>2.63</td><td>5 mg/mL PLpro (20 mM Tris, pH 7.5, 10 mM DTT), 1 mM inhibitor, 1 M (NH4)2SO4, 50 mM MES, pH 6.5, and 2.5% PEG 400. Cryo solution: well solution, 400 μM inhibitor, 16% glycerol</td><td>Vapor diffusion, sitting drop</td><td><italic>C2</italic></td><td>2</td><td><xref rid="b0130" ref-type="bibr">Ghosh et al. (2010)</xref></td></tr><tr><td>Inhibitor <bold>24</bold> complex</td><td align="char">3E9S</td><td>2.5</td><td>8 mg/mL PLpro (20 mM Tris, pH 7.5, 10 mM DTT) with 200 uM inhibitor, 1 M LiCl2, 0.1 M MES pH 6.0, 30% PEG 6000. Cryo solution: well solution, 400 μM inhibitor, 16% glycerol</td><td>Vapor diffusion, sitting drop</td><td><italic>I</italic> <italic>2</italic> <italic>2</italic> <italic>2</italic></td><td>1</td><td><xref rid="b0125" ref-type="bibr">Ghosh et al. (2009)</xref> and <xref rid="b0280" ref-type="bibr">Ratia et al. (2008)</xref></td></tr><tr><td>Inhibitor <bold>3k</bold> and <bold>3j</bold> complex</td><td>4OW0 and 4OVZ</td><td>2.1 and 2.5</td><td>6 and 12 mg/mL PLpro (25 mM Tris, pH 7.5, 100 mM NaCl, 10 mM DTT) with 200 μM inhibitor, 100 mM sodium citrate, pH 5.5, 40% (v/v) PEG 600. Cryo solution: well solution, 400 μM inhibitor, 20% glycerol</td><td>Vapor diffusion, sitting drop</td><td><italic>C2</italic></td><td>2</td><td><xref rid="b0015" ref-type="bibr">Baez-Santos et al. (2014a)</xref></td></tr><tr><td>Ubiquitin complex</td><td>4MM3</td><td>2.75</td><td>3–12 mg/mL PLpro–Ubals (20 mM Tris, pH 7.5)</td><td>Vapor diffusion, hanging drop</td><td><italic>P</italic> <italic>3<sub>1</sub>21</italic></td><td>1</td><td><xref rid="b0275" ref-type="bibr">Ratia et al. (2014)</xref></td></tr><tr><td>Ubiquitin-catalytic mutant complex</td><td>4M0 W</td><td>1.4</td><td>8 mg/mL PLproC112S-Ub (0.1 M CHES, pH 9.5, 18% PEG 3000)</td><td>Vapor-diffusion, sitting-drop</td><td><italic>P 2<sub>1</sub></italic></td><td>1</td><td><xref rid="b0085" ref-type="bibr">Chou et al. (2014)</xref></td></tr></tbody></table></table-wrap></p><p id="p0185">As more SARS-CoV PLpro inhibitors were developed, the need for less costly and labor-intensive crystallization methods increased. Therefore, new crystallization efforts were centered on finding conditions for the rapid and reliable co-crystallization of SARS-CoV PLpro–inhibitor complexes. These efforts included developing a new approach for enzyme preparation and crystallization screening and ultimately resulted in the identification of a new crystallization condition, which after optimization, yielded crystals with improved reproducibility and increased diffraction ability. The structure of SARS-CoV PLpro in complex with compounds <bold>3k</bold> and <bold>3j</bold> resulted from these new crystallization conditions (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>) (<xref rid="f0060" ref-type="fig">Fig. 12</xref>d). As observed for the SARS-CoV PLpro–<bold>15g</bold> complex, the co-crystals of PLpro–<bold>3k</bold>/<bold>3j</bold> complexes belong to space group <italic>C2</italic> with two molecules per asymmetric unit. Although poorer electron density is observed for the UBL domain, the SARS-CoV PLpro–<bold>3k</bold>/<bold>3j</bold> crystals diffracted to improved resolutions of 2.1 Å and 2.5 Å, respectively, and provided the basis for the crystallization and structure determination of SARS-CoV PLpro in complex with inhibitors from the same series of compounds. A summary of all SARS-CoV PLpro crystal structures and crystallization conditions reported to date is provided in <xref rid="t0005" ref-type="table">Table 1</xref>.</p><p id="p0190">The multiple X-ray crystal structures of SARS-CoV PLpro have consistently shown multiple conformations at the fingers domain and the BL2 loop (<xref rid="f0060" ref-type="fig">Fig. 12</xref>). The BL2 loop, which is observed in an open conformation in the unliganded SARS-CoV PLpro structure, closes upon inhibitor binding toward the catalytic cleft allowing the formation of intermolecular interactions with the inhibitors. The more closed conformation is observed with the smallest compound <bold>24</bold>, in which a flip of the peptide bond between Gln270 and Tyr269 allows the formation of a hydrogen bond between the backbone amide of Gln270 and the carbonyl at the center of the inhibitor (<xref rid="b0280" ref-type="bibr">Ratia et al., 2008</xref>) (<xref rid="f0060" ref-type="fig">Fig. 12</xref>c, right panel). A more open conformation is observed upon binding of the larger compounds <bold>15g</bold>, <bold>3k</bold> and <bold>3j</bold>, in which the BL2 loop retains the backbone orientation previously observed in the unliganded PLpro structure (<xref rid="f0060" ref-type="fig">Fig. 12</xref>c, right panel). While the closing motions of the BL2 loop are driven by the induced-fit mechanism of inhibitor binding, other inhibitor-independent motions are observed at the tip of the fingers domain, which may account for PLpro’s ability to interact with topologically different poly-Ub chains and the UBL modifier ISG15 (see Section <xref rid="s0035" ref-type="sec">2</xref>, <xref rid="f0010" ref-type="fig">Fig. 2</xref>c).</p><p id="p0195">In contrast to the motions observed outside the catalytic site, the catalytic triad undergoes limited movement. However, the active site cysteine Cys112 is highly reactive, as it has been observed in a fully oxidized state in the inhibitor bound structures (<xref rid="f0060" ref-type="fig">Fig. 12</xref>c, right panel). It remains unknown whether oxidation of Cys112 is enhanced by inhibitor-induced closure of the BL2 loop, which may restrict access of the active site to reducing agents but still allow molecular oxygen to enter the active site, or whether the inhibitor-induced conformation increases the reactivity of the cysteine to molecular oxygen. Nevertheless, the observed oxidation of SARS-CoV PLpro Cys112 in various crystal structures demonstrates the high reactivity of the SARS-CoV PLpro active site.</p></sec><sec id="s0150"><label>5.3</label><title>Structure of SARS-CoV PLpro in complex with ubiquitin</title><p id="p0200">Recently, crystal structures of SARS-CoV PLpro in complex with Ub were determined to resolutions of 1.4 Å (<xref rid="b0085" ref-type="bibr">Chou et al., 2014</xref>) and 2.75 Å (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>) (<xref rid="f0065" ref-type="fig">Fig. 13</xref>a). Two different approaches were used to stabilize the SARS-CoV PLpro ubiquitin complex for co-crystallization. One approach used an active site cysteine mutant, C112S, to form a noncovalent complex with ubiquitin. The C112S-PLpro–Ub complex crystallized in the monoclinic space group <italic>P 2<sub>1</sub></italic> with one PLpro–Ub complex per asymmetric unit (<xref rid="b0085" ref-type="bibr">Chou et al., 2014</xref>). The wild type SARS-CoV PLpro–ubiquitin complex is very weak as the <italic>K<sub>i</sub></italic> value of free ubiquitin cannot be determined from competitive inhibition studies (<xref rid="b0020" ref-type="bibr">Baez-Santos et al., 2014b</xref>, <xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>). The introduction of the serine side chain however appears to stabilize the interaction of ubiquitin with SARS-CoV PLpro through a hydrogen bond between the hydroxy group of serine and the carboxy terminus of ubiquitin. In the mechanism illustrated in <xref rid="f0025" ref-type="fig">Fig. 5</xref>, the C112S complex would represent the product (<bold>“E+Q”</bold>) complex.<fig id="f0065"><label>Fig. 13</label><caption><p>X-ray crystal structures of SARS-CoV PLpro in complex with ubiquitin. (a) An overlay of the crystallographic monomer from the two PLpro–Ub complex (PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4M0W" id="ir0105">4M0W</ext-link>, (<xref rid="b0085" ref-type="bibr">Chou et al., 2014</xref>) and 4MM3 (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>)) crystal structures. The PLpro–Ubal complex is shown in blue with Ubal in orange and the PLproC112S–Ub complex is shown in light blue with Ub in yellow. (b) The conformational difference of the BL2 loop and the different orientations of PLpro BL2 residues and the C-terminal residues (72–76) of Ub (italics labels). (c) The different orientations of the active site residues with the distances between the atoms indicated in angstrom (black for PLpro–Ubal and red for PLproC112S–Ub).</p></caption><graphic xlink:href="gr13"></graphic></fig></p><p id="p0205">Our lab utilized a different approach to capture a transient, covalent intermediate complex (“<bold>FQ</bold>”) that forms during the SARS-CoV PLpro catalyzed reaction cycle (<xref rid="f0025" ref-type="fig">Fig. 5</xref>). Ubiquitin-aldehyde (Ubal) was synthesized, using methodologies developed by <xref rid="b0340" ref-type="bibr">Wilkinson et al. (2005)</xref>, to form a stable, wild type SARS-CoV PLpro–Ubal complex (<xref rid="b0275" ref-type="bibr">Ratia et al., 2014</xref>). Ubiquitin-aldehyde can form a covalent but reversible complex that can increase the binding affinity of a protein for ubiquitin by as much as 300,000-times relative to unmodified ubiquitin (<xref rid="b0225" ref-type="bibr">Melandri et al., 1996</xref>). The SARS-CoV PLpro–Ubal complex crystallized in the trigonal space group <italic>P</italic>
<italic>3<sub>1</sub>21</italic> with one PLpro–Ubal complex per asymmetric unit. The crystals diffracted to well beyond 2.75 Å but the 342 Å unit cell dimension in the c-dimension and the synchrotron setup prohibited data collection beyond this resolution. Nonetheless, the X-ray structure of the SARS-CoV PLpro–Ubal complex clearly revealed the covalent bond between the cysteine 112 sulfur atom and the c-terminal carbonyl carbon of ubiquitin (<xref rid="f0025" ref-type="fig">Fig. 5</xref>, <xref rid="f0035" ref-type="fig">Fig. 7</xref>).</p><p id="p0210">A structural superposition of the wild type PLpro–Ubal and C112S PLpro–Ub complexes is shown in <xref rid="f0065" ref-type="fig">Fig. 13</xref> and both structures reveal that the binding of ubiquitin induces a significant shift in the zinc-binding motif toward the Ub molecule and alters the conformation of the BL2 loop so that it interacts with the C-terminal residues (residues 72–76) of Ub (<xref rid="f0065" ref-type="fig">Fig. 13</xref>b). The BL2 loop contributes the majority of PLpro–Ub intermolecular hydrogen bonds and plays an important role in aligning and orienting the C-terminus of ubiquitin toward the active site of PLpro. There are observable conformational differences of the BL2 loop between the two crystal structures of PLpro–Ubal and C112S–PLpro–Ub. For example, different orientations are observed for the side chain of Tyr269 (<xref rid="f0065" ref-type="fig">Fig. 13</xref>b). In addition, Arg72, Arg74 and Gly76 of ubiquitin are found in different positions (<xref rid="f0065" ref-type="fig">Fig. 13</xref>b and c). In the C112S PLpro–Ub complex, Gly76 is found within hydrogen bond distance of C112S and Trp107, an interaction that would not naturally occur with C112. However, in the PLpro–Ubal complex, Ub C-terminal Gly76 is found covalently bonded to Cys112 and hydrogen bonded to Typ107 and His273, corresponding to the intermediate (“<bold>FQ</bold>”) state (see also <xref rid="f0025" ref-type="fig">Fig. 5</xref>, <xref rid="f0035" ref-type="fig">Fig. 7</xref>). The subtle shift in the positioning of the Ub C-terminal Gly76 results from the formation of a covalent (1.6 Å) versus a hydrogen bond (2.5 Å) with PLpro, and perhaps, this shift translates to more prominent differences in the orientations of the preceding C-terminal residues Arg72 and Arg74 of Ub. Thus, these two PLpro–Ub complex crystal structures may provide a snapshot of the transition of PLpro from the “<bold>FQ</bold>” to the <bold>“E+Q”</bold> states revealing once again the high plasticity of the BL2 loop and its interactions with substrates, intermediates, products and inhibitors.</p></sec></sec><sec id="s0155"><label>6</label><title>Design of antiviral inhibitors targeting PLpro from other coronaviruses</title><p id="p0215">The success of designing inhibitors against SARS-CoV PLpro with antiviral activity and selectivity suggests that it will be possible to design inhibitors with similar properties against other coronaviruses. In addition, since the naphthalene-based inhibitors are potent, competitive inhibitors and bind within the active site of SARS-CoV PLpro, these scaffolds would appear to be excellent starting points. Toward this goal, we tested over 30 of the napthalene-based inhibitors (<xref rid="f0045" ref-type="fig">Fig. 9</xref>, <xref rid="f0050" ref-type="fig">Fig. 10</xref>) for their ability to inhibit the papain-like protease two (PLP2) from the human coronavirus (HCoV) NL63 and PLpro from the human Middle East Respiratory Syndrome coronavirus (MERS-CoV). We found that 6 compounds are able to inhibit the HCoV-NL63 PLP2 enzyme with potencies ranging from 18 to 60 μM (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>). However, none of the compounds inhibited PLpro from MERS-CoV (<xref rid="b0020" ref-type="bibr">Baez-Santos et al., 2014b</xref>).</p><p id="p0220">The lack of inhibition of the naphthalene-based inhibitors toward MERS-CoV PLpro and the lower inhibitory potency toward HCoV-NL63 PLP2 may be explained in part by the structural differences within the BL2 loop of these enzymes. In <xref rid="f0070" ref-type="fig">Fig. 14</xref>we show the X-ray structure of SARS-CoV PLpro in complex with inhibitor <bold>3k</bold> (<xref rid="b0015" ref-type="bibr">Baez-Santos et al., 2014a</xref>), which has been superimposed onto the homology models of HCoV-NL63 PLP2 and MERS-CoV PLpro (<xref rid="b0020" ref-type="bibr">Baez-Santos et al., 2014b</xref>). Although the X-ray structure of unliganded MERS-CoV PLpro was recently determined by Rolf Hilgenfeld’s group (<xref rid="b0200" ref-type="bibr">Lei et al., 2014</xref>), a number of amino acids in the β-turn/loop were not modeled in because of missing electron density. However, our energy-minimized homology models of MERS-CoV PLpro and HCoV-NL63 PLP2 included the BL2 β-turn/loop and a comparison of our MERS-CoV PLpro homology model and the X-ray crystal structure show that the overall structures were very similar (<xref rid="b0020" ref-type="bibr">Baez-Santos et al., 2014b</xref>).<fig id="f0070"><label>Fig. 14</label><caption><p>Structural comparisons of the active sites of SARS-CoV PLpro, HCoV-NL63 PLP2 and MERS-CoV PLpro. The X-ray structure of SARS-CoV PLpro in complex with compound <bold>3k</bold> (orange ball and sticks, PDB: <ext-link ext-link-type="uri" xlink:href="pdb:4OW0" id="ir0110">4OW0</ext-link>) is shown superimposed with homology models of MERS-CoV PLpro and HCoV-NL63 PLP2 (green). The amino-acid residues important for SARS-CoV PLpro–inhibitor interactions are shown (blue front) along with the predicted corresponding amino acid in HCoV-NL63 PLP2 (green font) and MERS-CoV PLpro (black font). Highlighted in bold are the non-conserved substitutions in MERS-CoV PLpro sequence. Shown at the bottom of the figure is a comparison between SARS-CoV, HCoV-NL63 and MERS-CoV PLpro’s amino acid composition of the BL2 <italic>β</italic>-turn/loop (highlighted with an arrow). This loop is essential for the inhibitor induced fit mechanism of association of compound <bold>3k</bold> and related inhibitors with SARS-CoV PLpro. This figure has been adapted from <xref rid="b0020" ref-type="bibr">Baez-Santos et al. (2014b)</xref>.</p></caption><graphic xlink:href="gr14"></graphic></fig></p><p id="p0225">The structural models reveal that Y269 in SARS-CoV PLpro, an essential residue for binding of the naphthalene-based inhibitors, is absent in MERS-CoV PLpro resulting in the removal of a T-shaped π–π interaction with the naphthalene group of the inhibitor as well as other van der Waals interactions (<xref rid="f0070" ref-type="fig">Fig. 14</xref>). In addition, the BL2 β-turn/loop of MERS-CoV PLpro is one residue longer which may alter the hydrogen-bonding interactions between the inhibitor and backbone atoms of Y269. In contrast, the length of the BL2 β-turn/loop is identical for HCoV-NL63 PLP2 and F271 is a conservative substitution for Y269 in SARS-CoV PLpro. The benzyl group of F271 is still fully capable of forming a T-shaped π–π interaction with the naphthalene group and the van der Waals interactions. However, subtle structural shifts surrounding the binding pocket are likely to cause the decreased inhibitory potency of the naphthalene-based series for HCoV-NL63 PLP2. The sequence and structural variability of the BL2 β-turn/loop among coronavirus suggests that design of a broad-spectrum coronavirus antiviral compound based on the naphthalene scaffold will be difficult, but a more narrow spectrum antiviral may be more readily achievable.</p></sec><sec id="s0160"><label>7</label><title>Conclusions</title><p id="p0230">Over the past 10 years, the papain-like protease domain of SARS-CoV nsp3 has emerged as a viable drug target for the development of anti-SARS therapeutics. The PLpro enzyme is essential for viral replication and small molecule inhibitors of this enzyme are capable of eliciting a SARS-CoV antiviral response in cell culture. The X-ray structures of SARS-CoV PLpro in complex with inhibitors provide a detailed map of the active site, which can be exploited for structure-based drug design (SBDD) of potent inhibitors. Analyses of the structural differences surrounding the active site BL2 loop indicate that the plasticity of this loop and the amino acid side chains within this loop will have a profound influence on the design of inhibitors against SARS-CoV PLpro and other coronavirus PLpro enzymes including the enzyme from the Middle East Respiratory Syndrome coronavirus. 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Chem.</source><volume>276</volume><year>2001</year><fpage>33220</fpage><lpage>33232</lpage><pub-id pub-id-type="pmid">11431476</pub-id></element-citation></ref></ref-list><ack id="ak005"><title>Acknowledgements</title><p>The authors wish to express their gratitude to Dr. Kiira Ratia from the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois, Chicago, Prof. Susan Baker from the Department of Microbiology & Immunology, Loyola University Chicago Stritch School of Medicine, Prof. Arun Ghosh from the Department of Chemistry, Purdue University, and Prof. Scott Larsen from the Department of Medicinal Chemistry, University of Michigan for their significant scientific contributions and collaboration in developing SARS-CoV PLpro inhibitors over the past decade. In addition, we would like to acknowledge financial support over the years for this project from the <funding-source id="gp005">National Institute of Allergy and Infections Diseases</funding-source> (NIAID) via R01AI085089 and P01AI060915. ADM is supported by a grant from the <funding-source id="gp010">Walther Cancer Foundation</funding-source>. Support for continued crystallographic and computational modeling of this project is being provided in part by the Purdue Center for Cancer Research, which is supported by a grant from the <funding-source id="gp015">NCI</funding-source>P30 CA023168. Finally, we would like to acknowledge the beamline support staff at LS-CAT, SER-CAT and LRL-CAT at the Advanced Photon Source, Argonne National Lab over the years for their assistance in our X-ray work, which provided the structures of SARS-CoV PLpro in complex with the antiviral inhibitors.</p></ack></back></pmc></record>Pour manipuler ce document sous Unix (Dilib)
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