2015 The SARS-coronavirus papain-like protease_ Structure, function and inhibition by designed antiviral compounds.pdf

1 2Review 4The SARS coronavirus papain like protease Structure function 5and inhibition by designed antiviral compounds 6 7 8Yahira M Baez Santos 1 Q1 Sarah E St John 1 Andrew D Mesecar 9 Department of Biological Sciences Purdue University West Lafayette IN USA 10 Department of Chemistry Purdue University West Lafayette IN USA 11 Center for Drug Discovery Purdue University West Lafayette IN USA Q2 12 Center for Cancer Research Purdue University West Lafayette IN USA 13 14 1 6 a r t i c l ei n f o 17 Article history 18 Received 29 August 2014 19 Revised 17 December 2014 20 Accepted 19 December 2014 21 Available online xxxx 22 Keywords 23 Papain like protease 24 3C like protease 25 Nsp3 26 SARS CoV 27 MERS CoV 28 Ubiquitin 29 3 0 a b s t r a c t 31Over 10 years have passQ4ed since the deadly human coronavirus that causes severe acute respiratory syn 32drome SARS CoV emerged from the Guangdong Province of China Despite the fact that the SARS CoV 33pandemic infected over 8500 individuals claimed over 800 lives and cost billions of dollars in economic 34loss worldwide there still are no clinically approved antiviral drugs vaccines or monoclonal antibody 35therapies to treat SARS CoV infections The recent emergence of the deadly human coronavirus that 36causes Middle East respiratory syndrome MERS CoV is a sobering reminder that new and deadly coro 37naviruses can emerge at any time with the potential to become pandemics Therefore the continued 38development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is 39warranted The coronaviral proteases papain like protease PLpro and 3C like protease 3CLpro are 40attractive antiviral drug targets because they are essential for coronaviral replication Although the pri 41mary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner PLpro 42has the additional function of stripping ubiquitin and ISG15 from host cell proteins to aid coronaviruses 43in their evasion of the host innate immune responses Therefore targeting PLpro with antiviral drugs may 44have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signal 45ing cascades in infected cells that may lead to cell death in surrounding uninfected cells This review pro 46vides an up to date discussion on the SARS CoV papain like protease including a brief overview of the 47SARS CoV genome and replication followed by a more in depth discussion on the structure and catalytic 48mechanism of SARS CoV PLpro the multiple cellular functions of SARS CoV PLpro the inhibition of SARS 49CoV PLpro by small molecule inhibitors and the prospect of inhibiting papain like protease from other 50coronaviruses This paper forms part of a series of invited articles in Antiviral Research on From SARS 51to MERS 10 years of research on highly pathogenic human coronaviruses 52 2014 Published by Elsevier B V 53 54 55 56 57Contents 581 Introduction 00 591 1 SARS CoV genome and replication 00 601 2 The multi domain protein nsp3 00 611 3 The SARS CoV PLpro domain within the nsp3 00 621 4 SARS CoV PLpro is a protease a deubiquitinating DUB and deISGylating enzyme 00 631 5 SARS CoV PLpro innate immune functions 00 642 SARS CoV PLpro structure and function 00 652 1 Active site structure and catalytic mechanism of SARS CoV PLpro 00 662 1 1 Active site structure 00 http dx doi org 10 1016 j antiviral 2014 12 015 0166 3542 2014 Published by Elsevier B V Abbreviations PLpro papain like protease CoV coronavirus nsp nonstructural protein USP ubiquitin specifi c protease DUB de ubiquitinating enzyme Corresponding author at Department of Biological Sciences Purdue University West Lafayette IN USA Tel 1 765 494 1924Q3 E mail address amesecar purdue edu A D Mesecar 1 These authors contributed equally to this work Antiviral Research xxx 2014 xxx xxx Contents lists available at ScienceDirect Antiviral Research journal homepage AVR 3565No of Pages 18 Model 5G 29 December 2014 Please cite this article in press as Baez Santos Y M et al The SARS coronavirus papain like protease Structure function and inhibition by designed anti viral compounds Antiviral Res 2014 http dx doi org 10 1016 j antiviral 2014 12 015 672 1 2 Catalytic mechanism 00 683 SARS CoV PLpro inhibitor reactions with Cys112 00 694 SARS CoV PLpro inhibitor classes 00 704 1 Inhibitors identified via a designed yeast based screen 00 714 2 Thiopurine compounds 00 724 3 Natural product inhibitors 00 734 3 1 Tanshinones 00 744 3 2 Diarylheptanoids 00 754 3 3 Geranylated flavonoids 00 764 4 Zinc Ion Zn2 and zinc conjugate inhibitors 00 774 5 Naphthalene inhibitors 00 784 5 1 Compound 24 00 794 5 2 Compound 15g 00 804 6 Second generation naphthalene inhibitors 00 814 6 1 Compounds 3k and 3j 00 824 6 2 Metabolically stable naphthalene based SARS CoV PLpro inhibitors 00 834 6 3 Selectivity of naphthalene based SARS CoV PLpro inhibitors 00 845 X ray crystal structures of SARS CoV PLpro 00 855 1 Structure of SARS CoV PLpro in an unbound state 00 865 2 Structure of PLpro in complex with inhibitors 00 875 3 Structure of PLpro in complex with ubiquitin 00 886 Design of antiviral inhibitors targeting PLpro from other coronaviruses 00 897 Conclusions 00 90Acknowledgements 00 91References 00 92 93 941 Introduction 951 1 SARS CoV genome and replication 96Coronavirus CoV replication is a highly orchestrated process 97involving complex replication machineries to protect the virus 98genome the largest known RNA genome and the viral proteins 99from the host s antiviral defense mechanisms Brian and Baric 100 2005 Before the zoonotic emergence of the fi rst human coronavi 101rus that caused the 2002 2003 epidemic of severe acute respiratory 102syndrome SARS CoV there was a paucity of information pertain 103ing to CoV genomes and mechanisms of replication Since then the 104numbers of CoVs sequenced and studied have increased dramati 105cally from which several potential drug targets have emerged 106Many of the SARS CoV drug targets are encoded in the 50 terminal 107two thirds of the genome within the two open reading frames 108 ORF that encode for the replicase or non structural proteins 109 nsps see Fig 1 Tong 2009 110Production of the replicase proteins is initiated by the transla 111tion of ORF1a and ORF1ab via a 1 ribosomal frame shifting mech 112anism Bredenbeek et al 1990 This mechanism produces two 113large viral polyprotein pp1a and pp1ab that are further process 114by two virally encoded cysteine proteases Fig 1 the papain like 115protease PLpro and a 3 chymotrypsin like protease 3CLpro 116which is sometimes referred to as main protease Mpro Thiel 117et al 2003 Ziebuhr 2004 Ziebuhr et al 2000 2001 Processing 118of the viral polyprotein is required for the release and maturation 119of 16 viral proteins non structural proteins or nsps involved in 120the formation of a membrane associated cytoplasmic enzyme 121complex the replicase complex which is responsible for directing 122the replication and transcription of the viral genome It is thought 123that the establishment of viral replication sites is initiated by the 124recruitment of replicase proteins to host membranes a process 125mediated by several viral transmembrane domain containing pro 126teins such as the nsp3 nsp4 and nsp6 Kanjanahaluethai et al 1272007 Oostra et al 2007 2008 van Hemert et al 2008 128Electron tomography and three dimensional reconstruction 129imaging studies have revealed that the replicase complex utilizes 130a reticulovesicular network of double membrane vesicles DMVs 131with interconnected outer membranes originating from the endo 132plasmic reticulum ER Angelini et al 2013 Gosert et al 2002 133Knoops et al 2008 As a result the viral replication mechanism 134is localized increasing the concentration and cooperation of viral 135macromolecules and forming a framework of RNA synthesis Most 136importantly this mechanism provides a microenvironment for the 137protection of viral RNA from host nucleases and of double 138stranded RNA intermediates from the host cell s innate immune 139surveillance Among the components of the replicase complex 140nsp3 is of special interest since it is believed to be part of the cen 141tral scaffolding protein of the replicase complex due to the large 142number of interactions with other nsp s Angelini et al 2013 143Imbert et al 2008 Snijder et al 2003 1441 2 The multi domain protein nsp3 145The SARS CoV nsp3 multi domain protein is the largest repli 146case subunit at 1 922 amino acids Snijder et al 2003 Thiel 147et al 2003 Nsp3 is thought to play an essential role during the 148formation of virus replication complexes via its insertion into host 149membranes and its numerous interactions with other nsps Imbert 150et al 2008 especially nsp4 and nsp6 Angelini et al 2013 151 Numerous domains have now been identifi ed in nsp3 Fig 1 and 152many are predicted to be conserved in all CoV Neuman et al 1532008 Due to the large size of the nsp3 multi domain protein 154in vitro and in cellular studies have mainly utilized truncated 155nsp3 constructs that represent the predicted domains boundaries 156 Fig 1 Using this approach the three dimensional structures of 157most of the domains from the nsp3 of SARS CoV have been deter 158mined by X ray crystallography or NMR spectroscopy Chatterjee 159et al 2009 Egloff et al 2006 Johnson et al 2010 Ratia et al 1602006 Saikatendu et al 2005 Serrano et al 2007 2009 Tan 161et al 2009 162The N terminal region of the nsp3 181 1000 is highly con 163served among CoV containing a ubiquitin like Ubl globular fold 164 followed by a fl exible extended acidic domain AC domain rich 165in glutamic acid 38 Serrano et al 2007 Next to the AC domain 166isacatalyticallyactiveADP ribose 100 phosphatase ADRP 167app 100 pase domain also called macro domain or X domain 2Y M Baez Santos et al Antiviral Research xxx 2014 xxx xxx AVR 3565No of Pages 18 Model 5G 29 December 2014 Please cite this article in press as Baez Santos Y M et al The SARS coronavirus papain like protease Structure function and inhibition by designed anti viral compounds Antiviral Res 2014 http dx doi org 10 1016 j antiviral 2014 12 015 168thought to play a role during synthesis of viral subgenomic RNAs 169 Egloff et al 2006 Saikatendu et al 2005 SARS Unique Domain 170 SUD a domain not yet identifi ed in other coronaviruses from 171alphacoronavirus and betacoronavirus follows next Tan et al 1722007 The SUD domain binds oligonucleotides known to form 173G quadruplexes Tan et al 2009 Downstream of the SUD domain 174is a second Ubl domain and the catalytically active PLpro domain 175 Barretto et al 2006 that proteolytically processes the nsp1 2 176nsp2 3 and nsp3 4 cleavage sites Harcourt et al 2004 Down 177stream of PLpro are found a nucleic acid binding domain NAB 178with a nucleic acid chaperon function Neuman et al 2008 which 179is conserved in betacoronavirus and gammacoronavirus and one 180uncharacterizeddomaintermedthemarkerdomain G2M 181 Neuman et al 2008 Following the G2M are two predicted dou 182ble pass transmembrane domains TM1 2 and TM3 4 Neuman 183et al 2008 Snijder et al 2003 a putative metal binding region 184 ZN and the Y domain of unknown function subdomains Y1 3 185 Snijder et al 2003 Thiel et al 2003 Ziebuhr et al 2001 Inter 186estingly comparative genome and proteome analyses of two 187bovine CoV BCoV isolates showed a predominant clustering of 188mutations within the nsp3 multi domain Chouljenko et al 1892001 Consequently the multi functionality of the nsp3 the fre 190quency of point mutations observed in nsp3 domains and the 191involvement of nsp3 in structural arrangements of the replicase 192complex and double membrane vesicles may engender pleiotropic 193effects not only in SARS CoV pathogenicity but also on future 194emerging coronaviruses Snijder et al 2003 1951 3 The SARS CoV PLpro domain within the nsp3 196 The SARS CoV PLpro catalytic domain is fl anked by numerous 197catalyticallyactiveenzymes transmembranedomains and 198domains of unknown function and the entire nsp3 is localized to 199the ER membranes where the majority of the domains reside in 200the cytosol of the cell Fig 1 Hagemeijer et al 2010 Oostra 201et al 2008 In the cytosol the membrane associated PLpro domain 202recognizes the P4 P1 consensus cleavage sequence LXGG found in 203the boundaries of nsp1 2 nsp2 3 and nsp3 4 where membrane 204association is required for cleavage of the nsp3 4 Han et al 2052005 Harcourt et al 2004 Proteolytic cleavage of the peptide 206bond after the glycine at position P1 results in the release of 207nsp1 nsp2 and nsp3 from the viral polyprotein Fig 1 left bottom 208panel a process that is essential for viral replication Therefore 209SARS CoV PLpro is proposed to be an excellent candidate as a drug 210target for the development of anti CoV therapeutics 2111 4 SARS CoV PLpro is a protease a deubiquitinating DUB and 212deISGylating enzyme 213Reminiscent of the overall architecture of human deubiquitinat 214 ing enzymes DUBs in the ubiquitin specifi c protease family USP 215the molecular structure of the PLpro catalytic domain consists of a 216 canonical right handed thumb palm fi ngers architecture which 217 is fl anked at the N terminus by an additional ubiquitin like UBL 218domain of unknown function Fig 2a and b Ratia et al 2006 219The in vitro characterization of PLpro enzymatic activities reveal 220that PLpro can recognize and hydrolyze the cellular proteins ubiq 221uitin Ub Barretto et al 2005 2006 Lindner et al 2005 2007 222and the UBL protein ISG15 interferon induced gene 15 Lindner 223et al 2007 Nicholson et al 2008 Ratia et al 2014 both bearing 224the LXGG recognition motif at their C terminus Fig 1 right bot 225 tom panel Ubiquitin and ISG15 are important cellular modifi ers 226that are covalently attached to target proteins via the formation 227of an isopeptide bond Fig 1 right bottom panel between their 228C terminus and thee amino group of a lysine side chain on a target 229protein These isopeptide bonds can be hydrolyzed by the isopep 230tidase activities of DUB and deISGlating enzymes to remove Ub and 231ISG15 from host cell proteins 232 Kinetic studies on the catalytic effi ciency of SARS CoV PLpro 233toward different substrates have shown that ISGylated and Fig 1 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 fi gure The replicase genes encoded by ORF1a and ORF1b are shaded in grey The nsp3 multi domain protein is shown with the amino acids defi ning 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 2GRI a N terminal Glu rich acidic domain AC ADP ribose 100 phosphatase ADRP domain PDB 2FAV the SARS unique domain SUD PDB 2WCT 2JZE 2KAF a papain like protease PLpro containing a second UBl 2 domain at the N terminus PDB 2FE8 followed by the nucleic acid binding domain NAB PDB 2K87 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 P0 sites from the nsps to the C terminal sequences of the cellular proteins ubiquitin Ub and ISG15 shown with an isopeptide bond at the P0 sites Y M Baez Santos et al Antiviral Research xxx 2014 xxx xxx3 AVR 3565No of Pages 18 Model 5G 29 December 2014 Please cite this article in press as Baez Santos Y M et al The SARS coronavirus papain like protease Structure function and inhibition by designed anti viral compounds Antiviral Res 2014 http dx doi org 10 1016 j antiviral 2014 12 015 234ubiquitinated substrates are more readily hydrolyzed than small 235peptide substrates such as RLRGG AMC which represents the C 236terminus sequence of Ub and ISG15 suggesting a more complex 237mechanism for substrate recognition that extends well beyond 238the S4 to S1 enzyme recognition subsites for the LXGG peptide 239 Baez Santos et al 2014b Lindner et al 2005 2007 Ratia et al 2402014 More recently a comprehensive analysis of PLpro substrate 241 specifi city by X ray crystallography and mutational analyses dem 242onstrated that two distinct Ub binding subsites SUb1 and SUb2 243exist distant from the catalytic site providing SARS CoV PLpro a 244 unique bivalent mechanism of interaction with Ub like modifi er 245substrates Ratia et al 2014 For most USPs and SARS CoV PLpro 246the primary Ub UBL binding subsite SUb1 is distal to the isopep 247 tide bond located at the boundaries of palm domain and fi ngers 248regions Fig 2c For SARS CoV PLpro a second distal Ub binding 249subsite SUb2 exists which is located in a ridge region of the 250thumb domain Fig 2c This region provides interactions for a 251second Ub molecule on a K48 linked di Ub chain and for ISG15 252which has structural resemblance to K48 linked di Ub molecules 253 Ratia et al 2014 2541 5 SARS CoV PLpro innate immune functions 255The DUB and deISGylating activities of SARS CoV PLpro have 256 signifi cant functional implications in the innate immune response 257during SARS CoV infection Both Ub and ISG15 are important sig 258naling elements of the host innate immune response against viral 259infection which can be negatively regulated by viral DUB and deIS 260Gylating enzymes Calistri et al 2014 SARS CoV PLpro has been 261shown to act as a strong antagonist of many Ub dependent cellular 262responses to viral infection Mielech et al 2014a Although the 263mechanism of PL