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ARTICLE IN PRESS G Model VIRUS 96420 No of Pages 11 Virus Research xxx 2014 xxx xxx Contents lists available at ScienceDirect Virus Research j ourna l h o mepa ge Middle East respiratory syndrome coronavirus inhibitors targeting spike protein Shuai Xia a 1 Qi Liu a c 1 Qian Wang a Zhiwu Sun a Shan Tianlei a University China b c University a Article Received Received Accepted Available Keywords Middle coronavirus MERS CoV Receptor binding domain Fusion inhibitor Entry inhibitor Six helix bundle to the entry fusion the applications with specific and nonspecific MERS CoV replication inhibitors for the treatment and prevention of MERS CoV infection 2014 Elsevier B V All rights reserved 1 SARS like breath he Saudi the named virus virus Viruses been syndrome WHO District http dx doi org 10 1016 j virusres 2014 10 007 0168 1702 Please cite this article in press as Xia S et al Middle East respiratory syndrome coronavirus MERS CoV entry inhibitors targeting spike protein Virus Res 2014 http dx doi org 10 1016 j virusres 2014 10 007 Introduction On June 13 2012 a 60 year old Saudi man suffering with a disease characterized by fever cough and shortness of was admitted to a hospital in Jeddah eleven days later died from respiratory and kidney failure Dr Ali Moh Zaki a Arabia virologist isolated a new coronavirus nCoV from patient s specimens It was later verified as a betacoronavirus as hCoV EMC Zaki et al 2012 In May of 2013 this was renamed as Middle East respiratory syndrome corona MERS CoV by the International Committee on Taxonomy of ICTV de Groot et al 2013 As of 23 July 2014 WHO had informed of 837 confirmed cases of Middle East respiratory MERS including 291 deaths a higher case fatality rate 35 than SARS 10 Drosten et al 2003 Lu et al 2013b 2014 The case numbers in Saudi Arabia spiked in April and Corresponding author at 130 Dong An Road Building 13 Room 611 Xuhui Shanghai 200032 China Tel 1 212 570 3058 fax 1 212 570 3099 Corresponding author E mail addresses lul L Lu shibojiang S Jiang 1 These authors contributed equally to this work May of 2014 with more than 25 of the reported cases being health care workers WHO 2014 raising a concern about the pandemic potential of MERS The major clinical manifestation of MERS CoV is rapidly pro gressive acute pneumonia which closely resembles SARS In some patients other severe complications may occur including acute renal failure diarrhea and abdominal pain consumptive coag ulopathy and pericarditis Assiri et al 2013 Most previously reported patients have been adults and the average age is around 47 WHO 2014 It should be noted that over three quarters of MERS CoV cases have occurred in patients with comorbidi ties Research Group 2013 The most common comorbidities for MERS CoV cases include some chronic diseases such as diabetes hypertension obesity cancer and chronic kidney heart and lung disease Assiri et al 2013 From the viewpoint of clinical mani festation and treatment options these chronic diseases are likely impact disease progression but the biased correlation between chronic disease and MERS CoV infection is still unknown MERS CoV is an enveloped virus with a positive sense single stranded RNA genome belonging to lineage C betacoronaviruses Zaki et al 2012 The genome contains 30 kb nucleotides and 10 plus open reading frames ORFs which encode the nonstructural replicase polyprotein and structural proteins including a number 2014 Elsevier B V All rights reserved Ying a Lu Lu a Shibo Jiang a b Key Lab of Medical Molecular Virology of MOE MOH Shanghai Medical College Fudan Lindsley F Kimball Research Institute New York Blood Center New York NY 10065 USA Department of Medical Microbiology and Immunology School of Basic Medicine Dali r t i c l e i n f o history 15 August 2014 in revised form 6 October 2014 6 October 2014 online xxx East respiratory syndrome a b s t r a c t The recent outbreak of Middle led to more than 800 laboratory confirmed serious threat to global public and prophylactic strategies studies on the structure of development of MERS CoV binding domain RBD and then look ahead to future MERS CoV entry Su a Lanying Du b 130 Dong An Road Xuhui District Shanghai 200032 Dali 671000 China East respiratory syndrome MERS coronavirus MERS CoV infection has MERS cases with a high case fatality rate 35 posing a health and calling for the development of effective and safe therapeutic treat and prevent MERS CoV infection Here we discuss the most recent MERS CoV spike protein and its role in virus binding and entry and the inhibitors targeting the S1 subunit particularly the receptor S2 subunit especially the HR1 region of the MERS CoV spike protein We of these viral entry fusion inhibitors either alone or in combination ARTICLE IN PRESS G Model VIRUS 96420 No of Pages 11 2 S Xia et al Virus Research xxx 2014 xxx xxx of enzyme proteins S protein E protein M protein and N protein Zaki et al 2012 van Boheemen et al 2012 MERS CoV is the sixth human coronavirus known to infect humans Phylogenetic analysis demonstrated that MERS CoV is more closely related to the bat coronaviruses HKU4 and HKU5 than it is to SARS CoV van Boheemen et al 2012 Lau et al 2013 Woo et al 2014 suggest ing that MERS CoV originated from bats which may serve as the natural the to in from Price of be thought from RNA Tunisia 2013 2014 believed milk airborne in et firm should For camels tidyl is on ney the demonstrated for DPP4 receptor and while able 2014 adapted into through fusion manner pH dependent with compartment membrane after efficient Belouzard ine fusion causing 2013 membrane lines syncytia treated with serine proteases such as TMPRSS2 becomes fusion activated suggesting that the plasma membrane fusion is mediated by the cleaved S1 and S2 subunits Qian et al 2013 Gierer et al 2013 The emergence of MERS CoV as a cause of severe respiratory disease highlights an urgent need for the development of effective therapeutic and prophylactic agents for treatment and prevention Please cite this article in press as Xia S et al Middle East respiratory spike protein Virus Res 2014 http dx doi org 10 1016 j virusres 2014 10 007 reservoir Annan et al 2013 Anthony et al 2013 Indeed coronaviruses isolated from bats in South Africa were found be genetically related to MERS CoV and a coronavirus identified a bat from Saudi Arabia with 100 nucleotide identity to virus the human index case patient Memish et al 2013 Milne et al 2014 Therefore the possibility of direct transmission MERS CoV or MERS CoV like virus from bats to humans cannot excluded Dromedary camels in the Middle East and Africa are now to be the intermediate hosts for transmission of MERS CoV bats to humans because MERS CoV specific antibodies and fragments are widespread in camels from Saudi Arabia Egypt Nigeria and Kenya Matsuyama et al 2005 Hemida et al Lau et al 2013 Reusken et al 2013a 2013b Barlan et al Human infection of MERS CoV from the infected camels was to occur through consumption of the animal s meat or However recent study suggests that MERS CoV may also be since the genetic fragments of MERS CoV were detected an air sample from a barn that housed an infected camel Azhar al 2014 Although further investigations are required to con this claim the results suggest that people working with camels take every precaution to stop the spread of MERS CoV example face masks should be worn by persons who handle particularly those that appear sick The cellular receptor for MERS CoV has been identified as dipep peptidase 4 DPP4 also named CD26 Raj et al 2014 DPP4 a type II transmembrane glycoprotein which is widely expressed nonciliated bronchial epithelium and the epithelial cells in kid small intestine liver parotid gland even in testis and prostate Lu et al 2013a The wide distribution is in good accordance with diversity of clinical manifestations Our recent studies have that the bat coronavirus HKU4 could also use DPP4 its entry into the target cell However HKU4 prefers to use bat while human MERS CoV prefers to utilize human DPP4 as its In the absence of exogenous proteases both MERS CoV HKU4 pseudoviruses can enter the DPP4 expressing bat cells only MERS CoV pseudovirus but not HKU4 pseudovirus is to enter human DPP4 expressing human cells Yang et al As suggested by these findings MERS CoV unlike HKU4 has to human DPP4 and human cellular proteases to gain entry host cells Like other coronaviruses MERS CoV enters the target cell two pathways either endocytosis or plasma membrane in a cathepsin L and low pH dependent or independent respectively Qian et al 2013 Shirato et al 2013 For endocytosis virus entry occurs after internalization fusion taking place in the acidic environment of endosomal Belouzard et al 2012 For pH independent plasma fusion the virus is able to fuse directly at the cell surface binding to the receptor which is about 100 to 1000 fold more than pH dependent endocytosis Matsuyama et al 2005 et al 2012 After MERS CoV S protein is cleaved by ser proteases during virus maturation MERS CoV enters the cell via between viral envelope and plasma membrane at neutral pH massive syncytia formation Qian et al 2013 Gierer et al MERS CoV seems to enter cells mainly through the plasma fusion pathway since several MERS CoV infected cell such as Calu 3 lower airway and Huh 7 liver cells form Chan et al 2013a Lu et al 2014 MERS CoV pseudovirus syndrome coronavirus MERS CoV entry inhibitors targeting of MERS CoV infection Currently no specific anti MERS CoV drug is available Some in vitro studies have shown that a nonspecific antiviral drug Ribavirin a nucleoside analog could inhibit MERS CoV replication Chan et al 2013b Mycophenolic acid MPA which is commonly used in clinics as an immunosuppressant drug to prevent rejection in organ transplantation is reported to be effective against a number of viruses including hepatitis E virus HEV Wang et al 2014 HCV Pan et al 2012 Ye et al 2012 influenza virus H1N1 Chan et al 2013b West Nile virus Morrey et al 2002 Chikungunya virus Khan et al 2011 yellow fever virus Leyssen et al 2005 and MERS CoV in cell culture Chan et al 2013b Hart et al 2014 Poly I C an immunostimulatory double stranded RNA analog can induce antiviral responses in vitro and in vivo Leyssen et al 2003 Kumar et al 2006 Perlman and colleagues have shown that treatment of mice expressing human DPP4 with poly I C before or after MERS CoV challenge significantly accelerated virus clearance Zhao et al 2014 Type I interferons IFNs including IFN H92521b IFN H92521a and IFN H92512b were reported to be effective in inhibiting MERS CoV replication in in vitro cell culture Chan et al 2013b Recently a number of compounds with inhibitory activity at low micromolar levels on MERS CoV replication in cell cultures in vitro have been identified from the FDA approved drug libraries Dyall et al 2014 de Wilde et al 2014 but their mechanisms of action have not been well defined Entry inhibitors are a class of antiretroviral drugs that prevent the virus from entering the cell Baldwin et al 2003 Liu et al 2007 Este and Telenti 2007 As exemplified by the anti HIV peptide T20 enfuvirtide entry inhibitors usually have good compatibility and complementarity with other kinds of drugs in cocktail therapies by increasing curative effect while decreasing side effects Lalezari et al 2003 Lazzarin et al 2003 Hence virus entry inhibitors could serve as promising candidate drugs against MERS CoV infection with good prospects in clinical application In this review we will summarize recent advances in studies reporting on the structure and function of the MERS CoV spike S protein leading to a better understanding of MERS CoV entry and fusion mechanisms Following this we will address the develop ment of MERS CoV entry fusion inhibitors targeting the S1 and S2 subunits of the MERS CoV spike protein particularly focusing on the viral fusion inhibitors interacting with the HR2 region in the spike protein and their future application for treatment and prevention of MERS 2 Structure of the MERS CoV S protein and its role in MERS CoV entry into the target cell Similar to SARS CoV MERS CoV spike S protein is also a type I transmembrane glycoprotein which is located at the viral envelope surface in a trimer state MERS CoV S protein contains 1353 amino acids and can be cleaved into two subunits S1 and S2 Fig 1 The S1 subunit is responsible for binding to the cellular receptor DPP4 and the S2 subunit mediates membrane fusion Lu et al 2013a Raj et al 2013 Mou et al 2013 Lu et al 2014 MERS CoV S1 protein is located within the N terminal 14 751 amino acids of S protein Lu et al 2013a Chen et al 2013 Wang et al 2013 containing the receptor binding domain RBD Fig 1 Although the crystal structure of the DPP4 RBD complex had not previously been solved we used multiple sequence alignment with Please cite this article in press as Xia S et al Middle East respiratory syndrome coronavirus MERS CoV entry inhibitors targeting spike protein Virus Res 2014 http dx doi org 10 1016 j virusres 2014 10 007 ARTICLE IN PRESS G Model VIRUS 96420 No of Pages 11 S Xia et al Virus Research xxx 2014 xxx xxx 3 Fig 1 Structure of MERS CoV S protein A The schematic representation of MERS CoV S protein compared with SARS CoV S protein SP signal peptide FP fusion peptide HR1 heptad repeat 1 domain HR2 heptad repeat 2 domain TM transmembrane domain CP cytoplasmic domain Residue numbers correspond to their positions in S protein of MERS CoV B The S1 subunit containing the receptor binding domain the target of mAbs which could block MERS CoV by binding to its receptor DPP4 The S2 subunit contains the HR1 domain the target of fusion inhibitors Fig 2 The structure of MERS CoV 6 HB A The cartoon of the MERS CoV fusion core structure with HR1 colored in gray and HR2 colored in green respectively B The helical wheel of HR1 and HR2 Most of the residues located at the a and d positions of the helical wheels are hydrophobic and more conserved than those in the b c and f positions which are hydrophilic C The interaction between HR1 and HR2 Through the interaction of the residues located at the a and d positions yellow three residues of HR1 form the internal trimer The residues of HR1 located at the e and g positions red interact with residues of HR2 at the a and d positions blue finally forming the 6 HB structure ARTICLE IN PRESS G Model VIRUS 96420 No of Pages 11 4 S Xia et al Virus Research xxx 2014 xxx xxx SARS CoV S1 subunit to predict that the RBD of MERS CoV might be located at residues 377 662 Du et al 2013b Later three indepen dent groups did solve the crystal structure of DPP4 RBD complex and determined that the RBD of MERS CoV is located at residues 367 606 Lu et al 2013a Chen et al 2013 Wang et al 2013 They also showed that the core structure of MERS CoV S protein RBD is a five stranded antiparallel H9252 sheet with several short H9251 helices harboring to Chen in between 2004 acting 2014 MERS CoV the Hotez that body Ying an as antibodies S harbors residues transmembrane lar SARS CoV ating S1 its Its conserved then structure proximity of in enter manner a that Liu and at to itions positions at face oligomeric in The manner eral portion hydrogen These of Cap with the 6 HB fusion core structure of SARS CoV S protein Liu et al 2004 Xu et al 2004 the MERS CoV S2 subunit contains stronger hydrogen bonds suggesting that the 6 HB of MERS CoV S protein is more stable than that of SARS CoV S protein For this reason the HR2 peptide derived from MERS CoV S2 is expected to be more effective against MERS CoV fusion than the HR2 peptide derived from SARS CoV S2 against SARS CoV fusion These findings confirm Please cite this article in press as Xia S et al Middle East respiratory spike protein Virus Res 2014 http dx doi org 10 1016 j virusres 2014 10 007 three disulfide bonds that connect C383 to C407 C425 C478 and C437 to C585 in order to stabilize the core structure Lu et al 2013a The RBD also contains 2 glycans Lu et al 2013a et al 2013 Wang et al 2013 The receptor binding motif RBD domain binds to the side surface of the DPP4 H9252 propeller Lu et al 2013a This interaction is very similar to the interaction adenosine deaminase ADA and DPP4 Weihofen et al Therefore ADA can compete with DPP4 for virus binding as a natural antagonist for MERS CoV infection Raj et al As an important domain to mediate the binding between and its receptor DPP4 RBD serves as a critical target for development of vaccines and therapeutics Jiang et al 2012 et al 2014 We and others have previously demonstrated the MERS CoV RBD could induce significant neutralizing anti responses in mice Du et al 2013a 2013b Ma et al 2014 et al 2014 providing a solid rationale for using the RBD as attractive target to develop MERS CoV entry inhibitors such soluble DPP4 sDPP4 and RBD specific neutralizing monoclonal mAbs By multiple sequence alignment with SARS CoV protein S2 subunit we found that the S2 subunit of MERS CoV an fusion peptide FP residues 943 982 an HR1 domain 984 1104 an HR2 domain residues 1246 1295 a domain residues 1296 1317 and an intracellu domain residues1318 1353 Fig 1A Like HIV 1 gp41 and S2 subunit MERS CoV S2 subunit is responsible for medi viral fusion with the target cell membrane After MERS CoV binding to DPP4 we hypothesize that the S2 subunit changes conformation by inserting its FP into the target cell membrane HR1 helices form a homotrimer with exposure of three highly hydrophobic grooves on the surface Its HR2 molecules bind to the HR1 trimer to form a six helix bundle 6 HB core which brings the viral and cell membranes into close to facilitate fusion Fig 2A Finally the genetic materials MERS CoV enter the host cell via the fusion pore for replication the cytoplasm Lu et al 2014 Alternatively MERS CoV can also cells via endocytosis in a cathepsin L and low pH dependent Qian et al 2013 Shirato et al 2013 Through further analysis we have found that MERS CoV uses fusogenic mechanism to enter the target cell which is similar to of HIV or SARS CoV to enter the target cell Chan and Kim 1998 et al 2003 2004 2007 Yu et al 2012 The residues at the a d positions in one of the HR1 helices interact with the residues the d and a positions in the adjacent HR1 helices respectively form the internal HR1 trimer Those located at e and g pos in the HR1 helices interact with the residues at e and g in the HR2 helices to form 6 HB Fig 2B The residues the b c and f positions in HR2 helices form a hydrophilic toward the solution to maintain the water solubility of the bundles Fig 2C In the crystal structure of MERS CoV 6 HB a notable difference length is observed between the HR1 and HR2 helices Fig 2A N and C terminal tails of the HR2 region pack in an orderly against hydrophobic grooves of the HR1 trimer with sev hydrophobic interactions mainly distributed at the N terminal and C terminal portion of the HR2 One HR2 helix formed 11 bonds to interact with its two neighboring HR1 domains hydrogen bonds located at N terminal or C terminal portion the HR2 helix constitute two anchoring points the N Cap and C conformation further stabilizing 6 HB construction Compared syndrome coronavirus MERS CoV entry inhibitors targeting that the HR1 region in MERS CoV S protein S2 subunit is a promising target of HR2 peptides based MERS CoV fusion inhibitors 3 MERS CoV entry inhibitors targeting RBD in the S protein S1 subunit MERS CoV infection is initiated by binding of the viral particle via the RBD in S protein on viral surface with the cellular receptor DPP4 on the cell surface Raj et al 2013 Targeting of the bind ing site between the RBD and the receptor could block the initial step of virus entry and thus might provide superior pharmacolog ical action to suppress MERS CoV infection In this regard sDPP4 could serve as an excellent blocker for the attachment and entry of MERS CoV to cells Indeed sDPP4 has been found to bind MERS CoV RBD with high affinity Du et al 2013b Ying