Therapeutics for filovirusinfection: traditional approachesand progress towards in silicodrug designAmy C Shurtleff†, Tam L Nguyen, David A Kingery & Sina Bavari†U.S. Army Medical Research Institute of Infectious Diseases, Integrated Toxicology Division,
Frederick, MD, USA
Introduction: Ebolaviruses and marburgviruses cause severe and often lethal
human hemorrhagic fevers. As no FDA-approved therapeutics are available
for these infections, efforts to discover new therapeutics are important,
especially because these pathogens are considered biothreats and emerging
infectious diseases. All methods for discovering new therapeutics should be
considered, including compound library screening in vitro against virus and
in silico structure-based drug design, where possible, if sufficient biochemical
and structural information is available.
Areas covered: This review covers the structure and function of filovirus
proteins, as they have been reported to date, as well as some of the current
antiviral screening approaches. The authors discuss key studies mapping
small-molecule modulators that were found through library and in silico
screens to potential sites on viral proteins or host proteins involved in virus
trafficking and pathogenesis. A description of ebolavirus and marburgvirus
diseases and available animal models is also presented.
Expert opinion: To discover novel therapeutics with potent efficacy using
sophisticated computational methods, more high-resolution crystal structures
of filovirus proteins and more details about the protein functions and host
interaction will be required. Current compound screening efforts are finding
active antiviral compounds, but an emphasis on discovery research to inves-
tigate protein structures and functions enabling in silico drug design would
provide another avenue for finding antiviral molecules. Additionally,
targeting of protein-protein interactions may be a future avenue for drug
discovery since disrupting catalytic sites may not be possible for all proteins.
Keywords: ebolavirus, filovirus, glycoprotein, library screening, marburgvirus, protein
interactions, rational drug design, small molecule, structure, structure-based drug design,
viral entry mechanism, viral hemorrhagic fever
Expert Opin. Drug Discov. (2012) 7(10):935-954
1. Introduction
Structure-based drug design, or the rational design of drugs using structural insightsgained from known three-dimensional structures of biological targets, is a sophisti-cated computationally enabled method which has been used to produce successfulnew treatments for diseases such as cancer, influenza and acquired immune defi-ciency syndrome (AIDS) [1-4]. The utility of this approach depends upon the deter-mination of the three-dimensional structures of proteins of interest, and for manyhighly virulent viruses, such protein structures have yet to be solved, making theimmediate application of this method impossible. For example, the bcr-abl fusionprotein, which is characteristic of chronic myelogenous leukemia and some other
types of malignancies was specifically targeted by imatinib, atyrosine kinase inhibitor designed through structure-baseddrug design and medicinal chemistry through a knowledgeof the structure of the protein target [5,6]. Zanamivir, a neur-aminidase inhibitor, which is approved for prophylaxis andtreatment of influenza Type A and B infections, was also dis-covered through structure-based drug design [7,8]. A paradigmfor structure-based drug design of anti-infective agents is thearray of approved drugs that have been developed to targetthe human immunodeficiency virus (HIV) protease [1]. HIVprotease cleaves the Phe(Tyr)-Pro bond for group-specificantigen and group-specific antigen polymerase precursor pro-teins. Structural studies of HIV protease function greatlyimproved the speed and efficiency of the rational design anddevelopment of the first protease inhibiting anti-HIV drugs:saquinavir, ritonavir, indinavir, and nelfinavir [1,9]. The deter-mination of the HIV protease structure and its active sitefacilitated the rational design and development of drugs thatblocked native substrate binding [10]. The anti-HIV drugsamprenavir and lopinavir, as well as those listed above, arepeptido-mimetics that were designed to bind to the activesite of the HIV protease dimer. A newer generation of anti-HIV drugs, characterized by atazanavir, fosamprenavir, anddarunavir, were designed to overcome mutations in HIVprotease that resulted in viral drug resistance.Such a wealth of rationally designed compounds is not avail-
able for many other highly pathogenic viruses. To date, thereexists only a handful of high-resolution crystal structures forintact filovirus proteins and consequently very few studies thatdescribe the interaction of a candidate therapeutic with a knownviral protein structure or functionally active site. Even fewer
studies describe an approach using classical in silico structure-based drug design [11,12]. Instead, filovirus researchers havehad to rely upon compound library screening to identify smallmolecules with drug-like properties, such as kinase inhibitorsgenistein and tyrphostin, as two examples [13-15]. A number ofcandidate therapeutics have been found through compoundlibrary screening or through design of antisense based products,and some of these candidates have been studied to delineatetheir mechanism of action [14,15]. For some of the available filo-virus candidate therapeutics, a description of how they mayinteract with a target protein is available, or can be predictedbased on published work [15-17]. In addition, an area with strongtherapeutic promise has been achieved through the design ofantisense-based products, thoroughly reviewed in [18].
The filoviruses are highly lethal zoonotic agents of viralhemorrhagic fevers that are of concern as emerging pathogensand potential bioweapons. These viruses emerge in unpredic-table sporadic outbreaks in certain areas of Africa and arepurported to have been weaponized in the former SovietUnion [19]. There are no available therapeutics which havebeen approved by the United States Food and Drug Admini-stration (FDA) for the treatment of filovirus infections, so thereis a great need for continued drug discovery efforts to protectagainst these lethal viruses. Due to their highly pathogenicnature, handling these viruses poses great risk for laboratorypersonnel. The lack of vaccines or therapeutics available fortreatment of laboratory personnel or the general public, drivestheir classification as Class 4 risk-group pathogens whichmust be handled at biosafety level 4 (BSL-4), the highest levelof biocontainment [20]. Research on BSL-4 agents is quitelimited because of constraints due to biosafety and biosecurity,and there is a short list of laboratories worldwide whereBSL-4 research can be performed [19,20].
Highly virulent filoviruses are endemic to parts of Africa,and some filoviruses with unknown human pathogenicityhave been found in the Philippines. Ebolaviruses were firstdiscovered during simultaneous outbreaks in Zaire (todaythe Democratic Republic of Congo) and Sudan in 1976.Today there are five known species in the Ebolavirus genus:Zaire ebolavirus (EBOV), Sudan ebolavirus (SUDV), Restonebolavirus (RESTV), Taı Forest ebolavirus (TAFV), andBundibugyo virus (BDBV) [19]. The Marburgvirus genusconsists of one only species, Marburg marburgvirus, whichhas two members: Marburg virus (MARV) and Ravn virus(RAVV) [21]. Marburgvirus was the first filovirus discovered,when in 1967 Yugoslavian laboratory workers manipulatingan African green monkey imported from Uganda becameinfected and initiated a 31-person outbreak of hemorrhagicfever in Marburg, Germany [22]. The case fatality rates ofEbola and Marburg hemorrhagic fevers range from 50 -- 90% and 23 -- 90 %, respectively [23,24].
Article highlights.
. The filoviruses are virulent, lethal human viruses forwhich FDA-approved therapeutics are unavailable.
. Rodent and nonhuman primate animal models offilovirus infection are in use for evaluation of efficacyof therapeutics.
. Drug discovery approaches for anti-filovirus compoundsinclude compound library screening against authenticvirus or pseudotyped viruses, and in silico structure-based drug design.
. General functions have long been known for most ofthe filovirus proteins, but many structural and functionaldetails are still unknown. The available structuralinformation may begin to inform in silico drug design,but more structural and functional information isneeded about viral proteins and any involvedhost proteins.
. Entry of filoviruses into host cells is an area of researchwhich has recently been addressed and some structure-based drug design techniques may be employed fordiscovery of new therapeutics directed against specifictarget processes such as entry.
This box summarizes key points contained in the article.
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Outbreaks in human populations are rare and oftenself-limiting as person-to-person transmission is only docu-mented to occur upon close contact with blood or otherbodily fluids [25]. In experimental models of infection, aerosolpreparations of filoviruses are highly infectious, and have ledto concerns that these agents could be used in events ofmass dissemination [26,27]. In humans, the incubation periodsfor EBOV and MARV infection are generally 4 -- 7 days witha reported range of 2 -- 21 days and death often occurs within6 days of symptom onset [28]. The disease progresses quickly,with symptoms such as fever, chills, headache, malaise, andmyalgia typically appearing within 4 days after infection [28-31].Nausea, vomiting, abdominal pain, diarrhea, anorexia, a dry,non-productive cough, chest pain and shortness of breathhave all been reported as the predominant gastrointestinaland respiratory symptoms [24,32,33]. Vascular abnormalitiesinclude conjunctival injection, hypotension and edema [33].Hemorrhagic signs include development of petechiae on thetorso and arms, ecchymoses, bleeding from venipuncture sitesand mucous membranes, gingival bleeding, epistaxis, andvisceral hemorrhaging [28,33]. Currently, only supportive careor experimental therapeutics administered under specialprotocols can be offered to individuals with suspected orconfirmed cases of EBOV or MARV infection.
Animal models of filovirus infection are available to aid thestudy of disease pathology and evaluate potential therapeuticsand vaccines (Table 1) [18,34,35]. Mice and guinea pigs do notsuccumb to infection with wild-type ebolaviruses or marburgvi-ruses unless the virus has been passaged and adapted to viru-lence for the species [36-39]. For EBOV and MARV mousemodels, there is rapid viremia, high viral burden in the spleen,liver and multiple organ tissues, lymphopenia, thrombocytope-nia, and liver damage resulting in high AST and ALT levels inserum [38]. For both mouse models, there is an early inflamma-tory cytokine response, and lymphopenia and neutropenia areobserved, similar to the observations in guinea pig and nonhu-man primate infections. Mice do not demonstrate disseminatedintravascular coagulation (DIC), fibrin deposits, or skin rashessuch as petechiae, but some coagulation abnormalities arefound, such as induction of fibrin degradation products.Rodent models are useful for early stage discovery, as com-pounds can be evaluated in these models for antiviral effects,optimal delivery route and dosing frequency, dose level,prophylactic versus therapeutic efficacy studies and some earlyin vivo toxicity assessments [40,41]. Generally, due to small size,ease of handling, and possibility of working with larger groupsizes (where generally n is at least 10), mice are used as the initialmodels for testing, and success in this species generally warrantsprogression into the guinea pig efficacy model [42,43]. Althoughthese rodent infection models are valuable screening tools forevaluating virus-specific therapeutic agents, the nonhuman pri-mate (NHP) models are considered the most reliable predictorof therapeutic efficacy.
NHP are the most suitable model species to satisfy theFDA’s Animal Rule of Efficacy for licensure of therapeutics
and vaccines for agents where it is unethical or unfeasible toconduct human efficacy evaluations [34,44,45]. Several speciesof NHP are susceptible to infection with filoviruses [26,46-48].However, the majority of research is performed using rhesusmonkeys and cynomolgus macaques, as these animal modelsreliably reproduce human disease [49]. Fever in monkeys ismeasureable 3 -- 4 days after infection, and is coincidentwith onset of viremia [50]. A macropapular rash generallyappears by day 3 -- 4. Dehydration, anorexia and diarrheabegin approximately 4 days after infection [50-52]. Nearly allinfected animals appear ill by day 5 or 6, exhibiting moderateto severe depression in activity, dehydration, and hunchedposture; rashes with hemorrhage appear around day6 [46,48,50,53-55]. White blood cell counts and various bloodchemistry parameters are drastically altered during humanand NHP infection with EBOV or MARV. Lymphocytope-nia occurs around days 3 -- 4 postinfection [53-57]. Neutrophilcounts increase and comprise the majority of the circulatingleukocyte population; concomitantly monocytes, macro-phages and natural killer (NK) cell numbers decline. Thehemorrhagic manifestations include alterations in bloodcoagulation, fibrinolysis, thrombocytopenia, consumptionof coagulation factors, deficiency of anticoagulation, andincreased levels of fibrin degradation products [46,48,50,53-55].The average time to death after intramuscular (i.m.) challengewith 1000 plaque forming units (pfu) of EBOV is ~ 8 days forrhesus monkeys and ~ 6 days for cynomolgus macaques [28],which points to a longer therapeutic window for treatmentin models utilizing rhesus monkeys. In fact, research in thefilovirus field has frequently taken advantage of this longertimeframe for rhesus monkeys to manifest symptoms andsuccumb to disease, and have preferentially used this speciesover cynomolgus macaques for testing of small-moleculetherapeutics [43,58]. Even though most but not all filovirusresearchers have perpetuated the practice of evaluatingtherapeutics in rhesus monkeys, there has been no guidancefrom the FDA that this is the definitive model species fortherapeutics evaluation. While the human and NHP diseaseappear to be very similar, there is still a great amount ofresearch that can be done to characterize all of the animalmodels, to prove they are each suitably useful animal modelsfor therapeutics discovery. Table 1 provides a summary of use-ful animal models of filovirus infection for efficacy evaluationof small molecules.
3. Drug discovery for filovirusesthrough library screening
In the face of the challenges stated above, significant discoveriesand advances in therapeutics development have been made. Tocircumvent research limitations using authentic viral agents,detailed molecular biology studies have been performed withcloned genes, expressed viral proteins, and pseudotyped virusparticles. Pseudotyped viruses are useful tools for studyingvirus entry or egress in some systems [59]. These pseudotyped
Therapeutics for filovirus infection: traditional approaches and progress towards in silico drug design
viruses take advantage of multiple parts of several differentviruses to study a specific process in the viral life cycle. Forexample, the core proteins of one virus packaging system suchas a lentivirus or vesicular stomatitis virus (VSV) are coexpressedin a producing cell line with the glycoprotein gene of the virusof interest plus a reporter gene like luciferase [59-61]. The endproduct is a single-cycle virion which is not replication compe-tent, but which can enter a target cell using the entry glycopro-tein of interest, such as filovirus GP1,2, thereby providing asystem in which to investigate virus-entry inhibitors. Thesepseudotyped viruses are useful tools for higher throughputscreening of libraries of small molecules for activity against viralentry and trafficking, and there are several hit compounds thathave been discovered using this approach [13,15,62]. There hasbeen a push to discover filovirus therapeutics using in vitrohigher throughput screens, since these methods could identifya possible lead candidate therapeutic, regardless of the
mechanism of action [63]. Once a hit candidate is found througheither library screens or structure-based drug design methods(Figure 1), it must be evaluated under rigorous in vitro condi-tions to determine its true specificity for and potency againstauthentic virus. Assays for cytotoxicity, ADMET and solubilitywill assess its chemical tractability as a molecule which could besuccessfully advanced as a safe and effective clinical candi-date [63,64]. Compounds with favorable activity and drug-likeproperties can proceed through in vivo efficacy studies in animalmodels. Mechanism of action studies are then performed tohelp define the antiviral activity of the compound and completethe data package to be evaluated by the FDA.
4. The filovirus proteins and their functions
Ebolaviruses and marburgviruses have 19kb genomes thatencode seven genes. For marburgviruses, these seven genes
Structure based approach
• Crystal structure of a protein/enzymatic site of importance is available • In-silico molecular dynamics and energy minimization algorithms identify candidate
small molecules with predicted binding • Synthetic chemistry is performed to produce
derivatives for screening
Small molecules found using these two approaches are screened in cell based assays againstpseudotyped viruses (BSL-2), GFP expressing or authentic viruses (BSL-4), in a high throughput format.
Hit candidates are identified andrescreened against authentic virus for
confirmation of antiviral activity
Mechanism of action studies (e.g.,):
• Viral entry/egress • Confocal microscopy • Effect of viral or host gene knockdown on viral growth, viral transcription/translation, etc.
• Mutational analysis
Continued screening: • In vitro assays for high antiviral potency • Specificity • Cytotoxicity • Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) • Favorable solubility properties • Agreement with Lipinski’s rule of 5 [59]. • Hits proceed through in vivo efficacy studies in animal models, and preclinical testing.
or pseudotyped virus
Authentic virus
Library based approach
• Libraries of thousands of small molecules are identified and purchased for execution of antiviral screening assays
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Figure 1. A general comparison of structure-based drug design and compound library screening approaches for the discovery
of new filovirus therapeutics.
Therapeutics for filovirus infection: traditional approaches and progress towards in silico drug design
are translated into seven unique proteins. However, alter-native mRNA translation results in a total of 9 ebolavirusproteins [65-67]. A significant body of literature is availablethat provides preliminary insights into how these proteinsare involved in the viral life cycle and pathogenesis (reviewedin [19]). Unfortunately, the crystal structures and functionaldomains for many of these proteins have not been describedin full detail. This lack of structural detail is a limitation tothe utility of structure-based drug design as an alternativemethod to rote library screening to find possible antiviralhits. Table 2 provides an overview of the filovirus proteinsand their functions.
4.1 NucleoproteinThe nucleoprotein (NP) is involved in nucleocapsid andinclusion body formation, encapsulation of RNA genomeand antigenome, and viral genome replication and transcrip-tion [68]. It is a central component of the ribonucleoproteincomplex (RNP) where it binds itself, another viral proteinVP35 at its interferon inhibitory domain, the secondarynucleoprotein known as VP30 and associates with L, the poly-merase [69]. The nucleoprotein gene is located upstream of allother genes in the filovirus RNA genome [70]. This gene sharesconserved sequence homology in central nucleotide sequenceregions with other members of the mononegavirales, para-myxoviruses and rhabdoviruses. There is variability in theC-terminal region, where there are multiple sites of phosphor-ylation that have been mapped for marburgvirus, variantMusoke (MARV-Mus) [19,65,71]. When phosphorylated byhost cell kinases, MARV-Mus NP can bind to itself, VP35,and VP30, and then incorporate into virions [19,72,73]. Studiesto map functional motifs and residues of importance toregions within the protein have been completed, and theregions responsible for NP--NP homodimerization, replica-tion and nucleocapsid-like structure formation have beenidentified [74,75]. Recent EBOV and MARV virus-like particle(VLP) and native virus studies using cryo-electron microscopyand tomography have described the nucleocapsid formationin detail, showing how NP, VP24, VP35, and VP40 arenecessary and sufficient to form a tight, structurally soundnucleocapsid [76,77]. At the time of this writing, neither thecrystal structure nor a description of any particular activesite of any filovirus NP was available. Continued investigationinto NP functions may reveal that there are specific sites thatmay be inhibited by specially designed small molecules.
4.2 VP30VP30 is thought to be a secondary nucleoprotein in filovi-ruses [78]. This protein colocalizes with NP in inclusion bodieswhen NP and VP30 are coexpressed in vitro [79]. VP30 andNP interact at VP30 C-terminal domain, through aminoacids (aa) 142-272, as shown through a crystal structure ofthe compact C-terminal domain [78]. VP30 oligomerizationis necessary for its function as a transcriptional activator butis not required for NP binding [80,81]. In vitro, the N-terminus
has been shown to bind single-stranded RNAs with a prefe-rence for filovirus RNAs [82,83]. The crystal structure of theC-terminal domain of VP30 demonstrates that this subunitfolds into a dimeric helical structure [78]. It is postulated thatVP30 dimers are the building blocks for the oligomerizationof VP30 into hexamers, and mutagenesis studies revealedseveral key interaction sites. The first site is Glu197, whichwas shown to be crucial for incorporation of VP30 into nucle-ocapsids. The second interaction site is a basic cluster ofthe amino acid residues Arg179, Lys180 and Lys183 inVP30 that are essential for nucleocapsid association and tran-scription activation [78]. Because this Lys180-centered, basiccluster is crucial for transcription activation, it may be feasiblethat a small molecule inhibitor that targets this site wouldinhibit transcription activation, and thus prove to be aneffective antiviral agent.
4.3 VP35Multifunctional VP35, a 35 kDa protein, is known to asso-ciate in RNP complexes and inclusion bodies, is likelyinvolved in replication, and functions as an innate immuneantagonist [69,84-86]. VP35 binds L protein to form the filo-viral replicase-transcriptase holoenzyme [85]. VP35 formshomodimers at aa 70 -- 120, and heterodimers withL protein at aa 70 -- 120 and 121 -- 219 [19,87].
One of the major functions of filovirus VP35 is to inhibitType-I interferon responses [84]. This protein is functionallyequivalent to HIV-1 Tat protein, which suppresses RNAi-based innate antiviral response of virus infected cells [88].Many reports describe that VP35 and VP35-NP complexesinhibit dsRNA and virus-mediated induction of interferonresponsive promoters leading to a general inhibition of thecellular immune response [19,84,89-91]. Crystal structures ofVP35 have been reported [69,92,93]. The structure of VP35 incomplex with an 8-base-pair dsRNA provides insights intothe functional role of VP35 in inhibition of Type-I interferonresponse in infection [69]. A small-molecule inhibitor couldpotentially be designed to block the binding of VP35to dsRNA targets, promoting downstream production ofinterferon. Mutagenesis studies showed that many aminoacid residues are crucial for the biological function ofVP35 [92]. The F239A, R312A, R322A, and K339A mutantswere shown to be unable to bind dsRNA and thus were alsounable to suppress IFN-b promoter activation relative towild-type VP35 [69]. Arg312, Arg322, and Lys339 form abasic patch on the VP35 protein surface. Presumably, asmall-molecule inhibitor that binds at this basic patch wouldprevent VP35 binding dsRNA and concomitantly allowexpression from the IFN-b promoter.
4.4 L proteinThe filoviral RNA-dependent RNA polymerase, the L protein,is the largest protein encoded in the 19kb genome. TheN-terminus of L protein binds to VP35 and together theymake up the replicase-transcriptase holoenzyme [94]. Bacterial
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expression system production and gel electrophoresis puri-fication have been reported [82]. Detailed protein functionand interaction studies have not been reported in the publishedliterature. L protein may be responsible for polyadenylation. Lis a high-fidelity polymerase, with self-correcting features [95,96].L mRNAs are recognized by the host zinc finger antiviral pro-tein (ZAP). ZAP has been shown to decrease production ofinfectious EBOV and MARV in vitro [97]. The functionaldomains of L which are responsible for polyadenylation, self-correcting activities, and the interactions of ZAP with LmRNAs could all be further studied as candidate targets fortherapeutic intervention, and if structures become available,rational drug design methods could be applied. Designing ther-apeutics against L protein would require exacting protocols toensure that host polymerases are not also down-regulated.
4.5 VP40As the most abundant viral protein, VP40 is the viral matrixprotein and is encoded by the most conserved filovirusgene [12,65,96,98-100]. VP40 is the filovirus protein with themost functional data partly due to tractable expression andpurification from cellular systems [19]. The crystal structurehas been solved [98,101,102]. The crystal structure of aa44 -- 321 at 2 A resolution helped to describe the N-terminaldomain structure, and the C-terminal domain at aa201 -- 326 [98,101,102]. The hydrophobic interaction ofVP40 with cellular membranes is reminiscent of M (matrix)proteins from paramyxoviruses, measles, and respiratory syn-cytial virus (RSV) [103]. In fact, VP40 accumulates in areasof cellular membrane proliferation, where protein sorting forthe lysosome or the plasma membrane may occur [104].VP40 associates with cellular protrusions or filopodia in theabsence of any other viral proteins, and this membrane inter-action has been mapped to the C-terminus [100,105]. Theprotein alone will accumulate at membranes and causebudding of virus-like particles, which may be mediated bythe c-Abl1 tyrosine kinase [106,107]. Two compounds, nocoda-zole and paclitaxel, have been shown to reduce the release ofVP40-based VLPs into cell culture medium [106]. These twocompounds are microtubule depolymerization inhibitors.There is a region of sequence for VP40 from EBOV-Mayinga,which has homology to a tubulin binding motif found inmicrotubule-associated protein 2 (MAP2), and deletion ofthis region resulted in loss of binding of VP40 to the cellmembrane [108]. Thus, it appears that microtubule associationwith VP40 may occur and is important for egress of virionsfrom cells [106]. VP40 could not stabilize microtubulesin Niemann-Pick C1 (NPC-1)-deficient cells, which aredeficient in cholesterol transport properties, or cells treatedwith the cholesterol trafficking inhibitor U18666A [109].This suggests that cholesterol and/or its proper handlingand/or transport proteins may also be required for VP40function [109].Recently, a study provided insights into the structure and
function of filovirus VP40 [12]. This study demonstrated
that if the structure and function of filovirus proteins werebetter defined, then in silico drug design could provide insightinto directed therapeutics. The filovirus VP40 proteincontains one or more short amino acid sequences called theL-domain, or late budding domain, which serve to mediaterelease of virus particles. The EBOV L domains are PTAPand PPxY motifs, whereas the MARV VP40 L domain isa PPxY motif [110-113]. EBOV VP40 PTAP motif binds hostprotein Tsg101, and this has been visualized in live cellsas a mechanism for virus budding from host cells [12].A fluorescence-based technique called biomolecular comple-mentation was applied to VLP budding assays to prove thedirect interaction of Tsg101with EBOV VP40 at the PTAPmotif. This heterodimer may traffic to the plasma membranevia the microtubule network [12,108]. More importantly, theinvestigators screened in silico a 2.4 million compound libraryagainst the UEV domain of human Tsg101 which containsthe PTAP binding pocket, and identified potential inhibitorsof EBOV or MARV VP40 interaction with human Tsg101 atthe PTAP or PPxY binding pocket, for each virus, respec-tively [12]. Out of six inhibitors tested for ability to blockEBOV VP40 interactions with Tsg101, one small molecule,compound 5539-0062, reduced egress of EBOV VLPsin vitro by greater than 90% at 80 to 100 µM concentrations.In contrast, compound 5539-0062 only reduced MARV VLPegress by twofold, due to the lack of PTAP L-domain inMARV VP40 [12]. The compound was also found to directlyinhibit the fluorescence produced in the biomolecular com-plementation assay based on the interaction of Tsg101 andthe VP40 PTAP motif. Therefore, the small molecule wasshown to specifically inhibit VP40 at that site, as originallypredicted in silico. The molecule was tested against a livevesicular stomatitis virus, VSV-M40, modified to possessthe EBOV PTAP motif [114]. Compound 5539-0062 wasable to reduce growth of VSV-M40 by 3 -- 10 fold [12]. Thesestudies are promising, but results should be viewed with therecognition that compound 5539-0062 was not tested inassays with authentic viruses, and the most active concentra-tion was fairly high (above 80 µM). These studies demon-strate a proof of concept that if substantial structural dataexist, then in silico screening can be performed and effectiveantiviral hits can be obtained.
4.6 VP24Described as a secondary matrix protein, VP24 colocalizeswith VP40 in virions [65,73,115]. VP24 has not been shown tobind directly to VP40, the primary matrix protein [115].VP24 appears to be important for intracellular nucleocapsidassembly, and it may bind directly to NP [19,68,106,116]. TheVP24 from MARV-Mus may bind directly to GP2 at thecytoplasmic tail [19,117]. Changes to the NP and VP24 genesare responsible for most of the virulence associated withmouse adaptation of EBOV to cause lethal disease inmice [118]. VP24 may modulate the conversion of viralgenome from a form used in replication and transcription,
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to a form ready to promote viral assembly and budding [119].VP24 appears to be involved in immunosuppression, throughinhibition of IFNa/b and IFNg signaling by sequesteringkaryopherin alpha proteins, and preventing movement ofphophorylated STAT1 to the nucleus [120-122]. A singleVP24 structure has recently been determined using x-ray crys-tallography and sheds light on this biological activity [123]. Itwas found that VP24 directly binds transcriptional activatorSTAT1, but how this binding exactly impacts function ofeither protein is unknown [123]. Structural differences inVP24 from RESTV and SUDV were observed [123]. Thesestructural differences led to postulation that perhaps RESTVVP24 may be less efficient at sequestering karyopherins andbinding STAT1, compared to the VP24 from the virulentSUDV species [123]. Now that these VP24 structures arereported, small molecules can be identified that bind to keyinteraction sites, and this may eventually lead to inhibitionof VP24 functions such as transcription, replication andimmunosuppression.
4.7 GlycoproteinThe glycoprotein genes of ebolavirus and marburgvirus differin their expression, but both function to produce the viral spikesurface glycoprotein that decorates the surface of virions, and isresponsible for virus entry into cells [67]. Only the ebolavirusglycoprotein gene encodes a frameshift reading site whichallows for a soluble glycoprotein (sGP) product to be madeand secreted from producing cells [67,124,125]. This sGP has notransmembrane domain or anchor. sGP released into thebloodstream may serve as an immune decoy during infec-tion [61]. The ebolavirus glycoprotein gene also encodes asecondary secreted glycoprotein of unknown function [126].The transmembrane spike GP1,2 is the full length transcript,and GPs differ by 37 -- 41 % nucleotide and 34 -- 43 % inamino acids across different ebolavirus species [67]. Ebolavirusand marburgvirus differ in glycoprotein sequence homologyby 55% at the nucleotide level and 67% at the amino acid level.The GP1,2 spike protein is a peplomer and a Type-Itransmembrane protein which is N- and O-glycosylated, phos-phorylated and post-translationally cleaved into two subunits,GP1 and GP2. The N-terminus of the glycoprotein precursorbecomes GP1 (about 160 -- 170 kDa) and theC-terminus becomes GP2 (about 38 -- 45 kDa). These twoproteins remain linked by a disulfide bond after host furincleavage [127]. N- and O-linked glycosylation accounts forabout one third of the molecular mass of the GP spike pro-teins [128]. GP1 is heavily glycosylated and responsible forattachment to host cells, but GP2 is responsible for fusion ofthe viral membrane with the host cell membrane, once thevirion has entered the lysosome [129-131].
GP1 and GP2 have functional domains which are reportedto have activities outside of binding and entry, and may beinvolved in pathogenesis. GP1 is reported to have about40 amino acid residues at the N-terminus which suppresslymphocyte blastogenesis in vitro, but the significance of this
activity in vivo has not yet been determined [132]. GP1 has amucin-like domain (MLD) that holds the glycans, and func-tions to prevent neutralizing antibodies from binding to theviral surface at GP1,2, and has been reported to have toxiceffects on cells [133-138]. GP2 is the transmembrane part ofthe spike glycoprotein and mainly functions as the fusion pep-tide, but an N-terminal immunosuppressive motif has alsobeen described [103,139,140]. This region may be cleaved andreleased to exert immunosuppressive activity [132,141].
A GP1,2 dependent model for filovirus entry has beendescribed through research involving studies of GP1,2 structureand through small molecule discovery [17,142-144]. External hostcell receptors have been evaluated and a variety of host cell fac-tors have been implicated in binding and entry: DC-SIGN/L-SIGN, hMGL, B-integrins, folate receptor A, and Tyrofamily receptors [145-149]. Once inside the endosomal/lysosomalcompartment, endosomal proteases cathepsins L and B arerequired for GP1 cleavage. This cleavage exposes the receptorbinding region and promotes fusion of viral membranes tocell membranes through GP2 fusion activities [142,143,147,150].Inside the lysosomal compartment, NPC-1 has been identifiedas the cellular factor required for entry of cells by GP2 throughthe cellular membrane [17,144]. Complete biological investi-gations such as these enable screening and mechanism ofentry studies like the ones presented below, and help directstructure-based drug discovery efforts.
A small molecule benzodiazepine derivative, named com-pound 7, was recently shown to inhibit filovirus entry [15].This molecule was discovered using a cell culture-basedhigh-throughput screen and a pseudotyped HIV virus express-ing EBOV-GP and containing a luciferase reporter gene(see Figure 1). Pseudotyped virus was screened against manydifferent compounds at a 25 µM concentration in a 96-wellplate format [59]. From a library of 52,000 compounds,18 compounds were found which had specific activity againstHIV/EBOV-GP, and no activity against HIV that had beenpseudotyped with VSV-G protein as a control for non-specific virus glycoprotein interaction [15]. These 18 hits hadgreater than 90% inhibitory activity against HIV/EBOV-GPand each compound was only cytotoxic to the indicatormonolayer at concentrations greater than 25 µM. Oncefound, these 18 compounds were tested against replication-competent EBOV expressing green fluorescent protein. Eightcompounds were identified with a 50% inhibitory concentra-tion (IC50) £ to 20 µM [15]. The cell cytotoxicity 50% level(CC50) was measured at > 50 µM for these compounds, there-fore the selectivity index (ratio of cytotoxicity to inhibitoryactivity) would be greater than 5. All eight molecules hadgood drug-like properties, but compound 7 demonstratedthe greatest potency, the most selective inhibition, andhad the most chemically “tractable” scaffold [64].
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The analysis was taken even further by investigation ofcompound 7 with GP1 and GP2 using molecular modeling.The 3.4 A crystal structure of EBOV GP1 and GP2, whichwas solved in complex with a human neutralizing antibody(PDB code 3CSY), was selected as the template [151].Structural analysis of the GP1 and GP2 subunits revealed ahydrophobic pocket at their interface that could be thebinding site for compound 7. This site is demarked byVal66, Leu68, Asn69, Leu70, Leu184, and Leu186 fromGP1, and by Tyr517, Met548 and Leu558 from GP2(Figure 2). Subsequently, mutational studies of GP1 and GP2were performed in order to confirm the binding of compound7 to this hydrophobic site at the GP1-GP2 interface. Themutation work showed that even at concentrations as highas 50 µM, compound 7 exhibited little or no inhibition ofN69A and L70A mutants, and similarly, weakly inhibitedby £ 30% at a 25 µM concentration the L184A, I185A,L186A, K190A, and K191A mutants. Consistent with themolecular models, the results suggest that compound 7 bindsat or near amino acid residues Asn69, Leu70, Leu184, Ile185,Leu186, Lys190, and Lys191. Unfortunately, Basu et al. do
not present a binding model showing compound 7 in com-plex with GP1-GP2 in identified site. Instead, Basu et al.delineate the structure-activity relationship for compound 7.Twelve different analogues of compound 7 were assayed.The most potent of these analogues had an IC90 value of3.7 µM. Preliminary structure-activity relationship analysisshowed four sites (R1, R2, R3, and R4) that play a role inactivity (Figure 3). For R1 and R2, activity was increasedwhen the substituents were halogen atoms, and lost whenR1 and R2 were bulky benzo fusions. For R3, activity wasretained when the substituents were halogenated alkyl groupsand lost when the substituents were aromatic groups. For R4,small substituents at various positions can be tolerated. Forimproved structure-based drug design for anti-EBOV com-pounds, it is essential to determine the binding modes of theseactive benzodiazepine derivatives in the GP1-GP2 complex.This understanding would provide a structural basis for theiractivity and thus, become the platform for the design ofdrug candidates.
6. Mechanistic protein functional studiesreveal inhibitors with structural importance
An elegant body of work has recently shown that theNiemann-Pick C1 (NPC-1) protein is an essential host cellu-lar factor that viral GP1,2 binds for entry into cells at thelysosomal stage [17,144]. This discovery was made through ascreening effort investigating host cellular targets which couldimpede virus entry, as determined through the use of pseudo-typed VSV expressing the EBOV GP1,2 [17]. A benzylpipera-zine adamantine diamide derivative compound, named 3.0,inhibited growth of the VSV/EBOV-GP on vero cells over96 h postinfection. Adding a methoxycarbonyl benzyl groupat the ortho position increased the molecule’s potency by
Figure 2. Interface of the GP1 and GP2 subunits extracted from the 3CSY crystal structure [151] shows the binding site for
benzodiazepine derivatives identified by Basu et al. [15]. GP1 and GP2 are rendered in cyan and purple ribbon, respectively.
The surface of the hydrophobic binding site is shown in yellow.
N
HN
R2
R1
R3
R4
Figure 3. Preliminary structure-activity relationship of the
twofold (compound 3.47), resulting in decreased infectivityfor the VSV/EBOV-GP1,2. Endosomal protease cathepsin Bis required for EBOV GP1 cleavage, therefore the interactionof these new candidate molecules with cathepsin B was inves-tigated [142,143]. Through cellular activity studies and in vitrocathepsin activity assays, it was determined that compounds3.0 and 3.47 do not target cathepsin B protease [17]. NPC-1and cathepsin B do have distinct roles in EBOV entry witheach having a distinct timing for their activity [17,142,143].
These compounds induce formation of cytoplasmic vacuoleswhich are full of cholesterol, indicating that cholesterol uptakealong with its normal trafficking and regulation are hinderedby 3.0 and 3.47 [17]. Proteins known to regulate cholesterolaccumulation in late endosomes were evaluated for involve-ment and only NPC-1 was shown to be both essential toEBOV-GP infection and involved in cholesterol transport.Further, CHO cells lacking the NPC-1 protein could not beinfected with pseudotyped VSV/EBOV-GP, and experimentsexpressing recombinant mutants of NPC-1 on CHO cells indi-cated NPC-1 must be expressed, but need not be able to trans-port cholesterol for interaction with EBOV GP1,2 [17]. It wasalso shown that the GP1 component of EBOV-GP1,2 mustbe cleaved by the cathepsin B protease to bind to NPC-1,because engineered proteins that are made to the final cleavagephenotype would bind NPC-1 but those that were engineeredto be uncleavable did not bind the protein.
The NPC-1 protein is directly targeted by compounds3.0 and 3.47, as neither compound binds cleaved or wholeEBOV GP1,2. Instead, these compounds inhibit NPC-1 inter-action with the cleaved GP1 binding site. This was demon-strated through the use of a competitor compound designedto compete with 3.47 for NPC-1 binding. In addition,
NPC-1 over-expression conferred resistance to compounds3.0 and 3.47 [17]. Other small molecules, U18666A and imip-ramine, are known to cause a cellular phenotype similar tothat of NPC-1 deficiency, possibly because treatment withthese compounds inhibits late endosomal or lysosomal choles-terol transport [152,153]. U18666A and imipramine have beenshown to inhibit infection of vero cells and HAP1 cells byVSV/EBOV-GP1,2 when given at early stages of cellular infec-tion, but not when administered to cells late during infection.U18666A and imipramine may function as entry inhibitorsdue to interference with cholesterol transport, but they donot directly bind cleaved GP1 or NPC-1.
Given these data, NPC-1 is the host cellular factor to whichthe exposed N-terminal domain of GP1 binds, after theheavily glycosylated residues are cleaved off by endosomalcathepsin B protease. These studies, together with the struc-ture of the GP1,2 [151], have given us a clearer understandingof the activities of GP1 and GP2 in the lysosome and howNPC-1 may participate in enabling viral membrane fusionwith the cell membrane. Residues in the N-terminal domainof GP1 are interspersed with residues that make stabilizingcontacts with GP2. GP1 binding to NPC-1 may allow forGP2 to be freed up to change conformation and promotefusion of the viral membrane to the cellular membrane. Alter-natively, GP2 must also change conformation after GP1 bindsNPC-1, in order for the second step of GP2 to fuse. This exactmechanism is not yet solved.
NPC-1 is a large transmembrane protein that has beenproposed to be a proton-coupled transporter of sterols andother amphipathic molecules. The topology of NPC-1 isbuilt around 13 transmembrane helices and three majordomains [154,155]. The sterol sensing domain of NPC-1 exists
Figure 4. Crystal structure of the N-terminal domain of Niemann-Pick C1 (NPC1) in complex with cholesterol (PDB code
3GKI) [156]. NPC1 is shown in cyan ribbon and cholesterol in yellow CPK. The cholesterol binding site may also be the binding
site for compound 3.47 [17].
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in the 5 transmembrane segment defined by helices 3-7. Thefirst domain is the N-terminal domain, which occurs onthe luminal side of the membrane and contains a Cys-rich leu-cine zipper motif as well as the sterol-binding site. The seconddomain is Loop C, which occurs on the luminal side and linkstransmembrane helices 2 and 3, and the third is Loop E whichlinks transmembrane 8 and 9, and is characterized by a Cys-rich ring finger motif. The concentration of Cys residues inthe N-terminal domain and Loop E makes NPC-1 susceptibleto suicide inhibitors. The structure of the N-terminal domainof NPC-1 in complex with cholesterol has been solved byx-ray crystallography (Figure 4) [156]. The 1.8 A structure ofNPC-1 (PDB code 3GKI) shows a deep, internal pocket forcholesterol binding. If the structure of NPC-1 is to be usedas a template for the structure-based design of more potentanalogues of compound 3.0 and 3.47, further biochemicalexperiments are necessary to determine the binding modesof these compounds in NPC-1.Furthermore, novel NPC-1 inhibitors may be gained from
the significant number of cholesterol-absorption inhibitors(CAI) that have been shown to target the closely relatedNiemann-Pick C1-Like 1 (NPC1L1) protein [157]. Whilehuman NPC-1 (uniprot code O15118) and human NPC1L1(uniprot code Q9UHC9) possess a sequence identity of36.9%, potential structural and functional similarity betweenNPC-1 and NPC1L1 may allow CAIs to also target NPC-1,providing a molecular basis for the development of anti-ebolavirus agents. The archetype among the CAI inhibitors isthe clinically approved drug ezetimibe (Zetia�) [158], which isan effective treatment for hypercholesterolemia. Biochemicaland proteomic studies delineated a mechanism of action forezetimibe, involving the binding of ezetimibe to the large extra-cellular loop C of NPC1L1, which prevents conformationalchanges in NPC1L1 that are essential for cholesterol transloca-tion across the membrane [159]. Potentially, as would ezetimibebind loop C in NPC1L1, compounds 3.0 and 3.47 bind tocorresponding loop C in NPC-1. The discovery of ezetimibewas further enriched by structure-activity relationship studiesof its azetidinone class of compound [160]. Moreover, thediscovery of ezetimibe opened the way for the developmentof other structural classes of NPC1L1 inhibitors, which arehighlighted by the recent disclosure of spiroimidazolidinoneNPC1L1 inhibitors [161,162].
7. Expert opinion
A key finding in this review is that there are many reportsdescribing solved filovirus protein structures (see Table 2). Inaddition, recent studies on host proteins such as NPC-1 andcAbl-1 highlight the crucial roles that host cellular factorsplay in the filovirus life cycle, making it possible that certainhost cellular factors are viable antiviral targets. Despite ourincreasing structural understanding of viral proteins VP30,VP35, VP40, VP24, and GP1,2, at present, a major weaknessin the field is that while some structures are available, many of
their functions and interactions remain to be fully elucidated.For example, it is known that NP, VP35, VP30, and Linteract to form the replication complex, but exactly howthese proteins interact and where might they physically bindone another is not yet known. If these interactions were betterunderstood, through a method such as bimolecular comple-mentation [16] compounds which could potentially block theassembly of the replication complex could be designed andtested in in vitro and in vivo models of filoviral replication.While there is a tremendous need for requisite biology studies,dissecting the biological process of viral proteins is slowed bythe rigors of a high biocontainment environment.
Continued screening of existing molecules is going toremain a critical activity in filovirus therapeutics discovery.To date, a variety of screening projects to look for new com-pounds with anti-filovirus activity, such as “compound 7” orto repurpose old ones (antidepressant imipramine or anti-malarial chloroquine) have been reported [15,144,163]. Screeningnot only provides lead compounds, but also may identifychemical probes that may be used to inform researchers aboutthe basic biology of filovirus infection [163]. As we continue toidentify ever increasing numbers of hits and lead compounds,and we are able to discern their potential mechanismsof action, we will be able to fully delineate the functions offiloviral proteins. These new functions may provide thebasis for rational drug design. As a classic example of this,Garcia M. et al. recently reported that VP40-mediated bud-ding of Ebola virus-like particles is mediated by phosphoryla-tion of Tyr13 on VP40 by the host cellular c-Abl1 tyrosinekinase [107]. In this work, viral release is decreased both byc-Abl1-specific small interfering RNA and by Abl-specifickinase inhibitors, imatinib (Gleevec) and nilotinib (Tasigna).Imatinib and nilotinib are approved drugs for the treatmentof chronic myeloid leukemia and are well known to specifi-cally target bcr-abl activity in leukemia. The ability of imati-nib and nilotinib to modulate the release of Ebola virus-likeparticles suggests that these two drugs may prove to be effica-cious short-term treatments against Ebola virus infection [107].Moreover, the anti-Ebola activity of two well-known anti-cancer drugs establishes a paradigm in which novel antiviraltherapeutics can be found in repurposed drugs and ininvestigational compounds.
In the coming years, research will continue to shed light onhow host proteins, such as cell-signaling molecules, stress-response proteins, or kinases and phosphatases, interact inthe virus life cycle. Small molecules with antioxidant proper-ties have shown anti-filovirus activity and may modulatemultiple host signaling pathways [164]. Other host proteins,such as Tsg101, Nedd4, actin, tubulin, some annnexins,heat-shock protein (HSP) A5, and ribosomal protein (RP)L18 have all been detected by proteomic analysis to be pack-aged inside EBOV and MARV virions [111,165-167]. siRNAstargeting HSPA5 and RP L18 in culture inhibited EBOVand MARV infection of 293T cells [167]. Continued drugscreening and viral biology studies will help to address
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knowledge gaps about host protein interactions, hopefullyidentifying active sites of host or viral proteins through mech-anism of action studies. As these details become available,inhibition of host targets, or specific blocking of the site(s)of interaction of host targets with viral proteins may be thekey to unlocking good therapeutics for infection with highlyvirulent viruses. It will be important to specifically block thesesites of interaction, and not just inhibit the host protein acti-vity or its production, because such non-specific blocking orreduction of host proteins may lead to toxicity and unwantedside effects.
As an alternative to targeting catalytic sites, it is temptingto propose exploring the rational design of compoundsthat block essential protein-protein interactions (PPI), anapproach which has gotten some attention at recent biode-fense conferences as a method that could be employed whenlittle is known about target protein structure [168,169]. Consid-ering the limited number of known catalytic functions forfilovirus proteins, this may prove to be an avenue for potentialtherapeutics. The buried surfaces in PPIs are generally large(~ 1600 A) and it is thermodynamically improbable that arandom small molecule would disrupt the favorable energeticsenough to dissociate the interacting partners [170]. However,recent research has demonstrated that a small area of the inter-acting surface may actually account for the high-affinitybinding [171-173]. These compact regions of increased affinityhave been termed “hot-spots” and have been proposed to bea common motif for PPIs since these hot-spots are involvedin the binding of multiple partners for the same protein [171].Some recent work using bimolecular complementation tocharacterize the protein-protein interactions of VP40-VP40,NP-VP40, and NP-NP for EBOV and MARV has enhancedthe understanding of the role of these proteins in virus assem-bly and budding, and viral replication [12,16]. Using deletionmutant analysis, bimolecular complementation helpedpinpoint the protein sequences necessary for VP-40 selfinteractions and oligomerizations, and maybe soon somemolecules can be designed which mimic the structure andfunction of the interacting protein surfaces [16]. While there
are no specific examples of proteomimetics that have success-fully disrupted PPIs for filoviruses, examples of proteomimeticsas protease inhibitors do exist [169].
Comparatively speaking, data relating the structure andfunction of filovirus proteins is certainly less voluminousthan data available for protein constituents of HIV, cancersand other human pathogens of mainstream clinical impor-tance [3,6,7,9]. The substantial research funding that is allocatedfor cancer and AIDS has enabled scientists to generate thestructural and biochemical data needed to employ modernstructure-based drug design, and as a result, these fieldshave progressed much further than fields that are less wellfunded [174]. This limited funding combined with researchconstraints due to biosafety issues have restricted the availableapproaches for discovering lead candidates for therapeuticsand vaccines for biothreat agents [20,175]. Compounding theissue is the fact that it has been difficult to attract large phar-maceutical companies with state-of-the-art drug discoverytechniques to the field of biodefense due to the limited marketfor sale and distribution of their products [175]. The deve-lopment of therapeutics for biodefense or for use in lowersocio-economic countries with an unmet medical need offersa very limited return on investment compared to profitsfrom developing therapeutics for diseases of human clinicalimportance typically seen in the developed world [175,176].
The authors thank numerous colleagues and peers who arestriving to develop filovirus medical countermeasures andregret that space limitations may have prevented inclusion ofsome articles. Support for filovirus evaluations has been pro-vided by the Joint Science and Technologies Office/ChemBio Defense 00048_RD_T. The opinions, interpretations,conclusions and recommendations are those of the authorsand are not necessarily endorsed by the US Army.
Declaration of interest
This paper was sponsored by the Defense Threat ReductionAgency and Transformational Medical Technologies. Theauthors declare no other conflicts of interest.
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