JOURNAL OF VIROLOGY, Apr. 2011, p. 3106–3119
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 85, No. 7
Identification of a Small-Molecule Entry Inhibitor for Filoviruses?†
Arnab Basu,1* Bing Li,1Debra M. Mills,1Rekha G. Panchal,2Steven C. Cardinale,1
Michelle M. Butler,1Norton P. Peet,1Helena Majgier-Baranowska,1
John D. Williams,1Ishan Patel,1Donald T. Moir,1Sina Bavari,2
Ranjit Ray,3Michael R. Farzan,4Lijun Rong,5and Terry L. Bowlin1
Microbiotix, Inc., Worcester, Massachusetts 016051; U.S. Army Medical Research Institute of Infectious Diseases, Fort Detrick,
Maryland 217022; Department of Internal Medicine, St. Louis University, St. Louis, Missouri 631103; Department of
Molecular Biology and Genetics, Harvard Medical School, Southborough, Massachusetts 017724; and
Department of Microbiology and Immunology, University of
Illinois at Chicago, Chicago, Illinois 606125
Received 12 July 2010/Accepted 22 December 2010
Ebola virus (EBOV) causes severe hemorrhagic fever, for which therapeutic options are not available.
Preventing the entry of EBOV into host cells is an attractive antiviral strategy, which has been validated for
HIV by the FDA approval of the anti-HIV drug enfuvirtide. To identify inhibitors of EBOV entry, the EBOV
envelope glycoprotein (EBOV-GP) gene was used to generate pseudotype viruses for screening of chemical
libraries. A benzodiazepine derivative (compound 7) was identified from a high-throughput screen (HTS) of
small-molecule compound libraries utilizing the pseudotype virus. Compound 7 was validated as an inhibitor
of infectious EBOV and Marburg virus (MARV) in cell-based assays, with 50% inhibitory concentrations
(IC50s) of 10 ?M and 12 ?M, respectively. Time-of-addition and binding studies suggested that compound 7
binds to EBOV-GP at an early stage during EBOV infection. Preliminary Schro ¨dinger SiteMap calculations,
using a published EBOV-GP crystal structure in its prefusion conformation, suggested a hydrophobic pocket
at or near the GP1 and GP2 interface as a suitable site for compound 7 binding. This prediction was supported
by mutational analysis implying that residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191 are
critical for the binding of compound 7 and its analogs with EBOV-GP. We hypothesize that compound 7 binds
to this hydrophobic pocket and as a consequence inhibits EBOV infection of cells, but the details of the
mechanism remain to be determined. In summary, we have identified a novel series of benzodiazepine
compounds that are suitable for optimization as potential inhibitors of filoviral infection.
Ebola viruses (EBOV) are enveloped, single-stranded, neg-
ative-sense RNA viruses and have been classified as category A
pathogens by the Centers for Disease Control and Prevention
(CDC). Together with Marburg virus (MARV), they constitute
the filovirus family. There are five species of EBOV, namely,
Zaire, Sudan, Ivory Coast, Bundibugyo, and Reston (61).
EBOV infection causes severe viral hemorrhagic fevers
(VHFs) in humans and nonhuman primates, with a mortality
rate of up to 90% (55). These outbreaks are infrequent and so
far have been restricted to small pockets of population in
Africa. The natural reservoir for the virus is still not known, but
fruit bats have been implicated (27, 34).
The EBOV genome contains seven genes that encode eight
viral proteins, NP, VP35, VP40, glycoprotein (GP), sGP, VP30,
VP24, and RNA-dependent RNA polymerase (L) (44, 56).
Transcriptional editing of the fourth gene results in expression
of a 676-residue EBOV envelope glycoprotein (EBOV-GP), as
well as a 364-residue secreted glycoprotein (sGP1) (44).
EBOV-GP mediates the viral entry into host cells and provides
a potential target for the design of vaccines and entry inhibi-
tors. EBOV-GP is posttranslationally cleaved by furin, to yield
disulfide-linked GP1 and GP2 subunits (63). GP1 is involved in
attachment of EBOV to host cells, whereas GP2 mediates
fusion of viral and host membranes (18, 59). EBOV is believed
to enter host cells by receptor-mediated endocytosis (44),
where further processing by endosomal cathepsin L (cat L)
and/or cathepsin B (cat B) (11, 31, 46) is required for entry. A
cellular receptor(s) for EBOV has not yet been identified, but
DC-SIGN/L-SIGN, hMGL, ?-integrins, folate receptor-?, and
Tyro family receptors have all been implicated as cellular fac-
tors in entry (10, 51, 52). EBOV-GP, apart from its role in virus
entry, also plays an important role in the pathogenicity of
infection. Expression of EBOV-GP induces a cytopathic effect
(CPE) in cell lines and human blood vessel explants (53, 62).
This cytopathic effect was mapped to the mucin-like region
present in the C terminus of GP1(62). EBOV-GP, when over-
expressed, also downregulates molecules involved in cell ad-
hesion and causes anoikis (39). Virus-like particles (VLPs)
containing EBOV-GP on the surface activate macrophages to
secrete several proinflammatory cytokines (6, 54).
Virus entry is an essential component of the viral life cycle
and an attractive target for therapy because inhibition of this
step can block the propagation of virus at an early stage,
minimizing the chance for the virus to evolve and acquire drug
resistance. Anti-infective drug discovery for EBOV presents
significant logistical and safety challenges due to the require-
ment for biosafety level 4 (BSL-4) containment and proce-
dures. The advent of replication-incompetent pseudotype vi-
* Corresponding author. Mailing address: Microbiotix, Inc., One
Innovation Drive, Worcester, MA 01605. Phone: (508) 757-2800. Fax:
(508) 757-1999. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jvi
?Published ahead of print on 26 January 2011.
ruses, which utilize the replication machinery of vesicular
stomatitis virus (VSV) (16, 48), murine leukemia virus (MLV)
(37), or human immunodeficiency virus (HIV) (29, 30) but
package the EBOV-GP on the virion surface, offers an oppor-
tunity to safely screen libraries of small molecules for antiviral
properties in a BSL-2 environment. In this study, we report the
discovery of a novel small-molecule entry inhibitor with spe-
cific inhibitory activity against both EBOV and MARV. A
benzodiazepine derivative (compound 7) was identified from a
high-throughput screen (HTS) of small-molecule compound
libraries utilizing the EBOV pseudotype virus. Compound 7
also specifically inhibited cell culture-grown EBOV in vitro,
and the potential binding site was mapped to a specific area
within a hydrophobic pocket at the EBOV GP1-GP2 interface.
(Part of this study was presented in 2009 at the 28th Annual
Meeting of the American Society for Virology, Vancouver,
MATERIALS AND METHODS
Cell lines, chemicals, and plasmids. 293T, Vero E6, A549, HeLa, MDCK, and
BHK cell lines were procured from ATCC. The cell lines were maintained in
either Dulbecco modified Eagle medium (DMEM) or minimal essential medium
(MEM) supplemented with 10% fetal bovine serum (FBS) and penicillin-strep-
tomycin (50 units/ml). Plasmid vectors expressing the envelope proteins of
EBOV Zaire (GenBank accession number L11365) (EBOV-GP), MARV
(Lake Victoria strain) (MARV-GP), vesicular stomatitis virus (VSV-G), lym-
phocytic choriomeningitis virus (LCMV), Lassa virus (LASV), and Machupo
virus (MACV) and the hemagglutinin [HA(H5)] gene from a highly patho-
genic H5N1 avian influenza virus (Goose/Qinghai/59/05) were described ear-
lier (14, 29, 30, 37).
Pseudotyping. Ebola pseudotype viruses (HIV/EBOV-GP) were produced by
cotransfecting 12 ?g of wild-type (wt) or mutant EBOV-GP with 12 ?g replica-
tion-defective HIV vector (pNL4.3.Luc-R–E-) (15) into 293T cells (90% conflu-
ent) in 10-cm plates with Lipofectamine 2000 (Invitrogen) according to the
supplier’s protocol (4, 29). The supernatants containing the pseudotype viruses
were collected at 48 h posttransfection, pooled, clarified from floating cells and
cell debris by low-speed centrifugation, and filtered through a 0.45-?m-pore-size
filter (Nalgene). The culture supernatants containing HIV/EBOV-GP were ei-
ther used immediately or flash frozen in aliquots and stored at ?80°C until use.
Each aliquot was thawed only once for use in a single round of replication.
Mutant HIV/EBOV-GP pseudotype viruses, along with pseudotype viruses bear-
ing VSV envelope protein (HIV/VSV-G), LASV envelope protein (HIV/LASV-
GP), LCMV envelope protein (HIV/LCMV-GP), MACV envelope protein
(HIV/MACV-GP), and HA(H5) from avian influenza A H5N1 virus (Quinghai
strain) [HIV/HA(H5)], were prepared in a similar fashion, using the same Env-
deficient HIV vector as previously described (4, 29, 37, 42).
Chemical libraries. The chemical libraries screened represent broad and well-
balanced collections of over 52,500 compounds. They were purchased from
Chembridge (San Diego, CA) and Timtec (Newark, DE), diluted in 96-well
master plates at 2.5 mM in dimethyl sulfoxide (DMSO), and stored at ?20°C.
Compounds were selected in the molecular mass range of 200 to 500 Da and are
within the size range of molecules considered small and “drug-like.” Compounds
were evaluated using numerous chemical filters, including Lipinski’s “rule of 5”
(28), to remove unwanted and known cytotoxic scaffolds, such as metal com-
plexes, highly conjugated ring systems, oxime esters, nitroso groups, and strong
Michael acceptors. The compounds have favorable cLogP values (calculated
logarithm of n-octanol/water partition coefficient), and encompass over 200 che-
HTS of chemical libraries. High-throughput screening (HTS) of chemical
libraries using pseudotype virus was performed in 96-well plates as described
previously (22, 24). The final concentration of test compound was 25 ?M, while
the final concentration of DMSO was maintained at 1% in all wells. Low-passage
293T cell monolayers were infected with 100 ?l of p24-normalized HIV/
EBOV-GP pseudotype virus containing 8 ?g/ml Polybrene in the presence of test
compounds. After 2 h, the inoculum was removed, the cells were washed briefly,
fresh medium was added, and the plates were incubated for 72 h. Prior to each
screening, each batch of the viral preparation was titrated to determine the
amount of virus required to infect the target cells, so that a relatively high
luciferase activity could be recorded while still remaining in a linear response
range (105to 106relative luciferase units [RLU]). Infection was quantified from
the luciferase activity of the infected 293T cells using the Britelite Plus assay
system (Perkin-Elmer, Waltham, MA) in a Wallac EnVision 2102 multilabel
reader (Perkin-Elmer). In the absence of compound, the assay showed an aver-
age luciferase signal of 1.2 ? 106? 0.6 ? 106RLU, a signal-to-background ratio
of ?103, and a calculated screening window coefficient (Z? factor) (63) of 0.5 ?
0.2. The luciferase signal standard error was ?50%, and greater than 90%
inhibition of luciferase activity at 25 ?M was used as the criterion for designating
a compound a “hit.” Test compounds were in DMSO solutions, with 80 com-
pounds per plate. Controls were also included in each plate: eight wells for 0%
inhibition (DMSO only, maximum signal) (positive control) and eight wells for
100% inhibition (e.g., E64 for EBOV, minimum signal) (negative control). The
percent inhibition was calculated as 100 ? [1 ? (RLU in the presence of
compound ? RLU of negative control)/(RLU of positive control [without any
inhibitor] ? RLU of negative control)].
Cell viability assay. Cells were plated in a 96-well format (4 ? 103cells/well)
in the presence or absence of serial dilutions of the test compound. Following 3
days of incubation in growth medium at 37°C, cell viability was determined using
the CellTiter 96 aqueous nonradioactive cell proliferation assay (Promega, Mad-
ison, WI) as previously described (5).
Infection assay with cell culture-grown recombinant GFP-ZEBOV. All exper-
iments with cell culture-grown Zaire EBOV expressing green fluorescent protein
(GFP-ZEBOV) were performed under BSL-4 conditions. Cell culture-grown
GFP-ZEBOV was used to screen compounds for inhibition of EBOV replication
using methods described previously (1, 33). Briefly, Vero E6 cells (40 ? 103
cells/well in a 96-well plate format) were infected with the GFP-ZEBOV at a
multiplicity of infection (MOI) of 5 in the presence of compounds. Cultures were
incubated for 48 h before fixation (10% neutral-buffered formalin, 72 h) and
removal from the BSL-4 facility. After nuclear (Hoechst dye) and cytoplasm/
nuclear (high-content screening [HCS] cell mask deep red stain) staining, viral
infection was imaged and quantified using an automated Opera confocal reader
(model 3842-quadruple excitation high sensitivity [QEHS]; Perkin-Elmer).
Images were analyzed within the Opera environment using standard Acapella
scripts. This procedure identifies the total number of adherent cells and the
fraction of infected cells, thereby providing an immediate assessment of efficacy
and toxicity. Cell viability assays were conducted as detailed previously (1, 33).
All assays were repeated at least three times, and representative findings are
shown. Percent inhibition values were calculated as follows: 100 ? [1 ? (average
GFP fluorescence from compound-treated wells/average fluorescence from wells
containing medium only)].
Antiviral assays. The activity of compound 7 against hepatitis C virus (HCV)
was evaluated using cell culture-grown HCV genotype 1a (H77) and immortal-
ized human hepatocytes (IHH) as previous described (4). The HCV titer was
calculated as ?105focus-forming units (FFU)/ml, and antiviral activity was
measured in a dose-dependent manner (4). Cells were incubated with the virus
in the presence of compounds for 3 days, and the fluorescent-focus formation
was determined by immunofluorescence using NS4-fluorescein isothiocyanate
(FITC)-conjugated monoclonal antibody (Biodesign). Nuclear staining was per-
formed with TO-PRO-3 (Molecular Probes). This procedure identifies the total
number of adherent cells and the fraction of infected cells, thereby providing an
immediate assessment of efficacy and toxicity. The percent inhibition was calcu-
lated based on the number of fluorescent foci in the treated sample compared to
the number in the untreated positive control. A selectivity index (SI) (50%
cytotoxic concentration [CC50]/50% inhibitory concentration [IC50]) of ?3 was
used as an indication of selective antiviral activity.
The activities of compound 7 against Tacaribe virus (strain TRVL 11573), Rift
valley fever virus (strain MP-12), Venezuelan equine encephalitis (VEE) virus
(strain TC-83), cowpox virus (strain Brighton), vaccinia virus (strains WR and
IHD), influenza A (H1N1) virus (strain California/07/2009), influenza A (H3N2)
virus (strain Brisbane/10/2007), and severe acute respiratory syndrome (SARS)
virus (strain Urbani) were determined in collaboration with NIAID’s Antimi-
crobial Acquisition and Coordinating Facility (AACF) using standard proce-
dures (2, 3, 49, 64). For toxicity studies different methods, including (i) visual
observation of the viable cells and (ii) neutral red uptake assay, were used to
determine the number of viable cells in the well. This procedure provided an
immediate assessment of efficacy and toxicity. An SI (CC50/IC50) of ?3 was used
as an indication of selective antiviral activity.
Time-of-addition experiment. The “time-of-addition” experiment was de-
signed to determine the mechanism of action of the antiviral compounds, and the
procedure is shown schematically in Fig. 2A. 293T cells were incubated with a 25
?M concentration of the test compound at 1 h before infection (?1 h), during
infection (0 h), and 1 h following infection (?1 h). Duplicate wells were used for
VOL. 85, 2011 EBOLA VIRUS ENTRY INHIBITOR3107
each time point. Control infected-cell cultures were treated with drug vehicle
(DMSO) only. At 72 h postinfection, cells were developed and infectivity was
measured as described above.
Compound-virus binding assay. The compound-virus binding assay, with slight
modifications, was performed as described earlier (13, 60). Microcon Ultracell
YM3 centrifugal devices (Millipore), which are capable of separating small
molecules from large macromolecules, were used to measure the binding of
compound 7 to EBOV-GP, as follows. HIV/EBOV-GP and HIV/VSV-G pseu-
dotype viruses were ultracentrifuged using a 10% sucrose cushion at 35,000 rpm
at 4°C in a Beckman SW41 rotor for 60 min. The viruses were resuspended in
DMEM, and the concentration of HIV capsid protein p24 was measured by
enzyme-linked immunosorbent assay (ELISA). Fifty microliters of either HIV/
EBOV-GP or HIV/VSV-G pseudotype virus supernatant (containing 300 ng/ml
of p24) was incubated with 25 ?M compound 7 at room temperature for 60 min.
The virus-compound mixture was then loaded onto the Microcon Ultracell YM3
centrifugal device, equilibrated for 5 min, and centrifuged for 2 min. After
centrifugation, the eluent was analyzed by high-pressure liquid chromatography
to measure the quantity of unbound compound 7. Signals from a reaction mix
containing no pseudotype virus were used as a positive control. The percent
binding was calculated based on the quantity of compound 7 in the eluent in the
presence of pseudotype viruses compared to that in the positive control.
EBOV-GP RBD cell binding assay. The EBOV-GP receptor binding domain
(RBD) cell binding assay was performed according to a protocol described by
Kuhn et al. (20, 21). The RBD of EBOV-GP (residues 54 to 201) was fused with
human IgG Fc to generate a fusion protein (GP-hIgG). The fusion protein gene
was expressed and the protein purified as previously described (20, 21). This
RBD fusion protein has been demonstrated to bind to cells that are permissive
for EBOV infection but not to lymphocyte cell lines, which are nonpermissive for
EBOV infection (20, 21). 293T cells were detached by treatment with phosphate-
buffered saline (PBS)–5 mM EDTA, resuspended in PBS–1% bovine serum
albumin (BSA), and kept on ice for 30 min. Then, 10 ?g of GP-hIgG protein was
incubated with 0.5 ? 106cells on ice for 1 h in the presence of different
concentrations of compound. Following the incubation, cells were washed with
PBS–1% BSA two times. Anti-human IgG antibody conjugated with FITC was
incubated with cells on ice for 45 min at a dilution of 1:100. Cells were washed
three times with PBS–1% BSA and two times with PBS and subjected to flow
cytometry. In this experiment, a SARS virus spike fusion protein (S1-hIgG) that
does not bind to Vero or 293T cells was used as a negative control.
Computational methods. A published crystal structure of EBOV-GP, at 3.4-Å
resolution, in its metastable trimeric, prefusion conformation in complex with the
neutralizing human antibody KZ52 (25), was used for the computational exam-
ination. To keep regions between protein monomers from generating unnatural
putative binding sites, which exist only in the crystal lattice, only the protein
monomer was retained and the antibody was edited out of the structure. The
protein structure was prepared in Maestro (Schro ¨dinger, San Diego, CA) using
the protein preparation wizard. The OPLS 2005 force field was employed as the
force field for the study. Appropriate bond orders and formal charges were
assigned using Maestro. To remove interactions between nonrelevant contacts,
the “impref” procedure was employed to relax the protein structure. This pro-
cedure carries out a series of short refinements until the root mean square
deviation (RMSD) is ?0.3 Å. Next, binding site prediction was performed using
the SiteMap (Schro ¨dinger, San Diego, CA) package (default setting). SiteMap
assigns numerical descriptors to evaluate predicted binding sites by a series of
physical parameters such as size, degree of enclosure/exposure, tightness, hydro-
phobic/hydrophilic character, and hydrogen bonding possibilities. A weighted
average of these measurements is then assigned to prioritize possible binding
HTS for the identification of EBOV entry inhibitors. A
chemically diverse small-molecule library (52,500 compounds)
was screened for ?90% inhibition of HIV/EBOV-GP infection
to identify viral entry inhibitors. As shown in Table 1, the initial
high-throughput screening (HTS) hit rate from the primary
screen was 2.2%, with a total of 1,146 “hit” compounds inhib-
iting the HIV/EBOV-GP. To assess the target specificity of the
HTS hits, we counterscreened these hits against a second pseu-
dotype virus (HIV/VSV-G) having the same HIV backbone
but expressing unrelated vesicular stomatitis virus envelope
glycoprotein (VSV-G). Based on this counterscreen, only 57
compounds were demonstrated to block EBOV-GP-mediated
infection specifically with little or no effect on infectivity of
HIV/VSV-G (Table 1). These EBOV-GP-specific hits were
tested for cytotoxicity, and 18 compounds exhibited CC50val-
ues of ?25 ?M (Table 1). These 18 specific hits were subse-
quently reordered from different batches from the original
vendors and retested. The reordered compounds were deter-
mined to have the correct mass and to be ?95% pure by
LC-MS. All 18 compounds were reconfirmed against HIV/
EBOV-GP, dose-response curves were generated, and 90%
inhibitory concentration (IC90) and CC50values of compounds
were determined. Table 2 shows results of the eight most
Validation of HTS hits against cell culture-grown GFP-ZE-
BOV. The anti-EBOV activities of the 18 HIV/EBOV-GP-
specific inhibitors were also evaluated against cell culture-
grown GFP-ZEBOV in a BSL-4 containment facility at
USAMRIID (Frederick, MD). As shown in Table 2, eight of
the HIV/EBOV-GP-specific hits, all from different chemical
families, were found to inhibit GFP-ZEBOV with IC50s of ?20
?M, as calculated from the linear portions of full dose-re-
sponse curves. Cytotoxicity was determined in the same assay.
Compounds 2 and 7 were found to be of most interest, with
CC50values of ?50 ?M and with calculated selectivity index
(SI ? CC50/IC50) values of ?5. SI values for the remaining
active compounds were ?4, and several exhibited CC50values
tracking close to their IC50s (Table 2). Based upon its overall
potency, significant selectivity for antiviral activity versus cyto-
toxicity (Fig. 1A), and chemically tractable benzodiazepine
backbone, compound 7 was selected as a suitable starting point
for further viral specificity studies, mode-of-action (MOA)
evaluation, and medicinal chemistry optimization.
Compound 7 exhibits antifilovirus activity. EBOV and
MARV are the two members of the filovirus family. The crit-
ical amino acid residues in EBOV-GP that are important for
virus entry are highly conserved in EBOV and MARV (21, 29).
Compound 7 displayed a concentration-dependent inhibitory
activity againstboth HIV/MARV-GP
(IC90? 3.1 ?M) and infectious MARV (IC50? 12.5 ?M) (Fig.
1B). These overall results demonstrate that compound 7 dis-
plays filovirus-inhibitory activity, inhibiting both infectious
EBOV and MARV with similar potencies (EBOV IC50? 10
?M) (Table 2; Fig. 1A).
TABLE 1. Results of the screening of compound libraries
Primary hits (inhibition of HIV/EBOV-GP
in the HTS screen)a.......................................................... 1,146 (2.2)
Primary hits inhibiting HIV/VSV-Ga,b............................... 1,089 (2.07)
Specific hits (primary hits inhibiting HIV/
Specific hits displaying a CC50of ?25 ?Mc......................
aHIV/EBOV-GP and HIV/VSV-G were generated by transfection of 293T
cells with pNL4.3.Luc.R–E– as the HIV-l expression vector and with EBOV-GP
or VSV-G, respectively.
bA total of 1,089 primary hits inhibited HIV/VSV-G by more than 90% at 25
?M, whereas the RLU values of the controls varied by ?50%.
cCC50values were determined using the CellTiter 96 aqueous nonradioactive
cell proliferation assay (Promega).
3108 BASU ET AL.J. VIROL.
TABLE 2. Anti-EBOV activities of the hit compounds
SI IC50, ?M
IC90, ?M (%)
1 18.4301.6 7.5 10152
7.6253.2810 2051.5 5.15
?20 29.5 2.95
1.1 3027.2 15.5
20.8 542.59 14.520 402.75
?20 (78) 48.5 3.2
12.1 756.19 10
?20 (70) 55.25.5
?20 (59) 22.61.1
aHIV/EBOV-GP was generated by transfection of 293T cells with pNL4.3.Luc.R–E– as the HIV-l expression vector and with EBOV-GP. IC90values were
determined using reordered compounds as different batches from the original vendors. For determination of CC50values, 293T cells were treated with compound alone,
and values were determined from linear portions of the dose-response curve. SI, selectivity index (CC50/IC90).
bAll experiments with GFP-ZEBOV were performed under biosafety level 4 conditions. GFP-ZEBOV was incubated with Vero E6 cells at a multiplicity of infection
of 5 for 1 h in the presence or absence of inhibitor compounds. Virus was removed after 1 h, cells were washed in PBS and incubated for 48 h, and the percentage
of GFP-expressing cells was measured. IC50and IC90values were determined from the linear portion of the full dose-response curve. For determination of CC50values,
Vero E6 cells were treated with compound alone, and values were determined from linear portions of the dose-response curve. SI, selectivity index (CC50/IC50).
VOL. 85, 2011 EBOLA VIRUS ENTRY INHIBITOR 3109
Antiviral spectrum of compound 7. We next investigated the
activity of compound 7 against arenaviruses and avian influ-
enza H5N1 virus since they bear type 1 envelope proteins
similar to that of EBOV. The pseudotype platform was used
because it can be performed in a BSL-2 facility and it pro-
vides a direct comparison of the activities of compound 7
against these viruses and the HIV/EBOV-GP. Compound 7
inhibited HIV/LASV-GP, HIV/LMCV-GP,
MACV-GP weakly, in a dose-dependent manner, exhibiting
inhibition of ?30 to 40% at 25 ?M (Fig. 1C), while almost
no inhibition (?10%) was observed against HIV/HA(H5) at
25 ?M in A549 cells (Fig. 1C). In contrast, compound 7
inhibited HIV/EBOV-GP and HIV/MARV-GP infection by
?90% at 12.1 ?M and 3.1 ?M, respectively (Table 2 and
The effect of compound 7 on several RNA and DNA viruses
was further investigated by the Antimicrobial Acquisition and
Coordinating Facility (AACF) of the National Institute of Al-
lergy and Infectious Diseases (NIAID). As shown in Table 3,
compound 7 did not inhibit cell culture-grown infectious Tac-
aribe virus, influenza A H1N1 virus (California/07/2009), in-
fluenza A H3N2 virus (Brisbane/10/2007), or SARS virus (Ur-
bani strain) at concentrations of up to 32 ?M, 25 ?M, 33 ?M,
and 36 ?M, respectively (the 50% cytotoxic concentration
[CC50] in each assay). The inhibitory activity of compound 7
against other infectious RNA viruses, including Rift Valley
fever virus, Venezuelan equine encephalitis (VEE) virus, and
hepatitis C virus (HCV), and DNA viruses, including cowpox
virus and vaccinia virus, was also investigated. None of these
viruses was inhibited by compound 7 at or below its 50%
cytotoxic concentration (Table 3). Clearly, compound 7 inhib-
its EBOV and MARV significantly more potently than it does
any of the other viruses tested, indicating that it is primarily
selective for filoviruses.
Compound 7 binds to HIV/EBOV-GP. EBOV entry can
broadly be divided into two major steps: (i) virus attachment
and receptor binding, followed by (ii) receptor-mediated en-
docytosis, pH-dependent GP processing, and membrane fu-
sion. Entry inhibitors can act at any stage of the entry process.
A time-of-addition experiment was performed with HIV/
EBOV-GP to determine the target stage of EBOV entry that
is blocked by compound 7 (Fig. 2). Compound 7 was added at
1 h before infection (?1 h), during infection (0 h), and at 1 h
postinfection (?1 h). Control infected cultures were treated
with either DMSO (no inhibition) or E64, an EBOV entry
inhibitor (complete inhibition) (11, 46). Compound 7, when
added either before (?1 h) or after (?1 h) virus infection,
displayed less than 18% and 27% inhibition of HIV/EBOV-GP
infection, respectively (Fig. 2B). Interestingly, when compound
7 was present during the virus adsorption process (0 h) at
either 37°C or 0°C, it inhibited more than 75% of the HIV/
EBOV-GP infection (Fig. 2B). These results suggest that com-
pound 7 is acting early during the virus attachment and recep-
tor binding stage, possibly by binding to HIV/EBOV-GP.
We further addressed the binding of compound 7 with HIV/
EBOV-GP directly by using a modification of the gel filtration
method of the compound-virus binding assay (13, 60). We used
a Microcon Ultracell YM3 centrifugal device (Millipore) that
is capable of separating small molecules from large macromol-
ecules. In the absence of virus, compound 7 will flow into the
lower chamber and will be detected in the flowthrough fraction
when analyzed by liquid chromatography-mass spectrometry.
In contrast, virus-bound compound 7 will not pass through a
Microcon YM3 centrifugal filter device upon centrifugation
and will not be collected in the flowthrough. As shown in Fig.
FIG. 1. Specificity of inhibition by compound 7. (A) Comparison of
antiviral activity and cell toxicity of compound 7. GFP-ZEBOV was
incubated with Vero E6 cells at an MOI of 5 for 1 h in the presence or
absence of compound 7 in a dose-dependent manner as described in
Materials and Methods. The blue diamonds represent anti-GFP-
EBOV activity, while the red squares represent cytotoxicity. (B) The
inhibitory effect of compound 7 on virus infectivity was investigated
using HIV/MARV-GP pseudotype and infectious cell culture-grown
MARV as described in Materials and Methods. Three independent
experiments were performed to determine the effect of compound 7 on
virus infectivity, and standard deviations are indicated. (C) Retroviral
pseudotypes of LASV (HIV/LASV-GP), LCMV (HIV/LCMV-GP),
and MACV (HIV/MACV-GP) were added to 293T cells, and influ-
enza H5 [HIV/HA(H5)] pseudotype virus was added to A549 cells,
in the presence of the indicated concentrations of compound 7.
Pseudotype infection was assessed after 72 h by luciferase reporter
assay. Three independent experiments were performed to deter-
mine the effect of compound 7 on virus infectivity, and standard
deviations are indicated.
3110 BASU ET AL. J. VIROL.
2C, only 30% of compound 7 (compared to untreated com-
pound 7) was observed in the flowthrough when it was prein-
cubated with HIV/EBOV-GP. When the experiment was re-
peated using HIV/VSV-G, 73% of compound 7 was observed
in the flowthrough. The results suggest that compound 7 ex-
hibits some affinity for both HIV/EBOV-GP and HIV/VSV-G
pseudotype virus. However, there is a higher degree of bind-
ing of compound 7 to HIV/EBOV-GP under similar exper-
imental conditions. Since HIV/EBOV-GP and HIV/VSV-G
have similar HIV backbones but different envelope glyco-
proteins at their surfaces, the results along with the time-of-
addition studies indirectly suggest that compound 7 is binding
Computational prediction of potential EBOV-GP binding
sites. Recently, Lee et al. (25) published a crystal structure
(3.4 Å) of EBOV-GP in its metastable trimeric, prefusion
conformation. Using this published crystal structure of
EBOV-GP and the Schro ¨dinger SiteMap program, we were
able to examine the EBOV-GP structure and identify potential
small-molecule binding sites on the basis of contiguous groups
of hydrophobic amino acids positioned in an orientation that
defined a hydrophobic pocket. For unbiased results, we set the
program parameters to cover the entire EBOV-GP structure
so that potential binding sites could be discovered.
The computational studies identified three potential binding
sites (S1 to S3) with site scores of ?0.9 (Fig. 3A). S1 and S3 are
hydrophilic in nature and seem unlikely binding sites for com-
pound 7, a small hydrophobic molecule which bears two hy-
drophobic groups (cLogP, ?5). Therefore, the hydrophobic
binding site S2, with a hydrophobic/hydrophilic ratio of 4.75,
appears to be the most likely compound 7 binding site. The S2
site is created by clamping of the GP1 base subdomain with the
GP2 internal fusion loop and the heptad repeat 1A helix (Fig.
3A and B). At site S2, amino acid residues Val66, Leu68,
Asn69, Leu70, Leu184, and Leu186 in the base and head sub-
domains of GP1 and residues Tyr517, Met548, Leu554, and
Leu558 on the GP2 internal fusion loop are predicted to be the
residues most likely involved in compound 7 binding to EBOV-
GP. Close to this hydrophobic pocket, amino acids Arg64,
Glu523, and Thr520 may act as potential hydrogen bond do-
nors and acceptors for ligand binding (Fig. 3B). An examina-
tion of the residues in the GP1 sequence suggests that (i) these
residues are located in and around the conserved charged and
hydrophobic residues of GP1 (Fig. 3C) that are important for
GP1 structure, folding, and receptor binding (29, 30) and (ii)
except for Lys190 and Lys191, all other amino acid residues
potentially involved in binding of compound 7 to EBOV-GP
are either nonpolar or aromatic and are involved in maintain-
ing the structural integrity of GP1 for receptor binding or
fusion. Therefore, single-amino-acid mutations of these non-
polar or aromatic residues would be expected to generate
escape mutants. In summary, the computational studies using
the Schro ¨dinger SiteMap program identified a putative binding
site on the basis of contiguous groups of hydrophobic amino
acids positioned in an orientation that defines a hydrophobic
Confirmation of binding of compound 7 in the hydrophobic
S2 site by mutational analysis. We performed mutational stud-
ies to confirm and validate the computational results. Work
with EBOV requires a BSL-4 facility, making it challenging to
generate EBOV escape mutants that are resistant to com-
pound 7. Moreover a selectivity index of less than 10 also
suggests that we may not be able to generate mutants without
considerable cytotoxicity. Therefore, to support the S2 site
binding hypothesis generated by the computational studies, we
examined HIV/EBOV-GP mutants carrying single-amino-acid
mutations in the S2 region for inhibition of infectivity by com-
pound 7. Previous studies by Manicassamy et al. (29) have
shown that single-amino-acid alterations at positions R64K,
N69A, L70A, D78A, K84A, R172A, L184A, I185A, L186A,
K190A/K191A, and Y225A cause no defect in viral entry. The
same study also indicated that mutations at amino acid posi-
tions Val66, and Leu68 in EBOV-GP1 generate noninfectious
virus. Mutants carrying K84A, R172A, and Y225A were used
as controls in the study, since these residues are unlikely to be
involved in binding with compound 7 but are conserved among
filoviruses. As shown in Fig. 4A, compound 7, even at concen-
trations of as high as 50 ?M, displayed little or no inhibition
of HIV/EBOV-GP N69A and L70A mutants. The HIV/
EBOV-GP L184A, I185A, L186A, and K190A/K191A mutants
TABLE 3. Inhibitory activity of compound 7 against different viruses
FamilyVirus Assay typeIC50(?M)a
Arenavirus Tacaribe virusNeutral red
?3232 Vero 76
Biodefense-related virusRift Valley fever virus
DNA virusCowpox virus
Respiratory virus Influenza A (H1N1) virus California/07/2009
Influenza (H3N2) virus Brisbane/10/2007
SARS virus Urbani
aIC50values were generated for cell culture-grown viruses from the linear portion of the dose response curve.
bCC50values were determined from linear portions of the dose-response curve.
VOL. 85, 2011EBOLA VIRUS ENTRY INHIBITOR 3111
were also less susceptible to inhibition, displaying ?30% inhi-
bition at a 25 ?M concentration of compound 7 (Fig. 4B). In
contrast, compound 7 inhibited the infectivity of the R64K,
D78A, K84A, R172A, and Y225A mutants by ?75% at a
concentration of 25 ?M (Fig. 4A and B). Together, these
results suggest that compound 7 interacts with the amino acid
residues Asn69, Leu70, Leu184, Ile185, Leu186, Lys190, and
Lys191 and therefore likely binds to EBOV-GP at or near the
S2 hydrophobic site.
We further investigated whether compound 7 binds to the
RBD of EBOV-GP and inhibits its binding to host receptors
and cofactors. We used a previously described fusion pro-
tein that contains EBOV-GP1 RBD (amino acid residues 54
to 201) fused with the human IgG Fc region (21). As a
negative control, we used the SARS virus spike protein
(S1-hIgG), which was previously shown not to bind to 293T
and Vero cells due to a very low level of the SARS virus
receptor (20). We found that compound 7 had no effect on
the binding of the EBOV RBD domain to 293T or Vero
cells (data not shown). Therefore, we hypothesize that com-
pound 7 binds to the S2 hydrophobic pocket and not to the
RBD of EBOV-GP.
Preliminary SAR analysis. We performed a preliminary
structure-activity relationship (SAR) analysis of the benzo-
diazepine structure without altering the benzodiazepine
backbone. The results are shown in Table 4 along with those
for the screening hit, compound 7. The initial SAR study
was designed to explore the effect of electronic properties
and to examine the effect of the sizes of substituents of the
benzodiazepine backbone. As shown in Table 4, a dichloro
substitution on the benzene ring of the benzodiazepine core
(compound 12) increased activity against the anti-HIV/
EBOV-GP pseudotype virus 3-fold. In contrast, a bulky
fused benzo ring (compound 9) or dimethyl substitution
(compound 10) decreased the potency. Compound 12 was
also found to inhibit GFP-ZEBOV in a dose-dependent
manner, providing over 90% inhibition at a concentration (5
?M) that displayed no cytotoxicity (Fig. 5B). Overall, small
substitutions on the diazepine ring of the benzodiazepine
are tolerated, while addition of a second aromatic or het-
eroaromatic group on the diazepine ring is detrimental to
the antiviral activity (Fig. 5A). These preliminary SAR re-
sults are consistent with a specific target-inhibitor interac-
tion in which the bulky aromatic substitutions on the
azepine ring introduce some size constraints on the inhibitor
and prevent it from occupying the binding pocket. Because
compound 12 exhibits increased antiviral potency and a
higher selectivity index than compound 7 (Table 4), it will be
the starting point for optimization of the benzodiazepine
series for identification of lead compounds using medicinal
As shown in Fig. 5C, compound 12 also failed to inhibit the
same HIV/EBOV-GP mutants, carrying N69A, L70A, L184A,
I185A, L186A, and K190A/K191A, that were resistant to com-
pound 7, suggesting that compound 12 localizes in the same
hydrophobic pocket (S2) in EBOV-GP as compound 7. Taken
together, these results confirm our earlier findings that these
novel benzodiazepine filovirus inhibitors bind to the S2 hydro-
phobic pocket and interact with amino acid residues Asn69,
Leu70, Leu184, Ile185, Leu186, Lys190, and Lys191.
FIG. 2. Compound 7 binds to EBOV-GP and inhibits virus infection.
(A) Schematic diagram of a “single-cycle time-of-addition experiment”
with HIV/EBOV-GP to determine the stage of virus entry blocked by
compound 7. This experiment was designed to characterize the mecha-
nism of action of the antiviral compounds. (B) A single-cycle time-of-
addition experiment was done with HIV/EBOV-GP to determine the
stage of EBOV entry blocked by compound 7. 293T cells were infected
with 100 ?l of p24-normalized HIV/EBOV-GP. Compound 7 was added
and left for 1 h before infection (?1 h), for 1 h during adsorption (0 h),
and for 1 h after infection (?1 h). Infected monolayers were washed with
PBS and incubated for 72 h. Inhibition of HIV/EBOV-GP pseudotype
infection was detected as a reduced luciferase signal. Error bars indicate
standard deviations. (C) A Microcon Ultracell YM3 centrifugal device
(Millipore) was used to study the binding of compound 7 to HIV/
EBOV-GP and HIV/VSV-G pseudotype viruses. Pseudotype viruses (or
a no-virus buffer control) were incubated with 25 ?M compound 7 at
room temperature for 60 min, and the virus-compound mixture was then
loaded onto the Microcon Ultracell YM3 centrifugal device. After cen-
trifugation, the flowthrough was analyzed for unbound compound by
high-pressure liquid chromatography. The percentage of bound com-
pound was calculated based on the peak area compared to that of a
no-virus control. Error bars indicate standard deviations.
3112 BASU ET AL.J. VIROL.
In recent years, entry inhibitors have emerged as a new class
of antiviral drugs, with the HIV fusion-blocking peptide enfu-
virtide being the first available fusion inhibitor applied in clin-
ical treatment of a viral infection in humans (57). Several
small-molecule entry inhibitors against other important vi-
ruses, such as measles virus (35, 36), influenza virus (43), re-
spiratory syncytial virus (12), and arenaviruses (22, 24), are
currently in development. EBOV-GP mediates binding of
EBOV with its cell surface receptor(s)/coreceptor(s) and sub-
sequent entry, involving endocytosis of virus and fusion be-
tween viral and host cell membranes (44). Therefore, we hy-
pothesized that blocking of EBOV-GP-mediated viral entry
will lead to inhibition of infection. Moreover, the aggressive
nature of EBOV infections, in particular the rapid and over-
whelming viral burdens in infected patients and EBOV-GP-
related cell cytotoxicity (44, 45), justify preferentially targeting
the entry process over subsequent downstream stages of viral
In this study, we used HIV/EBOV-GP pseudotype virus as a
surrogate model to screen libraries of 52,500 small-molecule
compounds for inhibitors based on the following rationale.
Since viral entry is determined solely by interaction of the virus
envelope proteins with cell receptors (32), the replication-in-
competent HIV/EBOV-GP mimics the EBOV entry process
(29, 30) and can be used in HTS of compound libraries in a
BSL-2 laboratory. The HTS “hits” include at least four possible
categories of inhibitors: (i) inhibitors of HIV/EBOV-GP entry,
(ii) inhibitors of HIV replication, (iii) inhibitors of luciferase
enzyme activity, and (iv) cytotoxic compounds. Therefore, to
FIG. 4. Effect of compound 7 on the infectivity of HIV/EBOV-GP
mutants. 293T cells were infected with mutant or wild-type HIV/
EBOV-GP pseudotype virus in the presence of increasing concentra-
tions of compound 7. Inhibition of HIV/EBOV-GP pseudotype infec-
tion was detected as a reduced luciferase signal. Error bars indicate
standard deviations. (A) Effect of compound 7 on the infectivities of
R64K, N69A, L70A, D78A, and K84A HIV/EBOV-GP mutants.
(B) Effect of compound 7 on the infectivities of R172A, L184A, I185A,
L186A, K190A/K191A, and Y225A HIV/EBOV-GP mutants.
FIG. 3. Computational studies locate a hydrophobic pocket at or near the GP1-GP2 interface in the GP prefusion crystal structure. (A) The
three proposed binding sites S1 to S3, calculated with the Schro ¨dinger Sitemap program, are shown in colored grids. Hydrophobic, ligand donor,
and ligand acceptor maps are shown in yellow, blue, and red, respectively. EBOV-GP1 is shown in yellow and red ribbons, and GP2 is displayed
in blue ribbons. (B) Magnified view of the S2 binding site circled in panel A. The putative binding site S2 was identified using Schro ¨dinger SiteMap.
Selected residues which are proposed to make contacts within the binding site are displayed as stick models. (C) The amino acid residues in the
EBOV-GP1 sequence that are important for viral entry are shown. The charged residues are underlined, while hydrophobic residues are shown
as gray shaded regions. The arrows indicate the positions of mutations in EBOV-GP1 of the HIV/EBOV-GP mutant pseudotype viruses utilized
in this study.
VOL. 85, 2011 EBOLA VIRUS ENTRY INHIBITOR3113
TABLE 4. IC90values for the benzodiazepine analogs
7 12.175 6.25.63
Continued on following page
3114 BASU ET AL. J. VIROL.
eliminate off-target hits such as the inhibitors of HIV replica-
tion and/or luciferase enzyme activity, we counterscreened the
HTS hits with HIV/VSV-G pseudotype virus. HIV/VSV-G has
the same HIV backbone as HIV/EBOV-GP but expresses
VSV-G, a member of a different class of viral fusion-active
protein (24, 40, 41), on its surface. The counterscreen with
HIV/VSV-G proved to be particularly useful since it elimi-
nated the off-target hits that compromise 95% of the total HTS
hits (Table 1). It is possible that the counterscreen may have
eliminated some HIV/EBOV-GP entry inhibitors, since the
cell receptors of both EBOV-GP and VSV-G are not known
and the wild-type EBOV and VSV infect a wide range of cells.
Nevertheless, the counterscreen is an important step to elim-
inate undesired off-target HTS hits, and it allows us to focus on
the most specific hits. Moreover, both EBOV and VSV enter
cells by endocytosis followed by pH-dependent membrane fu-
sion in endosomes (11, 31, 46). Therefore, this counterscreen
also eliminated compounds that modulate membrane traffick-
ing or endosomal pH and identified compounds that bind to
either EBOV-GP or specific components of the EBOV entry
pathway that are not shared with VSV.
We identified eight novel inhibitors of EBOV entry. The
IC90values for six of the eight compounds against GFP-ZE-
BOV were within ?2- to 3-fold of the values for HIV/EBOV-
GP, while IC90values for compounds 3 and 4 against GFP-
ZEBOV are significantly higher (?20-fold difference) (Table
2). At this time we do not know the reasons for the differences
in the IC90values between HIV/EBOV-GP and GFP-ZEBOV,
but they may be due to differences in the (i) virus morphology
(EBOV is cylindrical, while HIV/EBOV-GP is spherical), (ii)
aHIV/EBOV-GP was generated by transfection of 293T cells with HIV-l expression vector (pNL4.3.Luc.R–E–) and with EBOV-GP. IC90values were determined
using reordered compounds as different batches from the original vendors or freshly synthesized compounds.
b293T cells were treated with compound alone. CC50values were determined from linear portions of the dose-response curve.
cSI, selectivity index (CC50/IC90).
dcLogP values were calculated with ChemDraw v12.0.
eND, not done.
VOL. 85, 2011 EBOLA VIRUS ENTRY INHIBITOR 3115
GP density at the cell surface, (iii) GP modification (e.g.,
producer cell type-specific glycosylation patterns), and (iv) tar-
get cells (293T versus VeroE6). Compound 7 was selected for
further chemical optimization and mechanism studies because
(i) the benzodiazepine structure is a suitable “drug-like” start-
ing point for medicinal chemistry, (ii) the compound exhibited
good potency in both the HIV/EBOV-GP and cell culture
grown-infectious EBOV assay, and (iii) its selectivity index (SI
of ?5) indicates that the compound exhibits significant selec-
tivity for antiviral activity versus cytotoxicity (Fig. 1A). The
preliminary SAR evaluation so far has identified several addi-
tional benzodiazepine analogs that exhibit anti-EBOV activity,
including one more potent (3 times more) analog (compound
12) that also displays a selectivity index of greater than 10.
Although the sample size for the SAR analysis is small, the
increased activity of compound 12 suggests that more potent
inhibitors can be designed using the benzodiazepine backbone.
In addition, with a selectivity index of ?10 and higher antiviral
potency, compound 12 will be the starting point for optimiza-
tion of the benzodiazepine series to identify a lead inhibitor
using medicinal chemistry. The systematic synthesis of a larger
library of compounds (see the supplemental material), with the
substituents carefully controlled, will be necessary to identify
informative SAR trends, and this will be the first priority of the
synthetic plan for further development of the benzodiazepine
series to obtain lead compounds.
Virus entry is a multistep process, and target identification
for compound 7 may be challenging. Three approaches have
been taken so far to determine the antiviral target. First, we
examined the antiviral spectrum of compound 7 against vari-
ous filoviruses and nonfiloviruses (Table 3). It is reasonable to
hypothesize that a compound capable of inhibiting multiple
viruses from different families most likely inhibits or blocks
either host receptors or other host factors involved in virus
entry. However, compound 7 inhibited primarily only EBOV
and MARV, the two members of the filovirus family. The
critical amino acid residues that are important for virus entry
are conserved in both EBOV-GP and MARV-GP (29, 30).
Compound 7 exhibited very weak activity against other viruses
bearing similar type 1 envelope proteins but, in general, exhib-
ited no activity against other RNA and DNA viruses. The
results suggest that compound 7 is not inhibiting host factors
such as the endosomal pathways involved in infection of a
number of enveloped viruses.
Second, we performed “time-of-addition” studies, which in-
dicated that compound 7 blocks at an early stage of virus entry
into cells, possibly by binding to EBOV-GP. Preincubation of
HIV/EBOV-GP with compound 7 also blocked its infection of
293T cells (data not shown), further suggesting that compound
7 binds to HIV/EBOV-GP. Similarly, the differential binding
of HIV/EBOV-GP and HIV/VSV-G by compound 7 in the
virus-compound binding assay suggests that compound 7 binds
EBOV-GP, since both the viruses have the same HIV core.
Taken together, these studies indicate that compound 7 acts
early in the infection process, probably by binding to EBOV-
GP. From this experiment, it is not possible to stoichiometri-
cally measure the binding of EBOV-GP and compound 7.
Since the pseudotype virus randomly acquires EBOV-GP from
the surfaces of infected cells and since it is competent for only
one round of replication, it has not been feasible to determine
precisely the number of mature trimeric EBOV-GP molecules
on the viral surface, the number of viral particles carrying
the EBOV-GP, or the stoichiometry of the compound
7–EBOV-GP interaction. Nuclear magnetic resonance (NMR)
studies are planned to better understand the interactions of
compound 7 with EBOV-GP.
EBOV-GP is heavily glycosylated by both N-linked and O-
linked glycans. As a result, only a few sites are left exposed and
accessible for binding interactions. These sites include (i) a
region at the base of the chalice where GP1 meets GP2, (ii)
short linear stretches of polypeptide between glycans in the
FIG. 5. Analysis of analogs of compound 7. (A) Summary of pre-
liminary SAR results for benzodiazepine analogs of HIV/EBOV-GP
compound 7. (B) Comparison of antiviral activity and cell toxicity of
compound 12, as described in Materials and Methods. The blue dia-
monds represent anti-GFP-ZEBOV activity, while the red squares
represent cytotoxicity. (C) Effect of compound 12 on the infectivities of
N69A, L70A, L184A, I185A, L186A, and K190A/K191A HIV/
EBOV-GP mutants. 293 T cells were infected with HIV/EBOV-GP
pseudotype virus in the presence of increasing concentrations of com-
pound 12. Inhibition of HIV/EBOV-GP infection was detected as a
reduced luciferase signal. Error bars indicate standard deviations.
3116 BASU ET AL.J. VIROL.
mucin-like domain, and (iii) the HR2 region. Studies using an
EBOV-GP1 RBD peptide (amino acids [aa] 54 to 201) showed
that compound 7 has no effect on the binding of EBOV-GP to
293T and Vero cell lines. In addition, compound 7 inhibited
both wt HIV/EBOV-GP and HIV/EBOV-GP?mucin with sim-
ilar potencies (data not shown). In HIV/EBOV-GP?mucin,
the mucin domain is removed to expose the EBOV-GP1 RBD.
Taken together, these results indicate that the primary mech-
anism of action of compound 7 is not direct blocking of virus
attachment to the cells by binding the RBD of GP1. Instead,
we hypothesize that compound 7 binds to a region of the
EBOV-GP at the base of the chalice where GP1 meets GP2,
since this region is exposed in both EBOV-GP and EBOV-
Supporting evidence for this hypothesis comes from our
computational studies using the recently published crystal
structure (3.4 Å) of EBOV-GP in its metastable trimeric, pre-
fusion conformation (25), as confirmed by our mutational stud-
ies. The computational studies using the Schro ¨dinger SiteMap
program identified a putative hydrophobic binding pocket (S2)
for small-molecule ligand binding at the junction of the GP1
base subdomain with the GP2 internal fusion loop and the
heptad repeat 1A helix (HR1A). Amino acid residues Val66,
Leu68, Asn69, Leu70, Leu184, and Leu186 from the base and
head subdomains of GP1 together with residues Tyr517,
Met548, Leu554, and Leu558 on the GP2 internal fusion loop
in the hydrophobic S2 binding site appear to be involved in
interacting with bound ligands, according to the model. In-
deed, a significant reduction in the potency of compound 7
against HIV/EBOV-GP mutants N69A, L70A, L184A,
I185A, L186A, and K190A/K191A further implicates the S2
hydrophobic pocket of EBOV-GP as a potential binding site
for small-molecule ligands such as the benzodiazepine com-
The EBOV-GP1 base subdomain contains four discontinu-
ous sections (residues 33 to 69, 95 to 104, 158 to 167, and 176
to 189) which form two mixed beta-sheets, with strands ?3 and
?13 shared between the two beta-sheets. The head subdomain
is composed of residues from four discontinuous segments, i.e.,
residues 70 to 94, 105 to 157, 168 to 175, and 214 to 226, and
forms a four-stranded, mixed beta-sheet supported by an al-
pha-helix and a smaller, two-stranded antiparallel beta-sheet
(25, 26). As shown from the mutation studies, the HIV/
EBOV-GP N69A and L70A mutants are not inhibited by com-
pound 7, suggesting that the amino acid residues at the junc-
tion between the head and base subdomains of GP1 are
important for binding. Compound 7 weakly inhibited HIV/
LASV-GP, HIV/LMCV-GP, HIV/MACV-GP, and HIV/
HA(H5) at higher concentrations. These pseudotype viruses
also contain type 1 membrane proteins similar to EBOV-GP.
The type 1 membrane protein carries a similar cavity formed by
the clamping of the N-terminal membrane-proximal base of
the receptor binding subunit over the internal fusion loop
through hydrophobic interactions, including an interchain di-
sulfide bond (37, 38). Therefore, our results suggest that the
size and the conformation of the S2 hydrophobic pocket of
EBOV-GP are important and that compound 7 selectively
binds in this pocket to prevent viral activity. This hypothesis is
supported by limited preliminary SAR analysis of compound 7,
which showed that bulky aromatic substitutions on the
diazepine ring introduce some size constraints on the inhib-
itor and reduce its antiviral activity (Fig. 5A; Table 4). The lack
of activity for compounds with a bulky substituent(s) implies a
binding site with well-defined boundaries.
A limited number of small-molecule inhibitors of EBOV
have been discovered (7, 8). However, none of these inhibitors
are used in clinical settings, and none of them resemble com-
pound 7 structurally. The existing small-molecule inhibitors of
EBOV infections can be characterized by three general modes
of action: (i) impairment of viral replication, (ii) stimulation of
innate antiviral mechanisms, and (iii) prevention of virus entry
into the cells.
The carbocyclic adenosine analog 3-deazaadenosine (C-
c3Ado), inhibits cellular S-adenosylhomocysteine hydrolase
and inhibits the replication of EBOV Zaire in vitro, with an
IC50of 30 ?M (7, 9). The activity of this antiviral agent has
been attributed to diminished methylation of the 5? cap of viral
mRNA by (guanine 7) methyltransferase, which impairs the
translation of viral transcripts. Administration of C-c3Ado to
EBOV-infected mice has also been found to dramatically in-
crease production of alpha interferon (IFN-?), which may
serve to counteract the virus suppression of the innate antiviral
response. Unfortunately, C-c3Ado failed to promote IFN-?
production in Ebola virus-infected monkeys (7, 9). More re-
cently, other small-molecule EBOV inhibitors, such as FGI-
103, FGI-104, and FGI-106, were discovered (1, 50, 61) by
HTS screening. These inhibitors were found to be potent and
to provide protection against EBOV and Marburg virus in
vitro, and in vivo. While FGI-103 inhibits infection by an un-
known mechanism, FGI-104 targets EBOV vp40-tgs101-medi-
ated budding. In contrast, FGI-106 displays potent and broad-
spectrum inhibition oflethal
pathogens, including Ebola, Rift Valley and dengue fever vi-
ruses, in cell-based assays, suggesting that it interferes with a
common pathway utilized by different viruses (1).
The glycodendritic structure BH30sucMan (23, 58), which
contains 32 individual ?-mannose units linked to the hyper-
branched dendrimer BH30 through succinyl spacers, has re-
cently been shown to block the interaction between dendritic
cell-specific intercellular adhesion molecule 3-grabbing nonin-
tegrin (DC-SIGN) (58) and EBOV-GP. However, this com-
pound is not specific for EBOV infection. Endosomal pro-
teases CatB and CatL mediate viral entry by carrying out
proteolysis of the EBOV-GP1 subunit (11). Recently, the CatL
inhibitor tetrahydroquinoline oxocarbazate was reported to in-
hibit EBOV infection at nanomolar concentrations (47). It also
inhibits other viruses that use CatL for entry. Unfortunately,
given the demonstrated hypersensitivity of EBOV-GP1 to di-
gestion by other proteases (17, 46), such as thermolysin (17),
the clinical prospects for antiviral agents that solely target
CatB and CatL are not encouraging. Compound 7 and its
analogs were found to have no activity against CatL in in vitro
enzymatic assays (data not shown). Compound 7 and its ana-
logs differ from these previously reported small-molecule in-
hibitors by the specificity exhibited for filoviruses and the ap-
parent mechanism of action. Unlike the other entry inhibitors,
the benzodiazopenes may bind directly to EBOV-GP within a
hydrophobic pocket at the EBOV GP1-GP2 interface. More-
over, blocking of propagation of EBOV at an early stage will
VOL. 85, 2011EBOLA VIRUS ENTRY INHIBITOR 3117
minimize the chance for the virus to evolve and acquire drug
We conclude that compound 7 acts at an early stage of viral
entry, apparently by binding to a hydrophobic pocket (S2) in
the prefusion conformation of EBOV-GP and preventing in-
fection by an unknown mechanism. An analogy can be made
with several different classes of small-molecule HIV entry in-
hibitors, including maraviroc, that are thought to bind within a
pocket created by four transmembrane domains of CCR5, an
important HIV coreceptor (19). Furthermore, computational
and mutational studies suggest that the S2 hydrophobic bind-
ing pocket is a well-defined small-molecule binding site, which
may serve as a viable target for future antifilovirus drug dis-
We thank Krishna Kota, Dutch Boltz, Julie Tran, and Xiaoli Chi
from USAMRIID for assays using BSL-4 containment and procedures.
This research was supported by DHHS/NIH grants 1R43AI071450-
01, 1R01AI089590-01, and 5U01AI077767-02 from the National Insti-
tutes of Health.
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syncytial virus replication: effects of oligonucleotide structure modifications
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