Design of a small-molecule entry inhibitor with activity against primary measles virus strains

Article abstract of DOI:10.1128/AAC.49.9.3755-3761.2005

The incidence of measles virus (MV) infection has been significantly reduced in many nations through extensive vaccination; however, the virus still causes significant morbidity and mortality in developing countries. Measles outbreaks also occur in some d

Full text of DOI:10.1128/AAC.49.9.3755-3761.2005

Design of a Small-Molecule Entry Inhibitor
with Activity against Primary Measles Virus
Strains
Richard K. Plemper, Joshua Doyle, Aiming Sun, Andrew
Prussia, Li-Ting Cheng, Paul A. Rota, Dennis C. Liotta,
James P. Snyder and Richard W. Compans
Antimicrob. Agents Chemother. 2005, 49(9):3755. DOI:
10.1128/AAC.49.9.3755-3761.2005.
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ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Sept. 2005, p. 3755–3761
0066-4804/05/$08.00ϩ0 doi:10.1128/AAC.49.9.3755–3761.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 49, No. 9
Design of a Small-Molecule Entry Inhibitor with Activity against
Primary Measles Virus Strains
Richard K. Plemper,1* Joshua Doyle,1 Aiming Sun,2 Andrew Prussia,2 Li-Ting Cheng,1
Paul A. Rota,3 Dennis C. Liotta,2 James P. Snyder,2 and Richard W. Compans1
Department of Microbiology and Immunology, 3086 Rollins Research Center, 1510 Clifton Road, Emory University School
of Medicine, Atlanta, Georgia 30322,1 Department of Chemistry, 1515 Pierce Drive, Emory University,
Atlanta, Georgia 30322,2 and Measles Virus Section, National Center for Infectious Diseases,
Centers for Disease Control and Prevention, 1600 Clifton Road, MS-C22,
Atlanta, Georgia 303333
Received 20 May 2005/Returned for modification 15 June 2005/Accepted 5 July 2005
The incidence of measles virus (MV) infection has been significantly reduced in many nations through
extensive vaccination; however, the virus still causes significant morbidity and mortality in developing coun-
tries. Measles outbreaks also occur in some developed countries that have failed to maintain high vaccine
coverage rates. While vaccination is essential in preventing the spread of measles, case management would
greatly benefit from the use of therapeutic agents to lower morbidity. Thus, the development of new therapeutic
strategies is desirable. We previously reported the generation of a panel of small-molecule MV entry inhibitors.
Here we show that our initial lead compound, although providing proof of concept for our approach, has a
short half-life (<16 h) under physiological conditions. In order to combine potent antiviral activity with
increased compound stability, a targeted library of candidate molecules designed on the structural basis of the
first lead has been synthesized and tested against MV. We have identified an improved lead with low toxicity
and high stability (half-life ϾϾ 16 h) that prevents viral entry and hence infection. This compound shows high
MV specificity and strong activity (50% inhibitory concentration ؍ 0.6 to 3.0 ␮M, depending on the MV
genotype) against a panel of wild-type MV strains representative of viruses that are currently endemic in the
field.
Paramyxoviruses are nonsegmented negative-stranded RNA
viruses, most of which are highly contagious airborne patho-
gens that spread via the respiratory route. Members of this
viral family constitute major human and animal pathogens
such as measles virus (MV), human parainfluenza viruses
(HPIV), mumps virus, rinderpest virus, and Newcastle disease
virus (12). Despite the existence of an effective live-attenuated
vaccine (6), MV remains a serious threat to human health
globally, accounting for approximately 0.5 million deaths an-
nually (1). While most of these cases occur in developing
countries with limited access to vaccination, measles outbreaks
still occur in some developed countries that have failed to
maintain high vaccine coverage rates (4, 26). Recent out-
breaks, in particular in the United Kingdom, have been attrib-
uted to declining herd immunity as a result of reduced vacci-
nation coverage due to parental concerns about vaccination
safety (8). Furthermore, vaccine-induced immunity is less ro-
bust than naturally acquired protection, which may, in fully
vaccinated populations, result in a progressive loss of immunity
in adults due to the absence of natural boosting through cir-
culating virus (15, 16, 27). Taken together, these facts make
desirable the development of novel therapeutics that could be
produced cost-effectively and that could be used for the rapid
control of local outbreaks and improved case management to
limit severe outcomes of infection.
MV infects target cells through pH-independent fusion ei-
ther of the viral envelope with the plasma membrane of target
cells or of the plasma membrane of an infected cell with the
plasma membrane of neighboring uninfected cells (11, 12).
This is initiated by interaction of the hemagglutinin (H) enve-
lope glycoprotein with its cellular receptor, either the regulator
of complement activation CD46 or signaling lymphocyte acti-
vation molecule (SLAM/CD150w). While the MV vaccine
strains of the Edmonston lineage efficiently use CD46 as their
cellular receptor (3, 17), most wild-type strains of MV are
dependent on SLAM for efficient entry (19, 32, 33). Receptor
binding is thought to trigger H to activate the fusion (F) en-
velope glycoprotein, which through a series of conformational
changes mediates membrane merger, resulting in release of the
viral genome into the target cell (11, 12).
Interfering with virus entry is a novel and attractive thera-
peutic strategy to control virus infection and spread, and proof
of principle for the clinical benefit of this approach has most
notably come from the safe and efficacious peptidic human
immunodeficiency virus (HIV) inhibitor enfuvirtide (T-20)
(31). Paramyxoviruses against which peptides possess consid-
erable in vitro potency include HPIV type 2 (HPIV-2) and
HPIV-3 (13, 37), MV (13), respiratory syncytial virus (13),
Sendai virus (28), and Newcastle disease virus (38). While
confirming the therapeutic benefit of entry inhibitors for the
treatment of viral infections, T-20 has highlighted potential
obstacles that complicate large-scale production of peptide-
* Corresponding author. Mailing address: Department of Microbi-
ology and Immunology, 3086 Rollins Research Center, 1510 Clifton
Road, Emory University School of Medicine, Atlanta, GA 30322.
Phone: (404) 727-3228. Fax: (404) 727-5280. E-mail: rplempe@emory
.edu.
3755

3756
PLEMPER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
1H nuclear magnetic resonance (NMR) (600 MHz, CDCl3) data are as follows:
␦ 3.79 (2H, s), 7.33-7.41(5H, m), 7.62 (1H, d, J ϭ 8.4 Hz), 7.88 (1H, dd, J ϭ 2.4
Hz, 8.4 Hz), 9.55 (1H, d, J ϭ 1.8 Hz), 11.08 (1H, s). 13C NMR (400 MHz, CDCl3,
ppm) data are as follows: 170.93, 141.71, 130.25, 129.71, 128.77, 128.29, 117.74,
117.14, 46.52. Combustion was performed based on the elemental composition
based antivirals. Large heptad repeat-derived peptides such as
T-20 are often difficult to solubilize and purify, making man-
ufacture highly costly. Furthermore, such peptides usually
show poor absorption and bioavailability from the gastrointes-
tinal tract, necessitating delivery through injection, and virus-
derived peptides have the potential to be immunogenic in vivo
and may induce adverse events in some cases.
Considering these obstacles, we aimed to explore the inhib-
itory potential of nonpeptidic small molecules against MV
entry. Multiple routes of administration are conceivable for
these drug-like molecules, and highly cost-effective production
strategies can be easily achieved. Additional conceptual sup-
port for this approach comes from the previous identification
of small molecules that interfere with respiratory syncytial vi-
rus entry in vitro (2) and in vivo (2, 35).
In previous work (21, 25), we have reported the structure-
guided development of an MV entry inhibitor, N-(5-amino-2-
hydroxy-phenyl)-2-phenyl-acetamide (AM-4), with a 50% in-
hibitory concentration (IC50) of 260 nM against the MV
vaccine strain MV-Edmonston (MV-Edm). In the present
study, we further characterize this compound, describe the
development of a second improved therapeutic lead, and re-
port the testing of this lead against a representative panel of
wild-type MV isolates. Based on its in vitro inhibitory activity
against these strains, this compound warrants further testing
under in vivo conditions.
C
15H13N3O4. The calculated values based on the formula areas are as follows: C,
60.20; H, 4.38; N, 14.04. Values found experimentally are as follows: C, 60.29; H,
4.50; N, 13.27.
All compounds were dissolved in dimethyl sulfoxide (DMSO). The highest
final DMSO concentration used in experiments was 0.2% (vol/vol), at which no
solvent-related effect on cell viability or the degree of membrane fusion could be
detected. As a control, cells treated with DMSO at the highest concentration
used in each particular assay were routinely included.
Compound stability assays. Compound stability was evaluated chemically by
monitoring the NMR spectrum in CDCl3 under atmospheric oxygen every sev-
eral hours over a 48-hour period. While OX-1 and AS-48 retained both solution
clarity and spectrum integrity, AM-4 slowly darkened with some precipitation
and the spectrum gradually showed the presence of impurities. Stability under
physiological conditions was assessed by preincubation of different compound
concentrations in OPTIMEM at 37°C for different time periods, followed by
mixing with MV-Edm corresponding to an MOI of 0.1 PFU/cell and transfer to
5 ϫ 105 target cells in a six-well plate format. Yields of cell-associated viral
particles were determined 36 h postinfection by TCID50 titration.
Compound screen, cytotoxicity, and dose-response curves. To screen candi-
date compounds, 7,500 Vero-CD150w cells per well were infected in four rep-
licates per compound concentration in a 96-well plate format with wild-type
strain MVi/Kansas. USA/43.00 (MV-KS) at an MOI of 0.1 PFU/cell in the
presence of compound ranging from 75 ␮M to 4.6875 ␮M in twofold dilutions.
Subsequent to incubation at 37°C for 96 h, virus-induced cytopathicity was
measured using a cell proliferation assay (Promega) and results were calculated
according to the formula % proliferation ϭ [(experimental Ϫ background)/
(maximum Ϫ background)] ϫ 100. For comparison, the first-hit compound OX-1
was included in all assays.
A standard nonradioactive cytotoxicity assay (Promega) was used to determine
the cytotoxicity of the compounds. In a 96-well plate format, 12,000 cells per well
were incubated in four replicates per concentration tested in the presence of
compound at 37°C for 24 h. Reduction of MTT [3-(4,5-dimethylthiazol-2-yl]-5-
(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] as an indicator for
cell viability was then measured. Values were calculated according to the formula
provided by the manufacturer: % cytotoxicity ϭ [(experimental Ϫ background)/
(maximum Ϫ background)] ϫ 100. Due to the extreme fusogenicity of MV, in the
absence of fusion inhibition essentially all cells of a monolayer were recruited
into a single large syncytium at approximately 40 h postinfection in the conditions
used in the study. Because of loss of mechanical stability, these giant cells detach
from the plate and cease to be metabolically active. All infected cells were
routinely assessed microscopically prior to each assay, and microscopic observa-
tions were consistent with the quantitative results obtained.
MATERIALS AND METHODS
Cell culture, transfection, and production of MV stocks. All cell lines were
maintained at 37°C and 5% CO2 in Dulbecco’s modified Eagle’s medium sup-
plemented with 10% fetal bovine serum, penicillin, and streptomycin. Vero
(African green monkey kidney epithelial) cells (ATCC CCL-81) are non-contact-
inhibited primate tumor cells. Vero-CD150w cells were derived from Vero cells
by stably transfecting them with a human CD150w-expressing plasmid. They
were incubated in the additional presence of G-418 (Geneticin) at a concentra-
tion of 0.35 mg/ml to maintain selective pressure. Lipofectamine 2000 (Invitro-
gen) was used for transient-transfection experiments according to the manufac-
turer’s instruction. To prepare virus stocks, cells were infected at a multiplicity of
infection (MOI) of 0.01 PFU/cell and incubated at 37°C. Cells were scraped in
OPTIMEM (Invitrogen), and virus was released by two freeze-thaw cycles. MV-
Edm and HPIV-2 stocks were grown and titered by 50% tissue culture infective
dose (TCID50) determination on Vero cells according to the Spearman-Karber
method, using the formula log10 (TCID50/ml) ϭ L ϩ d (s –0.5) ϩ log10 (1/v) as
previously described (23), where L is the negative log10 of the most concentrated
virus dilution tested at which all wells are positive, d is the log10 of the dilution
factor, s is the sum of individual proportions (pi; each pi is the calculated
proportion of an individual dilution, and v is the volume of inoculum [ml/well]).
All wild-type MV strains were grown and titered on Vero-CD150w cells using the
same method.
To generate dose-response curves, 4 ϫ 105 Vero-CD150w cells per well were
infected in a six-well plate format with wild-type MV strains at an MOI of 0.1
PFU/cell in the presence of AS-48 in twofold dilutions (75 ␮M highest) and
incubated in the presence of compound at 37°C. Thirty-six hours postinfection,
cells were scraped in OPTIMEM, cell-associated virus was released by two
freeze/thaw cycles, and virus titers in cleared cell lysates (subsequent to centrif-
ugation at 5,000 ϫ g, 5 min, 4°C) were determined by TCID50 titration. IC50
values reflect the compound concentration at which virus yields were half that of
solvent-only-treated control samples.
Isolation of envelope glycoproteins from primary MV isolates. Total RNA was
prepared from Vero-CD150w cells infected with the wild-type isolate of interest
using the RNeasy minikit (QIAGEN) and subjected to reverse transcription
using Superscript II reverse transcriptase (Invitrogen) and random hexamer
primers. Genome fragments containing the F and H genes were then further
amplified using TaqHiFi DNA-polymerase (Invitrogen), followed by transfer
into TOPO 2.1 vectors (Invitrogen) and further cloning into pCG expression
plasmids containing the constitutive cytomegalovirus promoter.
Transient-inhibition assays. To determine the ability of candidate compounds
to inhibit cell-to-cell fusion, 6 ϫ 105 cells per well were transfected with 4 ␮g
plasmid DNA each encoding MV-KS H and F genes, and cells were transferred
4 h posttransfection to 96-well plates with twofold compound dilutions ranging
from 75 ␮M to 4.6875 ␮M in four replicates each. Fusion activity was assessed
microscopically 48 h posttransfection, and the extent of cytotoxicity was quanti-
fied using the cytotoxicity assay (Promega) described above.
Compound synthesis. AM-4 and 5-amino-2-benzylbenzoxazole (OX-1) were
synthesized and analyzed as previously described (21). For synthesis of 4-nitro-
2-phenylaceylamino-benzamide (AS-48), 4-nitroanthranilic acid (202 mg, 1.0
mmol) in neat thionyl chloride (10 ml) was heated at 60°C for 3 h under N2 and
concentrated by rotary evaporation. Residual SOCl2 was removed by three se-
quential additions of CH2Cl2 followed by rotary evaporation. Concentrated
aqueous NH3 (10 ml) was added, and the mixture was stirred overnight at room
temperature. The precipitates formed were collected by filtration and washed
with water three times. The aqueous filtrate was extracted with ethyl acetate
(three times, 5 ml) and dried over Na2SO4, and solvent was removed. The crude
filtrate and the solid from filtrate extraction were combined and purified by
recrystallization (methanol) to afford 2-amino-4-nitro-benzylamide (49 mg, 27%
yield). The latter was dissolved in tetrahydrofuran, combined with 1.2 eq pyri-
dine, and treated with 1.1 eq phenyl acetylchloride at room temperature for 3 h.
The reaction mixture was poured into aqueous ammonium chloride, extracted
with ethyl acetate, dried over Na2SO4, and chromatographed on silica gel (hex-
ane/ethyl acetate at 1:1) to give AS-48 as a light yellow solid (54 mg, 67% yield).
Membrane protection assays. For entry experiments, virus inocula were trans-
ferred to 5 ϫ 105 Vero-CD150w cells per well in a six-well plate format at an

VOL. 49, 2005
ENTRY INHIBITOR OF PRIMARY MEASLES VIRUS STRAINS
3757
MOI of 0.1 PFU/cell in the presence of 50 ␮M AS-48 and incubated at 37°C for
60 min. Extracellular virions were inactivated by a 2-minute acid treatment (40
mM sodium citrate, 10 mM potassium chloride, 135 mM sodium chloride, pH
3.0) at 25°C, followed by extensive washing of cells and incubation at 37°C for
30 h. Control cells were subjected to washing steps only, and yields of cell-
associated viral particles were determined by TCID50 titration.
RESULTS
In previous work, we described an inhibitor of MV cell entry,
AM-4 (21). Since this compound has promising antiviral activ-
ity against the MV-Edm strain (IC50 ϭ 260 nM) we further
investigated its potential as a therapeutic lead. In particular, a
chemical half-life of at least 16 h under physiological condi-
tions was considered a minimal requirement for compounds to
warrant further testing. However, when AM-4 was subjected to
in vitro stability testing at physiological pH and temperature, it
was observed to be unstable. Preincubation for 16 h under
these conditions fully ablated its antiviral activity (Fig. 1A).
This observation was corroborated by high-performance liquid
chromatography (HPLC) analysis of the compound subse-
quent to a 16-hour incubation period and by NMR sampling
over a 48-hour period (data not shown).
Development of a stable lead inhibitor. To develop an ap-
plicable MV inhibitor candidate, we therefore aimed to com-
bine the antiviral properties of AM-4 with increased stability.
Our approach was guided by speculation that the instability of
AM-4 is due to its facile oxidation in air. Although we have not
demonstrated the transformation, the para orientation of the
OH and NH2 groups on the heterosubstituted aromatic ring
(Fig. 1B) suggests ready oxidation to a quinone analog. Con-
sequently, subsequent synthetic efforts were pursued with two
principles in mind. First, substituent combinations on the ben-
zamido ring of AM-4 analogs must be compatible with long-
term exposure to air oxygen. Second, both substituent combi-
nation and orientation should provide steric and hydrogen
bonding properties similar to AM-4 in terms of the previously
developed MV homology model (21). These considerations led
to the synthesis of a panel of 39 inhibitor candidates (to be
described in detail elsewhere) by varying the substituent range
of the structural backbone of AM-4, as depicted in Fig. 1C.
To evaluate compound activity against a clinically relevant,
currently circulating MV strain (30), all candidate compounds
were tested against MV-KS, a virus from genotype D5, which
has been endemic in Thailand and Japan and was associated
with imported cases in the United States and Europe (30). As
a first screen, all compounds were assessed for their ability to
suppress cytopathicity induced by MV-KS, as quantified by
reduction of metabolic activity of cells using an MTT assay.
Since cytopathicity due to extensive syncytium formation fol-
lowed by giant cell detachment and death is a hallmark of MV
infection in vitro, the inhibitory activity of the candidate com-
pounds can be assessed in this assay by measuring increased
cell survival and hence metabolic activity. When the antiviral
activity of each candidate compound was compared with that
of the stable first-hit compound OX-1 (21), the basis for all
other compounds including AM-4, compound AS-48 (Fig. 1D)
revealed the greatest increase in activity (7.8-fold increase
compared to OX-1) and was therefore selected for further
characterization.
FIG. 1. Development of a stable therapeutic lead on the structural
basis of compound AM-4. (A) AM-4 has a short half-life under phys-
iological conditions. Serial dilutions of compound in growth medium
were preincubated at 37°C as indicated, followed by transfer to cells
and infection with MV. Virus yields were determined by TCID50 ti-
tration and are expressed in percentages of control cells infected in the
presence of 0.01% DMSO. Values represent averages of two experi-
ments performed in duplicate each. Standard deviations are shown.
(B) Structure of AM-4. (C) Structure template of AM-4 used for
structure-activity relationship development. (D) Structure of the im-
proved lead compound AS-48.
Characterization of inhibitor candidate AS-48. Structural
predictions suggested that AS-48 would be air stable by com-
parison with AM-4 but still retain considerable activity. In-
deed, preincubation of AS-48 at physiologic conditions for 16 h
did not alter its inhibitory effect against MV (Fig. 2A), and
HPLC analysis and NMR sampling over a 48-hour period
(data not shows) likewise confirmed this assumption.
AS-48 was thus further characterized and its potential cyto-
toxicity assessed in a nonradioactive cytotoxicity assay per-
formed in the presence of a range of different compound
concentrations. We observed minimal cytotoxicity in com-
pound-treated cells compared to solvent-only-treated control
cells after 24 to 36 h of incubation in the presence of the

3758
PLEMPER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 3. AS-48 prevents viral entry. (A) AS-48 inhibits cell-to-cell
fusion induced by transiently expressed MV H and F glycoproteins
derived from the MV-Edm and MV-KS strains, respectively. Values
represent averages of four experiments and show quantification of
cytopathicity as a consequence of membrane fusion. Standard devia-
tions are given (B) Membrane protection assay to assess the entry
status of viral particles. Particles were absorbed to target cells in the
presence of either solvent alone (DMSO), AS-48, or FIP, followed by
pH 3.0 treatment, two wash cycles, and incubation in the presence of
DMSO, AS-48, or FIP as indicated. Virus yields were determined by
TCID50 titration, and values represent averages of two experiments,
each performed in duplicate. Standard deviations are given.
FIG. 2. AS-48 is a chemically stable analog with low cytotoxicity.
(A) AS-48 half-life exceeds 16 h under physiological conditions. Serial
dilutions of compound in growth medium were preincubated at 37°C
as indicated, followed by transfer to cells, infection with MV, and virus
titration as described for Fig. 1A. Values represent averages of two
experiments, each performed in duplicate. Standard deviations are
shown. (B) AS-48 has low cytotoxicity, comparable to the first-hit
compound OX-1. Cells were incubated in the presence of serial dilu-
tions of compound for 24 to 30 h, followed by assessment of cytotox-
icity. Values represent averages of four experiments and indicate per-
cent cytotoxicity normalized for control cells treated with solvent
(DMSO) only. Standard deviations are shown. For comparison, the
first-hit compound OX-1 and a toxic candidate compound (AS-19) are
included.
and F glycoproteins. One would expect inhibition of mem-
brane fusion if the compound indeed interferes with virus
entry, while interference of AS-48 with downstream steps of
the virus life cycle such as genome replication would suggest
little inhibitory activity in a transient-fusion assay. We there-
fore isolated the genes encoding the H and F glycoproteins
from MV-KS through reverse transcription-PCR of infected-
cell lysates and transferred them into expression plasmids har-
boring the constitutive cytomegalovirus promoter. Vero cells
stably expressing human CD150w showed extensive cell-to-cell
fusion beginning at 12 h posttransfection when cotransfected
with these plasmids (data not shown). But when cotransfected
cells were mixed with a range of different AS-48 concentrations
4 h posttransfection and incubation was continued in the pres-
ence of different compound concentrations, cell-to-cell fusion
was increasingly suppressed with rising concentration of AS-
48. The extent of fusion inhibition was quantified based on the
reduction of fusion-induced cytotoxicity (Fig. 3A). Dose-re-
sponse curves are shown for AS-48-mediated inhibition of fu-
compound (Fig. 2B). Cytotoxicity levels remained below 10%
in the presence of compound concentrations as high as 300
␮M. As controls, the first-hit compound OX-1, which showed
no detectable cytotoxicity, and compound AS-19, which in-
duces strong cytotoxicity at low concentrations, were also in-
cluded in the assay. AS-48 did show some cytostatic effect at
concentrations of 75 ␮M and higher, however, when cells were
incubated in the presence of the compound for extended pe-
riods of time exceeding 4 days (data not shown).
Based on our previous characterization of the mechanism of
antiviral activity of the first-hit compound OX-1, we postulated
that the antiviral activity of AS-48 was also based on inhibition
of the entry step of the virus life cycle. To test this hypothesis
experimentally, we first examined whether AS-48 inhibits the
cell-to-cell fusion activity induced by plasmid-encoded MV H

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ENTRY INHIBITOR OF PRIMARY MEASLES VIRUS STRAINS
3759
sion induced by glycoproteins derived from MV-KS compared
with those derived from the Edmonston vaccine strain cloned
into otherwise identical expression plasmids (22).
If AS-48 prevents membrane fusion and hence viral entry
but not virus absorption to target cells or postentry steps of the
viral life cycle, virions incubated with target cells in the pres-
ence of compound should be resistant to removal of the inoc-
ulum and washing but should remain sensitive to low-pH in-
activation of extracellular MV particles. A brief pH 3.0 shock
has been previously demonstrated to completely abolish the
infectivity of many enveloped viruses, including MV, through
inactivation of their fusogenic envelope glycoproteins (10, 21),
while virions in a postentry state of their life cycle remain
unaffected. Indeed, three consecutive wash steps after incuba-
tion of cells in the presence or absence of AS-48 did not result
in a significant reduction in virus yields (Fig. 3B, samples A and
B). When cells were exposed to low pH following incubation in
the presence of compound, however, we found a strong reduc-
tion in virus yield when AS-48 was present (Fig. 3B, sample C).
Furthermore, addition of the compound after particle absorp-
tion and low-pH treatment resulted in only a small reduction in
virus yield, confirming that postentry steps of the viral life cycle
are not targeted by AS-48 (Fig. 3B, sample B). The reduced
extent of inhibition in sample C compared to control cells
treated with AS-48 before and after the pH shock (Fig. 3B,
sample D) is likely due to the escape of some particles from
compound inhibition prior to low-pH treatment, while the
reduction in virus yield in sample B compared to control in-
fections in the absence of AS-48 is likely due to secondary
infections being inhibited when cells are incubated in the pres-
ence of compound after low-pH treatment. These assumptions
were confirmed by control samples in which AS-48 was re-
placed by fusion inhibitory peptide (FIP), a peptidic inhibitor
of MV entry (18) that has been demonstrated previously to
interfere with MV infection (Fig. 3B, samples E and F). Taken
together, these findings support the conclusion that AS-48 acts
at the level of MV entry.
FIG. 4. AS-48 shows high target specificity. Dose-response curves
of AS-48 for MV-KS and HPIV-2 are shown. Cells were infected with
MV-KS or HPIV-2 in the presence of serial dilutions of AS-48 as
indicated, and virus titers were determined by TCID50 titration 36 h
postinfection. Average values of two experiments are shown.
genotypes that are currently active worldwide (30) and calcu-
lated IC50 values for each isolate (Fig. 5).
AS-48 consistently demonstrated strong activity against all
wild-type strains tested in this experiment, with IC50 values
ranging from 0.6 ␮M to 2.5 ␮M depending on the strain. The
lowest activity (IC50 ϭ 3.0 ␮M) was observed for the vaccine
strain MV-Edm (clade A), which has not been endemic for
several decades. Consistent with our previous observations for
the first-hit compound OX-1 (21), the lower sensitivity of the
vaccine strain to inhibition by AS-48 may be at least partially
due to a methionine-to-valine mutation at position 94 of the
MV-Edm F protein. All wild-type strains analyzed so far con-
tain a methionine residue at this position. Indeed, a recombi-
nant MV-Edm (rMV-Edm F 94 M) in which this change has
Activity of AS-48 against relevant wild-type MV isolates. To
determine the target specificity of AS-48, we infected cells in
the presence of a range of different compound concentrations
with either MV-KS or HPIV-2, another member of the family
Paramyxovirinae that is related to MV. Dose-response curves
showing virus yields obtained after incubation in the presence
of different compound concentrations reveal an IC50 for AS-48
against MV-KS of 0.6 ␮M, while yields of HPIV-2 are not
affected even at the highest compound concentrations tested
(Fig. 4). Likewise, AS-48 did not prevent membrane fusion
induced by HIV type 1 (HIV-1) (data not shown), a member of
the lentivirus family. These findings thus suggest the high tar-
get specificity of AS-48 and furthermore argue against residual
cytotoxicity contributing to its inhibitory activity, since com-
pound-mediated cytotoxicity would act nonspecifically and
hence reduce yields of other viruses such as HPIV-2.
FIG. 5. AS-48 is active against a representative panel of wild-type
MV isolates. Dose-response curves were generated in duplicate exper-
iments for a panel of currently endemic MV strains (MV-Y22, MVi/
Yaounde.CAM/83 [B1]; MV-JM, MVi/Maryland.USA77 [C2]; MV-
KS, D5; MV-NJ: MVi/New Jersey-1.USA [D6]; MV-Vic, MVi/
Victoria.AUS/16.85 [D7]; MV-Ill, MVi/Illinois.USA/50.99 [D3]; MV-
Ams, MVi-Amster.NET/49.97 [G2]; MV-Alaska, MVi/Alaska.USA/
16.00 [H2]) representing genotypes B, C, D, G, and H. For
comparison, the vaccine strain MV-Edm (genotype A) was included.
Values indicate IC50 concentrations that were calculated on the basis
of these inhibition curves.
An IC50 of 0.6 ␮M against MV-KS confirms that AS-48 is
more than seven times as potent as the first-hit compound
OX-1 and only about 2.5-fold less active than the unstable
compound AM-4. To further assess its potency against a rep-
resentative panel of primary MV strains, we generated dose-
response curves for viruses representing a relevant subset of

3760
PLEMPER ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
been reverted (20, 24) shows increased sensitivity to AS-48
(Fig. 5).
have previously described as important for the fusion process
(21, 25). In previous work, we identified several mutations in
this cavity and elsewhere in the F protein that conferred resis-
tance to inhibition. One of these residues is amino acid 94,
which is a valine in the MV-Edm vaccine strain and a methi-
onine in all other strains analyzed thus far. MV-Edm shows a
reduced susceptibility to inhibition with both AS-48 and OX-1,
suggesting a role for this residue in determining sensitivity to
inhibition by this family of inhibitors. Consistent with this ob-
servation, substituting valine for methionine in MV-Edm con-
ferred increased sensitivity to AS-48 on a recombinant virus.
We reason that the development of small-molecule measles
virus inhibitors that can be produced cost-effectively may be
highly beneficial in complementing the existing measles vacci-
nation program by rapidly controlling local outbreaks and aug-
menting management of severe cases of measles. Since MV
infects through the respiratory route and our compounds or
derivatives thereof could easily be aerosolized for oral admin-
istration, we furthermore consider a prophylactic application
as an attractive future possibility. We predict that viral spread
could be drastically reduced in a setting similar to the recent
outbreaks reported in the United Kingdom if an effective in-
hibitor were made available prophylactically to nonimmunized
individuals in direct contact with identified index cases. Indeed,
computer simulation models have demonstrated that an influ-
enza pandemic could be blocked at the onset if antiviral drugs
were administered to susceptible contacts of index cases (14).
Based on its activity against a range of measles virus strains
currently circulating worldwide, we hypothesize that AS-48
may have significant potential as a therapeutic lead inhibitor.
The potent in vitro antiviral activity of AS-48 reported here
supports further testing in vivo to assess the candidacy of
AS-48 for clinical development.
DISCUSSION
We describe the development of a potent and stable small-
molecule inhibitor of MV entry, AS-48. This compound was
designed on the basis of our previously reported MV entry
inhibitor AM-4, which has strong antiviral activity but lacks
stability. AS-48 demonstrates greatly improved stability under
physiologic conditions, shows low cytotoxicity, and has potent
antiviral activity, with an IC50 ranging from 0.6 to 2.5 ␮M
against a panel of wild-type MV isolates representing currently
circulating strains.
AM-4 is a masked hydroquinone with the NH2 group re-
placing the second OH on the aromatic ring. It is well known
that several 1,2-, 1,3-, and 1,4-dihydroxybenzene analogs (py-
rocatechol, resorcinol, and hydroquinone, respectively) are
powerful antioxidants in fruits (34, 36), oils (29), and wine (7).
Furthermore, the hydroquinone coenzyme Q (ubiquinol) is the
membrane-sequestered small-molecule mediator of electron
transport in the inner membrane of the mitochondria (5).
Similarly, para-hydroxyanilines are also strong reducing agents
that are readily air oxidized to quinones and related polymers
(9). Consequently, the short half-life of AM-4 in air is under-
standable by analogy. We have thus attempted to introduce
chemical stability, retain overall compound geometry, and
present similar pharmacophoric elements compared to AM-4
in alternative structures. The nonplanar geometry of AM-4
(21) arises from the CH2 group serving as a fulcrum in the
center of the molecule. It is retained in AS-48, as is the hydro-
phobic phenyl ring that we found to be critical for antiviral
activity (21). The NH2 and OH substituents in the benzamido
ring of AM-4 serve as proton donor and proton acceptor,
respectively. The former is replaced by an amide, the latter by
a nitro group in AS-48. As a result, the reducing capacity of the
ring has been blocked. Indeed, the stability of AS-48 was con-
firmed experimentally both by HPLC and NMR, and by the
fact that preincubation at physiologic conditions for 16 h
caused negligible reduction of its antiviral activity.
Our data support the conclusion that AS-48 acts at the level
of viral entry, as do its predecessors AM-4 and OX-1 (21).
AS-48 inhibits cell-to-cell fusion induced by the H and F pro-
teins of a currently prevalent genotype D5 strain, MV-KS (30),
and does not interfere with postentry steps of the virus life
cycle. Furthermore, our data suggest that AS-48, like AM-4
and OX-1, inhibits the membrane fusion step in the viral entry
process, rather than interfering with the attachment of virus to
target cells.
AS-48 demonstrated potent antiviral activity against a range
of MV strains from genotypes that are currently circulating
worldwide (30): B1, C2, D3, D5, D6, D7, G2, and H2. IC50
values ranging from 0.6 to 2.5 ␮M suggest that this compound
may have potential as a therapeutic lead with activity against a
broad range of MV strains. Additionally, AS-48 shows no ac-
tivity against entry of the related paramyxovirus HPIV-2 or of
the lentivirus HIV-1, demonstrating the target specificity of
this compound for MV.
ACKNOWLEDGMENTS
We are grateful to A. N. Vzorov for testing of compound activity
against HIV-1 and to L. K. White for critical reading of the manu-
script.
This work was supported by a pilot grant from the Southeastern
Center for Emerging Biological Threats (grant/cooperative agreement
number U38/CCU423095 from CDC), a research grant from the
American Lung Association, and Public Health Service grant
1R21AI056179-01A2 from NIH/NIAID (all to R.K.P.) and Public
Health Service grant AI057157 to the Southeastern Regional Center of
Excellence for Emerging Infections and Biodefense.
The contents of this publication are solely the responsibility of the
authors and do not necessarily represent the official views of CDC.
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