Nonnucleoside inhibitor of measles virus RNA-dependent RNA polymerase complex activity

Article abstract of DOI:10.1128/AAC.00289-07

Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of worldwide mortality and accounts for approximately

Full text of DOI:10.1128/AAC.00289-07

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, July 2007, p. 2293–2303
0066-4804/07/$08.00ϩ0 doi:10.1128/AAC.00289-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 51, No. 7
Nonnucleoside Inhibitor of Measles Virus RNA-Dependent
RNA Polymerase Complex Activityᰔ†
Laura K. White,1‡ Jeong-Joong Yoon,1 Jin K. Lee,1 Aiming Sun,2 Yuhong Du,3 Haian Fu,3
James P. Snyder,2 and Richard K. Plemper1*
Department of Pediatrics, Emory University School of Medicine, Atlanta, Georgia 303221; Department of Chemistry, Emory University,
Atlanta, Georgia 303222; and Department of Pharmacology and Emory Chemical Biology Discovery Center,
Emory University School of Medicine, Atlanta, Georgia 303223
Received 27 February 2007/Returned for modification 4 April 2007/Accepted 20 April 2007
Paramyxoviruses comprise several major human pathogens. Although a live-attenuated vaccine protects
against measles virus (MV), a member of the paramyxovirus family, the virus remains a principal cause of
worldwide mortality and accounts for approximately 21 million cases and 300,000 to 400,000 deaths annually.
The development of novel antivirals that allow improved case management of severe measles and silence viral
outbreaks is thus highly desirable. We have previously described the development of novel MV fusion inhib-
itors. The potential for preexisting or emerging resistance in the field constitutes the rationale for the
identification of additional MV inhibitors with a diverse target spectrum. Here, we report the development and
implementation of a cell-based assay for high-throughput screening of MV antivirals, which has yielded several
hit candidates. Following confirmation by secondary assays and chemical synthesis, the most potent hit was
found to act as a target-specific inhibitor of MV replication with desirable drug-like properties. The compound
proved highly active against multiple primary isolates of diverse MV genotypes currently circulating worldwide,
showing active concentrations of 35 to 145 nM. Significantly, it does not interfere with viral entry and lacks
cross-resistance with the MV fusion inhibitor class. Mechanistic characterization on a subinfection level
revealed that the compound represents a first-in-class nonnucleoside inhibitor of MV RNA-dependent RNA
polymerase complex activity. Singly or in combination with the fusion inhibitors, this novel compound class has
high developmental potential as a potent therapeutic against MV and will likely further the mechanistic
characterization of the viral polymerase complex.
Measles virus (MV), a member of the paramyxovirus family,
remains a principal cause of worldwide morbidity and mortal-
ity, being responsible for approximately 300,000 to 400,000
deaths annually, despite the existence of a live-attenuated vac-
cine (8, 50). Globally, measles is the leading cause of childhood
death from a vaccine-preventable disease (http://www.cdc.gov
/programs/global06.htm) and remains among the 10 most le-
thal human pathogens. Transmitted via the respiratory route,
the virus is highly communicable and one of the most infec-
tious pathogens identified (19, 21, 47). Complications associ-
ated with MV infection include acute encephalitis in approxi-
mately 0.1% of cases and subacute sclerosing panencephalitis,
a lethal late sequela that occurs years after the primary infec-
tion (19, 22).
Currently, no therapeutics are available for case manage-
ment of severe measles or the rapid silencing of local out-
breaks. Ribavirin, the only drug available for the treatment of
some paramyxovirus infections (9, 41), has been used experi-
mentally for the treatment of measles, but with limited efficacy
(2). This makes desirable the development of cost-effective
antivirals against MV that augment the existing vaccination
program.
MV infection is initiated by pH-independent fusion of the
viral envelope with the target cell plasma membrane (19). The
hemagglutinin (H) envelope glycoprotein mediates particle at-
tachment (13, 18, 32, 46), followed by membrane fusion or-
chestrated by the fusion (F) envelope protein (26). Viral-gene
expression and subsequent genome replication then take place
in the cytosol (19). Both processes are mediated by the viral
RNA-dependent RNA polymerase (RdRp) complex, which
consists minimally of a homotetramer of the viral phosphopro-
tein (P) and a single polymerase (L) protein (6, 25). The sole
target for RdRp is a ribonucleoprotein complex of viral RNA
encapsidated by the MV nucleocapsid (N) protein (6), mini-
mizing the presence of naked genomic RNA in the host cell.
Considering that human and animal tissues lack a known ho-
mologue of the RdRp or the fusogenic envelope proteins, the
polymerase complex and components of the entry machinery
constitute particularly attractive targets for virus-specific small-
molecule inhibitors.
Despite its critical role in the viral life cycle, our mechanistic
understanding of the MV RdRp is still limited and the struc-
tural characterization of its components is sparse. An abun-
dance of structural disorder has been found in the MV N and
P proteins (23, 27), and no paramyxovirus polymerase has been
purified yet (6). In addition to their therapeutic potential,
small-molecule compounds interfering with the function of the
MV RdRp complex may constitute viable tools for a better
* Corresponding author. Mailing address: Division of Infectious
Diseases, Emory University School of Medicine, 2015 Uppergate
Drive, Atlanta, GA 30322. Phone: (404) 727-1605. Fax: (404) 727-9223.
E-mail: rplempe@emory.edu.
† Supplemental material for this article may be found at http://aac
.asm.org/.
‡ Present address: The Scripps Research Institute, La Jolla, CA
92037.
ᰔ Published ahead of print on 30 April 2007.
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trations were 0.5%, at which no adverse effect on cell viability or virus growth
could be detected in control samples. All virus stocks used for screening were
subjected to dialysis against phosphate-buffered saline (PBS) to remove contam-
inating eGFP that had been synthesized during virus growth. Following a 64-hour
incubation period at 37°C, green fluorescence, indicating expression of viral
proteins, was quantified using an Analyst HT microplate reader (Molecular
Devices). To validate the assay, the MV fusion inhibitor AS-48 (35) was added
in an otherwise identical setting, and zЈ values were calculated according to the
following formula: zЈ ϭ 1 Ϫ (3SDC ϩ 3SDB)/(meanC Ϫ meanB), where C is the
control and B is the background (54). As a first-pass test to exclude false-positive
compounds, cytotoxicity was assessed microscopically for all wells that showed
low fluorescence intensity and, for selected compounds, photodocumented at a
magnification of ϫ200. The compound library used was a diversity set pur-
chased by the Emory Chemical Biology Discovery Center from ChemDiv (San
Diego, CA).
Chemical synthesis of compound 16677. For synthesis of compound 16677,
1-methyl-3-trifluoromethyl-5-pyrazolecarboxylic acid (see Fig. 4, step 3) was pre-
pared from commercially available compound 1 (see Fig. 4), as previously de-
scribed (39). Compound 3 (see Fig. 4) (820 mg; 4.2 mmol) in dichloromethane
(10 ml) was treated with oxalyl chloride (2.0 M in CH2Cl2; 8.5 mmol; 4.2 ml) and
a catalytic amount of dimethylformamide. The reaction mixture was incubated at
room temperature for 5 h. Evaporation of solvent delivered yellow acyl chloride
(see Fig. 4, step 4) in a quantitative yield. A portion of the latter in dichlorometh-
ane (0.55 mmol) was added to a cold solution of 4-amino-prolidinyl sulfonamide
(see Fig. 4, step 5) (113.1 mg; 0.5 mmol) and pyridine (48 ␮l; 0.6 mmol) in
dichloromethane (2 ml). The reaction mixture was warmed to room temperature
(18 h), poured into dilute hydrochloric acid (1 N), extracted with dichlorometh-
ane (three times 15 ml), and dried over anhydrous Na2SO4. The product was
purified by chromatography using hexane-ethyl acetate (3:1) to obtain compound
16677 as a white powder (110.2 mg; 55% yield). 1H nuclear magnetic resonance
(400 MHz, CDCl3) ␦1.76–1.80 (4H, m), 3.24–3.27(4H, m), 4.28 (3H, s), 7.75–7.77
(2H, m), 7.84–7.87 (3H, m). High resolution mass spectrometry calculated for
molecular and structural characterization of the viral replica-
tion machinery.
In contrast to the RdRp, considerable structural information
is available for the paramyxovirus attachment (12, 53) and
fusion proteins, including structures of the latter in both the
prefusion (52) and intermediate to postfusion (10, 51) confor-
mations. In previous work, we identified a new class of MV
fusion inhibitors, substituted anilides, in a structure-based drug
design approach (36, 38). The lead compound of this inhibitor
class, AS-48 (35, 44), shows activity in the low micromolar
range (50% inhibitory concentration, 0.6 to 3.0 ␮M) against a
panel of MV field isolates. A single sub-Saharan isolate is
resistant to inhibition by AS-48, however, and in vitro adapta-
tion has resulted in the appearance of characteristic escape
mutants after four to seven passages (14), suggesting that re-
sistance may emerge rapidly in the field. The identification of
additional drug candidates against MV with diverse target
characteristics is therefore imperative. In addition to counter-
acting preexisting resistance, combined administration of com-
pounds with different target sites may reduce the rate of viral
escape or result in impaired fitness of virions that develop
multiple resistance.
Toward this goal, we report here the development of a
robust, cell-based assay for high-throughput screening (HTS)
of MV inhibitor candidates. Implementation of this assay has
yielded several hit candidates, which were subsequently con-
firmed in manual secondary assays. The structure of the most
potent candidate was confirmed by independent synthesis. It
has desirable drug-like properties, does not block viral entry,
and is not subject to cross-resistance with the AS-48 class of
MV fusion inhibitors. Mechanistic characterization has re-
vealed that the compound acts late in the viral life cycle,
prompting us to address the question of whether it specifi-
cally interferes with the activity of the viral polymerase
complex.
C16H17 F3N4O3S, 402.0974, found 403.1044 (M ϩ 1). Analysis calculated for
C16H17 F3N4O3S: C, 47.76; H, 4.26; N, 13.92, found C, 47.71; H, 4.23; N, 13.81.
Subsequent to hit identification, the synthetic sample was used for all experi-
ments.
Quantification of compound cytotoxicity. Two independent assays, a nonra-
dioactive cytotoxicity assay (CytoTox 96 Non-Radioactive Cytotoxicity Assay;
Promega) and a trypan blue exclusion assay, were employed to determine the
cytotoxicities of compounds. For the cytotoxicity assay, 12,000 cells per well in a
96-well plate format were incubated at 37°C for 24 h in four replicates per
concentration tested in the presence of a range of compound concentrations in
twofold dilutions (highest, 150 ␮M; 300 ␮M for compound 16677). Conversion of
a tetrazolium salt into a colored formazan product by cellular lactate dehydro-
genase released into the culture supernatants was then measured at 490 nm using
a Bio-Rad plate reader. Values were calculated according to the following
formula: percent viability ϭ 100 Ϫ [(experimental Ϫ background)/(maximum Ϫ
background) ϫ 100]. For the trypan blue exclusion assay, 2 ϫ 105 cells per well
were seeded in a six-well plate format and incubated at 37°C for 30 h in three
replicates per concentration tested in the presence of a range of compound
concentrations in fivefold dilutions (highest, 500 ␮M). The cells were then
detached from the culture dishes, aliquots were incubated with trypan blue
solution for 15 min at room temperature, and the viable cells were counted using
a hemacytometer.
Dose-response inhibition curves based on virus yields. To generate virus
yield-based dose-response curves, 4 ϫ 105 cells per well were infected in a
six-well plate format with rMV-Edm, MV field isolates, CDV, or human para-
influenza virus type 2 (hPIV2) as specified at an MOI of 0.1 PFU/cell in the
presence of a range of compound concentrations in twofold dilutions (highest, 75
␮M) or equivalent volumes of solvent (DMSO) only and incubated in the
presence of compound at 37°C. For assessment of clinical MV isolates, com-
pound was added in threefold dilutions (highest, 37.5 ␮M). Thirty-six hours
postinfection, cell-associated viral particles were harvested and titered as de-
scribed above. Plotting virus titers as a function of the compound concentration
allowed the calculation of 50% effective concentrations (EC50s), at which virus
yields are 50% of DMSO-treated controls.
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-
SLAM cells, derived from Vero cells (African green monkey kidney epithelial
cells; ATCC CCL-81) and stably expressing human SLAM/CD150w; Vero-
dogSLAM cells (40) stably expressing dog SLAM; and BSR T7/5 cells (7) stably
expressing T7 polymerase were incubated at every third passage in the additional
presence of G-418 (Geneticin) at a concentration of 100 ␮g/ml. Lipofectamine
2000 (Invitrogen) was used for transient-transfection experiments. To prepare
virus stocks, cells were infected at a multiplicity of infection (MOI) of 0.001
PFU/cell and incubated at 37°C. The cells were scraped in OPTIMEM (Invitro-
gen), virus was released by two freeze-thaw cycles, and titers were determined by
50% tissue culture infective dose (TCID50) titration according to the Spearman-
Karber method (43) as previously described (37). MV-Edmonston (MV-Edm)
stocks were grown and titered on Vero cells, while for MV field isolates, Vero-
SLAM cells were used, and for canine distemper virus (CDV), Vero-dogSLAM
cells were used. All MV field isolates were originally derived from peripheral
blood mononuclear cell samples, and the viruses were isolated and minimally
passaged on SLAM-positive B95-a cells or Vero-SLAM cells.
High-throughput compound screening. For screening, Vero cells were seeded
in 96-well microtiter plates at a density of 7,500 cells per well in 100 ml growth
medium. After a 4-hour incubation period at 37°C and 5% CO2, compound was
added in 1.0-␮l/well doses (25 ␮M final concentration) with a Sciclone auto-
mated liquid handler system (Caliper, MA), followed by infection with a recom-
binant MV that harbors enhanced green fluorescent protein (eGFP) as an ad-
ditional transcription unit (rMV-eGFP) (15, 17) at an MOI of 0.25 PFU/cell in
100 ml serum-free medium. Final solvent (dimethyl sulfoxide [DMSO]) concen-
Compound specificity. To determine compound specificity, 4 ϫ 105 cells in
a six-well plate format were infected in serum-free growth medium at an MOI
of 0.1 or 0.5 PFU/cell, as specified, in the presence of a range of 16677
concentrations in fourfold dilutions (highest, 12.5 ␮M). Bovine serum albu-
min (BSA) was added to some samples at a final concentration of 10 mg/ml.
When virus-induced cytopathicity in DMSO control samples reached approx-
imately 75%, the complete series was harvested, titers of cell-associated viral

VOL. 51, 2007
NONNUCLEOSIDE INHIBITOR OF MV POLYMERASE ACTIVITY
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particles were determined by TCID50 titration, and EC50s were calculated for
each series.
Compound stability. To assess compound stability under physiological condi-
tions, inhibitor 16677 was dissolved in growth medium (15 ␮M final concentra-
tion) and incubated at 37°C and the physiological pH for different time intervals
(longest, 24 h). Control samples contained equal amounts of DMSO and were
likewise incubated for 24 h. Subsequent to preincubation, MV was added to the
compound aliquots, and the mixtures were transferred to 4 ϫ 105 target cells
seeded in a six-well plate format (resulting MOI, 0.1 PFU/cell). Thirty-six hours
postinfection, cell-associated viral particles were harvested and virus titers were
determined by TCID50 titration.
Transient fusion-inhibition assays. To assess the ability of compound 16677 to
inhibit cell-to-cell fusion induced by transiently expressed MV glycoproteins, a
previously established assay was employed (35). Briefly, 6 ϫ 105 cells per well
were transfected in a 6-well plate format with 4 ␮g plasmid DNA each encoding
MV H and F genes, and the cells were transferred 4 h posttransfection to 96-well
plates containing compound 16677 or AS-48 in a range of concentrations in
twofold dilutions (highest, 150 ␮M). Fusion activity was assessed microscopically
48 h posttransfection, and the extent of cytotoxicity as a consequence of extensive
syncytium formation was quantified according to the following formula, using the
cytotoxicity assay described above: percent cytotoxicity ϭ (experimental Ϫ back-
ground)/(maximum Ϫ background) ϫ 100. For some experiments, the cells were
photodocumented 24 h posttransfection.
FIG. 1. Development of a cell-based assay for the automated iden-
tification of MV inhibitors. Cells seeded in microtiter plates were
infected at an MOI of 0.25 PFU/cell with rMV-eGFP, and the fluo-
rescence intensity was determined at the indicated times postinfection.
For a control, cells were infected with rMV-eGFP in the presence of
37.5 ␮M AS-48, an MV fusion inhibitor. Average values of four rep-
licates are shown; the error bars represent standard deviations. zЈ ϭ 1 Ϫ
(3SDC ϩ 3SDB)/(meanC Ϫ meanB), where C is the control and B is the
background; S/B, signal to background ratio.
Dissociation assays. Viral particles (8 ϫ 104 PFU, equivalent to an MOI of 0.1
PFU/cell) were mixed with compound 16677 (final concentration, 15 ␮M) dis-
solved in 3 ml PBS. After a 10-minute incubation at 37°C to allow compound
binding, the sample was divided into two aliquots (1.5 ml each) that were either
subjected to dialysis against PBS (cutoff, 75 kDa; dilution factor, 100,000ϫ; 4°C;
10 h) or incubated for 10 h at 4°C without dialysis. A control sample was treated
with DMSO only and subjected to dialysis. All samples were then transferred to
4 ϫ 105 target cells seeded in a six-well plate, cell-associated viral particles were
harvested 36 h postinfection, and viral titers were determined by TCID50 titra-
tion.
Virus entry assays. For entry experiments, viral particles (MOI ϭ 0.5 PFU/
cell) were adsorbed to 4 ϫ 105 target cells in a six-well plate format at 4°C in the
presence of 10 ␮M 16677 or an equal amount of DMSO for 1 hour. The cells
were then shifted to 37°C for 30 min, followed by inactivation of adsorbed
extracellular virions by a 2-minute acid treatment (40 mM sodium citrate, 10 mM
KCl, 135 mM NaCl, pH 3.0) at 25°C as previously described (33, 35, 36).
Subsequent incubation for 30 h at 37°C in the presence or absence of 10 ␮M
16677, as specified, was followed by determination of cell-associated virus titers
by TCID50 titration.
RESULTS
Development of a primary assay suitable for automated
screening of MV antivirals. To identify novel MV inhibitor
candidates, we have developed a protocol for the automated
screening of compound libraries for their activities against live
MV. The assay relies on rMV-eGFP (15, 17) and detects
eGFP-mediated fluorescence as an indicator for expression of
the viral genome. Due to the positioning of the eGFP-encod-
ing transcription unit in the viral genome, every infectious cycle
results in eGFP synthesis prior to expression of the viral pro-
teins. Consequently, compounds that interfere with vial entry
or viral-gene expression should predominantly be identified.
To exclude compounds with substantial cytotoxicity prior to
secondary assays, cell viability was determined microscopically
in a second step for all wells in which the automated system
detected low fluorescence intensities.
When employing the MV entry inhibitor AS-48 to evaluate
this assay, we observed a time-dependent increase in signal
intensity, reflecting virus growth and genome expression. At
64 h postinfection, the signal-to-background ratio reached 6.43
and the overall zЈ value was 0.8 (Fig. 1). Robust screening
protocols typically yield zЈ values from 0.5 to 1, indicating the
broad suitability of an assay for automated screening (54).
These results therefore recommended our protocol as a suit-
able strategy for HTS-based hit identification.
Library screening yields four sulfonamide hit candidates.
We applied this assay to the automated screening of 34,000
compounds. The process yielded 12 potential hits that reduced
GFP fluorescence to background levels and showed low cyto-
toxicity when wells were microscopically assessed. Retesting of
these molecules in the same assay reduced the number of
reproducible hits to four compounds (Fig. 2), equivalent to a
hit identification rate of approximately 0.01%. The chemical
structures of the four hit compounds are shown in Table 1.
While they represent different structural classes overall, it is
remarkable that each of these compounds incorporates a sul-
fonamide unit. The compounds were not found in searches
Time of compound addition assays. Cells (3 ϫ 105/well in a 12-well plate
format) were infected with MV at an MOI of 1.0 PFU/cell, and compound 16677
(final concentration, 15 ␮M) or AS-48 (final concentration, 75 ␮M) was added at
the indicated time points. Control cells were infected in the presence of equal
amounts of DMSO. Twenty hours postinfection, when virus-induced cytopath-
icity exceeded 90%, cell-associated viral particles were harvested and subjected
to TCID50 titration.
Minireplicon assays. BSR T7/5 cells (5 ϫ 105 per well in a six-well plate
format) were transfected with plasmid DNA encoding MV L (0.24 ␮g), MV N
(0.94 ␮g), or MV P (0.29 ␮g) and 2 ␮g of the MV chloramphenicol acetyltrans-
ferase (CAT) minigenome reporter plasmid (42). For analysis of Nipah virus
polymerase activity, cells were transfected with plasmid DNAs encoding Nipah
virus L (0.4 ␮g), N (1.25 ␮g), or P (0.8 ␮g) protein and 3.5 ␮g of the Nipah CAT
reporter plasmid as previously described (20). Control wells included identical
amounts of reporter and helper plasmids but lacked the plasmids harboring the
respective L gene. Two hours posttransfection, compound 16677 was added in a
range of concentrations in threefold dilutions (highest, 30 ␮M), while some wells
received compound AS-48 or equal amounts of DMSO for comparison. Thirty-
eight hours posttransfection, the cells were lysed and CAT concentrations in the
lysates were determined using a CAT-enzyme-linked immunosorbent assay sys-
tem (Roche).
In vitro protein transcription/translation. Rabbit reticulocyte lysates were
mixed with 0.5 ␮g plasmid DNA encoding MV F under the control of the T7
promoter (pT7-MV F), 20 ␮Ci [35S]methionine, and compound 16677 (final
concentration, 50 ␮M) or an equal volume of DMSO. Samples were incubated
at 30°C for 90 min, mixed with urea buffer (200 mM Tris, pH 6.8, 8 M urea, 5%
sodium dodecyl sulfate, 0.1 mM EDTA, 0.03% bromphenol blue, 1.5% dithio-
threitol), and fractionated on 12% polyacrylamide gels. The dried gels were
exposed to XAR films.

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WHITE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 2. HTS identifies four hit candidates with potent anti-MV properties. Shown are phase-contrast and fluorescence micrographs of cells
infected with rMV-eGFP in the presence of 25 ␮M compound. Controls included cells infected with rMV-eGFP in the presence of equal volumes
of DMSO and mock-infected cells. Representative fields of view are shown.
within SciFinder Scholar (http://www.cas.org/SCIFINDER
/SCHOLAR) and PubChem (http://pubchem.ncbi.nlm.nih
.gov), however, revealing that this is likely the first report of
biological activity associated with them.
Secondary confirmatory assays identified compound 16677
as the most potent hit. To compare antiviral activities and
assess whether these compounds represent true hits that war-
rant hit-to-lead development, each was subjected to indepen-
TABLE 1. Structures of confirmed HTS hits and summary of secondary-assay results
Compound
15960
Structure
EC50 (␮M)a
CC50 (␮M)b
SIc
2.8 Ϯ 0.6
121 Ϯ 20.1 (ND)
43.2 (ND)
16677
0.24 Ϯ 0.01
Ͼ300 (Ͼ500)
Ͼ1,250 (Ͼ2,083)
68290
99920
15.7 Ϯ 9.6
2.0 Ϯ 0.6
Ͼ150 (71 Ϯ 22.3)
100 Ϯ 7.4 (ND)
Ͼ10 (4.5)
50 (ND)
a Concentration at which a 50% reduction of virus yields (MV-Edm strain) was observed. Averages and standard deviations (SDs) are based on three experiments.
b Determined for Vero cells using a CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega). Averages and SDs are based on four experiments. The highest
concentrations examined were 150 ␮M for compounds 15960, 68290, and 99920 and 300 ␮M for compound 16677. The values in parentheses reflect a 50% reduction
in the number of viable cells after a 30-hour incubation in the presence of compound, determined using a trypan blue exclusion assay. Averages and SDs are based
on three experiments. ND, not determined.
c SI, selectivity index (CC50/EC50). The values in parentheses are based on CC50s obtained using the trypan blue exclusion assay.

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dent secondary assays. We first quantified the inherent cyto-
toxicities of the four compounds using colorimetric
a
cytotoxicity assay (1). Candidates 99920 and 15960 revealed
50% cytotoxic concentrations (CC50s) of 100 and 121 ␮M,
respectively, while compounds 68290 and 16677 showed no
detectable toxicity at 150 ␮M and 300 ␮M, respectively, the
highest concentrations examined (Fig. 3A and Table 1).
These experiments were complemented for compounds
68290 and 16677 by an independent trypan blue exclusion
assay that determined the effects of the inhibitor candidates on
cell proliferation rates and viabilities. For compound 16677,
the number of live cells was reduced by only 20% after incu-
bation in the presence of compound concentrations as high as
500 ␮M, while for compound 68290, a 50% reduction was
observed at approximately 71 ␮M (Fig. 3B and Table 1).
Since the CC50s of all candidate compounds substantially
exceeded the concentrations used in the primary HTS assay
(25 ␮M), all four were subjected to a confirmatory assay that
measured the reduction of virus yields by the inhibitor. Using
as the viral target rMV-Edm, which was also employed for the
original HTS screen, compound 16677 was found to have the
greatest antiviral potency, with an EC50 of approximately 240
nM (Fig. 3C and Table 1). The EC50s for the other three
candidates are summarized in Table 1.
Taken together, these secondary assays recommended com-
pound 16677 as a particularly promising candidate MV inhib-
itor. Since compounds incorporated in HTS libraries, particu-
larly those dissolved in DMSO, are known to degrade and/or
rearrange upon prolonged storage (45), the integrity of 16677
was confirmed by chemical synthesis. The synthesis strategy is
summarized in Fig. 4. Pure synthetic 16677 proved highly ef-
fective against MV and was used for all experiments subse-
quent to hit identification.
Compound 16677 is a target-specific MV inhibitor. To ex-
amine the protein binding and viral specificity of candidate
16677, we first subjected the compound to two assays that were
reportedly suitable to determine nonspecific protein binding
(29, 30). For these and all following experiments, the MV field
isolate Alaska (MV-Alaska) was employed, since this strain
was found to be highly sensitive to 16677 (see below).
The first assay measured the effect of additional inert pro-
tein (BSA) on compound activity, and the second monitored
the outcome of an increase in the amount of available target.
BSA is thought to compete with the target protein for nonspe-
cific binding to compound, while an increase in the number of
target molecules would quickly saturate the absorption capac-
ity of compound aggregates. In either case, a sharp drop in
activity reflected by increased EC50s would result, while the
EC50s of well-behaved inhibitors would be essentially unaf-
fected. When EC50s were determined for 16677 after incuba-
tion of infected cells in the presence and absence of 10 mg/ml
BSA or after infection with a fivefold-higher virus inoculum, no
significant differences were observed (Fig. 5A), indicating little
nonspecific protein binding.
FIG. 3. Manual secondary assays confirm anti-MV activities of the
four hit candidates. (A) Quantification of the extent of chemical lysis
of cells incubated in the presence of compound. The values reflect
the percentages of signal intensity compared with cells incubated in the
presence of DMSO. Averages of four replicates are shown, and the
error bars represent standard deviations (SDs). (B) Quantification of
proliferation activities of cells incubated in the presence of compound.
The number of live cells was determined 30 h after compound addi-
tion. The values indicate the percentages of live cells compared with
DMSO-treated controls. Averages of three experiments are shown,
and the error bars represent SDs. (C) Virus yield assay to determine
the reduction of virus loads. Cells were infected with MV-Edm in the
presence of different compound concentrations, and titers of cell-
associated viral particles were determined by TCID50 titration 36 h
postinfection. Titers were normalized for DMSO-treated control in-
fections. Average values of two experiments are shown, and the error
bars represent standard errors of the mean.
Assessment of activities of 16677 against two related mem-
bers of the paramyxovirus family, CDV and hPIV2, corrobo-
rated these findings. Like MV, CDV belongs to the genus
Morbillivirus, and the two viruses share approximately 61%
protein identity, while hPIV2 is more distantly related and
shows only 21% protein identity with MV. When dose-re-
sponse curves were generated for these viruses, compound
16677 yielded an EC50 of 28 ␮M against CDV and was not
inhibitory against hPIV2 at 75 ␮M, the highest concentration
examined (Fig. 5B). These data thus demonstrate high speci-
ficity of compound 16677 for MV and to a lesser degree for the

2298
WHITE ET AL.
ANTIMICROB. AGENTS CHEMOTHER.
FIG. 4. Synthesis of 1-methyl-3-(trifluoromethyl)-N-[4-(pyrrolidinylsulfonyl)-phenyl]-1H-pyrazole-5-carboxamide (16677). Reagents: (a) MeOH, re-
flux; (b) n-BuLi/i-Pr2NH, then CO2; (c) (COCl)2, dimethylformamide(cat.), CH2Cl2, 0°C-rt; (d) py, CH2Cl2, rt.
intimately related CDV and argue against a nonspecific mech-
anism of activity.
Inhibitory activity is stable under physiological conditions,
and 16677 binding is reversible. A long half-life of antiviral
activity under physiological conditions and absence of chemical
reactivity are desirable properties of inhibitor candidates. To
assess its stability under physiological conditions, 16677 was
preincubated in cell culture medium at 37°C for different pe-
riods ranging from 0 to 24 h, followed by mixing with MV-
Alaska and infection of target cells. While virus grew efficiently
in solvent-only-treated control infections, no loss in antiviral
activity of 16677 could be detected even after preincubation for
24 h (Fig. 6A), indicating a favorable stability profile for the
compound.
To explore whether 16677 chemically reacts with its target or
whether compound docking is reversible, MV aliquots were
incubated with the compound in the absence of target cells for
10 min at 37°C, followed by dialysis at 4°C with a molecular
mass cutoff of 75 kDa, which ensured free diffusion of the
compound but not the viral particles (total dilution factor of
the compound, 100,000-fold). Controls included solvent-only-
treated particles that were similarly subjected to dialysis and
16677-treated virus samples that were, instead of being dia-
lyzed, held at 4°C for the same period. Subsequent infection of
cells with the different virus samples and titration of infectious
particles produced 30 h postinfection revealed that the inhib-
itory activity of 16677 is ablated by dialysis prior to infection
(Fig. 6B), indicating that compound docking is reversible and
is not based on a chemical reaction of the inhibitor with the
target.
Potent activity of 16677 against primary MV isolates, in-
cluding an isolate resistant to the MV entry inhibitor AS-48.
To explore the potential value of candidate 16677 as a clinically
relevant inhibitor of MV, we assessed its activities against a
panel of primary MV isolates that represented several geno-
types circulating worldwide (see Table S2 in the supplemental
material). This panel included the sub-Saharan isolate of ge-
notype B3, MV-Ibd, which we previously found to be resistant
to our series of MV entry inhibitors (14). Depending on the
genotype of the MV strain analyzed, we calculated EC50s rang-
ing from 35 to 140 nM for these isolates (Fig. 7). MV-Ibd was
likewise inhibited by 16677, indicating that no cross-resistance
exists between this compound class and the previously charac-
terized MV entry inhibitors. All primary isolates tested were
also more sensitive to compound 16677 than was the recom-
binant MV-Edm virus (genotype A), on which the original hit
FIG. 5. Compound 16677 is a well-behaved, target-specific MV in-
hibitor. (A) Unrelated protein material or variation of the amount of
target molecules does not affect 16677 activity. Virus yield-based dose-
response curves as described in the legend to Fig. 4B were generated
for 16677 upon infection of cells with MV-Alaska in the absence of
BSA, in the presence of BSA, and after infection with a fivefold-higher
virus inoculum. The values represent average EC50s of two experi-
ments, and the error bars represent standard errors of the mean
(SEMs). (B) 16677 is highly MV specific. Virus yield-based dose-
response curves were generated for MV, the closely related CDV, and
the more distantly related hPIV2. The titers were normalized for
DMSO-treated control infections to facilitate comparison of different
experiments. Average values of two experiments are shown, and the
error bars represent SEMs.

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2299
FIG. 7. 16677 is active against a panel of clinical MV isolates.
Dose-response curves were generated on the basis of virus yields de-
termined by TCID50 titration, and EC50s were calculated. Viral geno-
types are given above the graph. Averages of two experiments are
shown, and the error bars represent standard errors of the mean
(SEMs). For viruses MV-Alaska and rMV-Edm, the EC50s are based
on three experiments and the error bars represent standard deviations.
tification of the envelope glycoprotein-induced cytopathicity
and generation of dose-response curves for both 16677 and
AS-48 confirmed these microscopic observations, since MV H
and F protein-mediated cell-to-cell fusion was uninhibited
even at 16677 concentrations of 150 ␮M (Fig. 8B).
FIG. 6. Compound 16677 is stable under physiological conditions,
and inhibition is reversible. (A) Compound dilutions in growth me-
dium (final concentration, 15 ␮M) were preincubated at 37°C for the
indicated periods, followed by transfer to cells and infection with
MV-Alaska. For the control (untreated), equal dilutions of DMSO in
growth medium were preincubated for 24 h. The values represent titers
of cell-associated viral particles determined 36 h postinfection through
TCID50 titration. Averages of two experiments are shown, and the
error bars represent standard errors of the mean (SEMs). (B) Removal
of 16677 through dialysis fully restores viral replication. Virus dilutions
(equivalents of 0.1 MOI) were mixed with 16677 (final concentration,
15 ␮M) and subjected to dialysis against PBS. The cells were subse-
quently infected with the mixtures, and viral titers were determined
36 h postinfection by TCID50 titration. Controls include DMSO-
treated virions and compound-treated samples maintained under the
same conditions without (w/o) dialysis. Averages of two experiments
are shown, and the error bars represent SEMs.
A viral-entry assay was employed to assess whether the tran-
sient-expression assay accurately reflected the conditions of
virus infection. As demonstrated for several enveloped viruses
(24), the infectivity of MV particles is completely ablated by a
brief pH 3.0 wash (35, 36). Capitalizing on this, we adsorbed
MV particles (equivalent to an MOI of 0.5 PFU/cell) to target
cells at 4°C in the presence or absence of 16677, followed by
removal of unbound virus and a 30-minute incubation period
at 37°C to allow viral entry to proceed. Subsequently, particles
that were in a premembrane fusion state were neutralized
through a pH 3.0 wash, followed by incubation with or without
compound and determination of virus yields. If 16677 inter-
fered with viral entry, particle absorption in the presence of
compound followed by low-pH treatment should result in a
sharp drop in virus yields, as exemplified by the previous anal-
ysis of members of the AS-48 entry inhibitor class in this assay
(35, 36). However, virus yields were only slightly affected by the
pH 3.0 wash when 16677-treated samples were compared to
DMSO-treated cells (Fig. 8C). Substantial reduction of virus
yields by 16677 was observed only when the compound was
present during the incubation period subsequent to the wash
step, confirming its effectiveness (Fig. 8C).
To specify the time interval postentry in which the virus
remained sensitive to inhibition by 16677, the effects of com-
pound administration on replication efficiency at different
stages of the viral life cycle were assessed. For this assay, viral
growth was synchronized by infection of cells with MV at an
MOI of 1.0 PFU/cell, and all samples were harvested 20 h
postinfection, thus essentially excluding secondary infections.
Even when added 12 h postinfection, 16677 caused an approx-
imately 99.9% reduction in virus yields, thus demonstrating
high antiviral effectiveness (Fig. 8D). This was in contrast to
the entry inhibitor AS-48, which, as expected, had to be present
at the time of infection to achieve full inhibition. Taken to-
gether, these findings demonstrate that 16677 does not inter-
discovery was based. Viruses of genotype A have not been
endemic in several decades, underscoring the therapeutic po-
tential of the inhibitor class represented by 16677.
Compound 16677 targets a postentry step in the viral life
cycle. To gain insight into the mechanism of 16677 antiviral
activity, we subjected the compound to an initial mechanistic
characterization. We first addressed the question of whether
membrane fusion, and hence viral entry, is inhibited by the
compound. In the presence and absence of 16677, cells were
transiently transfected with expression plasmids encoding the
MV H and F envelope glycoproteins, and membrane fusion
activity was examined microscopically. Controls included trans-
fected cells treated with the lead entry inhibitor AS-48 and
16677-treated cells that were infected with MV rather than
plasmid transfected. In contrast to the strong inhibitory effect
of AS-48, cell-to-cell fusion mediated by transiently expressed
MV H and F proteins was uninhibited by 16677 (Fig. 8A).
However, virus-mediated cytopathicity was fully suppressed by
the compound, confirming the specificity of the assay. Quan-

2300
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ANTIMICROB. AGENTS CHEMOTHER.
FIG. 9. 16677 specifically inhibits MV RdRp complex activity.
(A) A CAT reporter-based minireplicon assay demonstrates inhibition
of MV polymerase complex activity by 16677. BSR T7/5 cells cotrans-
fected with the minireplicon plasmid and plasmids encoding MV nu-
cleoprotein (N), phosphoprotein (P) and polymerase (L), all under the
control of the T7 promoter, were incubated in the presence of 16677 or
the fusion inhibitor AS-48, followed by lysis and assessment of CAT
concentrations (conc.). Values were determined in quadruplicate and
are expressed as percentages of DMSO-treated controls. A compara-
ble minireplicon system derived from Nipah virus, a related member of
the paramyxovirus family, was not inhibited by 16677. (B) T7 polymer-
ase is fully active in the presence of 16677. Shown is in vitro MV F
transcription/translation in the presence of 50 ␮M 16677 or an equal
volume of DMSO. Controls (Co) included DMSO and a variant of
plasmid pT7-MV F that harbors the F-encoding gene in reverse ori-
entation to the T7 promoter.
fere with the viral entry machinery and point to a postentry
step as its mechanism of action.
Compound 16677 inhibits RdRp complex activity. Our ini-
tial HTS protocol does not favor the identification of inhibitors
of particle assembly or release. This suggests inhibition of the
RdRp machinery as a likely mechanism of antiviral activity of
16677. To evaluate the functionality of the polymerase com-
plex, a plasmid-based subinfection MV minireplicon reporter
assay under the control of the T7 promoter was employed (42).
In this assay, the amount of CAT reporter produced is pro-
portional to the activity of the MV polymerase complex. Com-
pound 16677 demonstrated a strong dose-dependent inhibition
of CAT expression when its effect on minireplicon activity was
examined (Fig. 9A). This was in contrast to the essentially
unchanged CAT levels found in control samples that were
treated with the entry inhibitor AS-48. The inhibitory activity
of 16677 was specific for the MV minireplicon, since the com-
pound had no effect on a comparable minireplicon that was
derived from the related Nipah virus (20). Nipah virus belongs
to the genus Henipavirus within the paramyxovirus family (5),
which is related to the genus Morbillivirus and shares approx-
imately 33% overall protein identify with MV.
FIG. 8. 16677 does not interfere with viral entry and is active late in
the replicative cycle. (A) Cell-to-cell fusion induced by transiently
expressed MV glycoproteins is not inhibited by the compound. Trans-
fected cells, treated with 15 ␮M 16677 or DMSO, were photographed
after a 24-hour incubation period. For comparison, cells treated with
the fusion inhibitor AS-48, cells infected with MV, and mock-trans-
fected/infected cells are shown. (B) Quantification of syncytium for-
mation of transfected cells treated as described for panel A. The values
represent averages of four replicates and are expressed as percentages
of the syncytium formation observed for DMSO-treated samples. The
error bars represent standard deviations. (C) Presence of 16677 during
adsorption of virions to target cells does not render particles sensitive
to neutralization by a pH 3.0 wash, indicating successful viral entry in
the presence of compound. Virions were adsorbed to target cells in the
presence of DMSO or 16677, followed by pH 3.0 treatment to neu-
tralize all particles that were in a premembrane fusion state and incu-
bation in the presence of DMSO or 16677 as indicated. Yields of
cell-associated viral particles were determined by TCID50 titration.
Average values of two experiments are shown, and the error bars
represent standard errors of the mean (SEMs). (D) Antiviral activity of
16677 persists to late phases of a replicative cycle. The compound
(final concentration, 15 ␮M) was added at the indicated times postin-
fection to a one-step replicative cycle. Cell-associated viral particles of
compound-treated samples and DMSO-treated controls were har-
vested 20 h postinfection, and virus yields were determined by TCID50
titration. For comparison, infected cells were treated with the fusion
inhibitor AS-48. Average titers of two experiments are shown, and the
error bars represent SEMs.
The specificity of the minireplicon assay was further con-
firmed when an effect of 16677 on the cellular transcription/
translation machinery or the T7 polymerase function was as-
sessed in a cell-free in vitro transcription/translation assay of
MV F. Equal amounts of F protein were detected in treated
and untreated samples (Fig. 9B), indicating that cellular pro-

VOL. 51, 2007
NONNUCLEOSIDE INHIBITOR OF MV POLYMERASE ACTIVITY
2301
tein biosynthesis was unimpaired by the compound. Taken
together, these findings argue against interference of 16677
with the cellular protein biosynthesis machinery or T7 poly-
merase function as an alternative explanation for the reduction
of reporter expression in the MV minireplicon assay. Our data
thus highlight 16677 as a first-in-class compound of nonnucleo-
side inhibitors of MV RdRp complex activity.
activity when 16677 and AS-48 were combined in vitro (data
not shown), combined administration of inhibitors with differ-
ent target structures, in analogy to the clinical experience with
highly active antiretroviral therapy (3, 4, 31), has the potential
to reduce the frequency with which resistant virions emerge in
vivo and/or lower the overall fitness of viral variants with mul-
tiple resistances.
Consistent with the absence of cross-resistance with the MV
fusion inhibitors and in contrast to the findings obtained for
AS-48, compound 16677 does not prevent cell-to-cell fusion
mediated by plasmid-encoded H and F MV glycoproteins and
shows no inhibitory activity in an MV entry assay. Time-of-
addition assays demonstrated that 16677 potently inhibits MV
even when added late in the replicative cycle. These observa-
tions suggest interference with RdRp complex function as the
mechanism of 16677 activity, since inhibitors acting down-
stream of RdRp function are not expected to interfere with
eGFP synthesis in rMV-eGFP-infected cells. A minigenome
reporter assay that monitors the activity of the viral RNA
polymerase complex confirmed this hypothesis, since it dem-
onstrated dose-dependent inhibition of MV minigenome ex-
pression. The fact that an analogous minigenome assay estab-
lished for Nipah virus, a related member of the paramyxovirus
family, was not sensitive to 16677 underscores the target spec-
ificity of the inhibitor. The EC50s of 16677 were on average 25
times higher in the minigenome assay than against the different
MV isolates. It is tempting to speculate that this could reflect
the approximately 24-fold difference in length between the
reporter gene and the viral genome. Longer template se-
quences, requiring more polymerization cycles, may increase
the likelihood of productive inhibitor binding when compound
concentrations are low and thus result in the lower EC50s
determined for 16677 against live virus.
Our functional characterization of HTS hit 16677 has re-
vealed that the compound represents a first in class of novel,
highly potent nonnucleoside inhibitors of MV RdRp complex
activity. Provided that these compounds exhibit favorable
safety and pK profiles, members of this inhibitor class may be
suitable for the treatment of persistent MV infections. Viruses
isolated from subacute sclerosing panencephalitis patients typ-
ically show defects in their envelope proteins (19, 22), which
may render entry inhibitors ineffective. While several nucleo-
side inhibitors of MV have been reported previously, on aver-
age these show at least 10- to 100-fold lower potency than
16677, with EC50s typically in the micromolar range, and none
has progressed to human trials (2). Nonnucleoside inhibitors of
viral polymerases have, however, proven highly beneficial in
antiretroviral therapy (11, 28, 34, 48).
DISCUSSION
With the goal of developing novel therapeutics against MV,
we have established a robust protocol for the automated iden-
tification of MV inhibitors. Our hit identification strategy com-
bines an automated screen with several confirmatory assays
that manually assess cytotoxicity, antiviral activity, and com-
pound structure. A screen of 34,000 compounds from a library
of the Emory Chemical Biology Discovery Center has yielded
four confirmed hits, providing proof of concept for the validity
of this assay for the discovery of MV inhibitors.
All four hit compounds harbor a sulfonamide group, al-
though chemically they belong to different structural classes.
Future testing will reveal whether this has a specific molecular
cause or merely reflects a bias of the compound library as-
sessed in this screening exercise. Comparative structural anal-
ysis of all four hit compounds may also provide valuable infor-
mation for the future generation of structure-activity relationships
for hit-to-lead development.
The identification of promising small-molecule inhibitors
can be hampered by promiscuous compounds that frequently
emerge as putative hits (29, 30). Rather than docking to de-
fined target areas, promiscuous compounds are thought to act
nonspecifically through adsorption or absorption of target
structures to larger compound aggregates. Nonspecific and
noncompetitive binding ultimately leads to flat structure-activ-
ity relationships, typically in the low micromolar range, that
usually render chemical efforts to improve biological activity
futile (29, 30). Our observation that the addition of a large
amount of BSA and an increase in the number of infectious
particles does not affect the EC50 determined for our most
potent hit compound, 16677, recommends this compound as a
well-behaved inhibitor of MV. This is further confirmed by its
high target specificity. Yields of hPIV2, a distantly related
paramyxovirus, are unaffected by 16677, and CDV, a closely
related member of the same genus as MV, is mildly inhibited.
Selective inhibition of MV also corroborates the results of our
cytotoxicity assays, since it argues against general interference
with cellular functions, which would likely be nonspecific, as
the underlying mechanism of antiviral activity of the com-
pound.
Prior to any future hit-to-lead development efforts, com-
pound 16677 shows activity in the low nanomolar range against
a panel of MV field isolates, thus underscoring the therapeutic
potential of the compound. Importantly, the panel of viruses
examined includes the MV-Ibd isolate of genotype B3, which
we have previously found to be naturally resistant to the AS-48
MV entry inhibitor class (14). The lack of cross-resistance of
16677 with the entry inhibitors suggests a different mechanism
of antiviral activity for 16677 and opens potential avenues to
counteract spontaneous viral resistance that may develop in
the field. While we have not observed an increase in antiviral
At present, it is not known whether 16677 binds directly to
the L polymerase, docks to one of the polymerase cofactors N
or P, or interferes with the interaction of these proteins with
each other, with a cellular cofactor specific for MV, or with the
RNA template. Generation of resistant viral variants through
stepwise adaptation, followed by mapping of characteristic
sites of resistance, will guide an in-depth molecular character-
ization of the compound’s mechanism of activity. Combined
with a ligand-based structure-activity relationship, this infor-
mation should constitute a viable platform for educated fur-
ther lead optimization.
Conceptually, our MV-based findings may be adaptable to

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other clinically relevant members of the paramyxovirus family,
such as the recently emerged, highly pathogenic henipaviruses
(16, 49). In this scenario, they prepare the path for a better
mechanistic understanding of these viruses and the develop-
ment of novel therapeutic strategies against pathogens for
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ACKNOWLEDGMENTS
We thank P. A. Rota for generously providing the MV and Nipah
virus replicon systems, Y. Yanagi for Vero-dogSLAM cells, N. Chan-
drakumar for help with compound synthesis, D. Li for technical assis-
tance with HTS, the Emory Chemical Biology Discovery Center for the
use of its screening system, and A. L. Hammond for critical reading of
the manuscript.
This work was supported by a research grant from the American
Lung Association and Public Health Service grants AI056179 and
AI071002 from NIH/NIAID (to R.K.P.).
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