Validation and Characterization of Five Distinct Novel Inhibitors of Human Cytomegalovirus

Article abstract of DOI:10.1021/acs.jmedchem.9b01501

The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants toward existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400"?000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains, and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low micromolar concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex virus (HSV1 and HSV2), and another compound was active against Epstein-Barr virus (EBV). Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on optimization of these new anti-HCMV compounds along with mechanistic studies.

Full text of DOI:10.1021/acs.jmedchem.9b01501

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Article
Validation and characterization of five distinct
novel inhibitors of human cytomegalovirus
Arun Kapoor, Ayan Kumar Ghosh, Michael Forman, Xin Hu, Wenjuan Ye, Noel Southall, Juan J
Marugan, Robert F Keyes, Brian C Smith, David J. Meyers, Marc Ferrer, and Ravit Arav-Boger
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01501 • Publication Date (Web): 19 Mar 2020
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Validation and characterization of five distinct novel inhibitors
of human cytomegalovirus
Arun Kapoor#1, Ayan K. Ghosh#1, Michael Forman2, Xin Hu3,
Wenjuan Ye3, Noel Southall3, Juan Marugan3, Robert F. Keyes4,
Brian C. Smith4, David J. Meyers5, Marc Ferrer3, Ravit Arav-
Boger1,6
1Department of Pediatrics, Division of Infectious Disease, Medical College of Wisconsin, Milwaukee, WI 53226,
USA
2Department of Pathology, Johns Hopkins University, Baltimore, Maryland 21287, USA
3National Center for Advancing Translational Sciences (NCATS), National Institute of Health, Bethesda, MD
20850, USA
4Department of Biochemistry, Program in Chemical Biology, Medical College of Wisconsin, Milwaukee, WI
53226, USA
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5Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,
Baltimore, MD 21287, USA
6Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD 21287, USA
#
Authors contributed equally to this work
Keywords: human cytomegalovirus, screening, entry inhibitor, immediate early-early inhibitors, early-late
inhibitors
Running title: New inhibitors of human cytomegalovirus
Corresponding Author:
Ravit Arav-Boger, MD
Department of Pediatrics
Division of Infectious Disease
Medical College of Wisconsin
Phone: 414 337 7070 FAX: 414 337 7093
Email: rboger@mcw.edu
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ABSTRACT: The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population
and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants
towards existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV
was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400,000
compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds,
several virus strains and detailed virology assays resulted in the identification of five structurally unique and
selective HCMV inhibitors, active at sub to low µM concentrations. Further characterization revealed that each
compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex
virus (HSV1 and HSV2) and another compound was active against Epstein Barr Virus (EBV). Drug combination
studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on
optimization of these new anti-HCMV compounds along with mechanistic studies.
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1. INTRODUCTION
Letermovir, a terminase inhibitor, is approved
for HCMV prophylaxis after hematopoietic
bone marrow transplantation17. Rapid
selection of UL56 mutations in cell culture16
and letermovir resistance in patients is being
reported18. Maribavir, a viral UL97 kinase
inhibitor, is in clinical trials19.
Within the world population, human
cytomegalovirus (HCMV) is one of the most
common pathogens. Seroprevalence rates
approach 90% in individuals over 80 years
old1. Although HCMV infection is usually
asymptomatic,
it
gravely
threatens
immunocompromised hosts, including those
with AIDS and transplant recipients. HCMV is
the most common infection transmitted
during pregnancy from mother to fetus,
leading to hearing impairment and global
There is a clear need to improve the anti-
HCMV drug armamentarium, either as
monotherapy or combination therapy.
Towards this goal, we completed a large
screening campaign of ~400,000 compounds
using our highly-sensitive pp28-luciferase
HCMV reporter and several collections of
chemical libraries20. In-depth validation
studies resulted in the identification and
characterization of five structurally distinct
and selective compounds active in vitro at sub
to low µM concentrations.
disabilities, in children2-6
.
Ganciclovir (GCV), a nucleoside analog, and its
oral formulation val-GCV have dramatically
improved the outcome of transplantations by
reducing sequelae from HCMV replication and
disease7, 8. GCV was also shown to prevent
hearing deterioration in congenitally infected
children9. However, prolonged therapy with
GCV or val-GCV often results in serious bone
marrow toxicities10-13. A phase III clinical trial
of oral val-GCV in infants with congenital
HCMV infection found that 6 months’ therapy
resulted in better neurological outcomes
compared to 6 weeks’ therapy; however GCV-
resistant mutants developed in study
2. RESULTS
2.1. High throughput screening for HCMV
inhibitors: The pp28 luciferase HCMV was
used for screening of the Molecular Library
Small
Molecule
Repository
(MLSMR)
collection of 370,000 compounds, the NIH
Chemical Genomic Center (NCGC) diversity
collection of 65,000 compounds and the NCGC
Pharmaceutical Collection of investigational
and approved drugs. The screening used two
compound doses plated before infection and
luciferase activity was quantified at 72 hours
post-infection (hpi) in lysates. A total of 2215
inhibitors of luciferase activity in our
engineered HCMV were selected using
published compound dose-response curve
participants14.
mutants emerge in transplant recipients15, 16
Similarly,
GCV-resistant
.
When GCV resistance arises, the limited
therapeutic alternatives, foscarnet and
cidofovir, are highly nephrotoxic and can only
be administered intravenously. Their use in
cases of resistant or refractory HCMV is
associated with high morbidity and
mortality13. Several new drugs have advanced
into FDA approval and clinical trials.
algorithms21.
performed to remove compounds that showed
A
secondary screen was
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toxicity in human foreskin fibroblasts (HFFs)
resulting in 847 confirmed, non-toxic HCMV
inhibitors (Fig. 1).
validation in several antiviral assays, using
compounds that were resynthesized or
obtained from vendors. Each of the five
compounds was tested in multiple virological
and molecular assays and is represented in a
unique color in each figure and the table of
contents. The last four digits of each
compound are used in all subsequent figures.
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Figure 1. Flow chart of HTS.
2.2. Validation of anti-HCMV activity:
Additional studies including luciferase
activity, plaque reduction and toxicity assays
were performed on 20 compounds which
passed the primary and secondary screen.
This iterative process resulted in the
identification of five chemotypes (Fig. 2) that
were selected for further characterization and
Figure 2. Chemical structure and MW of the five
compounds that resulted from HTS.
Compound
EC50 - Towne (µM)
EC50 - GCVR Towne (µM)
EC50 - TB40 (µM)
CC50 (µM)
Slope
MLS8969
NFU1827
MLS8554
MLS8091
NCGC2955
GCV
0.55±0.1*
0.85±0.1
0.64±0.1
0.39±0.0
4.61±1.4
2.1±0.3
0.25±0.1*
0.84±0.1
0.17±0.0
0.26±0.0
1.4±0.2**
29.7±1.2
0.12±0.0*
1.21±0.1
0.34±0.0
0.33±0.0
1.18±0.2
0.72±0.0
>500
70.9
0.64±0.1*
1.27±0.1
1.02±0.1
1.37±0.3
0.80±0.1
0.93±0.1
86.0
>250
>200
>200
* = Pre-treatment followed by
plaque reduction assay
** = Plaque reduction assay
Table 1. Anti-HCMV activity of the five compounds against different strains of HCMV, toxicity and slope.
HCMV inhibition was measured by luciferase
activity of pp28-recombinant Towne and pp28
GCV-resistant Towne strains of HCMV (72 hpi)
and by plaque reduction using HCMV Towne
or TB40 (at 8-10 days). Western blot was used
to measure viral protein expression.
A
colorimetric MTT assay was performed in
non-infected HFFs to determine cell
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cytotoxicity in parallel to the infectivity assay,
2.3. Inhibition of other herpesviruses:
Inhibition of herpes simplex 1 (HSV1) and
i.e. at 72 h and at 10 days, and is represented
as CC50. All five compounds were active at sub
to low µM concentrations against all the HCMV
strains used. Based on plaque reduction,
MLS8969 had the best activity against HCMV
followed by MLS8091, MLS8554, NCGC2955
and NFU1827 (Table 1). The slope parameter
(similar to the Hill coefficient) was calculated
for each compound. A slope equal to 1 signifies
either one ligand binding site or a lack of
cooperativity between multiple sites of
binding. Positive cooperativity indicates that
binding of a ligand to one site strengthens
affinity of that ligand at a secondary site
(slope>1). In contrast, negative cooperativity
(slope<1) indicates binding towards one site
weakens ligand affinity at a secondary site. In
HIV infection, modeling the slope parameter
showed that this parameter could distinguish
drug classes with distinct mechanisms of
action. Moreover, the slope was reduced even
in cases where the EC50 value was the same in
resistant virus mutants22, 23. Based on plaque
reduction assays we found that all compounds
except for MLS8969 had a slope near 1.
herpes simplex
2 (HSV2), mouse CMV
(MCMV), and Epstein Barr virus (EBV) was
tested with the five compounds. Plaque
reduction assays were used for HSV1, HSV2
and MCMV. Inhibition of lytic EBV replication
was quantified in AKATA cells following IgG
induction by real-time PCR. Only MLS8554
was active against HSV1, HSV2 and MCMV
(Table 2). NCGC2955 was active against MCMV
(EC50 of 1±0.0 µM, Table 2), but toxicity to
MEFs was observed at 10 µM and higher
concentrations. NFU1827 showed activity
against EBV (EC50 – 1.92±0.1 µM). The other
compounds, MLS8969 and MLS8091 had
specific activity against HCMV and did not
inhibit HSV1, HSV2, EBV or MCMV.
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Compound
MLS8969
NFU1827
MLS8554
MLS8091
NCGC2955
GCV
EC50 - HSV1-CI (µM)
EC50 – HSV2-CI (µM)
EC50 - EBV (µM)
EC50 - MCMV (µM)
NA
NA
>10
NA
NA
NA
1.92±0.1
NA
NA
1.78±0.2
NA
1.3±0.1
NA
11.67±2.0
NA
>10
NA
NA
NA
1.0±0.0
0.96±0.1
0.04±0.0
0.58±0.0
5.2±0.4
Table 2. Activity of the five compounds against other human herpes viruses and mouse CMV.
NA- Not active; CI- Clinical isolate
2.4. HCMV inhibition timing: Add-on and
removal assays were carried out at 0, 6, 12, 24,
36 and 48 hpi, and luciferase activity was
quantified at 72 hpi. Based on these assays
each compound displayed a specific time of
maximal activity. The compounds are
reported below based on the timing of
maximal virus suppression.
2.5. Inhibition of HCMV entry: Compound
MLS8969 was first tested after infection at
MOI of 1 PFU/cell using a luciferase assay at 72
hpi, but minimal reduction in luciferase
activity was observed (Fig. 3A). However, a
plaque assay showed good anti-HCMV activity
of MLS8969 (Fig. 3B).
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Figure 3. MLS8969 inhibits HCMV replication. A, Following infection of HFFs with HCMV pp28-luciferase Towne (MOI= 1
PFU/cell), cells were treated with MLS8969 or GCV. Luciferase activity was measured at 72 hpi. B, Following infection of
HFFs with HCMV TB40 (100 PFU/well), cells were treated with MLS8969 or GCV. Plaques were stained and enumerated
after 10 days. C, HFFs were pretreated with MLS8969 or GCV for 24 h followed by infection with HCMV Towne at MOIs 1,
0.1, and 0.01. Luciferase activity was quantified after 3 days. D, E, HFFs were pretreated with the specified concentrations
of MLS8969 or GCV for 24 h followed by infection with HCMV TB40 (D) or GCV-resistant HCMV Towne (E), at 100 PFU/well.
After 10 days plaques were stained and enumerated. A-E Data are average ± SD of triplicate values of a representative
experiment. F, HFFs were either pre-treated or infected followed by treatment. Infection (Towne) was performed at MOI 1,
0.1 and 0.01. The expression of viral proteins from cell lysates was quantified at 72 hpi. WB images are from a representative
experiment.
Figure 4. MLS8969 is an entry inhibitor of HCMV. HFFs were pretreated (24 h) with MLS8969 (3 µM) followed by
infection with HCMV Towne (MOI = 1, 0.1, 0.01). Heparin (30 µM) was used as a positive control for blocking viral entry
into the cells and GCV pretreatment (5 µM) was used as a negative control. IFA for HCMV-encoded pp65 was performed 2
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hpi to determine the amount of pp65 localized in the nucleus under the different conditions. The experiment was
repeated independently three times, and images from a representative experiment are shown.
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Because in the high-throughput screens HFFs
were treated with compounds before infection
(pre-treatment), and our plaque assay, in
which MLS8969 was added after infection,
revealed an EC50 of 0.12 ± 0.0 µM (Table 1), we
suspected lack of inhibition of luciferase
activity could represent a MOI-dependent
phenomenon, and thus suggestive of entry
inhibition24. The assay was therefore
performed using a range of MOIs (0.01, 0.1 and
1 PFU/cell) (Fig. 3C) and MLS8969 was added
before infection. MOI-dependent inhibition of
luciferase activity was observed (Fig. 3C).
TB40 and a GCV-resistant HCMV Towne were
also inhibited with MLS8969 pretreatment in
the plaque reduction assay (Fig. 3D&E).
Western blot analysis corroborated these
data, showing no inhibition of viral protein
expression at post or pretreatment at MOI of 1
PFU/cell, while pretreatment followed by
infection at low MOI (0.01) resulted in
decreased IE1/2, pp65, UL84 and UL44
protein levels (Fig. 3F). Further evidence for
MLS8969 being an entry inhibitor was
obtained by an indirect immunofluorescence
assay (IFA) for HCMV pp65 (Fig. 4). MLS8969
inhibited HCMV entry only at MOI of 0.1;
irrespective of MOI, GCV did not affect pp65
nuclear staining (Fig. 4). Heparin (positive
control) inhibited HCMV entry.
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Figure 5. NFU1827 is an immediate early-to early inhibitor of HCMV replication. A, Following infection of HFFs with HCMV
TB40 (100 PFU/cell), cells were treated with the specified concentrations of NFU1827. At 8 dpi plaques were stained and
enumerated. The experiment was repeated three times and data shown are average ± SD of triplicate values of a
demonstrative experiment. B, Following infection of HFFs with a GCV-resistant HCMV Towne (MOI = 0.1), cells were treated
with the specified concentrations of NFU1827. Luciferase activity was assayed in cell lysates at 72 hpi. Data shown are
average ± SD of triplicate values of a representative experiment. C, D, HFFs were infected with HCMV Towne (MOI = 1
PFU/cell) followed by addition or removal of compounds (0, 6, 12, 24, 48, 72 hpi). Luciferase activity was quantified in cell
lysates after 3 days. Data shown are average ± SD of triplicate values of a demonstrative experiment. E, Following infection
of HFFs with HCMV Towne (MOI = 1), cells were treated with the specified concentrations of compounds. The expression
level of viral proteins and cellular β-actin was determined at 72 hpi. The experiment was repeated twice, and WB images
are from a demonstrative experiment. F, G Following infection of HFFs with HCMV Towne (MOI = 0.1), cells were treated
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with NFU1827 (5 µM) or GCV (5 µM). Viral DNA replication in cells (F), and viral DNA load in supernatants (G) was
quantified by US17 real-time PCR. The experiment was repeated three times and data shown are average ± SD of triplicate
values of a demonstrative experiment.
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Figure 6. MLS8554 is an immediate early to early inhibitor of HCMV replication. A, Following infection of HFFs with HCMV
TB40 (100 PFU/well), cells were treated with the specified concentrations of MLS8554. Plaques were stained and
enumerated at 8 dpi. The experiment was repeated three times and data from a representative experiment are shown. B,
Following infection of HFFs with a GCV-resistant HCMV Towne (MOI = 0.1), cells were treated with the specified
concentrations of MLS8554. Luciferase activity was measured in cell lysates at 72 hpi. Data shown are average ± SD of
triplicate values from a single experiment. C, D Following infection of HFFs with HCMV Towne (MOI = 1) compounds were
either added or removed at the specified time points. Luciferase activity was quantified in the cell lysates after 3 days. The
experiment was repeated independently twice, and representative data from a single experiment are shown. E, Following
infection of HFFs with HCMV Towne (MOI = 1), cells were treated with MLS8554 or GCV at the indicated concentrations.
The expression level of viral proteins IE1/2, UL84, UL44, pp65 and cellular β-actin was determined after 3 days. WB data
are from a representative experiment. F, G HFFs were infected with HCMV Towne (MOI = 0.1) followed by treatment with
MLS8554 (2 µM) or GCV (5 µM). Supernatants were harvested and cells were lysed at the specified time points to isolate
DNA. Viral DNA replication in cells (F), and viral DNA load in supernatants (G) were quantified by US17 real-time PCR. Data
shown are average ± SD of quadruplicate values from two separate experiments.
2.6. Immediate early-to early stage
inhibitors of HCMV replication: Two
compounds showed anti-HCMV activity
between 0-24 hpi (NFU1827 and MLS8554).
NFU1827 was active against HCMV and GCV-R
HCMV, EC50 0.85±0.1 and 0.84±0.1 µM,
respectively (Table 1, Fig. 5 A&B), as well as
EBV (EC50 1.21±0.1 µM), but had no activity
against HSV1&2 or MCMV (Table 2). Add on
and removal assays revealed an immediate
early to early time of activity. When added to
infected cells after 24 h it lost its ability to
inhibit HCMV. Removal of NFU1827 after 6 h
already
achieved
significant
HCMV
suppression (Fig. 5 C&D). NFU1827 reduced
the level of both IE1 and IE2 (Fig. 5E). In
accordance with its immediate early to early
activity, viral DNA replication and DNA yield in
supernatants were significantly reduced (Fig.
5 F&G).
MLS8554 was active in most antiviral assays
inhibiting HCMV, GCV-R HCMV, HSV1&2 and
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MCMV (Fig. 6, Table 1&2). Its maximal activity
response against HCMV and GCV-resistant
HCMV with EC50 values of 0.39 ± .0.5 and 0.26±
0.0 µM, respectively (Table 1, Fig. 7), but was
not active against HSV1&2, EBV, or MCMV
(Table 2). The timing of MLS8091 activity
overlapped with that of GCV in the add-on
assay, but in the removal assay MLS8091
showed earlier effects (Fig. 7 C&D). Inhibition
of all viral proteins was observed at 72 hpi
(Fig. 7E). Inhibition of viral DNA replication
was higher than that achieved with GCV,
although the effects on DNA yield were not
significantly different from GCV (Fig. 7 F&G).
was measured at immediate early to early time
after infection. When added at 24 hpi or after,
its activity was significantly reduced, and
when removed at 6 h HCMV suppression was
already near complete (Fig. 6 C&D). Western
blot showed reduced IE2, UL44, UL84 and
pp65 protein levels at 72 hpi (Fig. 6E). In
contrast to NFU1827 which reduced both IE1
and IE2, MLS8554 mostly reduced IE2
expression. Both viral DNA replication and
DNA yield in supernatants were significantly
reduced (Fig. 6 F&G).
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2.7. Early to late stage inhibitors of HCMV
replication: MLS8091 showed
a
dose-
Figure 7. MLS8091 inhibits HCMV replication at early to late stage. A, Following infection of HFFs with HCMV TB40 (100
PFU/well), cells were treated with the specified concentrations of MLS8091. At 8 dpi plaques were stained and enumerated.
The experiment was repeated independently three times, and data from a single representative experiment are shown. B,
Following infection of HFFs with a GCV-resistant HCMV Towne (MOI = 0.1), cells were treated with the specified
concentrations of MLS8091. Luciferase activity was quantified in cell lysates (72 hpi). Data shown are average ± SD of
triplicate values from a single experiment. C, D HFFs were infected with HCMV Towne (MOI = 1) followed by addition or
removal of compounds at different time points after infection (0, 6, 24, 48, 72 h). Luciferase activity was quantified in the
cell lysates after 3 days. The experiment was repeated independently twice, and representative data from a single
experiment are shown. E, Following infection of HFFs with HCMV Towne (MOI = 1), expression level of viral proteins and
cellular β-actin was determined at 72 hpi. WB data are from a representative experiment. F, G HCMV-infected HFFs (MOI =
0.1) were treated with MLS8091 (1.5 µM) or GCV (5 µM). Supernatants were harvested and cells were lysed at specified
time points to isolate DNA. Viral DNA replication (F), and viral DNA load in supernatants (G) were quantified by real-time
PCR. Experiments were repeated independently twice. Data shown are average ± SD of triplicate values from a single
experiment.
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Figure 8. NCGC2955 is an early to-late inhibitor of HCMV replication. A, Following infection of HFFs with HCMV TB40 (100
PFU/well), cells were treated with specified concentrations of NCGC2955. Plaques were stained and enumerated at 8 dpi.
The experiment was repeated independently three times, and data from a single representative experiment are shown. B,
Following infection of HFFs with a GCV-resistant HCMV Towne (100 PFU/well), cells were treated with the specified
concentrations of NCGC2955. Plaques were stained and enumerated at 8 dpi Data shown are average ± SD of triplicate
values from a single experiment. C, D HFFs were infected with HCMV Towne (MOI = 1) followed by addition or removal of
compounds at different times after infection (0, 6, 24, 48, 72 h). Luciferase activity was quantified in the cell lysates after 3
days. Data shown are the average of three independent experiments (average ± SD). E, Following infection of HFFs with
HCMV Towne (MOI = 1), expression levels of viral proteins was determined at 72 hpi. The experiment was repeated
independently three times, and data from a single representative experiment are shown. F, G Following infection of HFFs
with HCMV Towne (MOI = 0.1), cells were treated with NCGC2955 (3 µM) or GCV (5 µM). Supernatants were harvested and
cells were lysed at specified time points to isolate DNA. Cellular viral DNA replication (F), and viral DNA load in supernatants
(G) were quantified by real-time PCR. Data shown are the average ± SD of quadruplicate values from two independent
experiments.
NCGC2955 inhibited HCMV and GCV-R HCMV
(Fig. 8 A&B). For this compound plaque assays
were preferred, since it did not show a full
dose response with the pp28-luciferase assay.
The timing of NCGC2955 activity overlapped
with that of GCV in the add-on assay, but in the
removal assay NCGC2955 appeared to have
longer activity than GCV (Fig. 8 C&D). At 72 hpi
there was a strong reduction in viral protein
expression including IE1 and IE2 (Fig. 8E).
Despite inhibition of viral progeny by plaque
assays and the observed decrease in viral
protein levels, the influence of NCGC2955 on
viral DNA replication and viral DNA yield was
modest (Fig. 8 F&G), suggesting inhibition via
mechanisms that do not involve the DNA
replication machinery.
2.8. Patterns of drug combination with GCV
and letermovir: The effect of each compound
by itself or combined with GCV or letermovir
was tested in HFFs infected with HCMV. An
additive or mild synergistic effect was
observed with GCV (Fig. 9) or letermovir (Fig.
10) for all five agents. A ratio of observed over
expected fold inhibition of > 1 was considered
drug synergy, < 1 as drug antagonism, and = 1
as additive. The Bliss coefficient of drug
combination
is provided in Table 3. For
letermovir and its combination with each of
the five compounds the EC50 was determined
based on plaque assay. The combination of
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GCV and 2955 was also tested by plaque assay.
All other combinations and Bliss coefficients
were measured by pp28-luciferase. All Bliss
coefficients were close to 1, suggesting an
additive effect between the compounds.
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Figure 9. Combination of newly identified HCMV inhibitors and GCV is additive. A-E HFFs were infected with HCMV Towne
(MOI = 1) and first treated with each individual compound, followed by combination of each compound with GCV at
different doses. In the drug combination experiments HCMV-infected HFFs were treated with an initial drug concentration
of twice the EC50 value of individual compound and two-fold serial dilutions. Luciferase activity was quantified in cell lysates
after 3 days and the Bliss Model was used to calculate the antiviral activity of compounds in combination. Solid lines indicate
observed HCMV inhibition (dose- response) and dotted lines indicate expected HCMV inhibition at each dose of drug
combination. A-D Experiments were repeated three times independently. Data from a single representative experiment are
shown. E, Data shown represent average ± SD of triplicate values from a single experiment.
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Figure 10. Combination of newly identified HCMV inhibitors with letermovir is additive. A-E HFFs were infected with TB40
(100 PFUs/well) and treated with each compound alone, and combination of each compound with letermovir at different
doses. For drug combination HCMV-infected HFFs were treated with starting concentration of twice the EC50 value of
individual compound followed by two-fold serial dilutions. The number of plaques in each condition was enumerated at 10
dpi and antiviral activity of compounds was calculated using the Bliss Model. Solid lines indicate observed HCMV inhibition
(dose-response) and dotted lines indicate expected HCMV inhibition at each dose of drug combination. Data shown are
average ± SD of triplicate values from a single experiment.
Compound 1
Letermovir
EC50
Compound 2
EC50 (μM)
Bliss Coefficient
1.1 ± 0.1 (nM)
MLS8969
NFU1827
MLS8554
MLS8091
NCGC2955
0.32 ± 0.0
0.61 ± 0.1
0.14 ± 0.0
0.13 ± 0.1
3.19 ± 0.2
1.2
1.2
1.0
1.3
1.1
GCV
GCV
GCV
GCV
GCV
1.33 ± 0.2 (μM)
1.63 ± 0.2
MLS8969
NFU1827
MLS8554
MLS8091
NCGC2955
0.72 ± 0.1
0.69 ± 0.1
0.55 ± 0.0
0.57 ± 0.1
1.51 ± 0.0
0.9
1.2
1.1
1.3
0.9
1.91 ± 0.6
1.25 ± 0.6
0.77 ± 0.1
Table 3. Bliss coefficient for drug combination. The EC50 was determined for each compound in the combination
experiment. For the letermovir combinations, a plaque reduction assay was performed with HCMV Towne. The GCV
combinations used the pp28-luciferase HCMV Towne except for GCV and NCGC2995 which was performed with
HCMV TB40. Letermovir combination assays were performed in a single experiment, whereas GCV combination with
the specified compounds was performed in separate experiments for which the EC50 was determined.
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3. DISCUSSION AND CONCLUSIONS
fluorescent intensity25. This screen used a
fixed drug concentration (10 µM) at time of
infection and a limited number of hits had no
apparent cell toxicity. Reporter lines have
previously been created to screen for anti-
HCMV compounds26, 27. One approach used a
promoter in front of a luciferase reporter that
was activated by IE proteins. Consequently,
inhibitors of later HCMV infection stages could
not be discovered using this reporter cell
line27. The Gray Kinase Inhibitor library (187
compounds) was screened against the HCMV
strain AD169, and cells were immunostained
for the HCMV antigen pp28. Three kinase
inhibitors were identified as inhibitors of IE2
production28. Recent drug repurposing
screening used the Spectrum Collection of
2560 compounds from Micro Discovery
System, Inc29, 30. Mercorelli used a mechanism-
based screen that identified compounds that
interfered with the transactivating activity of
IE2. Phenotypic assays measured the
expression of EGFP fused to IE231 or UL9932. A
major success of HTS was the identification of
letermovir (AIC246) from a screen of a
compound library33, which in subsequent
studies was shown to harbor a novel mode of
HCMV inhibition34.
We report the results of the largest HTS to date
for HCMV inhibitors using our pp28-luciferase
HCMV. This reporter virus has shown high
sensitivity and reproducibility for such
screening20. Its advantage is that HCMV-
encoded pp28 is a true late gene, thus its
activation represents a near complete cycle of
HCMV replication.
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Screening campaigns may result in false
positive hits and require intense validation.
Our combined approach using multiple HCMV
strains and several detailed anti-viral assays
resulted
in
the
identification
and
characterization of five distinct hits, each with
its unique characteristics. Of the five hits
MLS8969 inhibited HCMV entry, MLS8554 and
NFU1827 inhibited an immediate early to
early stage of HCMV replication (but were
different from one another in their effects on
other
herpesviruses),
MLS8091
and
NCGC2955 were active at an early to late stage,
but NCGC2955 did not have strong
suppression of viral DNA replication. All five
compounds demonstrated additive effects or
mild synergy when combined with GCV or
letermovir. No antagonism was found in any of
the drug combination experiments. In
addition, the five compounds were active
against GCV-resistant HCMV, indicating a
mechanism separate from the viral DNA
polymerase. Future studies should provide
insights into the mechanism of action of each
of five compounds. Based on data presented
here, we expect each compound to have a
unique mechanism for HCMV suppression.
In conclusion, our study provides several
novel compounds that inhibit HCMV
replication at sub to low µM concentrations.
Each of these compounds represents a good
candidate for future mechanistic studies and
drug development.
4. EXPERIMENTAL SECTION:
Screens with libraries containing a limited
number of small molecules, different HCMV
strains, cell types and timing of treatment have
been performed. Three recombinant HCMV
strains carrying enhanced yellow fluorescent
protein (EYFP) fusions of the immediate early
2 (IE-2), ppUL32 (pp150), and ppUL83 (pp65)
viral proteins were tested against a cellular
kinase library (80 compounds) as a pilot
screen. Several kinase inhibitors blocked
HCMV replication as shown by decreased
4.1. Compounds: The compounds were
identified from a high throughput screen
(HTS) and then validated using several HCMV
strains, multiple antiviral assays and new
compound preparations. In addition to
compounds obtained from the libraries used
in the screening, NFU1827 was synthesized in
the Department of Biochemistry, Program in
Chemical Biology at the Medical College of
Wisconsin, MLS8091 was purchased from
Princeton Biomolecular Research, Inc.,
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MLS8969 was purchased from ChemBridge
N-isopropylpiperidine-4-carboxamide
Corporation, San Diego, CA, MLS8554 was
purchased from InterBioScreen STOCK1N-
59867, and NCGC2955 was synthesized in the
Chemistry Core, Johns Hopkins University
School of Medicine. The purity of all
synthesized and purchased compounds used
in bioassays was determined by HPLC using
either a Phenomenex Luna C18 3.0 × 75 mm
column with a 7 min gradient of 4-100% ACN
in H2O with 0.05% v/v TFA or a Higgins
Analytical, Inc. Targa C18 5 µm 4.6 × 150 mm
column with a 30 min gradient of 0-100% ACN
in H2O with 0.01% TFA and either absorbance
detection at 254 nm or evaporative light
scattering detection. All compounds used in
bioassays exhibited NMR and MS data
consistent with their structures and purities of
>95% as determined by HPLC.
trifluoroacetic acid salt (2). To a solution of
compound 1 (2.95 g, 10.9 mmol) in CH2Cl2 (5
mL) was added triisopropylsilane (250 µL)
and trifluoroacetic acid (3 mL) at room
temperature. After stirring for 24 h at room
temperature, the volatiles were removed in
vacuo providing a viscous syrup used without
further purification. 1H NMR (500 MHz,
CDCl3) δ 4.06 (qd, J = 6.86, 13.38 Hz, 1H), 3.57
(d, J = 12.89 Hz, 2H), 3.10 (br. s., 2H), 2.45 -
2.63 (m, 1H), 2.05 - 2.16 (m, 4H), 1.18 (d, J =
6.60 Hz, 6H).
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methyl 4-(4-chlorobenzyl)-4H-thieno[3,2-b]
pyrrole-5-carboxylate (3). To a suspension of
methyl
4H-thieno[3,2-b]
pyrrole-5-
carboxylate36 (1.44 g, 7.99 mmol, 1.0 equiv.)
and cesium carbonate (3.91 g, 12.0 mmol, 1.5
equiv.) in anhydrous DMF (10 mL) was added
4-chlorobenzyl bromide (1.97 g, 9.59 mmol,
1.2 equiv.) in one portion at rt. After stirring at
rt until complete (ca. 4 h), the reaction was
diluted with water and extracted with EtOAc
(3 × 20 mL). The organic layers were
combined, washed with brine, dried with
anhydrous MgSO4, and concentrated in vacuo.
Purification by flash chromatography (3m 0%,
Dimethyl sulfoxide (DMSO) was used to
dissolve compounds, and stock solutions of 50
and 10 mM were stored at -80 °C. Ganciclovir
was purchased from Sigma Aldrich (St. Louis,
MO), a 10 mM stock solution was prepared in
ddH2O.
Synthesis of NCGC2955: tert-butyl 4-
(isopropylcarbamoyl) piperidine-1-carboxylate
(1). To a solution of 1-(tert-butoxycarbonyl)
piperidine-4-carboxylic acid35 (3.94 g, 17.2
mmol, 1.0 equiv.) in CH2Cl2 (39 mL) was added
N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (3.46 g, 18.0
mmol, 1.05 equiv.), 1-hydroxybenzotriazole
monohydrate (2.83 g, 18.5 mmol, 1.075
equiv.) and isopropylamine (5.1 mL, 60.15
mmol, 3.5 equiv.). After stirring at room
temperature for 21 h, the reaction was diluted
with CH2Cl2, and washed with 5% aq. HCl. The
aqueous layer was extracted with CH2Cl2 (3 ×
20 mL). The organic layers were combined,
6m gradient
0
to 50%, 2m 50%
EtOAc/hexanes) provided 2.40 g of methyl
ester 3 as a pale yellow solid in 98% yield. 1H
NMR (500 MHz, CDCl3) δ 7.33 (d, J = 5.34 Hz,
1H), 7.21 - 7.29 (m, 3H), 7.05 (d, J = 8.17 Hz,
2H), 6.85 (d, J = 5.50 Hz, 1H), 5.71 (s, 2H), 3.83
(s, 3H).
4-(4-chlorobenzyl)-4H-thieno[3,2-b] pyrrole-
5-carboxylic acid (4). To a mixture of methyl
ester 3 (3.58 g, 11.7 mmol, 1.0 equiv.) in THF
(12 mL) and MeOH (12 mL) was added a
solution of lithium hydroxide monohydrate in
water (12 mL) in one portion. After 2 d of rapid
magnetic stirring at rt, the starting material
was consumed, and the organic solvents were
removed in vacuo. The remaining aqueous
solution was diluted with water (ca. 20 mL)
and under vigorous magnetic stirring the pH
was adjusted to pH = 1 with conc. aq. HCl. A
precipitate formed during acidification, and
was collected via vacuum filtration, washed
with water and dried under vacuum to provide
3.34 g of compound 4 in 98% yield. 1H NMR
dried
with
anhydrous
MgSO4,
and
concentrated in vacuo. Purification by flash
chromatography (0 to 100% EtOAc/hexanes)
provided 2.95 g compound 1 as a solid in 63%
1
yield. H NMR (500 MHz, CDCl3) δ 5.28 (d, J =
6.45 Hz, 1H), 4.01 - 4.25 (m, 3H), 2.73 (br. s.,
2H), 2.16 (tt, J = 3.69, 11.63 Hz, 1H), 1.79 (d, J
= 11.32 Hz, 2H), 1.61 (dq, J = 4.24, 12.42 Hz,
2H), 1.45 (s, 9H), 1.14 (d, J = 6.60 Hz, 6H).
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(500 MHz, DMSO-d6) δ 12.57 (br. s., 1H), 7.56 (d,
1H), 8.69 (s, 1H), 8.63 (d, J = 1.2 Hz, 1H), 8.10
(dd, J = 1.6, 8.3 Hz, 1H), 8.01 (d, J = 8.4 Hz, 1H),
7.78 (d, J = 8.7 Hz, 1H), 6.99 (d, J = 8.8 Hz, 1H),
3.77 (s, 3H).
J = 5.19 Hz, 1H), 7.29 - 7.43 (m, J = 8.49 Hz, 2H),
7.17 - 7.29 (m, 2H), 7.05 - 7.17 (m, J = 8.49 Hz,
2H), 5.76 (s, 2H).
1-(4-(4-chlorobenzyl)-4H-thieno[3,2-b]
N-[3-(4-ethylpiperazin-1-yl)propyl]-4-(4-
methoxyphenyl)-7-thia-2,5-diazatricyclo
[6.4.0.02,6] dodeca-1(8),3,5,9,11-pentaene-
10-carboxamide (NFU1827). Compound 5
pyrrole-5-carbonyl)-N-isopropylpiperidine-4-
carboxamide (NCGC2955). To a mixture of
compound 4 (51.9 mg, 0.177 mmol, 1.0 equiv.)
in CH2Cl2 (1.7 mL) was added N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (37.5 mg, 0.19 mmol, 1.1
equiv.), 1-hydroxybenzotriazole monohydrate
(31.0 mg, 0.204 mmol, 1.15 equiv.) and
diisopropylethylamine (123 µL, 0.71 mmol, 4
equiv.). After stirring at room temperature for
5 min, TFA salt 2 (60.7 mg, 0.21 mmol, 1.2
equiv.) was added as a solution in CH2Cl2 (0.2
mL, plus 0.2 mL rinse) and the reaction stirred
at rt. After 24 h, the reaction was diluted with
CH2Cl2, and washed with 5% aq. HCl. The
aqueous layer was extracted with EtOAc (3 ×
10 mL). The organic layers were combined,
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(100
mg,
0.308
mmol), N-(3-
Dimethylaminopropyl)-N′-ethylcarbodiimide
hydrochloride (118.2 mg, 0.617 mmol), and 1-
hydroxybenzotriazole hydrate (83.3 mg, 0.617
mmol) were dissolved in DMF and stirred at
room temperature for 1 h. The reaction was
then treated with 3-(4-ethylpiperazin-1-yl)
propan-1-amine (0.2269 mL, 211.2 mg, 1.23
mmol) and stirred at room temperature for 18
h. The reaction was then poured into H2O (20
mL). This was extracted with EtOAc (3 × 20
mL). The combined extracts were washed with
H2O (4 × 20 mL) followed by brine (1 × 20 mL).
The organic layer was dried (Na2SO4), filtered,
and the solvent removed in vacuo. The crude
compound was purified by flash column
dried
with
anhydrous
MgSO4,
and
concentrated in vacuo. Purification by flash
chromatography (0 to 100% EtOAc/hexanes)
provided 48.9 mg of NCGC2955 as a white
solid in 62% yield. 1H NMR (500 MHz, CDCl3) δ
7.24 (d, J = 8.33 Hz, 2H), 7.18 (d, J = 5.19 Hz,
1H), 7.08 (d, J = 8.33 Hz, 2H), 6.83 (d, J = 5.19
Hz, 1H), 6.58 (s, 1H), 5.43 (s, 2H), 5.19 (d, J =
8.02 Hz, 1H), 4.44 (d, J = 12.89 Hz, 2H), 4.08
(qd, J = 6.77, 13.34 Hz, 1H), 2.92 (t, J = 12.34
Hz, 2H), 2.24 (tt, J = 3.62, 11.08 Hz, 1H), 1.80
(d, J = 14.93 Hz, 2H), 1.43 - 1.63 (m, 6H), 1.15
(d, J = 6.60 Hz, 6H).
chromatography
using
hexanes/EtOAc
(gradient elution from 90:10 to 0:100) to
afford 60 mg of NFU1827 in 41% yield. 1H
NMR (500 MHz, CDCl3) δ 8.71 (s, 1H), 8.21 (s,
1H), 7.99 (d, J = 8.4 Hz, 1H), 7.91 (s, 1H), 7.80
(d, J = 8.7 Hz, 2H), 7.62 (d, J = 8.4 Hz, 1H), 6.96
(d, J = 8.7 Hz, 2H), 3.85 (s, 3H), 3.60 (q, J= 5.2
Hz, 2H), 2.63 (t, J = 5.5 Hz, 2H), 2.46 (q, J = 7.3
Hz, 2H), 1.82 (m, 2H), 1.78 (broad s, 8H), 1.10
(t, J = 7.2 Hz, 3H). 13C NMR (126 MHz, CDCl3)
δ 162.72, 156.34, 145.14, 130.89, 128.49,
127.24, 123.49, 123.25, 122.78, 120.26,
111.13, 109.09, 102.92, 57.32, 55.69, 52.27,
50.25, 49.91, 49.39, 38.42, 26.62, 20.55, 17.98,
11.12. HRMS (ESI+) m/z calcd for C26H31N5O2S
[M + H]+ 478.2272, found 478.2271.
Synthesis of NFU1827: 4-(4-methoxyphenyl)-
7-thia-2,5-diazatricyclo [6.4.0.02,6] dodeca-
1(8),3,5,9,11-pentaene-10-carboxylic acid (5).
2-Aminobenzothiazole-6-carboxylic acid (2.0
g, 10.30 mmol, 1.0 equiv.) and 2-bromo-4’-
methoxyacetophenone (2.60 g, 11.33 mmol,
1.1 equiv.) were charged to a pressure bottle
and suspended in 2-methoxyethanol (50 mL).
The suspension was heated to 40 °C for 24 h
and at 140 °C for 19 h. The reaction cooled to
rt and concentrated in vacuo. The crude
compound was purified by flash column
chromatography (5 to 75% EtOAc/hexanes) to
4.2. Cells: Human Foreskin fibroblasts (HFFs),
passage 12 to 16 (ATCC, CRL-2088) were
grown in Dulbecco’s modified Eagle medium
(DMEM) containing 10% fetal bovine serum
(FBS) (Gibco, Carlsbad, CA) in a 5% CO2
incubator at 37 °C. Mouse embryonic
fibroblasts (MEFs; ATCC, CRL-1658) were
used for infection with mouse CMV. Vero cells
were obtained from the laboratory of Dr. Gary
1
afford 1.83 g of compound 6 in 55% yield. H
NMR (500 MHz, DMSO-d6) δ 13.17 (broad s,
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Hayward, Johns Hopkins University School of
Medicine.
and HSV2 replication in Vero cells, the
adsorption time was 60 min and plaques were
counted after 36 h. Plaque assays in MEFs
were completed after 3 days.
4.3. Cytotoxicity assay: MTT assay was
performed as described by manufacturer’s
instructions (Sigma-Aldrich). Non-infected
cells were treated with compounds for 72 h or
8-10 days (same time points as the antiviral
assay), and 20 μL/well of MTT [3-(4, 5-
Dimethyl-2-thiazolyl)-2, 5-diphenyl-2 H-
tetrazolium bromide], 5 mg/mL in phosphate
buffered saline (PBS)] was added to each well.
After shaking at 150 rpm for 5 min the plates
were incubated at 37 °C for 2-3 h. Conversion
of yellow solution to dark blue formazan by
mitochondrial dehydrogenases of living cells
was quantified by measuring absorbance at
560 nm.
4.7. Inhibition of lytic Epstein Barr Virus
(EBV) replication: AKATA cells (0.5 × 106/
well of a 24-well plate) latently infected with
EBV were induced with 100 μg/mL of goat
antihuman IgG (Sigma). Compounds and GCV
control were added to triplicate wells right
after addition of human IgG. Cells were
collected 48 h after lytic induction for DNA
quantification. Cellular DNA was purified
using the Wizard SV Genomic Kit (Promega,
Madison, WI). The effect of compounds on lytic
induction of EBV was analyzed by real-time
PCR of the EBV DNA polymerase gene,
BALF538.
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4.4 Viruses and antiviral assays: A pp28-
luciferase recombinant Towne HCMV strain
expressing luciferase under the control of the
UL99 (pp28) late promoter was reported to
provide a sensitive and reproducible reporter
for drug screening20. A GCV-resistant HCMV
4.8. Add-on and removal assays: These
assays were performed to identify at which
stage during HCMV replication the compounds
had maximal activity. In the add-on group,
compounds were added to infected HFFs at 0,
6, 24, and 48 hpi, and luciferase activity was
measured at 72 hpi. In the removal group,
compounds were added immediately after
virus infection and were subsequently
removed after 0, 6, 24, and 48 h; luciferase was
measured at 72 hpi.
pp28-luciferase that contains
a
C607Y
mutation in UL97 was reported37. The HCMV
TB40 strain was obtained from ATCC (VR-
1578). Clinical isolates of Human Herpesvirus
1 and 2 (HSV1, HSV2) were collected from the
Johns Hopkins Microbiology laboratory
without identifiers that could link them to a
specific patient. Mouse CMV (MCMV) Smith
strain (ATCC VR-1399) was used for infection
of MEFs.
4.9. DNA isolation and quantitative real-
time (qPCR): Total DNA was isolated from
non-infected control and HCMV-infected HFFs
using the Wizard SV genomic DNA isolation kit
(Promega, Madison, WI). To determine viral
load in the supernatant, total DNA was isolated
from supernatants using automated DNA
extraction on a BioRobot M48 instrument
(Qiagen, Valencia, CA). A US17 real-time PCR
assay that targets 151-bp from the highly
conserved US17 region of the CMV genome
was used39. The primers and probe used for
4.5. Luciferase activity: Cell lysates were
collected at 72 hpi and luciferase activity was
determined using the Glomax-Multi
+
Detection System (Promega, Madison, WI) as
described previously20.
4.6. Plaque reduction assay: HFFs were
seeded into 12-well plates (2 × 105 cells/well)
and infected with HCMV TB40 at
approximately 100 plaques/well. After 90
min, media were aspirated, and DMEM
containing 0.5% carboxymethyl-cellulose
(CMC), 4% fetal bovine serum (FBS), and
compounds were added into duplicate wells.
Following incubation at 37 °C for 8-10 days the
overlay was removed, and plaques were
counted after crystal violet staining. For HSV1
US17
were:
forward
5′-
GCGTGCTTTTTAGCCTCTGCA-3′, reverse 5′-
AAAAGTTTGTGCCCCAACGGTA-3′ and US17
probe FAM-5′ TGATCGGGCGTTATCGCGTTCT-
3′.
4.10.
HCMV
entry
and
indirect
immunofluorescence assay: MLS8969, GCV
and heparin control were used to determine
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inhibition of HCMV entry. Compounds were
Laboratories Inc. (Burlingame, CA). Mouse
monoclonal anti-HCMV UL44, mouse
diluted in serum-free media and combined
with HFFs seeded on chamber slides 24 h prior
to infection. After infection (MOI 1 or 0.1
PFU/cell) for 2 h, cells were fixed with 100%
chilled methanol, blocked for 1 h with
phosphate-buffered saline (PBS), 5% serum,
0.3% Triton X-100. Cells were then incubated
with mouse monoclonal anti-pp65 antibody,
monoclonal anti-HCMV UL84, and mouse anti-
β-actin anti-mouse IgG was from Santa Cruz
Biotechnology (Santa Cruz, CA). Horseradish
peroxidase (HRP)-conjugated anti-mouse IgG
was from GE Healthcare (Waukesha, WI).
Horseradish peroxidase (HRP)-conjugated
anti-rabbit IgG was from Cell Signaling
(Beverly, MA).
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1:50
dilution
(Vector
Laboratories,
Burlingame, CA) at 37°C in humidified
chambers for 1 h, washed three times with
TBST (0.1%), incubated with rhodamine-
conjugated anti-mouse IgG, 1:500 dilution
(Sigma) at 37 °C in humidified chambers for 1
h and washed with TBST (0.1%). A drop of
mount oil containing DAPI (Santa Cruz
Biotechnology, Santa Cruz, CA) was added to
the slides before visualization with a Zeiss ZI
fluorescence microscope. Images were
captured at 40× magnification.
4.13. Drug combination and analysis: These
experiments were performed as previously
reported40. The combination of GCV and each
compound was tested using the pp28-
luciferase. Briefly, 2×106 HFFs/plate were
seeded in 96-well plates and infected with the
pp28-luciferase Towne HCMV strain (MOI=1).
First, a dose-response curve was generated for
each compound individually to determine its
EC50 value. Then, compounds were combined
at twice their EC50 values, diluted in DMEM
with 4% FBS, followed by serial dilution and
added in combination after infection.
Luciferase activity of each compound
individually and in combination was
quantified at 72 hpi. The combinations of
letermovir and each of the new compounds
were tested by the plaque assay using TB40.
The same principle applied in this assay for the
drug combination effect, and plaques were
counted at 8 dpi. The Bliss model was used to
calculate the effect of each drug combination
on pp28-luciferase activity and plaque
reduction. In this model, the drug combination
is represented in the following equation as the
product of two probabilistically independent
events41:
4.11.
SDS-polyacrylamide
gel
electrophoresis and immunoblot analysis:
Cell lysates containing an equal amount of
protein were mixed with an equal volume of
sample buffer (125 mM Tris-HCl, pH 6.8, 4%
SDS,
20%
glycerol
and
5%
β-
mercaptoethanol) and heated for 10 min at
100 °C. Denatured proteins were resolved by
Tris-glycine polyacrylamide gels (8-10%) and
transferred to polyvinylidine difluoride
(PVDF) membranes (Bio-Rad Laboratories,
Hercules, CA) by electroblotting. Membranes
were incubated in blocking buffer [5% w/v
non-fat dry milk and 0.1% Tween-20 in PBS
(PBST)] for 1 h, washed with PBST, and
incubated with primary antibodies at 4 °C
overnight. Membranes were washed with
PBST and incubated with horseradish
peroxidase-conjugated secondary antibodies
in PBST for 1 h at room temperature.
Following washing with PBST, protein bands
were visualized by chemiluminescence using
SuperSignal West Dura and Pico reagents
(Pierce Chemical, Rockford, IL).
Where D is the compound concentration, m is
the slope parameter, and EC50 is the effective
concentration resulting in 50% virus
inhibition. The combined effect of two
compounds (FU1+2) is calculated from the
product of the individual effects of the two
inhibitors (FU1 and FU2). In this model,
synergistic compounds will yield a ratio > 1 of
4.12. Antibodies: Mouse monoclonal anti-
HCMV IE1 and IE2 (MAB810) was from
Millipore (Billerica, MA). Mouse monoclonal
anti-HCMV UL83 (pp65) was from Vector
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the observed fold inhibition divided by the
expected fold inhibition. Antagonistic
compounds will yield a ratio < 1. Additive
compounds will yield a ratio = 1.
7
4.14. Statistical analysis: Student t-tests
were performed using Sigmaplot (Systat
Software, San Jose, CA) and GraphPad Prism
(GraphPad Software, La Jolla, CA). All sample
groups were compared to the control group
using a One-way ANOVA. P-values were
adjusted for multiple comparisons. In all
figures * indicates p value < 0.05, ** indicates
p<0.01, and *** indicates p < 0.001. The curve
fitting toolbox, MATLAB software (v7.10),
MathWorks (Natick, MA) was used to
determine EC50 and CC50 values using a four-
parameter logistic regression.
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Table of Contents Graphics: Time of maximal activity of the five new inhibitors and GCV during HCMV replication
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Addon and removal assays were performed for each of the five compounds as described in the materials and methods
section and shown for each compound in Figs 5-8 C&D. The timing of 75% reduction in luciferase activity was calculated
for each compound both when added and when removed and is depicted in a unique color. The individual addon and
removal assays are provided in Fig 5,6,7,8 C&D.
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Author Contributions
AK, AKG, MF performed all the validation and characterization experiments and analyzed the data.
MF and WY performed the HTS. MF, NS, XH, JM and RAB analyzed the HTS data. RFK, BCS, DM
resynthesized several compounds and performed compound characterization. RAB wrote the
paper, conceived and supervised the project.
ACKNOWLEDGMENT
Funding: Supported by NIH R01DC013550 (RAB), NIH R35GM128840 (BCS), and the Flight
Attendant Medical Research Institute (DJM). Work at NCATS was funded by the NIH Intramural
Research Program. Competing interests: None.
We thank Dr. Francis Peterson for assistance with purity determination of compounds by HPLC. We
thank Dr. Vera Tarakanova for providing the AKATA cells.
ABBREVIATIONS
CMV- cytomegalovirus, MCMV- mouse cytomegalovirus, HSV- herpes simplex virus, EBV- Epstein
Barr Virus, HTS – high throughput screening, HPLC – high performance liquid chromatography.
.
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