Broad Spectrum Inhibitor of Influenza A and B Viruses Targeting the Viral Nucleoprotein

Article abstract of DOI:10.1021/acsinfecdis.7b00120

S119 was a top hit from an ultrahigh throughput screen performed to identify novel inhibitors of influenza virus replication. It showed a potent antiviral effect (50% inhibitory concentration, IC₅₀ = 20 nM) and no detectable cytotoxicity (50% c

Full text of DOI:10.1021/acsinfecdis.7b00120

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Article
A Broad Spectrum Inhibitor of Influenza A and
B Viruses Targeting the Viral Nucleoprotein
Kris M White, Pablo Abreu, Hui Wang, Paul D De Jesus, Balaji Manicassamy,
Adolfo García-Sastre, Sumit K Chanda, Robert J DeVita, and Megan L. Shaw
ACS Infect. Dis., Just Accepted Manuscript • DOI: 10.1021/acsinfecdis.7b00120 • Publication Date (Web): 21 Dec 2017
Downloaded from http://pubs.acs.org on December 26, 2017
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A Broad Spectrum Inhibitor of Influenza A and B Viruses Targeting the Viral
Nucleoprotein
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Kris M. White1, Pablo Abreu Jr.1, Hui Wang2^, Paul D. De Jesus3, Balaji Manicassamy1#,
Adolfo GarcíaꢀSastre1,4,5, Sumit K. Chanda3, Robert J. DeVita2,6 and Megan L. Shaw1*
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1Department of Microbiology, 2Department of Pharmacological Sciences, 3 Immunity and
Pathogenesis Program, Infectious and Inflammatory Disease Center, Sanford Burnham
Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, California
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92037, USA, Department of Medicine, Division of Infectious Diseases, Global Health
and Emerging Pathogens Institute, 6Drug Discovery Institute, Icahn School of Medicine
at Mount Sinai, New York, NY 10029, USA,
^Present Address: Suzhou Novartis Pharma Technology Co., Ltd. 18 Tonglian Road,
Riverside Industrial Park, Changshu Economic Development Zone, Changshu / Jiangsu
Province 215537, China.
#Present Address: Department of Microbiology, University of Chicago, Chicago, Illinois,
USA.
*Corresponding author
Department of Microbiology
Icahn School of Medicine at Mount Sinai
One Gustave L. Levy Place
New York, NY 10029, USA.
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Tel.: +1ꢀ212ꢀ241ꢀ8931
megan.shaw@mssm.edu
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S119 was a top hit from an ultraꢀhigh throughꢀput screen performed to identify novel
inhibitors of influenza virus replication. It showed a potent antiviral effect (IC50 = 20 nM)
and no detectable cytotoxicity (CC50 >500 ꢁM) to yield a selectivity index greater than
25,000. Upon investigation, we found that S119 selected for resistant viruses carrying
mutations in the viral nucleoprotein (NP). These resistance mutations highlight a likely
S119 binding site overlapping with, but not identical to that found for the compound
nucleozin. Mechanism of action studies revealed that S119 affects both the
oligomerization state and cellular localization of the NP protein which impacts on viral
transcription, replication and protein expression. Through a hitꢀtoꢀlead structureꢀactivity
relationship (SAR) study, we found an analog of S119, named S119ꢀ8, which had
increased breadth of inhibition against influenza A and B viruses accompanied by only a
small loss in potency. Finally, in vitro viral inhibition assays showed a synergistic
relationship between S119ꢀ8 and oseltamivir when they were combined, indicating the
potential for future drug cocktails.
Key Words: influenza, antiviral, nucleoprotein, aggregation, nucleozin
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Influenza virus is an important human pathogen, which accounts for significant morbidity
and mortality worldwide in annual epidemics. In addition, through genomic reassortment,
influenza A virus has and will continue to cause periodic global pandemics. The worst of
these was the 1918 “Spanish flu”, which caused an estimated 50ꢀ100 million deaths.32
During the most recent H1N1 pandemic in 2009, efforts to produce and deliver a vaccine
to protect the world population were outpaced by the spread of the virus. This failure of
the influenza pandemic vaccination program highlights the importance of antiviral
therapies under pandemic conditions. Moreover, coverage of seasonal influenza vaccines
is estimated at only 33ꢀ73%33, leaving a significant proportion of the population
vulnerable to infection every year.
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There is an urgent need for new antiviral therapies against influenza virus, as the two
current FDAꢀapproved drug classes have both suffered from significant issues of viral
resistance, either currently or in the recent past. The CDC no longer recommends the M2
inhibitors (amantadine and rimantadine) for use in the clinic due to high levels of
resistance in both H1N1 and H3N2 circulating strains1. Neuraminidase inhibitors, of
which the orally available oseltamivir is the most heavily prescribed, are currently the
only FDA approved antiviral drugs recommended for use against influenza virus.
Although oseltamivir resistance remains relatively low (0ꢀ0.8%) currently, during the
2007ꢀ2008 influenza season the circulating H1N1 strain gained nearly complete
resistance2. This oseltamivir resistant strain was fortunately replaced by the 2009 H1N1
pandemic strain, but the quick spread of this resistance, to our last line of antiviral
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defense, is highly concerning and highlights the necessity of finding new therapeutics
with different targets.
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The influenza nucleoprotein (NP) is a critical factor in many stages of the viral life cycle.
The NP protein coats the viral genomic RNA to form ribonucleoproteins (RNPs)3 and is
involved in transportation of RNPs to the nucleus4, viral transcription and replication5,6,
and virion assembly7. Therefore, NP is localized within the cytoplasm during viral entry,
the nucleus during viral replication and finally in the cytoplasm again during egress8.
Knowledge of the NP crystal structure20 and RNP cryoꢀEM structure3,9 has significantly
advanced our understanding of the multimeric nature of NP and how important its
oligomeric state is to viral replication. Recently, the NP protein was shown to be a valid
and efficient target for inhibition of influenza virus replication and pathogenesis.10,11,12
The compound, nucleozin, was found to inhibit influenza via blocking NP import into the
nucleus and causing abnormal aggregation within the cytoplasm11.
In a high throughꢀput screen which we have previously described13, 919,960 compounds
were screened for inhibition of influenza virus replication in a cellꢀbased assay. In this
screen, we identified a number of influenza specific inhibitors of viral replication with
subꢀmicromolar IC50’s. One of these compounds, designated S119, was found to inhibit
replication through alteration of the oligomeric state of the NP protein. Herein we discuss
the mechanism of action of S119 and a preliminary structure activity relationship (SAR)
study to improve the breadth of antiviral activity as proofꢀofꢀconcept for future drug lead
optimization.
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Results and Discussion
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S119 is a Specific Inhibitor of Influenza A Virus. The compound S119 (Figure 1a) was
identified as a potent hit from a previously published cellꢀbased screen of nearly one
million compounds utilizing a luciferaseꢀbased reporter influenza A/WSN/33 H1N1
(WSN) virus14. S119 potently inhibited both the reporter virus and wildꢀtype WSN virus
in MDCK cells with an IC50 of 60 nM (Figure 1b). When tested in the human lung cell
line, A549, S119 caused a 4 log reduction in WSN viral titers resulting in an IC50 of 20
nM (Figure 1c). Cytotoxicity of S119 was tested in A549 cells using the CellTiterꢀGlo
system (Promega) and the compound showed very little toxicity at concentrations as high
as 500 ꢁM, yielding a minimum SI of 25,000. When examined for activity against other
strains of influenza A virus, we found that S119 caused a 77% reduction in the pandemic
strain A/California/2009 (H1N1) infectious titer, but was ineffective against A/Puerto
Rico/8/1934 (PR8, H1N1), A/Panama/2007/1999 (H3N2) and A/Vietnam/1203/2004
(H5N1) (Table 2).
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S119 Inhibits a Post-Entry Step and Targets the NP protein. To begin to dissect the
mechanism of action of S119, we first sought to determine at which stage of viral
infection the compound was inhibiting. We performed a time of addition assay at an MOI
of 1 (A/WSN/33) with S119 treatments at ꢀ2, 0, 2, 4, 6, or 8 hours relative to infection.
We found that S119 was able to maximally inhibit virus replication when added before
infection and up to 2 hours after infection. The compound also showed significant virus
inhibition when added 4 hours after infection, while still maintaining a 1 log effect at 6
hours (Figure 2). This indicated that S119 was exerting its effect at a postꢀentry step of
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the viral life cycle, possibly during viral transcription and replication. To determine
whether S119 was targeting a viral protein, influenza virus was passaged in the presence
of S119 and S119ꢀresistant viruses were selected. Briefly, A549 cells were infected with
influenza A/WSN/33 virus at an MOI of 0.01 in the presence of 2 ꢁM S119, which
yielded enough viral progeny for subsequent passages at a low MOI as detected by viral
plaque assay. The remaining passages were tracked by hemagglutination assay to
determine if the virus had escaped inhibition by S119. Viral resistance to S119 was
detected after 3 to 4 passages and isolates were obtained from 7 independent experiments,
with 3 viruses plaque purified from each experiment and submitted for full genome
sequencing. Of the 21 sequenced viral genomes, 11 contained mutations leading to 7
unique amino acid changes all occurring within the nucleoprotein (NP). When these
resistance mutations were mapped onto the crystal structure of the NP protein, 6 of the 7
residues were found to form a single pocket on the surface of the protein (Figure 3a). The
remaining mutated residue, R98 was found to be within close proximity to this pocket as
well. We found this particularly illuminating due to the 246 amino acid distance between
the most distal residues (Y40 and A286). This indicated to us, that not only was NP the
likely target for S119 inhibition, but this pocket highlighted by resistance mutations is
also likely the S119 binding site within the NP protein. This putative binding pocket is
located in the "body" domain of NP where an important NPꢀNP interaction occurs in the
formation of the ribonucleoprotein (RNP) complex. Quite interestingly, a known inhibitor
of NP, nucleozin11, has been shown to have two binding sites on either side of the
putative S119 binding pocket15. Nucleozin is proposed to cause an atypical NPꢀNP
interaction through the "body" domains12, which subsequently leads to formation of
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large, replication deficient NP aggregates. S119 is structurally unrelated to nucleozin and
the binding sites of the two compounds appear to be distinct with a small amount of
overlap. Previous publications have shown that mutations at positions Y40, S50, D51,
and A286 (which confer S119 resistance) do not cause resistance to nucleozin, while a
mutation at position R55 causes resistance to both compounds12. The NP protein is highly
conserved amongst influenza viruses, so it is surprising that S119 did not have a broad
spectrum of inhibition, particularly in the case of the PR8 strain, which is closely related
to WSN. The NP proteins of WSN and PR8 share identical amino acids at each of the
resistance mutation sites identified, indeed these sites are also 99.8 to 100% conserved in
currently circulating influenza A viruses. Even currently circulating influenza B viruses
show 100% conservation of identical or similar amino acids in the corresponding sites of
the influenza B NP protein. Interestingly, there is a proline at position 283 of the PR8 NP
protein, whereas there is a serine at this position in WSN. This S283P substitution would
have a significant effect on the structure of the alphaꢀhelix in which the A286 resistance
site resides and therefore change the conformation of the putative S119 binding pocket. It
may be that the extended structure of the S119 molecule is highly intolerant of any
change to the size of its binding pocket.
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To further explore the impact of resistance to S119, the Y40F mutation was introduced
into a recombinant influenza A/WSN/33 virus using a reverse genetics system16. This
virus, referred to as rWSNꢀY40F, was not impaired for growth when compared to a wild
type recombinant virus in a multiꢀcycle growth assay (data not shown). When S119 was
tested for efficacy against rWSNꢀY40F, the mutant virus had a resistant phenotype, with
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an IC50 50ꢀfold higher than the wild type counterpart (Figure 3b). Conversely, the IC50 for
nucleozin was only 4.5ꢀfold higher with the Y40F NP mutation (Figure 3c). This minor
resistance phenotype is in agreement with the literature that this residue is adjacent to, but
not part of, the nucleozin binding site in NP.12
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S119 Causes Aggregation of the NP protein. The importance of the oligomeric state of
the influenza virus NP protein has been well defined through previous biochemical and
structural studies17, 18, 19, and the location of the S119 resistance mutations near to those
conferring nucleozin resistance indicated that alteration of the NPꢀNP interaction at this
interface may be the mechanism of S119 antiviral activity. To determine whether S119
has an effect on NP oligomeric state, purified baculovirusꢀexpressed NP was incubated
with and without compound for 30 minutes in the presence or absence of purified viral
RNA and analyzed by blue native PAGE. In the absence of RNA, the purified NP runs at
a low molecular weight, likely representing monomeric NP (Figure 4a, upper panel). The
band appears as a doublet because of a slightly truncated form of NP being coꢀexpressed
with the fullꢀlength protein during baculovirus expression. This was confirmed via
western blot analysis, where the truncated band is recognized by both antiꢀNP and antiꢀ
His antibodies. This indicated a Cꢀterminally truncated NP species, because the Hisꢀtag is
located on the Nꢀterminus of the recombinant protein. In Figure 4a (lower panel, lane 1),
both forms of NP are shown to be capable of oligomerizing on RNA and therefore the
truncated protein should not affect the experimental outcome. An active analog of the
published PB2 inhibitor VXꢀ78720 was used as a negative control. When treated with
S119 or nucleozin in the absence of RNA, the low molecular weight NP band is lost
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relative to controls. For nucleozin, it has been described that NP shifts into an
unresolvable higher order complex11, and this is likely the case for S119 treatment as
well. Interestingly, S119 appears to have a greater effect on the low molecular weight NP
than nucleozin at the same concentration, despite nucleozin having similar potency in
viral inhibition assays. In the presence of influenza vRNA, most of the NP runs at a
higher molecular weight (indicative of oligomers), which are clearly visible in the native
PAGE gel, but a small amount of low molecular weight NP remains present. In the case
of nucleozin treatment, this oligomer ladder becomes a smear on the gel, indicating the
formation of an aggregate. When treated with S119, a faint oligomer ladder still remains
and the low molecular weight band is completely lost. Our interpretation of this effect is
that S119 perturbs the NP oligomerization state both in the presence and absence of viral
RNA, but may be doing so in a manner different from nucleozin. These results could
indicate that S119 has a higher affinity for NP in the monomeric state as compared to
oligomerized NP within the RNP structure. While these experiments where suggestive of
the S119 mechanism of action, the inherent limitations of the in vitro system with a
tagged protein outside of the context of an influenza virusꢀinfected cell meant that
confirmation of these results would be required from alternative experimental systems.
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Due to the inability to resolve higher order aggregations in the native PAGE system it
was difficult to address the exact effect of S119 on NP oligomerization, so size exclusion
chromatography was employed to investigate this further. Lysates from WSN infected
A549 cells treated with S119 or nucleozin were applied to a Superose6 column and 1 ml
fractions were collected. S20, a compound that targets HA13, was used as a negative
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control. In infected cells, the vast majority of the expressed NP is present in RNP
complexes, which are quite large and appeared in the exclusion volume (fraction 8) in the
Superose6 column (Figure S1). However, some of the NP was maintained in a lower
molecular weight pool, which is likely to be monomers or small oligomers (fractions 17ꢀ
19). A large NP aggregate would be expected to appear in the exclusion fraction similar
to the RNP structure, making differentiation of these two states difficult in this
experimental set up. Therefore, lysates were RNase A treated following drug treatment to
disrupt the RNP complexes prior to being applied to the column for subsequent
experiments. Once the RNPs were RNase treated, a moderate molecular weight NP
oligomer pool was observed in fractions 11 through 14. A clear shift to smaller oligomers
(fractions 14ꢀ15) was observed in both S119 and nucleozin treated samples along with a
distinct loss of the low molecular weight NP pool (Figure 4b). While a reduction in the
oligomeric size of NP was unexpected, the fact that the known NP aggregator, nucleozin,
showed a similar effect indicated that S119 could be causing a similar formation of
abnormal NP complexes. The shift to smaller oligomers might be due to changes in the
exposure of viral RNA to RNase A in the presence of drug. For example, if the NP
aggregates caused by both S119 and nucleozin were to make the RNA more accessible to
enzymatic digestion than the RNA within the tightly packed RNP structure, one would
expect the observed reduction in oligomer size after RNase A treatment. Interestingly,
S119 appeared to cause a greater reduction in the low molecular weight NP pool than that
observed with nucleozin and this effect occurred in the absence of RNase treatment as
well (Figure S1), which aligns well with our assays using purified NP protein (Figure 4a).
When we examined NP from the rY40F S119ꢀresistant virus, we found that
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oligomerization was unaffected by S119 treatment, but was altered by nucleozin similarly
to wild type virus (Figure 4c).
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Finally, immunofluoresence microscopy was performed on WSN infected A549 cells to
determine NP localization at 2, 4, 6 and 24 hours after infection. The incoming NP
protein (as part of the RNP) is first found in the cytoplasm during viral entry into the cell.
Then, at around 4 hours post infection, newly synthesized NP is detected exclusively in
the nucleus, and later it is exported to the cytoplasm for packaging into virions. This
temporal pattern of NP localization was observed in the DMSO control experiment
(Figure 5). With nucleozin treatment, NP remains exclusively in the cytoplasm and
begins to form visible aggregates by 4 hours post infection, consistent with previous
publications.11 In the case of S119, while NP translocation to the nucleus was delayed, it
was not excluded. At 6 hours post infection NP was observed in the nucleus, though with
much weaker expression. Aggregation of NP was also observed under S119 treatment,
but not until the 24 hour post infection time point (Figure 5). The S119ꢀinduced
aggregates also appeared to be larger than those from nucleozin, possibly due to an
overall higher expression of NP in the presence of S119 compared to nucleozin. All of
these data taken together suggest that S119 is causing an abnormal oligomerization of the
NP protein, likely resulting in the formation of large aggregates.
NP Aggregation by S119 Causes a Segment Specific Inhibition of Viral
Transcription. The nucleoprotein is essential for both viral transcription and replication.
Therefore, we wanted to determine whether S119ꢀinduced NP aggregation caused any
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downstream effects on the production of mRNA and vRNA from the various influenza
genomic segments. We utilized primer extension assays to analyze mRNA and vRNA
production and western blot to detect protein expression from the PB2, NP and NS
segments as representatives of long, medium and short segments respectively. Treatment
of WT WSN infected A549 cells with nucleozin caused a complete loss of both mRNA,
vRNA and viral protein expression from all three segments, regardless of size (Figure 6a,
b, c). In contrast, S119 inhibition resulted in a segment specific effect. In the presence of
S119, mRNA expression from the PB2 segment was reduced to undetectable levels
(Figure 6a), although the small amount of PB2 protein indicates that there is a minimal
level of mRNA expression. In the case of the NP segment, mRNA expression was
severely reduced by S119, but still observable and NP protein levels were moderately
reduced (Figure 6b). Finally, mRNA expression from the NS segment was slightly
reduced, but NS1 protein expression was unaffected (Figure 6c). vRNA could not be
detected from any segment under S119 treatment, indicating a block in replication. In
contrast, the rY40FꢀWSN virus was completely resistant to S119 inhibition of
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transcription and replication, while nucleozin exhibited efficient inhibition of this virus
(Figure 6d, e, f). The loss of global vRNA expression could be explained through two
possible mechanisms. First, the loss of protein expression of the polymerase subunits and
the nucleoprotein would make RNP formation impossible, which is the functional unit for
both transcription and vRNA replication. Secondly, the accumulation of the NP
monomeric pool has previously been shown to be a key molecular trigger in the switch
from transcription of mRNA to replication of vRNA in the viral life cycle.5,21,22 The loss
of this monomeric NP pool, evident in the size exclusion chromatography data (Figure
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4b), could prevent this switch from occurring and thereby block vRNA synthesis from all
segments. These two mechanisms are not mutually exclusive, with each likely playing a
role in S119 inhibition of influenza virus replication. The observed differential inhibition
of mRNA expression by S119 from the three tested segments indicates a possible lengthꢀ
dependent effect of the compound. An explanation for this phenomenon could again be
due to the loss of the monomeric NP pool. It has been well established that the viral
polymerase requires an NPꢀcoated template for full processivity and that the lack of NP
will cause the polymerase to dissociate prematurely.23 Absence of monomeric NP could
cause a reduction in NPꢀencapsidation of viral vRNAs resulting in reduced processivity
of the viral polymerase. It would be reasonable to infer that a loss of processivity would
disproportionately affect longer segments, as we observe in our experiments.
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Broad Spectrum Influenza A Virus Inhibition by S119 Analogs. Our initial analysis
of the breadth of S119 antiꢀinfluenza virus activity indicated that it was only effective
against some H1N1 viruses with maximum potency observed against the A/WSN/33
laboratory strain and no effect on another H1N1 laboratory strain A/PR/8/34 (Table 1).
Due to this specificity, we performed a structure activity relationship study with a focus
on improving the antiviral breadth. Analogs of S119 were synthesized and screened for
activity against an influenza A/PuertoRico/8/34 virus expressing GFP24, and to ensure we
were not selecting for H1N1 specific compounds, all analogs were counter screened
against an A/Vietnam/1203/2004 (H5N1) HALO GFP virus. Successive rounds of analog
production (66 in total), using an iterative process, resulted in increased potency against
the PR8 and H5N1 viruses. We found that activity against both the H1N1 and H5N1
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viruses was also predictive of the ability to inhibit a representative H3N2 virus
(A/Panama/2007/1999). Therefore, we were confident that this screening process could
identify S119 analogs that had broad spectrum antiviral activity against influenza A
viruses, as listed in Table 1.
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S119 is a symmetrical bis(4ꢀtꢀbutylphenyl)carboxamide of 1,4ꢀdiaminobenzene that
showed no detectable antiviral activity on the two influenza strains that were screened,
and a related symmetrical 1,3ꢀbenzenediamine analog S119ꢀ6 also showed narrow
spectrum activity. It is interesting to note that the unsymmetrical analogs, as in the
shortened monoꢀ4ꢀtertꢀbutylphenyl carboxamide S119ꢀ2, the diphenyl ether bearing
carboxamide S119ꢀ3 or the diphenylamino analog S119ꢀ8, all possessed broader
spectrum antiꢀinfluenza activity as compared to S119. The analogs were designed to
explore the nature of the influenza activity pharmaocophore as well as to improve
calculated physical chemical properties of the lipophilic (cLogP: 6.81), poorly soluble
S119 parent hit molecule. We plan to expand this SAR and will report on our progress in
future manuscripts.
Of these analogs, S119ꢀ8 demonstrated that it had acquired enhanced broadꢀspectrum
activity (Figure 7, Table 2) with improved calculated physical properties (i.e. lower clogP:
5.95), while maintaining significant potency (IC50 = 1.43ꢁM) at nonꢀtoxic (CC50 =
66.10ꢁM) concentrations, albeit somewhat higher (7ꢀfold) than that of the parent S119
against WSN virus. Most interestingly, S119ꢀ8 also showed activity against multiple
influenza B viruses and an oseltamivirꢀresistant influenza A virus (Figure 7d, Table 2),
but did not inhibit a nonꢀinfluenza virus, VSV (Figure S2). As evidence that S119ꢀ8 was
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still targeting NP, we showed that the rWSNꢀY40F virus maintained resistance to S119ꢀ8
(Figure S3).
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S119-8 Synergizes with oseltamivir. Finally, future antiꢀinfluenza therapies are likely to
contain a cocktail of small molecules to deter the development of resistance25. Therefore
it is important to understand how S119 interacts with currently licensed drugs and
potential influenza drugs in development. To this end, we tested the effect of the S119
analog S119ꢀ8 in combination with either nucleozin or oseltamivir. A549 cells were
infected with influenza PR8ꢀGFP virus in the presence of the two compounds which were
titrated against each other in 3ꢀfold serial dilutions starting with a concentration above the
IC90 of each compound alone. Isobolograms of viral inhibition were generated (Figure 8)
and based on these data, the combination index for the S119ꢀ8ꢀnucleozin interaction was
determined to be 0.79, suggesting an additive to slightly synergistic interaction. This was
somewhat surprising as we anticipated that the close proximity of the resistance
mutations of the two compounds could have potentially caused an antagonistic
interaction. When we examined the interaction between S119ꢀ8 and oseltamivir, we
found a combination index of 0.57 which indicates a synergistic relationship between the
two compounds. This implies that if the in vivo PK and safety profile of S119ꢀ8 is
acceptable, this compound would be a good candidate to include in a potential antiꢀ
influenza drug cocktail by enhancing the potency of the current standard of care,
oseltamivir, and lowering the chances of developing resistance. Studies to improve the
antiviral and drugꢀlike properties26 of the S119 series are onꢀgoing.
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Conclusion
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The discovery of nucleozin has highlighted the viral nucleoprotein as a promising target
for novel therapeutics against influenza virus. The highly conserved nature of the
nucleoprotein27,28 and its critical importance in multiple stages of the viral life cycle make
it an ideal target. Here we present S119 and its analog S119ꢀ8 as antiviral drug leads for
the development of a panꢀinfluenza NP inhibitor with a defined mechanism of action.
Through a SAR study, S119ꢀ8 was shown to extend its breadth to multiple influenza A
virus subtypes and even to influenza B viruses, therefore not suffering from the same
specificity limitations as nucleozin. Furthermore, the rise in resistance to currently
approved influenza antivirals has emphasized the potential benefits of antiviral cocktails
for future therapeutics. The observed synergistic relationship between S119ꢀ8 and
oseltamivir is encouraging for incorporation of such a compound (or improved
derivatives) in future influenza antiviral drug cocktails.
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Methods
Cell culture and reagents. MadinꢀDarby canine kidney (MDCK) epithelial cells, human
alveolar epithelial (A549) cells, and human embryonic kidney 293T (293T) cells, were
obtained from the American Type Culture Collection (ATCC, Manassas, VA). MDCK,
A549, and 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM)
(Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS) (HyClone,
South Logan, UT), and 1% penicillinꢀstreptomycin (P/S) (Gibco). All cells were grown at
37°C, 5% CO2. Transfection of DNA was performed in OptiꢀMEM Iꢀreduced serum
medium (OptiꢀMEM) (Gibco) with Lipofectamine LTX (Invitrogen) in A549 cells
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according to manufacturer’s specifications.
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Expression plasmids and cloning. The influenza virus plasmid pPolIꢀNP Y40F was
generated by exchanging one nucleotide in the parental plasmid pPolIꢀNP16 using the
QuickChange siteꢀdirected mutagenesis kit (Agilent Technologies, Wilmington, DE)
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using
specific
primers
(forward:
5’ꢀ
GATGGAATTGGACGATTCTTCATCCAAATGTGCACCGAAC ꢀ3’; reverse: 5’ꢀ
GTTCGGTGCACATTTGGATGAAGAATCGTCCAATTCCATC ꢀ3’). Presence of the
mutation was confirmed by sequencing (Macrogen, Rockville, MD). The mammalian
expression vector pCAGGS containing a chicken βꢀactin promoter has been previously
described.29 Proper insertion and presence of the mutation was confirmed by sequencing
(Macrogen).
Viruses. The influenza A/WSN/1933 (H1N1) virus (WSN) was propagated in MDCK
cells for 2 days at 37°C. Influenza A/California/04/ 2009 (H1N1) virus was propagated in
MDCK cells for 3 days at 35°C. Influenza viruses A/Puerto Rico/8/1934 (H1N1) (PR8),
A/Brisbane/59/2007ꢀS,
A/Brisbane/59/2007ꢀR,
A/Wyoming/03/2003,
A/Panama/2007/1999 (H3N2) and A/Vietnam/1203/2004 (H5N1) bearing a mutated
polybasic cleavage site in the HA segment (HAlo) were propagated in 10ꢀday old
embryonated chicken eggs for 2 days at 37°C. The A/Brisbane/59/2007ꢀS and
A/Brisbane/59/2007ꢀR viruses are oseltamivir sensitive (S) and resistant (R) versions of a
clinical isolate generated using a reverse genetics system as previously described39 The
NS segment (A/Vietnam/1203/2004) carrying GFP was generated by overlapping fusion
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PCR using standard molecular biology techniques. Briefly, the NS1 ORF without the stop
codon was fused to the Nꢀterminal of a codonꢀoptimized maxGFP (Amaxa) via a GSGG
linker region (NS1ꢀGFP). The maxGFP was followed by a short GSG linker, a 19ꢀaa 2A
autoproteolytic site (ATNFSLLKQAGDVEENPG⇓P)34 derived from porcine
teschovirus–1 and by the NEP ORF. Also, silent mutations in the endogenous splice
acceptor site in the NS1 ORF were introduced to prevent splicing.35 The engineered NSꢀ
GFP segment was cloned in the pDZ IAV rescue plasmid.36 Influenza
B/Yamagata/16/1988 and B/Brisbane/60/2008 viruses was propagated in 8ꢀday old
embryonated chicken eggs for 3 days at 33°C. All influenza viruses were titered by
standard plaque assay in MDCK cells. Vesicular stomatitis virus was grown and titered
by plaque assay in VERO cells. Recombinant influenza viruses were generated using the
influenza virus rescue protocol as previously described.16 Briefly, 293T cells were
transfected with eight pPolI constructs expressing the PB1, PB2, PA, NP (or NP Y40F),
HA, NA, M, and NS genomic segments as well as pCAGGS expression plasmids
encoding the PB1, PB2, PA, and NP proteins. Twenty four hours post transfection,
MDCK cells were coꢀcultured with the transfected 293Ts for an additional 24–48 hours,
until cytopathic effects were observed. Newly generated viruses were collected and
plaqueꢀpurified, and the presence of the mutation was confirmed by sequencing.
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Small molecular weight compounds. Nucleozin was purchased from Sigma Aldrich (St.
Louis, MO). The active VXꢀ787 analog and oseltamivir carboxylate were kindly
provided as gifts by Roche (Basel, Switzerland). Compounds were purchased through
eMolecules (La Jolla, CA) and dissolved in 100% DMSO. The final concentration of
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DMSO in the culture medium did not exceed 0.5%.
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Cell viability assay. The CellTiterGlo Cell Viability Assay (Promega) was used to detect
ATP levels as a function of cell viability, according to manufacturer’s specifications.
A549 cells were seeded into 96ꢀwell plates (1250 cells/well) and incubated at 37°C, 5%
CO2 for 24 hours. Culture medium was then replaced with 100 uL of fresh medium
containing compound (serially diluted), and this was further incubated for 24 hours. Cell
viability was measured by adding 50 uL of CellTiterGlo reagent to each well, and the
luminescence signal was read using a plate reader (Beckman Coulter, Brea, CA). The
CC10 and CC50 for each experiment were determined using the Prism (GraphPad
Software) software.
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Viral growth assays in the presence of inhibitors. 100,000 A549 cells were seeded into
24ꢀwell plates and incubated for 24 h at 37 °C, 5% CO2. Two hours before infection, the
medium was replaced with DMEM containing the compound of interest at the indicated
concentrations. Compounds were absent during the 1ꢀh virus incubation but were present
in the DMEM postꢀinfection medium. Infections were performed at a low MOI (0.01ꢀ0.1)
for 24 or 48 hours, depending on virus used. For infections with influenza viruses, postꢀ
infection medium also contained 1 ug/mL TPCKꢀtreated trypsin (SigmaꢀAldrich, St.
Louis, MO). The infected cells were incubated at 37 °C with the exception of influenza B
virusꢀinfected cells, which were incubated at 33 °C. Viral titers were determined by
standard plaque assay in MDCK cells. The IC50 and IC90 for each experiment were
determined using the Prism (GraphPad Software) software.
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Selection of S119-resistant influenza viruses.
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The concentration of S119 required for maximum virus inhibition (3 logs), while
maintaining enough virus production for subsequent passages was determined (2ꢁM
S119). A549 cells were infected with WSN at an MOI of 0.01 for 24 hours at 37°C under
S119 treatment. The supernatant was then collected and titered by plaque assay. If the
recovered S119 treated virus did not show increased viral titer similar to that of the
DMSO treated control, the virus was passaged again by same method. Once increased
titers in the presence of S119 were detected for two consecutive passages, the viruses
were plaque purified. Following plaque purification, all 8 genome segments were
sequenced and compared to DMSO treated control virus to detect escape mutations.
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Blue native PAGE
Blue Native PAGE was used to examine the effect of S119 on the multimeric state of the
NP proteins. Purified recombinant wildꢀtype WSN NP protein was kindly produced by
Florian Krammer as previously described37, 38 and 5 ꢁg of purified protein was incubated
with 10 ꢁM S119 at 25° C for 30 min. 1 ꢁg of purified influenza virus RNA or Nucleaseꢀ
free water was added, up to 10 ꢁl. After an additional incubation of 15 minutes at 25° C,
the samples were mixed with 2.5ꢁl of 5% Coomassie brilliant blue Gꢀ250 and loaded into
sample wells of nondenaturing 4–20% gradient MiniꢀProtean TGX gel (BioꢀRad).
Samples were separated by electrophoresis at a constant voltage of 150 V for 60 min at
25° C in a blue native Tris/Glycine buffer system (Coomassie brilliant blue Gꢀ250 0.02%
Cathode Buffer). Gels were destained with 25% methanol/10% acetic acid solution and
visualized using the BioꢀRad (Hercules, CA) ChemiDoc MP Imaging System.
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Size exclusion chromatography
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To determine the oligomerization state of NP, A549 cells were infected with WSN virus
(MOI = 1) in the presence or absence of 10ꢁM S119 or 1ꢁM Nucleozin for 24 hours.
Cells were then lysed in 50 mM TrisꢀHCl, 100 mM KCl, 5 mM MgCl2 and 0.5% NP40
containing protease and phosphatase inhibitor cocktails. Total cell extract was clarified
by centrifugation, treated with 50 ꢁg/ml of RNase A for 2 hours at room temperature, and
fractionated through a Superoseꢀ6 column preꢀequilibrated in lysis buffer. Fractions were
probed by western blotting with an antiꢀNP (HT103) antibody.
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Immunofluorescence microscopy
A549 cells were grown to 70–80% confluency on coverslips. Cells were infected for 2 h
at MOI = 5 in the presence or absence of 10ꢁM S119 or 1ꢁM Nucleozin and washed.
S119 was maintained in culture throughout the experiment. Infections were stopped at
indicated time points by fixation in methanol for 30 min at 4C. Cells were blocked with
1% BSA for 30 minutes and then were incubated for 1h with primary antibodies against
NP (HT103) in PBS (dilution 1:2000), washed and stained with Alexa Fluor 488ꢀ
conjugated secondary antibodies (Invitrogen) (dilution 1:1000) for 1h. Coverslips were
then washed and counterstained with 4′,6ꢀdiamidinoꢀ2ꢀphenylindole, dihydrochloride
(DAPI) (Invitrogen) for nucleus localization and mounted on slides using Prolong Gold
antifade mounting medium (Invitrogen) before image analysis by fluorescence
microscopy.
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Fluorescent primer extension assay
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Primer extension of influenza RNA was performed using 5’ꢀend labeled fluorescent
primers with either Alexa Fluor 488 for vRNA detection or Alexa Fluor 546 for m/cRNA
detection of the PB2 (vRNA: 5’ꢀ TGCTAATTGGGCAAGGAGAC ꢀ3’ or m/cRNA: 5’ꢀ
GCCATCATCCATTTCATCCT ꢀ3’), NP (vRNA: 5’ꢀ TGATGGAAAGTGCAAGACCA
ꢀ3’ or m/cRNA: 5’ꢀ TGATTTCAGTGGCATTCTGG ꢀ3’), and NS (vRNA: 5’ꢀ
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TGATTGAAGAAGTGAGACACAG
ꢀ3’
or
m/cRNA:
5’ꢀ
CGCTCCACTATTTGCTTTCC ꢀ3’), segments. An Alexa Fluor 488 5’ꢀlabeled primer
targeting the human 5S rRNA (5’ꢀ TCCCAGGCGGTCTCCCATCC ꢀ3’) was used as a
loading control. A549 cells were pretreated for 2 hours with S119, VXꢀ787, or DMSO
and infected with the WSN virus for 6 hours (MOI 5) under continued compound
treatment. Infected A549 total RNA (100 ug) was added to the fluorescent primer (10
pmol), heated to 90°C (3 min), and cooled on ice. Reverse transcription (RT) reactions
were performed in 50 mM TrisꢀHCl (pH 8.3), 75 mM KCl, 5 mM MgCl2, and 10 mM
DTT with 0.5 mM each dNTP. 50U SuperScript III (Invitrogen) was added and reactions
were incubated at 45°C for 60 min. cDNA samples were mixed with an equal volume of
loading dye solution (Formamide, 5mM EDTA pH 8, 1% bromophenol blue) and heated
to 95°C for 3 minutes to stop the reaction. Samples were loaded on a 6% polyacrylamide
gel containing 7M Urea in TBE buffer. Gel Fluorescence was detected using the Bioꢀ
Rad (Hercules, CA) ChemiDoc MP Imaging System.
Antiviral combination assay
The combined antiviral effect of S119ꢀ8 (S119 Analog) and nucleozin or oseltamivir
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carboxylate was tested against the A/Puerto Rico/8/34 strain expressing GFP from
segment 8 (PR8ꢀGFP), which has been previously described.24 A549 cells were infected
with PR8ꢀGFP (MOI = 0.01) in the presence or absence of multiple drug combinations
for 24 hours. The concentration of the drugs used was based on their respective 50%
inhibitory concentrations (IC50) against PR8ꢀGFP in A549 cells. The assays were
performed in 96ꢀwell format using a liquid handling robot (Biomek NXP, Beckman) to
ensure accuracy. Infected cells were detected through GFP fluorescence, quantified by
laser scanning cytometry (Acumen, TTP Labtech). Isobolograms and combination
indices were calculated as previous described.30
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Synthesis of S119 and related analogs
N,N'-(1,4-phenylene)bis(4-(tert-butyl)benzamide) (S119): To a solution of 1,4ꢀ
diaminoꢀbenzene 2c (0.07g, 1 equiv.), triethylamine (3.0 equiv.) in CH2Cl2 was added 4ꢀ
(1,1ꢀdimethylethyl)benzoyl chloride 1 (2.2 equiv.) at 0℃. The resulted white suspension
was stirred at room temperature for 12 h and then filtered. The white cake was washed
with CH2Cl2 (10 mL), EtOH (10 mL) and dried under vacuum to give the desired product
8 as white powder 0.2g (72% yield). 1HꢀNMR (600 MHz, d6ꢀDMSO): δ 10.17 (s,2H),
7.90ꢀ7.89 (d, 4H), 7.74 (m, 4H), 7.56ꢀ7.54 (d, 4H), 1.33 (s, 18H); 13C NMR (125 MHz,
d6ꢀDMSO): δ 165.2, 154.3, 134.9, 132.3, 127.4, 125.1, 120.5, 34.7, 30.9; LCMS (tof+)
for C28H32N2O2 [M] 428.2464; Found [M+ H]+ for 429.2538
Synthesis of S119-8: 4-(tert-butyl)-N-(4-(phenylamino)phenyl)benzamide (S119-8):
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Step 1: N-(4-aminophenyl)-4-(tert-butyl)benzamide: To a solution of 1,4ꢀdiaminoꢀ
benzene (0.42g, 1.2 equiv.), triethylamine (1.5 equiv.) in CH2Cl2was added 4ꢀ(1,1ꢀ
dimethylethyl)benzoyl chloride 1 (1.0 equiv.) at 0℃. The resulted white suspension was
stirred at room temperature for 12 h and then filtered. Product was purified via flash
chromatography (gradient elution from 40% EtOAc in Hexanes to 60% EtOAc in
Hexanes) to give product as light yellow powder 0.6g (70% yield). 1HꢀNMR (600 MHz,
CDCl3): δ 7.97 (s,1H), 7.80ꢀ7.79 (d, 2H), 7.46ꢀ7.45 (d, 2H), 7.40ꢀ7.39 (d, 2H), 6.67ꢀ6.66
(d, 2H), 3.58 (s, 2H), 1.35 (s, 9H); 13C NMR (125 MHz, CDCl3): δ 165.2, 154.5, 142.8,
131.8, 129.0, 126.4, 125.1, 121.9, 115.1, 34.5, 30.7; LCMS (tof+) for C17H20N2O [M]
268.1576; Found [M+ H]+ for 269.1654.
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Step 2: To a solution of Nꢀ(4ꢀaminophenyl)ꢀ4ꢀ(tertꢀbutyl)benzamide (0.115g, 1.0
equiv.), BiPh3 (0.19 g, 1.01 equiv.) and Cu(OAc)2 (0.08g, 1.0 equiv.) in CH2Cl2 (20 mL)
was added with triethylamine (0.07mL, 0.48 mmol, 1.1 equiv.) at room temperature.31
The resulted suspension was stirred at room temperature for 16 h before diluted with
CH2Cl2 (20 mL), followed by addition of 1N HCl (12 mL) and kept stirring for 1 h. The
organic layer was separated, washed with 1N HCl (20 mL) and dried over K2CO3. After
filtration, the filtrate was concentrated and purified via flash chromatography (gradient
elution from 10% EtOAc in Hexanes to 25% EtOAc in Hexanes) to give product as
brown solid 0.1g (71 % yield). 1HꢀNMR (600 MHz, CDCl3): δ 8.00 (s, 1H), 7.84ꢀ7.83 (d,
2H), 7.55ꢀ7.54 (d, 2H), 7.23 (t, 2H), 7.08ꢀ7.04 (dd, 4H), 6.94 (t, 1H), 1.37 (s, 9H); 13C
NMR (125 MHz, CDCl3): δ 165.3, 154.8, 143.0, 139.2, 131.6, 131.2, 128.9, 126.4, 125.2,
121.5, 120.2, 118.4, 116.7, 34.5, 30.7 ; LCMS (tof+) for C23H24N2O [M] 344.1889;
Found [M+ H]+ for 345.1956.
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Similar procedures as for S119 were used to prepared related analogs.
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S119ꢀ2: 4-(tert-butyl)-N-(4-(tert-butyl)phenyl)benzamide (S119-2) To a solution of 4ꢀ
tertꢀbutylaniline (0.23g, 1.54 mmol, 1.0 equiv.) and triethylamine (0.16 g, 1.54 mmol, 1.0
equiv.) in CH2Cl2 (20 mL) was added with 4ꢀ(1,1ꢀdimethylethyl)benzoyl chloride (0.28g,
1.54 mmol, 1.0 equiv.) at 0℃. The resulted white suspension was stirred at room
temperature for 12 h and then filtered. The filtrate was concentrated and purified via flash
chromatography (gradient elution from 0% EtOAc in Hexanes to 4% EtOAc in Hexanes)
to give product as white powder 0.46g (95% yield). 1HꢀNMR (600 MHz, CDCl3): δ 7.98
(s,1H), 7.83ꢀ7.82 (d, 2H), 7.60ꢀ59 (d, 2H), 7.49ꢀ7.48 (d, 2H), 7.40ꢀ7.39 (d, 2H), 1.36 (s,
9H), 1.35 (s, 9H); 13C NMR (125 MHz, CDCl3): δ 165.2, 154.8, 146.9, 135.0, 131.7,
126.4, 125.4, 125.2, 119.5, 34.5, 33.9, 30.9, 30.7; LCMS (tof+) for C21H27NO;[M]
309.2093; Found [M+ H]+ for 310.2158
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S119ꢀ3: 4-(tert-butyl)-N-(4-phenoxyphenyl)benzamide (3b): Prepared as above from
4ꢀphenoxyꢀphenylamine 2b (0.10g, 1.0 equiv.), triethylamine (1.0 equiv.) and 4ꢀ(1,1ꢀ
dimethylethyl)benzoyl chloride 1 (1.0 equiv.) Product was purified via flash
chromatography (gradient elution from 0% EtOAc in Hexanes to 8% EtOAc in Hexanes)
to give product as white powder 0.18g (96% yield). 1HꢀNMR (600 MHz, CDCl3): δ 8.05
(s, 1H), 7.84ꢀ7.82 (d,2H), 7.63ꢀ7.62 (d, 2H), 7.50ꢀ7.48 (d, 2H), 7.35 (t, 1H), 7.11 (t, 1H),
7.03ꢀ7.01 (m, 3H), 1.37 (s, 9H); 13C NMR (125 MHz, CDCl3): δ 165.3, 157.1, 154.9,
153.1, 133.1, 131.5, 129.3, 126.5, 125.2, 122.6, 121.6, 119.2, 117.9, 34.5, 30.7; LCMS
(tof+) for C23H23NO2 [M] 345.1729; Found [M+ H]+ for 346.1787.
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S119ꢀ6: N,N'-(1,3-phenylene)bis(4-(tert-butyl)benzamide) (S119-6): Prepared as
above from 1,3ꢀdiaminoꢀbenzene (0.10g, 1.0 equiv.), triethylamine (3.0 equiv.) and 4ꢀ
(1,1ꢀdimethylethyl)benzoyl chloride 1 (2.2 equiv.). Product was purified via flash
chromatography (gradient elution from 0% EtOAc in Hexanes to 25% EtOAc in
Hexanes) to give product as white powder 0.3g (77% yield). 1HꢀNMR (600 MHz,
CDCl3): δ 8.13 (s, 1H), 8.02ꢀ7.94 (m, 2H), 7.82ꢀ7.81 (m, 4H), 7.51ꢀ7.48 (m, 6H), 7.38ꢀ
7.34 (m, 1H), 1.38ꢀ1.38 (d, 18H); 13C NMR (125 MHz, CDCl3): δ 165.3, 155.1, 138.3,
131.4, 129.2, 126.4, 125.3, 115.3, 110.9, 34.5, 30.7; LCMS (tof+) for C28H32N2O2 [M]
428.2464; Found [M+ H]+ for 429.2523.
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Supporting Information
ꢀ
ꢀ
ꢀ
Figure S1 presents size exlcusion chromatography data from infected cell extracts
treated with S119 or nucleozin in the absence of RNase treatment.
Figure S2 shows that S119ꢀ8 does not inhibit VSV and is a specific inhibitor of
influenza viruses.
Figure S3 shows that S119ꢀ8 retains the same Y40F resistant phenotype as the
parental S119.
Abbreviations
VSV ꢀ Vesicular Stomatitis Virus, SAR ꢀ Structure Activity Relationship, IC50 ꢀ 50%
Inhibitory Concentration, IC90 ꢀ 90% Inhibitory Concentration, CC50 ꢀ 50% Cytotoxic
Concentration, CC10 ꢀ 10% Cytotoxic Concentration
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Author Information
1Department of Microbiology, 2Department of Pharmacological Sciences, 3 Immunity and
Pathogenesis Program, Infectious and Inflammatory Disease Center, Sanford Burnham
Prebys Medical Discovery Institute, 10901 North Torrey Pines Road, La Jolla, California
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92037, USA, Department of Medicine, Division of Infectious Diseases, Global Health
and Emerging Pathogens Institute, 6Drug Discovery Institute, Icahn School of Medicine
at Mount Sinai, New York, NY 10029, USA,
^Present Address: Suzhou Novartis Pharma Technology Co., Ltd. 18 Tonglian Road,
Riverside Industrial Park, Changshu Economic Development Zone, Changshu / Jiangsu
Province 215537, China.
#Present Address: Department of Microbiology, University of Chicago, Chicago, Illinois,
USA.
*Corresponding author
Department of Microbiology
Icahn School of Medicine at Mount Sinai
One Gustave L. Levy Place
New York, NY 10029, USA.
Tel.: +1ꢀ212ꢀ241ꢀ8931
megan.shaw@mssm.edu
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Conflict of Interest
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None to Report
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Acknowledgements
We thank Dr. Florian Krammer for producing the purified NP proteins. JChem for Excel
was used for structure database management, search, and prediction, JChem for Excel
6.1.1, 2013, ChemAxon (http://www.chemaxon.com). This work was supported in part
by National Institutes of Health (NIH) Grants U01AI1074539, R21AI102169 and
U19AI106754 and a Mount Sinai seed fund to the Devita laboratory. This work was also
partially supported by CRIP (Center for Research on Influenza Pathogenesis), an NIAIDꢀ
funded Center of Excellence for Influenza Research and Surveillance (CEIRS,
HHSN272201400008C).
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