Receptor Tyrosine Kinase Inhibitors That Block Replication of Influenza A and Other Viruses

ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2011, p. 5553–5559 Vol. 55, No. 120066-4804/11/$12.00 doi:10.1128/AAC.00725-11Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Receptor Tyrosine Kinase Inhibitors That Block Replication ofInfluenza A and Other Viruses�

Naveen Kumar,† Nishi R. Sharma, Hinh Ly, Tristram G. Parslow, and Yuying Liang*Department of Pathology and Laboratory Medicine, Emory University School of Medicine, Atlanta, Georgia 30322

Received 25 May 2011/Returned for modification 11 July 2011/Accepted 13 September 2011

We have previously reported that two receptor tyrosine kinase inhibitors (RTKIs), called AG879 andtyrphostin A9 (A9), can each block the replication of influenza A virus in cultured cells. In this study, wefurther characterized the in vitro antiviral efficacies and specificities of these agents. The 50% effectiveconcentration (EC50) of each against influenza A was found to be in the high nanomolar range, and theselectivity index (SI � 50% cytotoxic concentration [CC50]/EC50) was determined to be >324 for AG879 and50 for A9, indicating that therapeutically useful concentrations of each drug produce only low levels ofcytotoxicity. Each compound showed efficacy against representative laboratory strains of both human influenzaA (H1N1 or H3N2) and influenza B viruses. Importantly, no drug-resistant influenza virus strains emergedeven after 25 viral passages in the presence of AG879, whereas viruses resistant to amantadine appeared afteronly 3 passages. AG879 and A9 each also exhibited potent inhibitory activity against a variety of other RNA andDNA viruses, including Sendai virus (Paramyxoviridae), herpes simplex virus (Herpesviridae), mouse hepatitisvirus (Coronaviridae), and rhesus rotavirus (Reoviridae), but not against Pichinde virus (Arenaviridae). Theseresults together suggest that RTKIs may be useful as therapeutics against viral pathogens, including but notlimited to influenza, due to their high selectivity indices, low frequency of drug resistance, and broad-spectrumantiviral activities.

Despite annual vaccination programs, influenza virus con-tinues to be a major public health concern. Due to the highlyerror-prone nature of the viral RNA polymerase, influenzavirus variants resistant to drugs that target viral componentsarise commonly and spread rapidly. Indeed, viral variants re-sistant to each of the currently available anti-influenza drugs,i.e., to M2 ion channel inhibitors (amantadine and rimanta-dine) and neuraminidase inhibitors (zanamivir and oseltami-vir), are readily isolated (2, 3, 14, 22, 27). Targeting host sig-naling pathways or other host cell factors important forinfluenza virus replication, therefore, represents a potentialalternative approach that can minimize the emergence of drug-resistant viruses.

Receptor tyrosine kinases (RTKs) are a group of growthfactor receptors that regulate a variety of cellular activitiesrelated to growth, metabolism, and differentiation (15). Dueto their critical roles in the development and progression ofvarious cancers, RTKs such as epidermal growth factor re-ceptor (EGFR) and erythroblastosis oncogene homolog2/hergulin receptor 2 (ErbB2/HER2) have been studied ex-tensively as targets for anticancer therapeutics (16). RTKsand other tyrosine kinases have also been shown to playimportant roles in virus replication. For example, the ty-rosine kinase inhibitor genistein was found to block repli-

cation of type-1 human immunodeficiency virus (HIV-1),herpes simplex virus type 1 (HSV-1), and certain arenavi-ruses (24, 25, 28), whereas Src family kinases were shown tobe important for the assembly and maturation of denguevirus and West Nile virus (1, 9). Similarly, two of the majorsignaling pathways (i.e., the Raf/MEK/ERK and PI3K/Aktpathways) that are activated by RTKs were found to playimportant roles in influenza virus replication (4, 5, 7, 21),and a recent report (6) suggests that EGFR signaling isimportant to promote influenza A virus uptake by humantarget cells. Our laboratory recently identified two tyrphos-tin-type RTK inhibitors (RTKIs), called AG879 and tyr-phostin A9 (A9), that each strongly inhibit influenza A virusreplication in cultured cells. Specifically, each agent wasfound to block at least three postentry steps of the influenzavirus life cycle, including viral RNA synthesis, Crm1-depen-dent nuclear export of viral ribonucleoprotein (vRNP), andvirus assembly and budding (12). A9 is a selective inhibitorof the platelet-derived growth factor receptor (17), whereasAG879 is known to inhibit tyrosine kinase activity of thenerve growth factor receptor (TrkA) and the heregulin re-ceptor erbB-2 (HER-2) (19). Using additional small-mole-cule inhibitors and small-hairpin RNA (shRNA)-mediatedspecific-gene knockdown, we verified that, indeed, the TrkAsignaling pathway plays important roles in influenza A virusreplication (12).

In the current study, we have extended our previous findings(12) by quantitatively assessing the anti-influenza efficacy andspecificity of AG879 and A9, evaluating the evolution of viraldrug resistance, and testing the activities of these compoundsagainst several distinct RNA and DNA virus families. Collec-tively, our studies suggest that RTKIs may have significantpotential as broad-spectrum antiviral drugs.

* Corresponding author. Mailing address: Department of Pathologyand Laboratory Medicine, 615 Michael St., Suite 177, Rm. 175, White-head Biomedical Research Bldg., Emory University, Atlanta, GA30322. Phone: (404) 727-3243. Fax: (404) 727-8538. E-mail: [email protected].

† Present address: Division of Animal Health, Central Institute ofResearch on Goats, Indian Council of Agricultural Research, Makh-doom, PO-Farah-281122, District-Mathura, UP, India.

� Published ahead of print on 19 September 2011.

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MATERIALS AND METHODS

Compounds, viruses, and cells. All chemical compounds were purchased fromSigma. A549 cells (human lung epithelial cells) were grown in Dulbecco’s mod-ified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetalbovine serum (FBS). Madin-Darby canine kidney (MDCK) cells were main-tained in Eagle’s minimal essential medium (MEM) supplemented with 5% FBS.Vero (African green monkey kidney epithelial) cells were grown in MEM sup-plemented with 5% fetal bovine serum. Rhesus monkey kidney MA104 cellswere grown in MEM supplemented with sodium pyruvate, sodium bicarbonate,nonessential amino acids, and 10% FBS. Rat lung epithelial (L2) cells weregrown in DMEM supplemented with 10% FBS. Baby hamster kidney epithelial(BHK-21) cells were grown in MEM with 10% FBS.

Sendai virus and influenza virus A/Aichi �31 were obtained from S.-M. Kangand R. Compans (Emory University). Influenza virus B/Victoria was purchasedfrom ATCC. Sendai virus and various influenza virus strains (A/WSN/33, A/PR8/34, A/Aichi �31, and B/Victoria) were grown in 10-day-old embryonated chickeneggs. The viral titers were determined by plaque assay on MDCK (influenzavirus) or Vero (Sendai virus) cells. Herpes simplex virus (HSV) Kos strain wasprovided by B. Rouse (University of Tennessee) and was grown in Vero cells.Mouse hepatitis virus (MHV) was obtained from D. Brian (University of Ten-nessee) and was grown in L2 cells. Rhesus rotavirus (RRV) was obtained fromM. Vijay-Kumar and A. Gewirtz (Emory University) and was grown in MA104cells. Pichinde virus was derived from our reverse genetics system (13) and wasgrown in BHK-21 cells, and viral titers were determined by plaque assay on Verocells.

After infection with influenza A virus, MDCK cells were grown in low-serumL-15 medium, which consists of 15 mM HEPES (pH 7.5), nonessential aminoacids, 0.75 g of sodium bicarbonate per liter, and 0.125% (wt/vol) bovine serumalbumin (BSA). With the exception of A/WSN/33, infection of cultured cells withinfluenza viruses was conducted in the presence of trypsin at a concentration of2.5 �g per ml. Both HSV and MHV infections were conducted in low-serumDMEM medium supplemented with 0.1% BSA. RRV infection of MA104 cellswas conducted by treating RRV with 10 �g/ml of EDTA-free trypsin at 37°C for30 min followed by the infection of cells in serum-free medium with 10 �g/ml oftrypsin. Infections with Sendai virus and Pichinde virus were conducted in serum-free DMEM medium.

CC50 determination. A549 cells in 96-well plates were treated with 10-folddilutions of chemicals or dimethyl sulfoxide (DMSO) control, in triplicates, in atotal of 100 �l growth medium for 48 h. An amount of 20 �l of freshly made 5mg/ml MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide]solution was added to each well, and cells were incubated at 37°C for 5 h. Afterremoval of the medium, 200 �l of DMSO was added to each well to dissolve thepurple formazan product and the plates were incubated at 37°C for another 5min to remove any air bubbles. MTT signals were measured photometrically atan absorbance of 550 nm. The 50% cytotoxic concentration (CC50) was calcu-lated as the concentration needed to reduce cellular viability to 50%.

EC50 determination. A549 cells in triplicates were infected with influenza virusA/WSN/33 at a multiplicity of infection (MOI) of 0.1 for 1 h, washed three timeswith phosphate-buffered saline (PBS), and replaced with fresh L-15 mediumcontaining either DMSO or serial 3-fold dilutions of chemical compounds induplicates. Viral titers in the supernatants at 24 h postinfection (h.p.i.) weredetermined by plaque assay on MDCK cells. The 50% effective concentration(EC50) was calculated as the concentration required to reduce viral yield by 50%in the compound-treated cultures when compared to the viral yield in DMSO-treated cultures.

Virucidal activity determination. Virus suspensions containing approximately1 � 106 PFU of A/WSN/33 viruses were incubated in serum-free medium con-taining either DMSO or serial 5-fold dilutions of compounds for 1.5 h at 37°C.The mixed samples were chilled at 4°C and diluted by 104-, 105-, and 106-foldwith serum-free medium before being applied onto MDCK cells in 6-well platesfor plaque assaying. Plaques were scored at 48 h.p.i. after crystal violet staining,and the results plotted against the concentrations of compounds used. The 50%inactivating concentration (IC50) was calculated as the concentration required toinactivate the cell-free virions by 50%.

Inhibitory effects of RTKIs on influenza virus replication. MDCK or A549cells were infected in triplicate with influenza virus strains at the stated molarratio of infection (MOI) for 1 h, washed with PBS three times, and replaced withfresh L-15 medium with either vehicle control DMSO, 10 �M AG879, 4 �M A9,or 10 �M negative control AG494. Virus production at different time points wasquantified by plaque assay.

Selection of drug-resistant influenza virus strains. Influenza A/WSN/33 wassequentially passaged in MDCK cells at an MOI of 0.01 in the presence of

DMSO, 1 to 5 �M AG879, or 50 �M amantadine for 48 to 72 h. After eachpassage, viruses released into the supernatant were quantified by plaque assay.

Effect of RTKIs on replication of different viruses. The antiviral effects ofRTKIs on different viruses were studied by comparing the levels of viral repli-cation in target cells in the absence or presence of RTKIs. We characterized theeffects of RTKIs on the infection of Sendai virus in A549 cells, HSV in Verocells, MHV in L2 cells, RRV in MA104 cells, and Pichinde virus in A549 cells.To do this, target cells in triplicate were pretreated with DMSO, AG879 (10�M), A9 (4 �M), or AG494 (10 �M) for 30 min prior to virus infection at anMOI of 0.1 for 1 h. After being washed three times with PBS, the infected cellswere grown in serum-free medium containing DMSO or the respective com-pound. Viral titers in the supernatants at 16 to 24 h.p.i. were quantified by plaqueassay. Alternatively, RRV viral particles were analyzed by enzyme-linked immu-nosorbent assay (ELISA) (26). Briefly, 2-fold serial dilutions of supernatantswere added to the 96-well plates coated with anti-RRV polyclonal serum andincubated with guinea pig anti-RRV antibody followed by horseradish peroxi-dase (HRP)-conjugated anti-guinea pig antibody. After being washed with PBS,the HRP substrates were added to the plates and the samples were measured atan optical density of 490 nm (OD490).

Examination of anti-influenza activity of AG879 in the influenza virus-infectedmouse model. All experimental procedures were approved by the InstitutionalAnimal Care and Use Committee (IACUC) of Emory University. Briefly, 6- to8-week-old female BALB/c mice were housed for 3 to 5 days for acclimation andthen infected intranasally with 10 50% mouse lethal doses (MLD50) of A/PR8virus in a 50-�l volume. DMSO-PBS (16%, vol/vol) or AG879 at a dosage of 5mg/kg of body weight was given daily for 5 days to the infected animals byintraperitoneal injection. Mice (n � 5 per group) were monitored daily forclinical symptoms and body weight loss until day 21. Mice were euthanized if theyreached prespecified terminal points as previously described (18). Three miceper group were euthanized at day 3, and the viral titers in their lungs wereanalyzed by plaque assay.

Statistical analyses. Statistical analysis of the survival curve by log-rank (Man-tel-Cox) �2 test was conducted using GraphPad Prism 5 software. Statisticalcomparison of viral titers among different treatments presented throughout thepaper was performed using Student’s t test.

RESULTS

In vitro efficacy of AG879 and A9 against influenza A virus.We previously screened a small library of protein kinase inhib-itors for anti-influenza activities and identified two tyrphostin-type RTKI compounds, AG879 and A9 (Fig. 1), that exhibitedstrong inhibitory effects on influenza A replication in vitro (12).To evaluate their potentials as anti-influenza therapeutics, wetherefore set out to quantify more precisely their cytotoxicconcentrations (CC50) in cultured A549 human lung epithelialcells and their effective concentrations (EC50) against influ-enza A viral replication. The CC50 (i.e., the concentrationrequired to produce cytotoxic effects in 50% of target cells) wasdetermined by using an MTT assay to estimate the viability ofA549 cells grown in the presence of increasing concentrations(up to 81 �M) of each tested compound. As shown in Fig. 2A,no cytotoxicity was observed even after 48 h of incubation ofA549 cells with AG879 at 81 �M (CC50 � 81 �M), whereascell viability was noticeably affected by exposure to A9 overmuch of the range of concentrations we tested (CC50 � 8 �M).

FIG. 1. Chemical structures of AG879 (A), tyrphostin A9 (B), andAG494 (C).

5554 KUMAR ET AL. ANTIMICROB. AGENTS CHEMOTHER.

To determine the half-maximal effective concentration (EC50)of each compound alone, we measured the yield of influenzavirus infectious units in the presence of inhibitor concentra-tions ranging from 0.032 �M to 10 �M. The EC50, defined asthe concentration required to inhibit infectious viral yield by50%, was found to be 250 nM for AG879 and 160 nM for A9(Fig. 2B). Therefore, the selectivity indices (SI), defined asCC50/EC50, were calculated to be �324 for AG879 and 50 forA9 (Fig. 2D), providing one measure of the potential thera-peutic utility of each compound. To determine whether theinhibitory effects of these RTKIs are partially due to directinactivation of cell-free virions, we incubated infectious vi-rions with increasing concentrations of each compound for1.5 h and then tested their infectivity on cultured targetcells. As shown in Fig. 2C, neither AG879 nor A9 signifi-cantly inhibited virion infectivity even at high concentrations(i.e., each showed an IC50 of �81 �M). This supports ourearlier conclusion that the anti-influenza activities of AG879and A9 are due to their inhibitory effects on viral replicationwithin the target cells.

AG879 and A9 block influenza A virus infection in variouscell types and virus-to-cell ratios. To determine to what extent

the anti-influenza activity of AG879 and A9 is affected by celltype or virus-to-cell ratio, we conducted viral inhibition assaysin two different cell types (A549 lung and MDCK canine kid-ney epithelial cells) with various multiplicities of infection(MOI, defined as the ratio of input infectious viral particles pertarget cell). Both cell types were infected with A/WSN virus atan MOI of 0.01, 0.1, or 1 in the presence either of AG879 orA9, of vehicle control (DMSO), or of an unrelated RTKIcalled AG494 that we had previously shown (12) to lack anti-influenza activity in this assay. AG494 is a potent inhibitor ofepidermal growth factor receptor (EGFR) signaling in cell-free kinase assays, but it cannot inhibit EGFR in intact cells(20) and thus served as a negative control. As influenza virusreplication peaks at various time points with different MOIs,we quantified viral yield in the supernatants at 48 h (MOI �0.01), 18 h (MOI � 0.1), or 9 h (MOI � 1) after infection. Asshown in Fig. 3, both RTKIs strongly blocked infectious virusproduction in both cell lines and at all tested virus-to-cellratios, with the degree of inhibition ranging from 1 to �4 log.These findings imply that the inhibition of influenza A repli-cation by AG879 or A9 is not unique to A549 cells and iseffective against relatively high levels of input virus. Additional

FIG. 2. Characterization of AG879 and A9 for cytotoxicity and anti-influenza efficacy. (A) Determination of the 50% cytotoxic concentrations(CC50) of AG879, A9, and AG494. A549 cells were incubated with various concentrations of the compounds for 48 h and measured for cell viabilityby MTT assay. (B) Determination of the 50% efficacy concentration (EC50) of AG879, A9, or AG494 in blocking influenza A virus replicationin vitro. A549 cells were infected with influenza virus at an MOI of 0.1 in the presence of various concentrations of compounds. Viral titers at24 h.p.i. were quantified by plaque assay. (C) Determination of the virucidal activities of AG879, A9, and AG494. The 50% inactivationconcentrations (IC50) of AG879, A9, and AG494 to directly inactivate cell-free influenza virions were determined. (D) Summary of CC50, EC50,IC50, and selective indices (SI) for AG879 and A9. The results shown in panels A to C are the averages of at least three independent experiments,with error bars showing standard deviations.

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evidence for the efficacy of these compounds in a wide range ofcell types is presented below.

AG879 and A9 are effective against diverse strains of influ-enza virus. To evaluate the inhibitory effects of these com-pounds against various influenza virus strains, we infectedA549 cells with laboratory strains of H1N1 influenza A (A/WSN/33 or A/PR8/34), H3N2 influenza A (A/Aichi X31), orinfluenza B (B/Victoria) at an MOI of 0.01 in the presence ofthe tested compounds. As shown in Fig. 4, each of these fourinfluenza strains replicated to high titers at 48 h.p.i. in thepresence of vehicle control (DMSO) or of the inactive controlcompound AG494. For each of the influenza A and B strains,AG879 and A9 strongly inhibited viral production, by 2 to 3 log(Fig. 4). As A/H1N1, A/H3N2, and influenza B virus are thepredominant circulating human influenza strains, our data sug-gest that RTKIs with activity profiles similar to those of AG879

and A9 could have therapeutic value in treating all of thecirculating seasonal influenza viruses that are currently extant.

Failure to generate AG879-resistant influenza viruses. Onemajor theoretical advantage of developing antiviral drugs thattarget host components compared to antiviral drugs againstviral targets is that host-targeting compounds are less likely tofavor the evolution of resistant viral strains. This is particularlyimportant for anti-influenza drug development, as drug-resis-tant influenza strains are known to emerge and spread veryquickly. We evaluated the ability of H1N1 influenza virus todevelop resistance to RTKIs during long-term in vitro culture(up to 25 passages at an MOI of 0.01) of A/WSN virus inmedium containing AG879, vehicle control DMSO, or aman-tadine, which is an FDA-approved drug that inhibits the viralM2 ion channel protein. Viral supernatants were harvested at48 h following each passage and were then used to infect freshA549 cells, and the viral titer in each successive supernatantwas quantified, as shown in Fig. 5A. Throughout the long-termculture, DMSO-treated cells consistently supported high levelsof virus production, at titers of �107 PFU/ml. The presence ofamantadine significantly reduced virus production, by �2 log,at the earliest passages (first passage [P1] and P2), consistentwith its known potent anti-influenza activity. However, viralproduction began to increase detectably after the third passagein medium containing amantadine and regained the level seenin the DMSO control after the sixth passage, indicating thatamantadine-resistant virus strains had successfully emerged.This is consistent with previous reports that amantadine-resis-tant mutants can be isolated after as few as two passages (8). Incontrast, even after 25 passages in AG879, virus productionwas still significantly lower than that in DMSO (by �2 log),indicating the absence of highly AG879-resistant viruses. In-deed, when we compared the original (P0) and P25 viral stocksfor their sensitivity to AG879 inhibition, both showed compa-rably strong sensitivity to AG879; in particular, AG879 re-duced virus production by either stock to a level �4 log lowerthan that of the DMSO control (Fig. 5B). This suggests thatthe P25 stock does not contain significant quantities of AG879-resistant mutants. Moreover, the 15th amantadine-passaged

FIG. 3. AG879 and A9 inhibit influenza virus replication in differ-ent cell types and with different virus-to-cell molar ratios. Inhibition ofvirus production was conducted on MDCK cells (A) or A549 cells (B).Viral yield in the supernatants was quantified at 48 h (for an MOI of0.01), 18 h (for an MOI of 0.1), or 9 h (for an MOI of 1) after infection.The results shown are the averages of at least three independentexperiments, with error bars showing standard deviations. Pairwisestatistical comparisons to the DMSO control group were performedusing Student’s t test. ���, P � 0.001.

FIG. 4. AG879 and A9 inhibit replication of various influenza virus strains. Inhibition of virus production was conducted using the influenzavirus strains A/WSN, A/PR8, A/Aichi �31, and B/Victoria at an MOI of 0.01 as described in Materials and Methods. Viral production at 48 h.p.i.was quantified by plaque assay. The results shown are the averages of at least three independent experiments, with error bars showing standarddeviations. Pairwise statistical comparisons to the DMSO control group were performed using Student’s t test. ���, P � 0.001.

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virus supernatant was found to be completely resistant toamantadine but remained as sensitive to AG879 as the originalvirus (Fig. 5C), suggesting that AG879 is able to inhibit eventhose viruses that have already developed resistance to anotherantiviral drug. Taken together, our data clearly show that therate at which influenza viruses evolve resistance to RTKIs isextremely low, suggesting that RTKIs might possibly be usefulas salvage therapy in clinical settings where the virus has de-veloped resistance to other available drugs.

Anti-influenza activity of AG879 in an infected mousemodel. We used a well-established lethal influenza virus-mousemodel to assess the antiviral activity of RTKIs (18). Mice wereinfected with a lethal dosage of influenza virus A/PR8 and thentreated with either DMSO-PBS or AG879 at 5 mg per kg ofbody weight for 5 days, commencing on day 0. As expected, themice treated with the DMSO-PBS vehicle control all suc-cumbed to infection within 8 days, with 100% mortality (Fig.6A). In contrast, 60% of mice in the AG879 treatment groupsurvived (Fig. 6A), which is significantly higher than the sur-vival rate in the control group (P � 0.05). To further verify theantiviral activity of AG879 in vivo, we quantified viral titers inthe lungs from both groups at day 3, as the peak of viralreplication occurs between day 2 and day 4 postinfection (18).

Influenza viruses replicated to a mean titer of 4.3 � 105

PFU/mg in the lungs of DMSO-PBS-treated mice, which isnearly 2 log higher than the titers seen in the AG879-treatedanimals (Fig. 6B), indicating that AG879 can effectively sup-press influenza virus replication in vivo. In summary, we haveshown that AG879 can significantly reduce influenza virus rep-lication and the associated mortality in infected animals, sup-porting the potential of RTKIs as antiviral therapeutics.

AG879 and A9 have broad-spectrum antiviral activities. AsRTK signaling pathways might in principle play importantroles in regulating the life cycles of diverse viruses, we thenasked whether AG879 and A9 could inhibit viruses other thaninfluenza. The initial MTT assays verified that neither com-pound produced cytotoxicity at the tested concentrations inany of the target cell lines used for these studies (data notshown). To examine the effects of the compounds on the rep-lication of Sendai virus, a member of the Paramyxoviridae fam-ily, A549 cells were infected with Sendai virus at an MOI of 0.1in the presence of either the vehicle control DMSO, AG879,A9, or the negative control AG494. Virus production at24 h.p.i. was compared by plaque assay. As shown in Fig. 7A,both AG879 and A9 strongly reduced Sendai virus replication,by �3 log. We then determined whether the RTKIs couldinhibit herpesvirus replication using the HSV-1 Kos strain.Vero cells were infected with HSV-1 at an MOI of 0.1 for 16 h.Viral production was found to be reduced by �3 log in cellstreated with either AG879 or A9 (Fig. 7B), suggesting thatboth RTKIs can strongly inhibit HSV-1 replication. We alsonoticed that the negative control AG494 slightly reduced theHSV-1 yield, by �0.5 log, though the significance of this re-mains uncertain. Replication of the prototypic coronavirusmouse hepatitis virus (MHV) was then examined in L2 cellstreated with either DMSO or RTKIs, revealing that AG879and A9 could significantly block MHV production, by �2 logat 16 h.p.i. at an MOI of 0.1 (Fig. 7C). We also evaluated theeffect of AG879 on the replication of the rhesus rotavirus(RRV) in MA104 cells after infection at an MOI of 0.1. RRVviral production in the supernatants was evaluated by ELISA

FIG. 5. Selection of drug-resistant influenza viruses in vitro. (A) In-fluenza viruses were passaged in medium containing DMSO, amanta-dine, or AG879 as described in Materials and Methods. Viral titers inthe supernatants at each passage were determined. (B) The virusescollected from either the original (P0) or the 25th (P25) AG879 pas-sage were used to infect A549 cells at an MOI of 0.01 with and without10 �M AG879. Virus production at 48 h.p.i. was quantified by plaqueassay. (C) The 15th amantadine-passaged virus was used to infect A549cells at an MOI of 0.01 in the presence of DMSO, amantadine (50�M), or AG879 (10 �M). Virus production at 48 h.p.i. was quantifiedby plaque assay. The results shown in panels B and C are the averagesof at least three independent experiments, with error bars showingstandard deviations. Statistical analyses were performed using Stu-dent’s t test. ���, P � 0.001.

FIG. 6. In vivo anti-influenza activity of AG879 in the lethal mousemodel. Mice were infected with a lethal dosage of A/PR8 viruses andwere treated with either DMSO-PBS (16%, vol/vol) or with AG879 at5 mg/kg daily for 5 days from day 0. (A) Survival curves of infectedmice (n � 5) treated with DMSO-PBS or AG879. (B) Influenza virustiters in the lungs of mice at day 3 postinfection. The results shown arethe averages (n � 3), with error bars showing standard deviations.Pairwise statistical comparison was performed using Student’s t test.��, P � 0.01.

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at 48 h.p.i. using anti-RRV polyclonal serum as previouslydescribed (26). To ensure that the RRV particles were withinthe linear detection range of the ELISA, we tested 2-fold serialdilutions of the supernatants. Based on the OD490 values (Fig.7D), the yield of released RRV particles was significantly lowerin cells treated with AG879 than in those treated with DMSO.In contrast, AG879 and A9 did not appreciably affect the invitro replication of Pichinde virus (Arenaviridae): although theviral titers in A549 cells treated with DMSO or with the testedcompounds were statistically different (P � 0.05), the differ-ence between the mean viral titers was only �0.2 log (Fig. 7E).In summary, AG879 and A9 show broad-spectrum antiviralactivity against various RNA and DNA viruses, including in-fluenza virus, Sendai virus, HSV-1, MHV coronavirus, androtavirus, with the notable exception of arenavirus.

DISCUSSION

Here, we have shown that at least one tyrphostin-classRTKI, AG879, exhibits high potency and a high selectivityindex (SI � 324) in blocking influenza A virus replication invitro (Fig. 2). Moreover, in initial animal studies, we have now

shown that AG879 has significant ability to protect mice fromlethal infection by influenza A/PR8 (Fig. 6). These findingstogether underscore the great potential of RTKIs that targetspecific host-cell kinases to serve as novel therapeutic agentsagainst influenza virus.

Both AG879 and A9 can inhibit the replication of diverseinfluenza A (H1N1 and H3N2) and B viral strains, which arethe seasonal human influenza strains (Fig. 4). More impor-tantly, we did not detect the emergence of resistant viruseseven after 25 passages of influenza A virus in AG879-contain-ing medium, whereas amantadine-resistant variants quicklyemerged (Fig. 5A). At least two major factors may account forthe extremely low frequency of emergence of AG879-resistantinfluenza strains. First, AG879 targets host RTK signalingrather than viral components themselves. Second, AG879 haspreviously been shown by us to block several independent stepsof the influenza virus life cycle (12). Thus, RTKIs with prop-erties similar to those of AG879 may have great potential asanti-influenza drugs, as they minimize the emergence andspread of drug-resistant mutants. Indeed, our data also indi-cate that such RTKIs could be used to inhibit the replication ofviruses that have already developed resistance to the current

FIG. 7. AG879 and A9 have broad-spectrum antiviral activities. (A) A549 cells were infected with Sendai virus at an MOI of 0.1 for 24 h inthe presence of either DMSO or compound. Viral yield was determined by plaque assay. (B) Vero cells were infected by HSV-1 at an MOI of 0.1for 24 h in the presence of either DMSO or compound. Viral yield was determined by plaque assay. (C) L2 cells were infected with MHV at anMOI of 0.1 for 24 h in the presence of either DMSO or compound. Viral yield was determined by plaque assay. (D) MA104 cells were infectedwith RRV at an MOI of 0.1 for 48 h in the presence of either DMSO or compound. Viral particles in various dilutions of supernatants werequantified by ELISA. (E) A549 cells were infected with Pichinde virus at an MOI of 0.1 for 48 h in the presence of either DMSO or the indicatedcompound. Viral yield was determined by plaque assay. The results shown are the averages of at least three independent experiments, with errorbars showing standard deviations. Pairwise statistical comparisons to the control group were performed using Student’s t test. �, P � 0.05; ��, P �0.01; ���, P � 0.001.

5558 KUMAR ET AL. ANTIMICROB. AGENTS CHEMOTHER.

FDA-approved drugs, such as amantadine (Fig. 5C), thus en-hancing their possible utility and value in clinical settings.

We have recently shown that the anti-influenza activity ofAG879 is probably due to its inhibition of TrkA signaling (12),while the precise inhibitory mechanism of A9 remains as yetunknown. In addition to TrkA, other specific RTK signalingpathways may also play important roles in influenza virus rep-lication. Several independent genome-wide small-interferingRNA (siRNA) screens have implicated at least 5 differentRTKs, including TGFR (23), FGFR-1 through -4 (11),NTRK2/TrkB (11), EphB6 (10), and EphB2 (11), and many oftheir downstream targets as important host factors required forinfluenza virus replication. Therefore, we propose that usingRTKIs to target these specific RTK signaling pathways couldprovide significant therapeutic value against influenza infec-tion.

An expected difficulty of using antivirals targeting host cellfunctions, however, is their potential toxicity in vivo. Indeed,we observed that, although AG879 at 5 mg per kg significantlyreduced viral replication and the associated mortality in amouse influenza model (Fig. 6), higher dosages of AG879 ledto shorter survival than was seen in a PBS-treated controlgroup, indicating toxic effects at the higher dosages in vivo(data not shown). Further studies will be necessary to deter-mine the optimal dosage and duration of RTKI treatmentwhen used for antiviral therapeutics. Due to the high selectivityindex (SI � 324) of AG879 in anti-influenza activity (Fig. 2),we expect that a useful range of concentrations can indeed beidentified, though the optimal therapeutic strategies may varyfor different viruses and in various clinical settings. It is tempt-ing to speculate that antiviral compounds targeting host RTKsmight prove especially useful for treating acute, life-threaten-ing infections, such as those caused by the highly pathogenicH5N1 and pandemic influenza viruses, as well as other virusesof biodefense interest.

In summary, our data suggest that certain classes of RTKIshave the potential to be developed as broad-spectrum antiviraldrugs that could be used to treat various viral infections, in-cluding influenza. In recent years, significant progress has beenreported in clinical and preclinical studies of RTKIs as anti-cancer therapeutics, which may facilitate the development ofsuch compounds for use in antiviral therapies.

ACKNOWLEDGMENTS

We thank M. Vijay-Kumar and A. Gewirtz (Emory University) forproviding RRV and MA104 cells, S. Kang and R. Compans (EmoryUniversity) for Sendai virus and influenza virus A/Aichi, B. Rouse(University of Tennessee) for HSV, and D. Brian (University of Ten-nessee) for MHV.

This work was supported in part by NIH grants AI067704 to T.G.P.and AI083409 to Y.L.

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