The small molecule AZD6244 inhibits dengue virus replication in vitro and protects against lethal challenge in a mouse model
Leonardo C. de Oliveira1,2 · Aryádina M. Ribeiro1,2 · Jonas D. Albarnaz1,3 · Alice A. Torres1,3 · Luís F. Z. Guimarães1 ·
Amelia K. Pinto2 · Scott Parker2 · Konstantin Doronin2 · James D. Brien2 · Mark R. Buller2 · Cláudio A. Bonjardim1
Received: 24 July 2019 / Accepted: 6 December 2019
© Springer-Verlag GmbH Austria, part of Springer Nature 2020
Abstract
Dengue virus (DENV) is the most common mosquito-borne viral disease. The World Health Organization estimates that 400 million new cases of dengue fever occur every year. Approximately 500,000 individuals develop severe and life-threatening complications from dengue fever, such as dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF), which cause 22,000 deaths yearly. Currently, there are no specific licensed therapeutics to treat DENV illness. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. In this study, we demonstrate that the MEK/ERK inhibitor AZD6244 has potent antiviral efficacy in vitro against DENV-2, DENV-3, and Saint Louis encephalitis virus (SLEV). We also show that it is able to protect AG129 mice from a lethal challenge with DENV-2 (D2S20). The molecule is currently undergoing phase III clinical trials for the treatment of non-small-cell lung cancer. The effect of AZD6244 on the DENV life cycle was attributed to a blockade of morphogenesis. Treatment of AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented the animals from contracting dengue hemorrhagic fever (DHF)-like lethal disease upon intravenous infection with 1 × 105 PFU of D2S20. The effectiveness of AZD6244 was observed even when the treatment of infected animals was initiated 1-2 days postinfection. This was also followed by a reduction in viral copy number in both the serum and the spleen. There was also an increase in IL-1β and TNF-α levels in mice that were infected with D2S20 and treated with AZD6244 in comparison to infected mice that were treated with the vehicle only. These data demonstrate the potential of AZD6244 as a new therapeutic agent to treat DENV infection and possibly other flavivirus diseases.
Handling Editor: Zhenhai Chen.
Leonardo C. de Oliveira and Aryádina M. Ribeiro contributed equally to this study.
In memoriam: Mark R. Buller.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00705-020-04524-7) contains supplementary material, which is available to authorized users.
Introduction
The family Flaviviridae includes major human pathogens, including yellow fever virus (YFV), dengue virus (DENV), Saint Louis encephalitis virus (SLEV), Japanese encepha- litis virus (JEV), West Nile virus (WNV), and Zika virus (ZIKV), which belong to the genus Flavivirus, and hepatitis C virus (HCV), which belongs to the genus Hepacivirus. Flaviviruses are predominantly arthropod-borne viruses that
*
[email protected]
are mainly transmitted by mosquitoes of the genera Aedes and Culex. They are thus classified as arboviruses [1, 2].
1Grupo de Transdução de Sinal/Flavivírus, Laboratório de Vírus, Department of Microbiology, Institute of Biological Sciences, Universidade Federal de Minas Gerais, Av. Antônio Carlos, 6627, Campus Pampulha, Belo Horizonte, MG 31270-901, Brazil
2Molecular Microbiology and Immunology, Saint Louis University School of Medicine, Saint Louis, MO, USA
3Present Address: Department of Pathology, University
of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK
The genus Flavivirus comprises a group of positive- sense, single-stranded RNA viruses with a genome approxi- mately 11 kb in length. The genome encodes a polyprotein from a single open reading frame that is cleaved by host and viral proteases into 10 polypeptides. The 5´ end of the genome encodes the structural proteins C, prM/M, and E. The non-structural (NS) proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 are encoded by the remaining 3´
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portion of the genome and are required for virus-host inter- actions, viral assembly, and replication [2–4]. DENV is a human pathogen and the most common mosquito-borne viral disease. There are four antigenically distinct serotypes (DENV-1 to 4), which share approximately 70% amino acid sequence identity with each other [2]. Dengue fever is an acute, self-limiting febrile illness, and as a result of the host’s innate and adaptive immune response, patients recover within 4–7 days [5, 6].
Dengue shock syndrome (DSS) and dengue hemorrhagic fever (DHF) are severe and life-threatening complications of dengue fever that occur in less than 1% of dengue infec- tions. The World Health Organization (WHO) estimates that 400 million new cases of dengue fever occur every year, mostly in equatorial regions of the world. Among those, approximately 500,000 cases develop into severe dengue, with 22,000 deaths yearly [2, 7, 8].
The current candidates for treatment of DENV infection are inhibitors that target DENV proteins, such as the E pro- tein [9], NS3 protease [10], NS5 methyltransferase [11], NS3 helicase [12], and NS5 polymerase [13], or host proteins, such as glucosidase [14] and enzymes involved in choles- terol synthesis [15]. However, there are currently no specific licensed therapeutics to treat DENV illness [16].
For DENV as well as other flaviviruses, new therapeu- tics are urgently needed. We have previously shown that the MEK/ERK inhibitor U0126 inhibits the replication of the flavivirus yellow fever virus. Here, we provide evidence that a new generation of MEK/ERK inhibitor, the small molecule AZD6244 (selumetinib), shows potent antiviral activity in vitro against DENV-2, DENV-3, and SLEV, as well as anti- DENV activity in vivo. Our data demonstrate that AZD6244 has potential for use as a new therapeutic agent to treat dis- eases associated with flaviviral infections.
Materials and methods
Cell culture and chemicals
BHK-21 and C6/36 cells were cultured in autoclaved Eagle’s minimum essential medium (MEM, Auto Pow, Gibco, USA) and Leibowitz (L-15) medium (Gibco, USA), respectively. The cultures were supplemented with 5% (v/v) heat-inac- tivated fetal bovine serum (FBS) (Cultilab, Campinas, SP, Brazil), 200 mM L-glutamine (Invitrogen, São Paulo, Bra- zil), and antibiotics (40 μg of gentamicin, 200 U of penicil- lin, and 1.5 μg of Fungizone per ml) and incubated in a humidified atmosphere containing 5% CO2 at 37 °C (BHK- 21) or 28 °C (C6/36).
U0126 (1,4-diamino-2,3-dicyano-1,4-bis[2-aminophe- nylthio] butadiene), a noncompetitive inhibitor of MEK1/2 with a 50% inhibitory concentration (IC50) of 70 nM, was
purchased from Cell Signaling Technology (Beverly, MA, USA). JNK inhibitor (JNKi) VIII (N-(4-amino-5-cyano- 6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide) and the pan-caspase inhibitor z-VAD-FMK (L-alaninamide, N-[(phenylmethoxy)carbonyl]-L-valyl-N-[(1S)-3-fluoro- 1-(2-methoxy-2-oxoethyl)-2-oxopropyl]) were purchased from Calbiochem–Merck (Darmstadt, Germany). AZD6244 (6-(4-bromo-2-chlorophenylamino)-7-fluoro-N-(2- hydroxyethoxy)-3-methyl-3H-benzo[d]imidazole-5-carbox- amide) is a potent, highly selective noncompetitive inhibitor of MEK1/2 with an IC50 of 14 nM for MEK1. It also inhib- its ERK1/2 phosphorylation with an IC50 of 10 nM [17]. AZD6244 was purchased from Selleckchem (Houston, TX, USA). Although both U0126 and AZD6244 are noncom- petitive inhibitors of MEK1/2, the latter is five times more effective than the former. Also, while AZD6244 is currently undergoing clinical trials, U0126, due to its higher cytotox- icity, has never entered clinical trials.
Viruses and viral infection
To produce stocks of the viruses, DENV-2 genotype III (PI- 59/2006 – Piauí-59) [18] and DENV-3 genotype I (MG- 20/2004) [19] were propagated in C6/36 cells, and SLEV (BeH 355964) [20] was propagated in BHK-21 cells. Briefly, C6/36 or BHK-21 cells were cultured in the presence of 10% FBS until they reached ~ 90% confluence and then infected with the indicated virus at a multiplicity of infection (MOI) of 0.01. Incubation was carried out for 72 h, after which the supernatants were collected, clarified by centrifugation (900 g, 4 °C, 15 min), and stored at -80 °C.
Viral infectivity was determined by plaque assay with BHK-21 cells as described previously [21]. Pharmacological inhibition tests were performed by incubating BHK-21 cells with a specific pharmacological inhibitor (PI) for 30 min prior to viral infection (MOI of 1.0) and then incubating in its continued presence for the indicated times. The DENV-2 strain D2S20 [22], which was derived from DENV-2 PL046 (an AG129-mouse-adapted virus), was kindly provided by Dr. Sujan Shresta (La Jolla Institute for Allergy and Immu- nology, California, USA). This DENV-2 strain was propa- gated in C6/36 cells as described above.
Virus infectivity assays
Assays were carried out essentially as described previously [21]. Briefly, BHK-21 cells (5 × 105 cells per well in a 6-well culture dish) were cultured as described above, serum- starved (1% FBS) for 12 h, and then infected at an MOI of 1.0 with the indicated virus. Infections were performed in either the presence or absence of the indicated PI (U0126, AZD6244, JNK VIII or z-VAD-FMK) using the indicated times and concentrations. Virus titers were determined after
collecting the supernatant from virus-infected and centri- fuged cells and assayed for infectivity as described above. The results are presented as the average values from experi- ments conducted in triplicate, and the data were confirmed by three independent experiments with very similar results.
Cytotoxicity assays
BHK-21 cells (5.0 × 105 cells per well in a 6-well culture dish) were exposed to increasing concentrations of U0126 (5, 10, 15, and 20 μM), JNKi VIII (0.4, 4, and 40 μM), AZD6244 (5, 10, 20, and 40 μM), and z-VAD (10, 20, 40, and 80 μM) and incubated for 48 h. Next, an equal volume of trypan blue was added to each well, and the cells were stained for 10 min at 25 °C. The stain was then removed, and the cells were observed with an optical microscope to determine whether the stain had entered the cells, which is indicative of cell death or membrane permeability. The analysis revealed that ~ 90% of the cells were not stained when they were treated with the following PI concentrations: U0126, 15 μM; JNKi VIII, 4 μM; AZD6244, 20 μM; z-VAD, 40 μM. Thus, these concentrations were chosen to be used throughout the experiments.
Electron microscopy
BHK-1 cells were left uninfected or infected with DENV-3 (MG-20) at an MOI of 1.0 for 36 h. The cells were then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) for 1 h at room temperature. Next, the cells were processed and examined using a Tecnai G2-Spirit FEI 2006 transmis- sion electron microscope operating at 80 kV at the Micros- copy Center, UFMG, Brazil, as described previously [21].
RNA extraction and quantitative PCR
Fluorogenic quantitative RT-PCR (qRT-PCR) was used to determine the viral genome copy number and amount of cytokine mRNA. Total RNA extraction was performed using TRIzol Reagent (Ambion) according to the manu- facturer’s instructions. DENV RNA was detected using the pan-DENV primer probe set [23], which recognizes a con- served sequence in the 3′ untranslated region of all DENV serotypes. The viral genome copy number was determined using a defined positive single-stranded RNA generated in vitro using T7 polymerase containing the DENV and 18S target sequences [24].
The copy number of DENV-2 RNA amplified from the serum and spleen of mice was determined by comparison with a plasmid standard curve. The levels of IL-1β, IL-6, and TNF-α mRNAs in splenocytes were determined by qRT-PCR. Briefly, RNA from splenocytes was extracted using TRIzol and treated with Turbo DNase (Life Technologies) for 2 h at
37 °C. Next, qRT-PCR was performed using One-Step RT- PCR Master Mix and detected using a 7500 Fast Real-Time PCR System (Applied Biosystems). All reactions were carried out in a final volume of 25 μl with 1 × Prime Time mix (Inte- grated DNA Technologies) and 12.5 μl of TaqMan Master Mix (Applied Biosystems).
Mice
Experiments were performed with 129/Sv mice that were doubly deficient in interferon (IFN)-α/β and IFN-γ receptors (AG129). The experiments were conducted at the Department of Molecular Microbiology and Immunology, Saint Louis Uni- versity School of Medicine in the laboratories of Drs. Buller, Brien, and Pinto. The mice were bred and maintained under specific-pathogen-free conditions at the animal facility at Saint Louis University. All experiments were approved by the Ani- mal Care and Use Committee at Saint Louis University (Ani- mal Welfare Assurance: D16-00141, IACUC protocol: 2667).
Age- and sex-matched mice at 12 to 14 weeks of age were used, and the experimental groups contained six to eight animals. Experimental manipulation was performed under anesthesia, which was induced and maintained using keta- mine hydrochloride and xylazine. AG129 mice were mock infected or infected intravenously with the indicated dose of DENV-2 D2S20 and treated with vehicle or oral doses of AZD6244 twice daily (100 mg/kg/day). This dosage was defined based on antitumor activity against mouse xenograft models [25]. A study was conducted to determine the tolera- bility of AZD6244. Doses of 25, 50 and 100 mg/kg/day were administered orally to AG129 mice (groups of 4 animals) for 18 consecutive days. All doses were well tolerated and had no effect on the physical condition or body weight of the mice when compared to untreated animals (data not shown).
The animals were monitored daily for signs of body weight loss and disease, such as facial edema, hunched posture, light sensitivity, and ruffled fur. Once the animals reached 30% body weight loss, they were anesthetized (300 mg/kg ketamine, 30 mg/kg xylazine). Blood was col- lected by cardiac puncture to obtain serum, and the mice were humanely euthanized according to IACUC procedures. The mice were then perfused with 30 to 50 ml of phosphate- buffered saline (PBS), and tissue samples (spleen and intes- tine) were harvested, processed, and tested to determine the DENV-2 genome copy number.
Results
In vitro analysis of antiviral activity
The possible role played by specific cellular signaling pathways in flavivirus replication was investigated using a
pan-caspase inhibitor (zVAD), a JNK inhibitor (JNKi VIII), and the MEK/ERK inhibitors U0126 and AZD6244. BHK- 21 cells were treated with the indicated concentrations of
inhibitor for 30 min prior to infection with DENV-3 MG-20, DENV-2 Piauí 59, or SLEV at an MOI of 1.0 for 48 or 72 h. Next, supernatants containing DENV-3 MG-20 (Fig. 1A-D),
◂Fig. 1 (A-D) In vitro analysis of antiviral activity carried out with diverse inhibitors against dengue virus serotype 3 (DENV-3). (A) Treatment of BHK-21 cells with potential antiviral candidates, JNK inhibitor VIII, Z-VAD, U0126, and AZD6244. Cultures of BHK-21 cells were individually treated with each compound 30 min prior to and throughout infection with DENV-3 MG-20 at an MOI of 1.0. Virus yield was determined through plaque assay after 48 and 72 h.p.i. (A, B, and C). (B) Dose-response analysis carried out with U0126. BHK-21 cells were treated with increasing concentrations of U0126 and infected with DENV-3. After the indicated times, the supernatant was assayed for infectivity. (C) Same as in B, except BHK-21 cells were treated with AZD6244. (D) Growth curve analy- sis of DENV-3 replication at 12, 24, 36, 48, 72, and 96 h.p.i. in BHK- 21 cells treated or not treated with the indicated concentrations of U0126 or AZD6244. Experiments were carried out with three techni- cal and three biological replicates, with similar results. Graphs were plotted using Graphpad Prism 5.0 software. Statistical analysis was done using two-way ANOVA with the Bonferroni post-test. Asterisks denote statistically significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001). (E-J) In vitro analysis of antiviral activity carried out with U0126 or AZD6244 against dengue virus serotype 2 (DENV-2) or SLEV. (E and H) Treatment of BHK-21 cells with potential anti- viral candidates, JNK inhibitor VIII, Z-VAD, U0126, and AZD6244. Cultures of BHK-21 cells were individually treated with the indicated inhibitors for 30 min prior to and throughout infection with DENV-2 Piauí 59 (E) or with SLEV (H) at an MOI of 1.0. Virus yield was determined using a plaque assay at 36 and 48 h.p.i. Dose-response analysis carried out with AZD6244 (F and I). As indicated, cultures were treated with increasing concentrations of inhibitor prior to infec- tion with DENV-2 (F) or with SLEV (I). After the indicated times, the supernatant was assayed for infectivity. (G and J) Growth curve analysis of DENV-2 (G) or SLEV (J) replication after 6, 12, 24, 36, 48, and 72 h.p.i. in BHK-21 cells treated or not treated with U0126 or AZD6244. Experiments were carried out with three technical and three biological replicates, with similar results. Graphs were plotted using Graphpad Prism 5.0 software. Statistical analysis was done using two-way ANOVA with the Bonferroni post-test. Asterisks denote statistically significant differences (*, p < 0.05; **, p < 0.01; ***, p < 0.001)
DENV-2 Piauí 59 (Fig. 1E-G), or SLEV (Fig. 1H-J) were collected and assayed for infectivity.
U0126 and AZD6244 reduced virus growth more effec- tively, as shown in Fig. 1A, E, and H. They caused a reduc- tion of at least 2- to 3-log10 units in the viral titers, whereas the other pharmacological inhibitors tested (JNKi VIII and zVAD) did not inhibit viral growth with similar effective- ness. We also analyzed whether viral growth inhibition was dose dependent. DENV-3 infections were carried out in the presence of U0126 (ranging from 3.75 to 15 µM), and the viral titers were then assayed. The results showed that viral growth inhibition was indeed dose dependent (Fig. 1B). This assay was also performed with AZD6244 (5, 10, 20, and 40 µM), and the decrease in virus titer was again dose dependent (Fig. 1C). This assay was also carried out with DENV-2 and SLEV, and similar results were obtained (Fig. 1F and I).
To gain some insight into the extent to which U0126 and AZD6244 impact the life cycle of DENV-3, DENV-2, and SLEV, BHK-21 cells were infected at an MOI of 1.0
for 6, 12, 24, 48, 72, and 96 h in the continuous presence or absence of U0126 (15 µM) or AZD6244 (20 µM), and the viruses were collected and assayed for infectivity. The results are consistent with the idea that AZD6244 indeed plays an important role in the growth of DENV-3 (Fig. 1D), DENV-2 (Fig. 1G), and SLEV (Fig. 1J), since the block imposed by the inhibitor affected viral multiplication, with a decrease in viral yields reaching 2- to 3-log10 units.
Treatment with AZD6244 affects DENV‑3 morphogenesis
To investigate whether AZD6244 affects the morphogen- esis of DENV-3, BHK-21 cells were infected with DENV-3 (MG-20) at an MOI of 1.0 for 36 h in either the presence or absence of AZD6244 (20 µM). Cells were then prepared and analyzed by electron microscopy (Fig. 2A-E). Panel A shows the uninfected cells at 36 h post-infection (h.p.i.), where N indicates the nucleus, M indicates the mitochon- dria, and rER indicates the rough endoplasmic reticulum. After infection, DENV-3 stimulated the invagination of the ER-derived membranes. This led to formation of diverse cisternae of rER, where viral RNA replication occurs along with the morphogenesis of enveloped virions that bud into the lumen of the ER (panel B).
An expanded cisterna of the rER contains a number of “vesicle packets.” The electron-dense material observed within the vesicles is thought to contain the replication com- plex (RC) and serve as a site for viral RNA replication (panel C) [26, 27]. Cisternae containing electron-dense material are consistently found in untreated cells infected with DENV-3 (panels B and C). However, in remarkable contrast, they were rarely observed at 36 h when the infection was carried out in the presence of AZD6244 (panels D and E). Viral particles could also be observed in the absence of AZD6244 (indicated by arrows in panel B).
Disease susceptibility and weight change of AG129 mice infected with DENV‑2
Next, we evaluated the antiviral potential of AZD6244 in vivo. To this end, we chose the DENV-2 strain D2S20, which is able to reproduce the key feature of the severe form of dengue infection (i.e., increased vascular permeability) [28]. AG129 mice were used to carry out the infections, and the experimental groups contained six to eight animals.
Age- and sex-matched AG129 mice at 12 to 14 weeks of age were infected intravenously with 6 × 105 PFU of DENV-2 D2S20 (lethal infection) and were either left untreated (blue) or treated twice daily with oral doses of AZD6244 (100 mg/kg/day) (red). The treatment commenced at 12 hours prior to infection and during the subsequent 8 days after infection. The untreated and uninfected control
Fig. 2 (A-E) Treatment with AZD6244 affects DENV-3 morpho- genesis. Representative micrographs of the effects of AZD6244 on DENV-3 morphogenesis in BHK-21 cells. (A) Uninfected cells were cultured for 36 h. N, nucleus; M, mitochondria; rER, rough endoplas- mic reticulum. (B-C) Cells were infected with DENV-3 (MG-20) at an MOI of 1.0 for 36 h. Arrows, viral particles; dashed square inset: virus-induced cisternae of rER. (C) Arrowheads: enveloped virions inside rER. Ribosomes are sometimes absent from rER membranes. (D-E) BHK-21 cells were pre-treated with AZD6244 (20 µM) and
then infected with DENV-3 (MG-20) at an MOI of 1.0 for 36 h. (D) Dashed square: viral structures can be seen inside the rER, but with- out the electron-dense core. (E) Dashed square inset: vesicle packets containing enveloped viral structures without the electron-dense core. Ribosomes are sometimes absent from rER membranes. Asterisk, vesicle packets without the electron-dense core; arrows, viral par- ticles. Panels are representative images of at least three independent experiments
mice were injected intravenously with 100 µl of supernatant from uninfected C6/36 insect cells.
As shown in Fig. 3A, 100% of the untreated and infected animals succumbed to infection at 5 days postinfection (d.p.i.). In contrast, only 40% of the AZD6244-treated and infected animals succumbed to infection at 12 d.p.i., which was 4 days after the last administration of AZD6244. Nota- bly, 60% of the animals survived up to 28 d.p.i. in spite of terminating treatment at 8 d.p.i. These results are consist- ent with the effects of AZD6244 observed in vitro on the replication of DENV and other flavivirus and confirm the therapeutic potential of this molecule as an anti-DENV drug.
We also monitored the effect of AZD6244 on the weight of infected animals over the course of infection. AG129 ani- mals were infected and treated as shown in Fig. 3A. The animals were weighed prior to drug treatment and then daily until 8 d.p.i. As shown in Fig. 3B, the infected groups (blue and red) lost weight in comparison to the uninfected group (black), regardless of whether they were treated with AZD6244. In the infected and untreated group (blue), the median weight loss was about 8% at day 4 when the animals
succumbed to infection, while for the infected and AZD- 6244-treated group, the median weight loss was around 12% at 8 d.p.i., with 100% survival. The disease signs observed in live AG129 mice that were infected with DENV-2 strain D2S20 with and without AZD6244 treatment are shown in Supplementary Fig. S1.
DHF‑like disease caused by DENV‑2 in AG129 mice
To determine whether AZD6244 can prevent DHF-like lethal disease, we infected AG129 mice with the mouse- adapted DENV-2 strain D2S20. To this end, a representa- tive male mouse and a female mouse were mock infected (Fig. 4A) or infected intravenously with 1 × 105 PFU of DENV-2 (Fig. 4 B-C). At 5 d.p.i., the mice were euthanized, and a gross pathology analysis was carried out. As shown in Fig. 4B, an infected male mouse developed macroscopic signs of hepatic damage (white arrowhead), stomach edema (black arrowhead), and hemorrhage of the small intestines (white asterisks).
Fig. 3 (A-B) Survival rates and weight change of AG129 mice infected with DENV-2 strain D2S20. (A) Survival rates of male and female AG129 mice infected intravenously with
6 × 105 PFU of DENV-2 D2S20, untreated (blue) or treated
twice daily with oral doses of AZD6244 (100 mg/kg/day) (red) commencing at 12 hours prior to infection and during the subsequent 8 days. (B)
Weight change curve of animals infected in A. The animals were weighed daily prior to drug treatment until 8 d.p.i. After
8 d.p.i., animals were evalu- ated daily for disease signs and survival until 30 d.p.i. (n = 9 in both vehicle- and AZD6244- treated groups). Graphs were plotted using Graphpad Prism 5.0 software. Statistical analysis was done with the log-rank (Mantel-Cox) test
Fig. 4 DHF-like syndrome caused by DENV-2 strain D2S20 in AG129 mice. Gross pathology analysis of selected mice. (A) Unin- fected male mouse. (B) Male mouse infected intravenously with 6 × 105 PFU of DENV-2 D2S20 at 5 d.p.i. showing macroscopic signs of hepatic damage (white arrowhead), stomach edema (black arrow- head), and small-intestine hemorrhage (white asterisks). (C) Female mouse infected intravenously with 6 × 105 PFU of DENV-2 D2S20 at 5 d.p.i. showing similar signs of DHF-like syndrome. (D) Gross
pathology of the whole intestinal tract of the infected animal in C showing an accumulation of bloody contents in the small intestines (black arrows). (E) AG129 mouse infected intravenously with 6 × 105 PFU of DENV-2 D2S20 and treated twice daily with oral doses of AZD6244 (100 mg/kg/day). The animals were euthanized based on weight loss and disease signs. Panels A-E are representative of exper- iments conducted with one of five animals per group for each experi- mental condition, with similar results
Fig. 4C shows an infected female mouse with signs similar to those of DHF syndrome (white asterisks). The intestinal tract was removed, and a pathological analysis revealed an accumulation of bloody contents in the small intestines (black arrows, Fig. 4D). However, treatment of the AG129 mice twice daily with oral doses of AZD6244 (100 mg/kg/day) prevented them from contracting DHF-like lethal disease upon intravenous infection with 1 × 105 PFU of DENV-2 (Fig. 4E).
Therapeutic use of AZD6244 prevents severe DENV disease in AG129 mice infected with DENV‑2
Next, we investigated whether AZD6244 can protect mice from a lethal infection with DENV-2 D2S20 when the treat- ment is initiated after infection. To address this question, AG129 mice were infected intravenously with 5 × 104 PFU of DENV-2. Starting at either 1 d.p.i. or 2 d.p.i., mice were treated twice daily with oral doses of AZD6244 (100 mg/
kg/day). The treatment was continued for the subsequent 10 days (blue line). The survival curve presented in Fig. 5A indicates that about 70% of the vehicle-treated mice (black
line) succumbed to the infection, whereas the survival rate of post-treated mice was about 75% (green and red lines). These results show a considerable increase in the survival rates and demonstrate that AZD6244 can protect AG129 mice even when administered after infection.
The levels of viral RNA in the serum and spleen were measured, along with the cytokine mRNA in the spleen of D2S20-infected mice, as shown in Fig. 5A. To this end, the serum (Fig. 5B) and spleen (Fig. 5C) were harvested at 5 d.p.i., and viral RNA was quantified by real-time RT-PCR. As shown in Fig. 5B-C, viral RNA levels in the serum were significantly reduced in animals that received AZD6244 therapy at 1 d.p.i. (p = 0.057) and 2 d.p.i. (p = 0.036) when compared to infected animals treated with vehicle. This corresponds with the survival data presented in Fig. 5A. AZD6244 therapy initiated on day 2 was also able to reduce the number of viral RNA copies within the spleen.
Levels of IL-1β, IL-6, and TNF-α mRNAs were measured in splenocytes isolated from DENV-infected animals treated with vehicle orAZD6244. The levels of IL-1β mRNA were significantly elevated in the splenocytes of the infected ani- mals that received the AZD6244 therapy at day 2 (p = 0.04)
Fig. 5 Therapeutic treatment of dengue-virus-infected AG129 mice. AG129 mice were infected with 5 × 104 PFU of DENV-2 D2S20 and then given AZD6244 (100 mg/kg/day) orally starting on day 1 or 2. Vehicle-treated mice were started with the day 1 group. (A) During treatment, mortality was monitored; the data reflect two independ- ent experiments with 8-9 mice per experiment per group. Additional mice were treated as in A except that they were sacrificed on day 5, and organs were harvested to determine viral RNA and cytokine
RNA levels. Levels of viral RNA in the (B) serum and (C) spleen were determined. Data are shown as log10 DENV genome equiva- lents (GE) per 18S of tissue or per ml of serum from three to five mice per condition. (D-F) Proinflammatory cytokine analysis. IL-1β (D), TNF-α (E), and IL-6 (F) levels in the spleen were evaluated by quantitative real-time PCR. Statistical analysis was done using the Mann-Whitney test
when compared to the animals treated with vehicle (Fig. 5D). Notably, we did not observe a significant difference in the IL-1β mRNA levels in splenocytes between animals treated with AZD6244 at 1 d.p.i. and vehicle-treated animals. How- ever, there was a significant difference in the IL-1β mRNA levels between animals that received AZD6244 therapy at 1 and 2 d.p.i. (p = 0.02).
We observed a similar trend when we analyzed mRNA levels of TNF-α (Fig. 5E). For IL-6, we were unable to detect measurable amounts of cytokine mRNA in the RNA isolated from splenocytes following AZD6244 therapy at day 2 (Fig. 5F). However, we noted similar levels of IL-6 in animals that received the AZD6244 therapy at day 1 in comparison to vehicle-treated animals. In the case of TNF- α, AZD6244 therapy at day 2 resulted in increased levels of cytokine compared to animals treated with AZD6244 therapy at day 1. Taken together, the results of these studies demonstrate the protective efficacy of AZD6244 therapy in controlling both cytokine levels and viral titers in DENV- infected animals.
Discussion
Dengue is a growing disease worldwide that affects 400 mil- lion people yearly and can cause life-threatening complica- tions (DHF/DSS) in some patients. Therefore, there is an urgent need for effective anti-DENV therapeutics [7, 29]. We have previously shown that the MEK/ERK inhibitor U0126 has a strong effect on YFV replication [30]. However, due to its high toxicity, U0126 did not enter clinical trials. In contrast, AZD6244 (selumetinib), a new generation of MEK/ERK inhibitor, has entered phase III clinical trials for the treatment of non-small-cell lung cancer (NSCLC) [31]. Here, it is being repurposed as a potential new anti-DENV therapeutic. In support of this, AZD6244 was effective in vitro against tested strains of DENV-2, DENV-3, and SLEV. A substantial reduction in viral yields from 2- to 4-log10 units was consistently observed (Fig. 1A-J).
The inhibition of virus growth was followed by an appar- ent block of virion morphogenesis (Fig. 2). This was attrib- uted to the arrest of viral replication complex formation at the ER, which is very similar to what we have previously shown with the blockade of YFV replication [30]. However, it cannot be ruled out that the observed difference in mor- phogenesis could be due to the low level of viral replication. The FDA-approved MEK antagonist trametinib (Meknist) was employed as an effective inhibitor for controlling the infection by polyomavirus ??Merkel cell polyomavirus?? (MCPyV) [32]. This provided the opportunity to repurpose this licensed drug to treat Merkel cell carcinoma (MCC), which is a lethal form of skin cancer caused by MCPyV.
Two other MEK antagonists (AZD-8330 and RDEA-119) were also tested against influenza A virus in combination with oseltamivir, which showed synergistic antiviral activ- ity. The same anti-influenza effectiveness was also verified with MEK inhibitors (PD-0325901 or AZD6244) when used either alone or in combination with oseltamivir [33]. Trametinib has also been shown to be effective against influ- enza A virus [34]. A number of potential anti-DENV thera- peutics based on host target inhibitors have been successfully tested in animal models. These include peptide-conjugated phosphorodiamidate morpholino oligomers (PPMOs) [35], a cellular glucosidase inhibitor [14], the host cholesterol syn- thesis inhibitor lovastatin [15], and fenretinide, a nuclear transport inhibitor [36, 37].
Here, we report on the antiviral activity of AZD6244 in vivo against lethal infection of AG129 mice with DENV-2 (D2S20). Notably, 60% of animals survived through the 28 day study period, in spite of the treatment being dis- continued at 8 d.p.i., (Fig. 3A). This protective effect was also reflected by the weight loss of AZD6244-treated and infected group, which was around 12% at 8 d.p.i. with 100% survival. The weight loss of the untreated and infected ani- mals was about 8% at day 4 when the animals succumbed to infection (Fig. 3B).
Remarkably, treatment of AG129 mice twice daily with oral doses of AZD6244 prevented them from contracting DHF-like lethal disease (Fig. 4). The protective effect of AZD6244 was also observed even when the treatment was started at 1-2 d.p.i., with the survival rate reaching 75%. This strengthens the potential for the use of this small molecule to treat DENV infections (Fig. 5A).
Consistent with these data, AZD6244 reduced the lev- els of viral RNA in both the serum and spleen (Fig. 5B-C), whereas the mRNA levels of the pro-inflammatory cytokines IL-1α and TNF-α increased following AZD6244 therapy at day 2 (Fig. 5D-E). Strikingly, this increment coincides with increased survival rates (100%) observed at up to 14 d.p.i. In contrast, survival rates of ~ 85% were observed at the same period of time with the therapeutic treatment started at 1 d.p.i. The reason for higher survival with therapy initiated at 2 d.p.i. remains to be clarified.
Taken together, AZD6244 appears promising as a poten- tial anti-DENV therapeutic based on its effectiveness both in vitro and in vivo. The repurposing of the drug to treat chronic disease as a potential anti-DENV therapy could be advantageous with respect to the expected decrease in side effects usually associated with long-term treatments.
Acknowledgements The authors are grateful to Dr. Michael S. Dia- mond, Washington University, St. Louis, MO, USA, for providing us with the AG129 mice. We also thank Dr. Erna G. Kroon from Labo- ratório de Virus – UFMG for the kind gift of DENV-2 and -3 viruses, and Dr. Maurício L. Nogueira from FAMERP – SJRP - SP, Brazil, for the gift of SLEV. This work was supported by grants awarded to CAB
from FAPEMIG - CBB – APQ-01670-11; CBB – AUC-00071-15; CAPES – AUXPE/PROEX/2015; CNPq - 476288/2012-6; FAPEMIG/
PPSUS – CBB – APQ -04178-17.
Funding FAPEMIG - CBB – APQ-01670-11; CBB – AUC-00071-15; CAPES – AUXPE/PROEX/2015; CNPq - 476288/2012-6; FAPEMIG/
PPSUS – CBB – APQ -04178-17.
Compliance with ethical standards
Conflict of interest The authors declare no conflict of interest.
Ethical approval Animal Care and Use Committee at Saint Louis Uni- versity (Animal Welfare Assurance: D16-00141, IACUC protocol: 2667).
References
1.Gardner CL, Ryman KD (2010) Yellow fever: a reemerging threat. Clin Lab Med 30:237–260
2.Lindenbach BD, Murray CL, Thiel HJ, Rice CM (2013) Flaviviri- dae: the viruses and their replication. In: Knipe D M, Howley PM (eds) Fields virology. Lippincott Williams & Wilkins, Philadel- phia, pp 712–746
3.Fernandez-Garcia MD, Mazzon M, Jacobs M, Amara A (2009) Pathogenesis of flavivirus infections: using and abusing the host cell. Cell Host Microbe 5:318–328
4.Boldescu V, Behnam MAM, Vasilakis N, Klein CD (2017) Broad-spectrum agents for flaviviral infections: dengue, Zika and beyond. Nat Rev Drug Discov 16:565–586
5.Whitehorn J, Simmon CP (2011) The pathogenesis of dengue. Vaccine 29:7221–7228
6.Guzman MG, Harris E (2015) Dengue. Lancet 385:453–465
7.Bhatt S, Gething PW, Brady OJ, Messina JP, Farlow AW, Moyes CL et al (2013) The global distribution and burden of dengue. Nature 496:504–507
8.Halstead SB, Cohen SN (2015) Dengue hemorrhagic fever at 60 years: early evolution of concepts of causation and treatment. Microbiol Mol Biol Rev 79:281–291
9.Poh MK, Yip A, Zhang S, Smit JM, Wilschut J, Priestle JP et al (2009) A small molecule fusion inhibitor of dengue virus. Antivir Res 84:260–266
10.Nitsche C, Behnam MA, Steuer C, Klein CD (2012) Retro pep- tide-hybrids as selective inhibitors of the Dengue virus NS2B- NS3 protease. Antivir Res 94:72–79
11.Lim SP, Sonntag LS, Noble C, Nilar SH, Ng RH, Zou G et al (2011) Small molecule inhibitors that selectively block dengue virus methyltransferase. J Biol Chem 286:6233–6240
12.Mastrangelo E, Pezzullo M, De Burghgraeve T, Kaptein S, Pas- torino B, Dallmeier K et al (2012) Ivermectin is a potent inhibi- tor of flavivirus replication specifically targeting NS3 helicase activity: new prospects for an old drug. J Antimicrob Chemother 67:1884–1894
13.Noble CG, Lim SP, Chen YL, Liew CW, Yap L, Lescar J et al (2013) Conformational flexibility of the Dengue virus RNA- dependent RNA polymerase revealed by a complex with an inhibi- tor. J Virol 87:5291–5295
14.Rathore AP, Paradkar PN, Watanabe S, Tan KH, Sung C, Con- nolly JE et al (2011) Celgosivir treatment misfolds dengue virus NS1 protein, induces cellular pro-survival genes and protects against lethal challenge mouse model. Antivir Res 92:453–460
15.Martinez-Gutierrez M, Correa-Londoño LA, Castellanos JE, Gallego-Gómez JC, Osorio JE (2014) Lovastatin delays infection and increases survival rates in AG129 mice infected with dengue virus serotype 2. PLoS One 9:e87412
16.De Clercq E (2013) Antivirals: past, present and future. Biochem Pharmacol 85:727–744
17.Huynh H, Soo KC, Chow PK, Tran E (2007) Targeted inhibition of the extracellular signal-regulated kinase kinase pathway with AZD6244 (ARRY-142886) in the treatment of hepatocellular car- cinoma. Mol Cancer Ther 6:138–146
18.Figueiredo LB, Sakamoto T, Coelho LFL, Rocha ESO, Cota MMG, Ferreira GP et al (2014) Dengue virus 2 American-Asian genotype identified during the 2006/2007 outbreak in Piauí, Brazil reveals a Caribbean route of introduction and dissemination of dengue virus in Brazil. PLoS One 9:e104516
19.Figueiredo LB, Cecílio AB, Ferreira GP, Drumond BP, de Oliveira J, Bonjardim CA et al (2008) Dengue virus 3 genotype 1 associ- ated with dengue fever and dengue hemorrhagic fever, Brazil. Emerg Infect Dis 14:314–316
20.Vedovello D, Drumond BP, Marques R, Ullmann LS, Fávaro EA, Terzian AC et al (2015) First genome sequence of St. Louis encephalitis virus (SLEV) isolated from a human in Brazil. Arch Virol 160:1189–1195
21.Pereira AC, Leite FG, Brasil BS, Soares-Martins JA, Torres AA, Pimenta PF et al (2012) A vaccinia virus-driven interplay between the MKK4/7-JNK1/2 pathway and cytoskeleton reorganization. J Virol 86:172–184
22.Makhluf H, Buck MD, King K, Perry ST, Henn MR, Shresta S (2013) Tracking the evolution of dengue virus strains D2S10 and D2S20 by 454 pyrosequencing. PLoS One 8:e54220
23.Gurukumar KR, Priyadarshini D, Patil JA, Bhagat A, Singh A, Shah PS et al (2009) Development of real time PCR for detection and quantitation of Dengue viruses. Virol J 6:10
24.Pinto AK, Brien JD, Lam CY, Johnson S, Chiang C, Hiscott J et al (2015) Defining new therapeutics using a more immunocompetent mouse model of antibody-enhanced Dengue virus infection. MBio 6:e01316-15
25.Yeh TC, Marsh V, Bernat BA, Ballard J, Colwell H, Evans RJ, Parry J, Smith D, Brandhuber BJ, Gross S, Marlow A, Hurley B, Lyssikatos J, Lee PA, Winkler JD, Koch K, Wallace E (2007) Biological characterization of ARRY-142886 (AZD6244), a potent, highly selective mitogen-activated protein kinase kinase 1/2 inhibitor. Clin Cancer Res 13(5):1576–1583
26.Welsch S, Miller S, Romero-Brey I, Merz A, Bleck CK, Walther P et al (2009) Composition and three-dimensional architecture of the dengue virus replication and assembly sites. Cell Host Microbe 5:365–375
27.Chatel-Chaix L, Bartenschlager R (2014) Dengue virus and Hepa- titis C vírus induced replication and assembly compartments: the enemy inside—caught in the web. J Virol 88:5907–5911
28.Shresta S, Sharar KL, Prigozhin DM, Beatty PR, Harris E (2006) Murine model for dengue virus-induced lethal disease with increased vascular permeability. J. Virol 80:10208–10217
29.Neufeldt CJ, Cortese M, Acosta EG, Bartenschlager R (2018) Rewiring cellular networks by members of the Flaviviridae family. Nat Rev Microbiol 16:125–142
30.Albarnaz JD, de Oliveira LC, Torres AA, Palhares RM, Casteluber MC, Rodrigues CM et al (2014) MEK/ERK activation plays a decisive role in yellow fever virus replication: implication as an antiviral therapeutic target. Antivir Res 111:82–92
31.Zhao Y, Adjei AA (2014) The clinical development of MEK inhibitors. Nat Rev Clin Oncol 11:385–400
32.Liu W, Yang R, Payne AS, Schowalter RM, Spurgeon ME, Lambert PF et al (2016) Identifying the target cells and mecha- nisms of merkel cell polyoma virus infection. Cell Host Microbe 19:775–787
33.Haasbach E, Hartmayer C, Planz O (2013) Combination of MEK inhibitors and oseltamivir leads to synergistic antiviral effects after influenza A virus infection in vitro. Antivir Res 98:319–324
34.Dudek SE, Schreiber A, Ehrhardt C, Planz O, Ludwig S (2018) The clinically approved MEK inhibitor Trametinib efficiently blocks influenza A virus propagation and cytokine expression. Antivir Res 157:80–92
35.Stein DA, Huang CY, Silengo S, Amantana A, Crumley S, Blouch RE et al (2008) Treatment of AG129 mice with antisense mor- pholino oligomers increases survival time following challenge with dengue 2 virus. J Antimicrob Chemother 62:555–565
36.Fraser JE, Watanabe S, Wang C, Chan WK, Maher B, Lopez-Den- man A et al (2014) A nuclear transport inhibitor that modulates
AZD6244
the unfolded protein response and provides in vivo protection against lethal dengue virus infection. J Infect Dis 210:1780–1791
37.Carocci M, Hinshaw SM, Rodgers MA, Villareal VA, Burri DJ, Pilankatta R et al (2015) The bioactive lipid 4hydroxyphenyl reti- namide inhibits flavivirus replication. Antimicrob Agents Chem- other 59:85–95
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