AAC Accepted Manuscript Posted Online 16 May 2016 Antimicrob. Agents Chemother. doi:10.1128/AAC.00282-16 Copyright © 2016, American Society for Microbiology. All Rights Reserved.
1 Discovery of a broad-spectrum antiviral compound that inhibits pyrimidine biosynthesis
2 and establishes a type 1 interferon-independent antiviral state
3
4 Dong-Hoon Chung,a,b # Jennifer E. Golden,c,* Robert S. Adcock,b Chad E. Schroeder,c
5 Yong-Kyu Chu,b Julie B. Sotsky,b Daniel E. Cramer,b Paula M. Chilton,a,† Chisu Song,d
6 Manu Anantpadma,e Robert A. Davey,e Aminul I. Prodhan,f Xinmin Yin,f Xiang Zhangf
7
8 Departments of Microbiology and Immunology a, and Center for Predictive Medicine for
9 Biodefense and Emerging Infectious Diseases b, University of Louisville, KY, USA;
10 University of Kansas Specialized Chemistry Center, Lawrence, KS, USAc; Division of
11 Infectious Diseases and Northwestern HIV Translational Research Center, Department
12 of Medicine, Northwestern University Feinberg School of Medicine, IL, USAd; Texas
13 Biomedical Research Institute, San Antonio, TX, USAe; CREAM Center, University of
14 Louisville, KY, USA f.
15
16 # Address correspondence to Dong-Hoon Chung, [email protected]
17 * Current address: School of Pharmacy, 777 Highland Ave., University of Wisconsin-
18 Madison, Madison, WI, USA
19 † Current address: Christine M. Kleinert Institute for Hand & Microsurgery, 225
20 Abraham Flexner Way, Louisville, KY, USA 21 Abstract
22 Viral emergence and re-emergence underscores the importance of the developing
23 efficacious, broad-spectrum antivirals. Here we report the discovery of
24 tetrahydrobenzothiazole-based compound 1, a novel, broad-spectrum antiviral lead,
25 which was optimized from a hit compound derived from a CPE-based antiviral screen
26 using Venezuelan equine encephalitis virus. Compound 1 showed antiviral activity
27 against a broad-range of RNA viruses including alphaviruses, flaviviruses, influenza
28 virus, and ebolavirus. Mechanism of action studies with metabolomics and molecular
29 approaches revealed that the compound inhibits host pyrimidine synthesis, and
30 establishes an antiviral state by inducing a variety of interferons-stimulated genes (ISG).
31 Notably, the induction of the ISGs by compound 1 was independent of the production of
32 type 1 interferons. The antiviral activity of 1 was cell-type dependent with a robust effect
33 observed in human cell lines and no observed antiviral effect in mouse cell lines. We
34 herein disclose tetrahydrobenzothiazole 1 as a novel lead for the development of a
35 broad-spectrum, antiviral therapeutic and as a molecular probe to study the mechanism
36 of the induction of ISGs independent of type 1 interferons.
37 38 Introduction
39 Despite the economic and healthcare burden posed by viral infections, current
40 treatments for associated diseases are limited mostly to prophylactic vaccines. Only a
41 small number of viral diseases (e.g., Human immunodeficiency virus and Hepatitis C
42 virus, HCV) can be treated with virus-specific therapeutics (e.g., sofosbuvir) (1, 2) .
43 These agents, so-called direct acting antivirals (DAA), target viral gene products for
44 their activities. In general, DAAs are prone to develop resistant mutants and have a
45 narrow antiviral spectrum. Given the emergence of new viruses and the rapid spread of
46 emerging viral diseases to previously unaffected geographic areas, there is an urgent
47 need for the identification of agents that more efficiently target a broad range of viral
48 diseases, which DAA approaches may not be able to deliver in time.
49 While broad-spectrum antivirals may overcome these limitations, the development of
50 these agents has been hindered due to low efficacy or undesirable toxic effects, which
51 are intrinsic characteristics of most broad-spectrum antivirals. For example, ribavirin has
52 been studied since 1972 and tested against many RNA viruses; however, its useful
53 antiviral spectrum is relatively narrow(3). Many RNA viruses, such as alphaviruses, are
54 not susceptible to ribavirin, and patients may not benefit from the treatment due to its
55 limited therapeutic window (4-6). T-705, a RNA-dependent RNA polymerase inhibitor,
56 was also reported with antiviral activity against a variety of RNA viruses. It is under
57 development as a therapeutic candidate; however, its potency (IC50 value) falls in the
58 few hundred micromolar range for most viruses, with the exception of influenza viruses
59 (7). 60 Previously, we reported the discovery of new anti-Venezuelan equine encephalitis virus
61 (VEEV) inhibitors from a high-throughput screening (HTS) campaign(8). VEEV is an
62 RNA virus that causes encephalitis in humans and equids, and effective therapeutics for
63 the disease have not yet been developed. We screened a library of 348,000 small
64 molecule compounds with a cell-based assay measuring protection of cells from VEEV-
65 induced cytopathic effect (strain TC-83) and discovered five active compounds ('hits')
66 with EC50 values better than 15 μM. One of these hits and the resulting optimized lead,
67 ML336, turned out to be a DAA that inhibits viral RNA synthesis by targeting the amino
68 terminal domains of viral nonstructural proteins 2 and 4 (8, 9).
69 In this current study, we investigated whether the HTS had identified a broad-spectrum
70 antiviral inhibitor. Since the screen was based on a functional read-out, i.e. reduction in
71 virus-induced cell death, we hypothesized that the screen could identify a broad-
72 spectrum antiviral compound as well. Indeed, we found that one of our hit compounds,
73 CID 847035, did show antiviral effects in many cell-based antiviral assays, including
74 Marburg virus assay (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=540276),
75 Lassa virus assay (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=540256) and
76 RSV assay (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid=2391). Based on
77 these observations, we undertook a study aimed at the development of novel, broad-
78 spectrum antiviral inhibitors based on hit compound CID 847035 and the mechanism of
79 action underlying their activity against multiple viruses.
80 Herein we present the broad-spectrum antiviral behavior and mechanism of
81 tetrahydrobenzothiazole 1 (Figure 1), an analogue of our primary hit compound, CID 82 847035. Using metabolomics and genomics approaches, we found that compound 1
83 inhibits pyrimidine biosynthesis and establishes an antiviral state by activating genes
84 involved in the innate immunity, including retinoic acid-inducible gene I protein (RIG-I,
85 encoded by DDX58), interferon-induced protein with tetratricopeptide repeats 1(ISG56,
86 encoded by IFIT1) and 2'-5'-oligoadenylate synthetase-like (OASL, encoded by OASL)
87 genes. Interestingly, the antiviral status induced by compound 1 was independent of
88 type 1 interferons (IFNs) or exogenous RNA. Importantly, we also determined that
89 mouse cell lines are not capable of establishing antiviral status when treated with
90 compound 1.
91 92 Materials and Methods
93 Cells and viruses : Vero 76 (ATCC CRL-1587), BHK (ATCC CCL-10), HEp-2 (ATCC
94 CCL-23), Neuro 2A (ATCC CCL-131), SH-SY5Y(ATCC CRL-2266), MRC-5 (ATCC
95 CCL-171) and HEK 293T (ATCC CRL-3216) were obtained from ATCC and maintained
96 in Minimum Essential Medium with Earl's modification (MEM-E) containing 10% fetal
97 bovine serum (FBS) and 1X GlutaMAX (Gibco 35050-061) at 37 °C with 5% CO2.
98 MDCK (Sigma-Aldrich 84121903), RD (ATCC CCL-136) and NIH 3T3 (ATCC CRL-
99 1658) were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% FBS
100 and 2 mM L-glutamine at 37 °C with 5% CO2. TZM-bl cells were obtained through the
101 NIH AIDS Research and Reference Reagent Program from John C. Kappes, Xiaoyun
102 Wu, and Tranzyme, Inc. The TZM-bl indicator cell line, used for infectivity assays of
103 HIV-1, is a genetically engineered HeLa cell clone expressing CD4, CXCR4, CCR5, and
104 Tat-responsive firefly luciferase and Escherichia coli β-galactosidase under the control
105 of an HIV-1 long terminal repeat. TZM-bl cells were cultivated in DMEM (containing 4.5
106 g/liter glucose, L-glutamine, and sodium pyruvate) medium with 10% fetal calf serum,
107 50 IU/ml penicillin, and 50 μg/ml streptomycin at 37 °C with 5% CO2.
108 VEEV TC-83 (lyophilized vaccine from USAMRIID) and V3526 were amplified in and
109 titrated in BHK-21cells. V3526-luc was rescued from the BHK cells transfected a full-
110 length viral RNA derived from pV3526-luc as described elsewhere(10). VEEV TrD (gift
111 from Dr. R. Tesh, World Reference Center for Emerging Viruses and Arboviruses),
112 herpes simplex virus type-2 (HSV-2, gift from Dr. Steinbach, University of Louisville)
113 were grown in Vero 76 cells that were maintained in Dulbecco’s-modified essential 114 media (DMEM) with 10% FBS. Lymphocytic Choriomeningitis Virus ARM strain (LCMV-
115 ARM) was a gift from Dr. Lukashevich (University of Louisville). Chikungunya virus S-27
116 (BEIR NR-13220), West equine encephalitis virus California (WEEV California, ATCC
117 VR-70), Japanese Encephalitis virus SA14 ( BEIR NR-2335), West Nile virus NY-99
118 (gift from Dr. R. Tesh, World Reference Center for Emerging Viruses and Arboviruses),
119 Yellow Fever virus 17D (BEIR VR-1506) were grown in Vero76 cells in a virus infection
120 media (MEM-E with 10% FBS, 1X GlutaMAX and 25 mM of HEPES pH 7.3). Human
121 enterovirus D71 (strain MP4, BEIR NR-472) and human encephalomyocarditis virus MM
122 (BEIR NR-19846) were amplified in RD cells maintained in DMEM with 10% FBS.
123 To generate wild-type HIV-1 virus HEK293T cells were plated at a density of 6 x 106
124 cells/100 mm culture dish 24 hours prior to transfection. Cells were transfected with 10
125 μg of wild-type HIV-1 proviral clone, pNL4.3, using linear polyethylenimine (PEI; 25 kDa;
126 Polysciences, Inc.), as describe elsewhere (11). Culture supernatants were collected 48
127 hours after transfection and cellular debris was removed by filtration through a 0.45 μm
128 filter. Viral p24 was quantitated using standard p24 ELISA.
129
130 Dose-response studies EC50 and CC50 were evaluated in a dose-response format
131 starting from 50 µM by a 2-fold dilution, triplicates for each, in a 96-well format. For a
132 CPE-based assay, cells were seeded in white well plates at a cell density of 12,000
133 cells per well in a volume of 45 microliters and incubated in an actively humidified
134 incubator with 5.0% CO2 at 37 °C and 95% humidity for 18 hours. Test compounds
135 diluted in thirty microliters of cell culture media was added to each well. After a two- 136 hours incubation at 37 °C with a 5% CO2, a 600 pfu of virus (or cell culture media for
137 cytotoxicity assay) was added to the wells in a volume of fifteen microliters then
138 incubated two days in an actively humidified incubator with 5.0% CO2 at 37 °C and 95%
139 humidity. Cell viability was measured with 90 microliter per well of CellTiter-Glo™
140 reagent (Promega). Vero 76 cells were used for alphaviruses and RD cells were used
141 for HEV and EMCV assays. For a luciferase-tagged virus assay (i.e., V3526-luc and
142 pVSV-luc), an optimized amount of virus and cell numbers were used for each cell line
143 tested. For HEK 293T, Neuro 2A and SH-SY5Y cells, 24,000 cells and 2,400 TCID50
144 units of virus per well was used. For Vero 76 and BHK cells, 12,000 cells and 1200
145 TCID50 units of virus per well was used. For NIH3T3, 24,000 cells and 20,000 TCID50
146 units of virus per well was used. After an 18-hours incubation with virus, the plates were
147 developed with Bright-Glo™ reagent (Promega) and the luciferase activity was
148 measured as a readout for the virus replication.
149 For measurement of inhibition of Ebolavirus infection, a recombinant Ebolavirus with a
150 green fluorescent protein (GFP) gene inserted into the genome (Ebola-eGFP virus, a
151 kind gift of Dr. Heinz Feldmann) was used(12). The virus stock was generated by
152 infecting Vero E6 cells with Ebola-eGFP virus followed by pelleting the culture
153 supernatant through a 20% sucrose cushion. HeLa cells were pretreated for 2 hour with
154 two fold-dilutions of 10 to 0.005 µM of compound and incubated with virus for 24 hours
155 in the presence of the compound. Fixed cells were imaged by a fluorescent microscope
156 . Total and infected cells were counted by Cell Profiler image analysis software (Broad
157 Institute, MIT, Boston, MA), detecting nuclei stained with DAPI and virus encoded GFP 158 expression(13). This work was performed in a biosafety level 4 (BSL4) laboratory at
159 Texas Biomedical Research Institute.
160
161 Titer reduction assay To measure virus titer reduction, 12-well plates with 100,000
162 Vero 76 cells grown overnight were pre-treated with compound diluted in virus infection
163 medium at 37 °C with 5% CO2 for 8 hours, unless denoted. For virus adsorption, cell
164 plates were incubated on ice for a 15 min, and then the cell culture supernatant was
165 removed. Virus diluted at an MOI of 0.05 (or 3 for a time of addition study) in 250
166 microliter of virus infection medium was added to the cell and the virus was allowed to
167 absorb to the cells on ice for one hour. The unabsorbed virus was washed with 1 mL of
168 PBS once and the wells were replenished with virus infection medium with 5 µM of
169 ML416 or DMSO (0.25% vol/vol). The progeny virus was harvested after 26 hours for
170 VEEV, CHIKV, WEEV, HSV-2 or Influenza virus; 48 hours for JEV, YFV17D or WNV; 72
171 hours for RSV or LCMV virus. The progeny viruses in the supernatants were
172 enumerated by using either virus infection center assay or TCID50 assays. Virus
173 infection center assay was done with Vero76 cells grown confluent in 24-well plates.
174 The cells were infected with 167 µL of the serially diluted virus samples for one hour at
175 37 °C with 5% CO2. Wells were washed with PBS and replenished with virus infection
176 medium with 0.75% methylcellulose. Three or four days after virus infection, virus
177 infection centers were visualized with crystal violet staining (0.2% crystal violet, 4%
178 paraformaldehyde, and 10% ethanol). For influenza virus and HSV-2, a TCID50 assay
179 used for the titration. 180 HIV-1 Viral Infectivity Assay TZM-bl indicator cells were plated at a density of
181 10,000 cells/well in a 96 well culture plate 24 hours prior to HIV-1 infection and
182 incubated at 37 °C (5% CO2). On the day of infection, the culture medium was removed,
183 and the cells were inoculated in triplicate with 100 μL of 2-fold serial dilutions of viral
184 supernatants in culture medium containing 20 μg/mL DEAE-dextran and incubated at 37
185 °C (5% CO2). At the time of infection DMSO as a control or 10μM ML461 were added to
186 test effect of the compound on HIV-1 infection. After a 24 hours of incubation, culture
187 medium was removed from each well and replaced with 100 μL of Britelite Plus
188 luciferase assay substrate (PerkinElmer). Following 5 min of incubation at room
189 temperature, 70 μL of each cell lysate was transferred to a 96-well OptiPlate 96
190 (PerkinElmer) and luminescence was measured in a VICTOR X2 Multilabel Reader
191 (PerkinElmer). Relative infectivity was calculated by plotting luciferase activity of viral
192 particle with treated DMSO as 100 %.
193
194 Microarray
195 HEp-2 cells were treated for 18 hours either with 5 μM of CID 70698683 or with DMSO
196 for the control in cell culture medium. 100 ng total RNA was amplified and labeled
197 following the Affymetrix (Santa Clara, CA) standard protocol for their 3’IVT Plus
198 Labeling Kit, followed by hybridization to Affymetrix’ Primeview® Human Gene
199 Expression arrays. The arrays were processed following the manufacturer
200 recommended wash and stain protocol on an Affymetrix FS-450 fluidics station and
201 scanned on an Affymetrix GeneChip® 7G scanner using Command Console 4.0 . The 202 resulting .cel files were imported into Partek Genomics Suite 6.6 and transcripts were
203 normalized and summarized using RMA as normalization and background correction
204 method. A 1-way ANOVA was set up to compare the treatment of 5 μM of
205 CID:70698683 to the control. False Discovery Rate (FDR) was chosen as multiple test
206 correction for the resulting p-values.
207
208 RealTime PCR Total RNAs from cells in a 12-well plate were isolated with RNAzol ®
209 RT (Molecular Research Center, Inc) reagent as per the manufacturer’s protocol and
210 were dissolved in 50 μL of The RNA storage solution (Life Technologies). One
211 microgram of RNA samples were subjected to a cDNA synthesis with Maxima™ H
212 Minus Reverse Transcriptase (Life Technologies), random hexamers, and oligo-dT by
213 following the manufacturer’s protocol. For quantitation of gene expression, we used a
214 real-time PCR with 2(-Delta Delta C(T)) method in a total of twenty microliters per well
215 with 2 μL of 2-fold diluted cDNA mixture in a multiplex mode in conjunction with TaqMan
216 chemistry. Information on the primers and probes are described in Supplementary
217 Information (Table S2). The copy number of viral genome was quantitated as described
218 elsewhere(8). 18S rRNA (Cat. No. 4319413E, Life Technologies) and human GAPDH
219 (Cat. No. 4326317E Life Technologies) were used as the endogenous controls to
220 quantitate the relative viral and human gene RNA copy numbers, respectively. Three
221 biological replicates, each with two technical replicates, were used for the quantitation.
222 223 Interferon assays Interferon-α and -β in the cell culture supernatant were detected by
224 using LumiKine™ Xpress hIFN-α and LumiKine™ hIFN-β kits (InvivoGen) as per the
225 manufacturer’s protocol. One-day-old HEK 293T cells cultured in a 12-well plate were
226 treated with 5 µM of compound 1 or DMSO in virus infection medium for 18 hours and
227 the cell culture supernatants were harvested and cleared by centrifugation at 3000 x g
228 for 10 min. For each treatment, six replicates were used per group and three technical
229 replicates were used for the controls, HEK293-expressed human IFN-α2 and CHO-
230 expressed human IFN-β.
231 HEK-Blue™ IFN-α/β cell reporter assay (InvivoGen) was done followed by the
232 manufacturer’s protocol. One-day-old HEK-Blue™ IFN-α/β cells plated in a 96-well plate
233 were treated with two fold-dilutions of 50 to 0.4 µM of compound 1 or DMSO for 24
234 hours. The expression of SEAP under the control of ISRE9, which is activated by Type
235 1 interferons, was measured by the absorbance at 620 nm after 20 min incubation with
236 100 µL of SEAP substrate. Three replicates were used for each data points. 237 Metabolomics analysis Metabolites were extracted from cell samples using a solvent
238 mixture of acetonitrile, water, and chloroform (2:1.5:1 by volume). The metabolite
239 extract from each sample was then derivatized using N-methyl-N-(trimethylsilyl)
240 trifluoroacetamide (MSTFA). All derivatized samples were further analyzed on a LECO
241 Pegasus 4D GC×GC–TOF MS instrument (St. Joseph, MI). The instrument data were
242 first processed using LECO’s instrument control software ChromaTOF for peak picking
243 and tentative metabolite identification. MetPP software was used for retention index
244 matching, peak merging, peak list alignment, normalization, and statistical significance
245 test (14). The abundance test was performed using pairwise two-tail t-test with sample
246 permutation, to standardize the abundance variation of each metabolite between
247 sample groups. The presence-absence test was performed using Fisher’s exact test.
248 249 Results
250 Discovery of tetrahydrobenzothiazole 1
251 Our lead, compound 1, resulted from a rigorous medicinal chemistry optimization effort
252 focused on a hit compound (CID 847035) that was prioritized from our high-throughput
253 screen measuring the protection of Vero 76 cells from a VEEV- induced CPE (Figure 1A
254 and 1B) (8). Medicinal chemistry optimization of the this hit compound, which will be
255 described separately in due course, was executed in parallel to the development of a
256 structurally- and mechanistically-distinct anti-VEEV probe, ML336. Unlike ML336,
257 compound 1 showed a potent antiviral effect towards multiple alphaviruses with
258 cytopathic effect (CPE) EC50 values of 0.35 µM, 0.29 µM, and 0.96 µM for VEEV TC-83,
259 CHIKV S27, and WEEV, respectively (Table 1).
260 To verify the antiviral activity of compound 1 against multiple alphaviruses, we
261 employed various assays with different read-outs. In a luciferase-based anti-VEEV
262 assay (V3526-luc,Table 1), compound 1 showed promising antiviral activity with an EC50
263 of 0.17 µM. In a quantitative real-time PCR (qRT-PCR) assay with VEEV TC-83 strain
264 (Figure 1C), compound 1 (20 μM) decreased viral RNA copy numbers greater than
265 1000-fold compared to the control. Finally, we employed a titer reduction assay with
266 variety of alphaviruses to confirm the antiviral activity of compound 1. For VEEV TC-83,
267 the progeny virus production decreased by greater than 15,000-, 2,600-, or 340-fold at
268 20, 10, or 5 μM, respectively (Figure 1D). VEEV TrD and CHIKV S17 strains were more
269 sensitive to compound 1. At a concentration of 5 μM, the compound decreased the level
270 of viral replication more than 10,000-fold (4 Log) for the both viruses. From these 271 experiments, we concluded that tetrahydrobenzothiazole 1 potently inhibits multiple
272 alphaviruses.
273
274 Time of addition assay
275 To understand at what stage of virus replication compound 1 exerted its antiviral
276 activity, we employed a time of addition assay with Vero76 cells synchronously infected
277 with TC-83 at multiplicity of infection (MOI) of 3 (Figure 1E). Compound 1 was added to
278 cells at various time-points in regards to the virus replication cycle and maintained until
279 the progeny virus was harvested. The experiment showed that pre-treatment was
280 necessary in order to observe full antiviral activity. Treatment at time 0, right after the
281 virus adsorption to cells on ice, resulted in only 65% reduction in progeny virus titer;
282 however, pre-treatment for six hours resulted in ~ 400-fold reduction in virus titer.
283 Antiviral efficacy of compound 1 was dependent on the length of pre-treatment time
284 between 4 and 0 hours prior to infection. This result clearly shows that compound 1
285 requires the induction of a cellular response for its activity.
286
287 Cytotoxicity of tetrahydrobenzothiazole 1
288 The cytotoxicity of compound 1 was evaluated, as a cytotoxic compound may
289 misleadingly exhibit broad-spectrum antiviral activity. Vero76 cells were plated in 96-
290 well plates and incubated 48, 72, and 96 hours in the presence of 1 at various 291 concentrations. Compound 1 did not show apparent cytotoxicity up to 12.5 μM (Figure
292 S1 A). The CC50 values were 74.1, 31.0 and 34.6 μM after 2, 3, and 4 days exposure
293 respectively, resulting in a selective Index 50 (SI50) greater than 100 (CC50 at Day3/
294 EC50 of CHIKV = 106.9). This indicated that the agent is not toxic to cells at the effective
295 concentrations, and that the inhibition of virus replication is not be due to a non-specific
296 cytotoxicity. Cells treated with compound 1, however, looked larger and more stretched
297 compared to the control group under visual observation. The compound was further
298 tested in viability, cytotoxicity and/or apoptosis assays with HEK 293T cells for four
299 days. No significant cytotoxicity or apoptosis was induced with up to 25 μM of
300 compound 1 (Figure S1 B).
301
302 Antiviral spectrum of tetrahydrobenzothiazole 1
303 As compound 1 showed similar antiviral effects on all alphaviruses we tested, we
304 questioned whether the compound could inhibit a broader-spectrum of viruses. To
305 address this question, we tested compound 1 against various viruses including:
306 Vesicular stomatitis virus (pVSV-luc), Respiratory syncytial virus (RSV), ebolavirus
307 (EBOV), HIV-1, Japanese Encephalitis virus (JEV), Herpes simplex virus type-2 (HSV-
308 2), Yellow Fever virus 17D(YFV 17D), West Nile virus (WNV), Influenza virus (IFNV),
309 human enteroviruses 71 (EV-71), and Encephalomyocarditis virus (EMCV).
310 As shown in Table 1, compound 1 showed an antiviral effect against a broad-spectrum
311 of viruses with different sensitivity to each virus. Alphaviruses, LCMV, and IFNV were 312 most sensitive. Treatment with compound 1 (5 µM) resulted in a greater than 4 Log
313 reduction in the progeny virus titers of alphaviruses tested, and greater than 3 Log
314 reduction for IFNV and LCMV. The RSV, WNV, YFV 17D viruses were sensitive as well
315 with ~ 2 Log titer reduction; however, JEV was less sensitive than the others tested
316 (~1.3 Log reduction). HIV-1 infection was sensitive as well, with 94% reduction in viral
317 infectivity using TZM-bl reporter system measuring luciferase activity with 10 μM
318 concentration of compound 1. Interestingly, the replication of HSV-2 was not inhibited
319 by 1, resulting in no changes in progeny virus titer.
320 The compound’s antiviral activity was also assessed using a dose-response format
321 (EC50 determination). The replication of pVSV-luc or EBO-GFP was sensitive to the
322 compound treatment with EC50 values of 0.19 μM and 0.30 μM, respectively. The agent,
323 however, did not inhibit the replication of the picornaviruses efficiently, (i.e. EC50 values
324 of EV D71 and EMCV MM were 11.1 μM and > 25 μM, respectively). From these
325 experiments, we found that compound 1 is a broad-spectrum antiviral effect with
326 different sensitivities depending on virus. This result implies that the antiviral
327 mechanism of compound 1 has specificity to certain type of viruses.
328
329 Pyrimidine synthesis inhibition by tetrahydrobenzothiazole 1
330 To test if our lead compound could interfere with cellular metabolism for its antiviral
331 activity, we measured the difference in cellular metabolites using a metabolomics
332 approach. HEK 293T cells were treated either with 5 μM of compound 1 or with DMSO 333 for 18 hours and the cellular metabolites were analyzed and compared using GC-MASS
334 spectrophotometry. The treatment with compound 1 changed the abundance of cellular
335 metabolites significantly (Table S1). For example, the amounts of L-glutamic acid and
336 fumaric acid differed by 0.09- and 11-fold when compound 1-treated group was
337 compared to mock-treated cells. For some metabolites, the difference was more evident
338 (Table 2). For example, dihydroorotic acid (DHO) and orotic acid were found only in
339 compound 1-treated groups. DHO and orotic acid are metabolites that are related with
340 de novo pyrimidine synthesis, suggesting that compound 1 inhibits pyrimidine synthesis
341 after the synthesis of orotic acid from DHO. Consistent with this result, free uridine was
342 not detected in the compound 1-treated groups.
343 To validate this finding, we tested to see if the antiviral effect of compound 1 could be
344 overcome by the addition of exogenous pyrimidines. We measured the virus replication
345 in HEK 293T cells treated with mycophenolic acid (MPA) or compound 1 in the
346 presence of various exogenous nucleosides, and then compared the virus replication to
347 the controls (Figure 2). In MPA-treated groups, the antiviral effect of MPA was
348 completely reversed when guanosine was supplemented in the culture: from 47% to
349 105% and 17% to 62% for pVSV-luc and V3526-luc, respectively. This result was
350 consistent with our expectation that MPA inhibits inosine-5′-monophosphate
351 dehydrogenase (IMPDH) as its antiviral mechanism, which is a key enzyme to
352 synthesize guanosine de novo. Similarly, the antiviral effect of compound 1 greatly
353 decreased when the cells were supplemented with pyrimidines, cytidine or uridine. For
354 example, while 5 μM of compound 1 decreased the replication of V3526-luc to 1.9% 355 compared to the control, the addition of cytidine or uridine restored the viral replication
356 83.4 or 77.5 % compared to the control, respectively.
357 Two pyrimidine synthesis intermediates, DHO and orotic acid, were also tested to
358 confirm the metabolomics results with the accumulation of the molecules in the
359 compound 1-treated group. The addition of DHO did not affect the antiviral activity of
360 compound 1 at all, suggesting that compound 1 inhibits a downstream step from DHO.
361 Orotic acid showed a moderate reversion effect to compound 1 (21.9% to 55.0% and
362 1.9% to 7.1% for pVSV-luc and V3526-luc, respectively).
363
364 Induction of Innate immune genes by tetrahydrobenzothiazole 1 without virus
365 infection or type 1 interferons
366 It has been reported that the inhibition of pyrimidine biosynthesis could amplify the
367 cellular response to ssRNA via type 1 IFN system (15) ; however, we questioned this as
368 Vero 76, the primary cell line for the antiviral activity testing for compound 1 and its
369 derivatives, is known to be deficient in type 1 IFN production(16).
370 To test if compound 1 induced cellular immune response without type 1 IFN, we
371 examined the changes in host gene expression after the treatment with CID 70698683
372 without addition of ssRNA. CID70698683 is an N-phenyl, thiophene derivative of
373 compound 1 with similar antiviral effect towards multiple alphaviruses with CPE EC50
374 values of 0.60 µM, 0.66 µM, and 0.93 µM for VEEV TC-83, CHIKV S27, and WEEV,
375 respectively. Human HEp-2 cells were treated with 5 µM of CID 70698683 or DMSO for 376 18 hours and then the cellular mRNAs were subjected to a DNA microarray assay.
377 Ninety-two genes were up-regulated and one hundred forty five genes were down-
378 regulated by greater than two-fold difference (GEO accession ID: GSE72167). Among
379 these changes, certain sets of genes that are involved in the interferon pathways were
380 clearly upregulated (Figure 3), including RIG-I (4.63-fold) and OASL (3.76-fold
381 increase). We found that some ISGs (e.g., GBP2, ISG20, IFI44, IRF9 or IFIT1) were
382 also upregulated significantly (3.5, 2.85, 2.83, 2.4 or 2.3, respectively). While the ISGs
383 were upregulated, the expressions of interferons were not (0.9 ~ 1.1 fold changes);
384 rather, the expression of IFN-ε decreased in half. These findings are consistent with our
385 hypothesis that induction of those ISGs occurs without functional IFNs.
386 We further validated this finding with a real-time PCR and ELISA assay. First, we
387 sought to understand whether compound 1 simply amplified cellular innate immune
388 responses after virus infection or if the compound was able to establish an antiviral state
389 without an external interferon inducer, such as virus infection. To test this, we measured
390 the induction of innate immune response genes by compound 1 in mock or pVSV-luc
391 infected HEK 293T cells.
392 As shown in Figure 4, the induction of innate immune genes by compound 1 was
393 independent of virus infection. First, we confirmed that the treatment with compound 1
394 induced only certain genes in the interferon pathway. For example, the expressions of
395 MYD88 and OAS1 did not show any changes by compound 1; however, RIG-I, IFIT1,
396 IRF7, and OAS2 genes were upregulated more than 10-fold (solid bars in blue). More
397 interestingly, the induction of the genes by compound 1 was not amplified by virus 398 infection. Treatment of mock- or virus-infected cells (solid vs. spotty blue bars) with
399 compound 1 resulted in a same level of gene expression for the tested genes. We also
400 found that the induction of the ISGs by compound 1 was much stronger than by pVSV-
401 luc infection. pVSV-luc increased the expression of innate immune genes, such as
402 IFIH1, IFIT1, OAS2, OASL and IFNB1 by about 2~4 fold changes, indicating that HEK
403 293T cells are responsive to the infection of the virus. However, the effect was much
404 weaker compared to that of compound 1. Interestingly, compound 1 repressed the
405 expression of IFIH1 and IFNB1; whereas, these genes were induced rather than
406 repressed in the virus-infected groups (orange spotty bars).
407 The lack of induction of type 1 IFNs by compound 1 was confirmed at the cytokine level
408 with ELISA and a cell-based reporter assay. HEK 293T cells were treated with 5 μM of
409 compound 1 for 18 hours then the IFN α/β in the cell culture supernatant was detected
410 in an IFN ELISA assay (Figure 5A) or in a reporter assay (HEK-Blue™ IFN-α/β cells).
411 HEK-Blue™ IFN-α/β cells directly respond to IFN α/β and express the secreted alkaline
412 phosphatase (SEAP) via the activation of IRF9. In both assays, no measurable quantity
413 of IFNs was detected compared to the controls, indicating the lack of IFN α/β induction
414 by the treatment with compound 1. We also treated HEK-Blue™ IFN-α/β cells directly to
415 test whether compound 1 could directly activate the IFNAR and IRF9 pathway or
416 produce IFN as in an autocrine manner (Figure 5B). Treatment with compound 1 did
417 not induce SEAP activity compared to the control as well, indicating no direct activation
418 through IFNAR or any other receptor that can use IRF9. 419 In summary, these data indicate that compound 1 induces expression of certain sets of
420 innate immunity genes, creating an anti-viral state in cells in the absence of type 1 IFN
421
422 Cell line specificity of tetrahydrobenzothiazole 1
423 Since the supporting data strongly suggested that 1 inhibits pyrimidine synthesis and
424 induces the antiviral state of the host cells, we questioned if the antiviral effect and
425 mechanism of 1 is cell type-dependent. To address this question, we measured the
426 antiviral activity of 1 in several cell lines by determining an EC50 value for each. The cell
427 lines include HEK 293T (human embryonic kidney), SY-SH5S (human bone marrow
428 derived neuroblast), Vero76 (African green monkey kidney fibroblast), BHK C21
429 (hamster kidney fibroblast), Neuro2A (mouse neuroblast), and NIH3T3 (mouse
430 fibroblast).
431 The antiviral activities of compound 1 in various cell lines are summarized in Table 3 as
432 a function of EC50 value. As a control for the experiment, we used monensin, which is
433 known to inhibit virus replication by hampering the acidification of endosome during
434 virus entry. The antiviral activities of monensin were very close to each other in all cell
435 lines tested in this experiment. The EC50 values were within a range between 0.02 and
436 0.25 for V3526-luc, and between 0.17 and 0.56 μM for pVSV-luc. This result shows that
437 endocytosis is a critical pathway for the viruses in the cell lines and monensin worked
438 equally in the cell lines. 439 In contrast to monensin, the antiviral activities of compound 1 were cell line and/or
440 species-dependent. The EC50 values of compound 1 in HEK 293T or Vero76 cells were
441 between 0.15 μM and 0.68 μM using either V3526-luc or pVSV-luc, thus implying a
442 strong antiviral activity in these cell lines. Contrary to this, no antiviral activity of
443 compound 1 was detected in the mouse cell lines (Neuro 2A and NIH 3T3) we tested
444 (EC50 > 50 μM for both viruses). Interestingly, compound 1 still showed a moderate
445 effect in BHK cells, a hamster cell line. These data clearly indicate that the antiviral
446 activity of compound 1 is cell line-dependent and much less effective in mouse cells.
447 448 Discussion
449 This report discloses the discovery and mechanistic characterization of
450 tetrahydrobenzothiazole 1, a broad-spectrum antiviral agent that inhibits pyrimidine
451 biosynthesis and induces antiviral responses. This structural series was identified from
452 an anti-VEEV HTS with a mechanism-independent readout, offering the possibility that
453 multiple antiviral agents with different mechanisms of action could be identified from the
454 same screen. In fact, our pursuit of hits from the VEEV HTS led us to develop a
455 structurally distinct inhibitor, ML336, which targets a novel domain of VEEV nsP2 and
456 highlights the domain’s importance for viral replication and as a unique antiviral target.
457 In this study, we revisited our CPE-assay hit compounds with the intent of finding a
458 mechanism of action orthogonal to that of ML336. This effort ultimately led to the
459 development of compound 1. There have been many HTS campaigns that employed
460 the same strategy with different viruses (PubChem assay IDs: 1635, 2310, 2440,
461 540278, 463114, 540249, 588723, 651637, 1074, 51831 and 504781) with each
462 resulting in the discovery of novel hit compounds. As evidenced with our study, those
463 hits may include compounds targeting diverse antiviral mechanisms as well.
464 As Lucas-Hourani, M. et al. indicated, several antiviral HTS campaigns independently
465 discovered pyrimidine synthesis inhibitors targeting DHODH as an antiviral hit (15, 17-
466 19). This fact may indicate that the inhibition of de novo pyrimidine biosynthesis can
467 elicit antiviral activity without hampering cell proliferation. In fact, our experiment
468 addressing the effect of compound 1 on growing cells showed no or minimal impact to
469 cell growth or cytotoxicity at a concentration 10-fold higher than EC50 (Figure S1). We 470 believe that the cellular pyrimidine level that initiates the activation of the ISGs is higher
471 than the concentrations affecting cell proliferation or viability. Unlike the DHODH
472 inhibitors discovered by others, however, compound 1 seems to inhibit an enzyme
473 downstream from DHODH. Our metabolomics analysis clearly showed that treatment
474 with 1 caused the accumulation of orotate as well as DHO (Table 2), which indicates an
475 inhibition at a post-orotate step (Figure S2). The accumulation of orotidin-
476 monophosphate was not monitored; therefore, we hypothesize compound 1 could inhibit
477 orotate phosphoribosyltransferase (OPRTase) activity, which is executed by a dual
478 functional enzyme, uridine monophosphate synthetase (Umps) (20). Nevertheless, the
479 addition of orotate lessened the antiviral effect of compound 1 (Figure 2B), which could
480 indicate DHODH as the inhibitory step. We believe that the inhibition of OPRTase by
481 compound 1 may not be potent enough; therefore, high concentrations of exogenous
482 orotate (substrate) could facilitate the reaction even in the presence of 1. Alternatively,
483 exogenous orotate could affect the gene expression of ISGs through an unknown
484 pathway. A follow-up study is needed to understand the detailed mechanism of
485 compound 1 in the context of target enzyme.
486 Our assay matrix showed that compound 1 has a broad antiviral activity towards most of
487 the viruses tested but the effect was virus species-dependent (Table 1). While RNA
488 viruses seemed more sensitive than DNA viruses (e.g., HSV-2) to compound 1, not all
489 RNA viruses exhibited equal responsiveness. For example, enteroviruses were resistant
490 to the treatment with compound 1. While the most of RNA viruses we tested are
491 recognized by RIG-I in the infected cells, the infection of HSV-2 and enteroviruses are
492 known to be recognized by melanoma differentiation-associated gene 5 (MDA5, 493 encoded by the IFIH1 gene) and TLR/MYD88, respectively(21, 22). Consistent with this
494 finding, MDA5 and MYD88 were not induced by the treatment with compound 1 (Figure
495 4). A detailed study will be required to elucidate the exact mechanism; however, these
496 results imply that it may be associated with RIG-I pathway.
497 We also found that JEV was less susceptible to compound 1 compared to other
498 flaviviruses tested. Assuming that the viral RNA syntheses were similar to each other
499 (based on titers of the viruses), this finding may indicate a potential difference in the
500 sensitivity to the ISGs that are activated by pyrimidine synthesis inhibition. It is known
501 that the antiviral effect of pyrimidine synthesis inhibitors is through functional ISGs
502 driven by IRF1 transcription factor (15). Therefore, the difference in sensitivity may
503 indicate the resistance to ISGs by each virus. Interestingly, some dengue virus mutants
504 that are resistant to brequinar, a DHODH inhibitor, have been reported (23). In that
505 case, the authors proposed a resistance mechanism based on enhancement of
506 polymerase activity or viral assembly; however, our study with compound 1 as a probe
507 suggests that those mutants may exert resistance by evading the ISGs expressed by
508 pyrimidine deprivation. We have attempted to isolate compound 1-resistant mutant
509 VEEV, strain TC-83 by serial passages in the presence of compound 1 for three times;
510 however, we have not detected the emergence of resistant mutants (Figure S3). A
511 future study for isolation of resistant mutants with more passages would be informative
512 to determine resistant mechanisms for alphavirus to this class of compounds, which
513 could include an enhancement of polymerase activity or evasion of ISG. 514 One of the most interesting findings in our study is the induction of ISGs by compound
515 1, including OASL, in the absence of sensitizing the cells with virus, exogenous ssRNA,
516 or interferons (Fig. 4). OASL is an antiviral gene that is known to be expressed under
517 the control of IRF-3 by interferon and ISRE upon virus infection. However, our data
518 showed that OASL could be induced more than 100-fold without exogenous RNA or
519 induction of IRF-3. In our experiment, however, IRF-3 was downregulated after the
520 treatment. Further, the infection of compound 1-pretreated cells with pVSV-luc did not
521 change the cellular innate immune response. This could be explained by the strong
522 antiviral activity of compound 1, which may prevent viral RNA accumulation to a level to
523 trigger the RIG-I/MAVS pathway. Alternatively, the result may imply that the induction of
524 ISGs by compound 1 is independent of viral infection. Nonetheless, our study could
525 explain the mechanism of the amplification of cellular innate immune response by
526 pyrimidine synthesis inhibitors reported by others (15, 24). Our results clearly show the
527 amplification of the response is due to the IFN-independent induction of ISGs (See
528 below for further discussion), which will prime or amplify the antiviral state of the cells
529 upon viral infection. Our findings confirm the previous reports and further highlight the
530 antiviral mechanism of nucleoside synthesis inhibitors in detail.
531 In fact, our study showed that the expression of the ISGs is not associated with the type
532 1 IFNs. In our gene expression assays, no type 1 IFNs genes were induced by
533 compound 1; rather, they were downregulated. Expression of interferon could be
534 transient so the lack of interferon mRNA could not rule out a potential expression at an
535 early time point in the process. To avoid this pitfall, we validated our findings using an
536 ELISA-based assay in addition to a reporter-based assay to detect the interferons 537 produced by compound 1; however, no interferon-α/β activities were detected. Our
538 argument is also supported by the antiviral activity of compound 1 in Vero 76 cells,
539 which allow a variety of virus replication due to the lack of functional type 1 IFNs
540 production systems (16). Considering the antiviral activity of compound 1 in Vero76
541 cells was similar to that in other human cell lines (Table 3), this observation strongly
542 supports the idea that the presence of interferon is not essential for the antiviral activity
543 of compound 1 or other pyrimidine synthesis inhibitors, and the expressed ISGs are not
544 involved in the production of interferons. Our results are differ from the current view on
545 the antiviral mechanism of many ISGs such as OASL, which is believed to induce type I
546 interferons by assembling RIG-I complex in the response to viral infection(25).
547 Therefore, our results clearly highlight new insights on novel antiviral mechanisms of
548 ISGs.
549 The host cell type-dependent antiviral activity was particularly interesting in many
550 aspects. The species-dependent activity could be a universal characteristic of any host-
551 targeting compound, including 1. Study of host-dependent activity would be important to
552 choose an animal model to test those compounds. Compound 1 showed the strongest
553 antiviral effect in human cells but no antiviral activity in mouse cell lines we tested. We
554 also found brequinar does not show antiviral effect in mouse cell lines(26) and Clément
555 Grandin et al. mentioned that GAC50, a potent novel DHODH inhibitor, showed antiviral activity
556 only in primate cells (27). Interestingly, our finding is consistent with the lack of antiviral
557 activity of pyrimidine synthesis inhibitors against RNA viruses in mice as reported by
558 others. Some pyrimidine synthesis inhibitors have been evaluated for their in vivo
559 antiviral activities in mouse-based animal models (except cotton rat model for RSV) (17- 560 19, 28). Those tests have failed to show antiviral efficacy even with a significant
561 decrease in the pyrimidine concentration in the animals’ sera. Due to these results,
562 pyrimidine synthesis inhibitors have not been not well accepted as an antiviral strategy.
563 Our findings, however, conversely suggest that such failure may be due to the use of
564 non-responsive animal models for the inhibitors. Leflunomide, a pyrimidine synthesis
565 inhibitor, has been reported to have an antiviral effect in humans, suggesting pyrimidine
566 synthesis inhibition as a potential broad-spectrum antiviral target for humans (29-31).
567 Therefore, we believe that a worthy goal includes the development of an animal model
568 for this class of inhibitors and to evaluate them for antiviral activities. A recent study
569 using rhesus macaques, however, failed to show anti-RSV effect of a pyrimidine
570 synthesis inhibitor when it was administrated post infection. It has been proposed that
571 the level of circulating pyrimidines in the serum produced through the salvage pathway
572 could be sufficient to reverse the antiviral effect, or the interference with cell-mediated
573 immunity by the inhibitors could be proviral (27). In our study, a pre-treatment was
574 necessary to maximize the antiviral effect in the IFN-deficient cell line. It is not clear,
575 however, whether a pyrimidine synthesis inhibitor would successfully mount an antiviral
576 state and amplify the IFN-driven host innate immune response even at a post-infection
577 stage in IFN-competent cells. More in vivo antiviral studies to test pyrimidine synthesis
578 inhibitors in various animal models would be informative to evaluate their potential of
579 pyrimidine biosynthesis inhibitors as antiviral therapeutics.
580 As the antiviral activity of compound 1 is based on the pyrimidine synthesis inhibition
581 and the subsequent expression of certain ISGs, it is difficult to determine whether the
582 cell type-specificity is related to pyrimidine synthesis inhibition, or to inducing the ISGs. 583 Difference in the sequences of the protein targeted by compound 1, presumably Umps,
584 could account for this phenomenon. The sequence identity between human and mouse
585 Umps is high (88.8%); however, this cannot rule out the potential difference in the
586 binding residues in Umps. A future study is necessary to identify the molecular target.
587 An alternative hypothesis is that the mouse lacks the mechanism to induce the ISGs
588 upon the pyrimidine synthesis inhibition. This is based on the observation that
589 pyrimidine synthesis inhibitors with various chemical structures have failed to
590 demonstrate antiviral activity in mice as mentioned above. Currently, the molecular
591 mechanism of ISGs induction by pyrimidine depression is completely unknown. Lead
592 compound 1 could shed light on the antiviral mechanism involving innate immune
593 responses through the deprivation of pyrimidine.
594 In summary, tetrahydrobenzothiazole 1 is a molecular probe that enabled the study of
595 innate cellular immune responses involved with viral infection. More specifically,
596 compound 1 permits mechanistic scrutiny demonstrating that antiviral ISGs, induced by
597 the inhibition of pyrimidine synthesis, leads to broad-spectrum antiviral activity
598 independent of type 1 IFNs.
599 600 Acknowledgements
601 We thank Dr. Igor Lukashevich for LCMV, Dr. Jill Steinbach for HSV-2, and Dr. Robert
602 Tesh at World Reference Center for Emerging Viruses and Arboviruses for VEEV TrD
603 and WNV. We also thank BEI resources for CHIKV, JEV, YFV 17D, EV D71, and HCMV
604 MM. Microarray experiment was performed with assistance of the UofL Genomics
605 Facility, which is supported by NIH, the J. G. Brown Foundation, and user fees. The
606 funders had no role in study design, data collection and interpretation, or the decision to
607 submit the work for publication.
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735
736 737 Figure legend
738 Figure 1. Antiviral activity of compound 1 against VEEV.
739
740 The structures of CID 847035 (A) and compound 1 (B). (C) Viral RNA analysis. Viral
741 RNA was quantified using a quantitative real-time RT-PCR method with the total RNA
742 from the cells. RNA amounts were compared to the DMSO-treated controls. (D) Titer
743 reduction assay results for compound 1. Vero 76 cells grown in 12-well plates were
744 infected with 0.05 MOI of TC-83 and then incubated in the presence of compound 1 at
745 the denoted concentrations. Forty hours later the supernatant was harvested and the
746 titer of the progeny virus was determined. Each point represents the mean from three
747 biological replicates. (E) Time of addition study. Test compound, compound 1, was
748 added to the designated wells by replenishing the culture media with fresh culture
749 media containing 5 µM of the compound at the time points denoted on the x axis. The
750 cells were infected with 3 MOI of virus and the virus titers at 16 hours post-infection
751 from various time of addition points were depicted. Each data point is the mean from
752 two independent replicates with triplication in titration.
753 754 Figure 2. Reversion of antiviral effect of compound 1 by exogenous nucleosides.
755
756 One day-old HEK 293T cells were treated with DMSO, MPA (1 μM) or ML416 (1 μM) in
757 the presence of the denoted supplements for two hours, then cells were infected with
758 V3526-luc (A) or pVSV-luc (B). After eighteen hours of incubation, the luciferase activity
759 from the infected cells was measured. The values represent the means and their
760 standard deviations of 4 replicates samples as a percentage of the values for DMSO
761 control wells. 762 Figure 3. Induction of innate immune genes by CID 70698683.
763
764 * Immunity Volume 40, Issue 6, 19 June 2014, pages 936–948
765 Values in red : fold-change by the treatment of 5 μM of CID 70698683
766 767 Figure 4. Compound 1 induces innate immune response genes independently of virus
768 infection.
769
770 One-day-old HEK 293T cells were treated with 5 µM of compound 1 or DMSO (Control)
771 for 8 hours, then infected with mock or pseudotypeVSV-luc (pVSV-luc) at a MOI of 3.
772 Cells were further incubated for 16 hours in the presence of compound 1 or DMSO then
773 the host gene expression level was measured using quantitative real-time RT-PCR.
774 775 Figure 5. The treatment of compound 1 does not induce the production of type 1 IFNs.
776
777 (A) HEK 293T cells were treated with compound 1 or DMSO for 18 hours and the cell
778 culture media were subjected to IFN α/β ELISA assay to detect interferons. 125 pg/mL
779 of IFN-α and - β were used as controls. (B) HEK-Blue™ IFN-α/β reporter cells
780 incubated with 5 μM of compound 1 or DMSO for 18 hours and then the expressed
781 SEAP, which is controled by IRF9 was measured.
782
783 A C D E 1010 10 1010 109 109 N O 1 108 108 S N S 0.1 H 107
6 7 B 0.01 10 10 5 F 10 6
Relative vRNA copy 0.001 10 S O 104 Progeny virus titer (PFU/mL)
Progeny virus titer (PFU/mL) 3 N N S 0.0001 10 105 0 1.25 5 20 0 5 10 20 -8 -6 -4 -2 0 Ctrl CH3 Compound 1 (µM) Time of addition (hours to infection) A B V3526-luc pVSV-luc DMSO 150 150 A (50 µM) C (50 µM) C (50 µM) U (50 µM) 100 100 DHO (2 mM) Orotate (2 mM)
50 50 Relative luminescence (%) 0 0 DMSO MPA Compound A DMSO MPA Compound A Treatment Treatment CARD CARD CTD CID 70698683 Helicase N RIG-I 4.63 N S S O viral RNA CTD CTD
Helicase NEMO K63 Ub 1.16 1.43 NAP1 IRF3 or IRF7 * TRIM25 TBK1 IKKε homodimers CARD 0.95 Type I IFN transcription OASL Ub Helicase 0.8 Ub Ub Ub Ub 3.76 CARD Ub UbUb Ub Ub Ub IFN-α,β IFNs : 0.9 - 1.1 OASL Ub Ub OASL UbUb IFN-ε : 0.5 Ub
OASL CARD TRADDosome ISGs mean: 1.23 CARD ISGs CARD
* CARD TRADD NEMO GBP2 : 3.5 CARD CARD ISG20 :2.85
CARD 1.26 α β CARD IKK IKK MAVS IFI44 : 2.83 CARD IRF9 : 2.4 1.13 NF-kB Proin amatory IFIT1 : 2.3 cytokines IFI27 : 2.25 IL18 : 0.42 Control Control + pVSV-luc Compound 1 Compound 1 + pVSV-luc
100
10
1 (fold-increase) Gene expression
0.1 MYD88 DDX58 TRADDSTAT1 IFIH1IFIT1 IRF1 IRF3 IRF7 OAS1 OAS2 OASL IFNB1 A B
20000 1.5 IFN-α IFN-β 15000 1.0
10000 620 nm (IFN α / β ) OD 0.5 5000 Luminescence unit
0 0.0 IFN Background DMSO Compound 1 3.5 0.4 DMSO Compound 1 controls IFN-β (U/mL) Table 1. Antiviral activity of compound 1.
Log titer reduction at Virus family Virus EC50 (μM) 5 μM **
VEEV TC-83 0.35 -2.54
VEEV TrD 0.46 -4.77 Togaviridae VEEV V3526-luc 0.17* NT
CHIKV S17 0.29 -4.024
WEEV 0.96 NT
Rhabdovidae pVSV-luc 0.19* NT
Filroviridae Ebola virus-GFP 0.26 NT
Picornaviridae EV-71, MP4 11.1 NT
EMCV MM >25 NT
Influenza virus A Orthomyxoviridae NT -3.03 (H1N1)
Arenaviridae LCMV-ARM NT -3.466
Paramyxoviridae RSV Long NT -2.27
94% inhibition at Retroviridae HIV-1 NT 10 μM JEV NT -1.3
Flaviviridae YFV 17D NT -2.32
WNV NT -2.05
Herpesviridae HSV-2 NT 0.01
For titration reduction assays, the progeny virus titers were compared to the controls
(DMSO treated). For dose-response assays, cells were pretreated for two hours prior to infection and the luciferase activity was measured 16 hours later.
* luciferase-tagged virus assay.
** Negative values mean a decrease in progeny virus titer compared to the mock treated controls. The data represent the means of at least three replicates.
Table 2. Difference in cellular metabolites by the treatment of compound 1.
Groups Name p-value * The dl-Dihydroorotic acid 4.11 x10-4
-3 Metabolites detected Orotic acid 2.26 x10
only in compound 1- l-Serine 1.52 x10-2 treated cells (not -5 detected in mock- Citrulline 4.11 x10 treated cells) Alloxanoic acid* 4.11 x10-5
2-Ketoisovaleric acid* 4.11 x10-5
Uridine 4.11 x10-5
-4 D-(-)- Erythrofuranose,(isomer)* 4.16 x10
(3R,4R)-tetrahydrofuran-2,3,4- 4.11 x10-5 triol*
Metabolites detected Dihydromuconic acid; trans-3- 1.52 x10-2 Hexenedioic acid* only in mock-treated Uridine phosphate* 4.11 x10-4 cells (not detected in compound 1-treated Methanesulfinic acid* 4.11 x10-4 cells) N-Acetyl-L-glutamic acid* 4.11 x10-5
Putrescine; 1,4-Diaminobutane* 4.11 x10-5
Pidolic acid* 3.30 x10-3
2-Hydroxyglutaric acid 2.32 x10-3 compounds that have not been confirmed by using standard compounds.
Table 3. Antiviral activity of compound 1 in various cell lines.
μ EC50 ( M) of
Monensin compound 1
V3526-luc pVSV-Gp V3526-luc pVSV-Gp
0.08 0.19 HEK 293T 0.15 0.17 0.03 0.74
0.17 SY-SH5S 0.02 0.49 3.82 0.48
0.56 0.17 0.20 Vero76 3.87 2.57 0.25 0.68
0.09 0.50 1.12 8.26 BHK 0.24 0.19 0.92 7.16
0.22 0.25 Neuro2A 0.23 >50 >50 0.19 0.29
0.24 NIH 3T3 0.02 >50 >50 0.67
Each number represents EC50 evaluated from a dose-response study with concentrations starting from 25 µM by a five-fold dilution, triplicates for each, in a 96- well format. For assays with HEK 293T, Neuro 2A, and SH-SY5Y cells, 24,000 cells and 2,400 TCID50 units of virus per well was used. For Vero 76 and BHK cells, 12,000 cells and 1200 TCID50 units of virus per well was used. For NIH3T3, 24,000 cells and 20,000 TCID50 units of virus per well was used. EC50 values were calculated using XLfit
(IDBS) formula 205, a 4-parameter Levenburg-Marquardt algorithm with maximum and minimum limits set at 100 and 0, respectively.