Article pubs.acs.org/journal/aidcbc
Allosteric Regulation of Phosphatidylinositol 4‑Kinase III Beta by an Antipicornavirus Compound MDL-860 Minetaro Arita,*,† Georgi Dobrikov,‡ Gerhard Pürstinger,§ and Angel S. Galabov∥ †
Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Academician Georgi Bonchev Street, Bl. 9, 1113 Sofia, Bulgaria § Institute of Pharmacy, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria ∥ The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Academician Georgi Bonchev Street, 1113 Sofia, Bulgaria
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‡
S Supporting Information *
ABSTRACT: MDL-860 is a broad-spectrum antipicornavirus compound discovered in 1982 and one of the few promising candidates effective in in vivo virus infection. Despite the effectiveness, the target and the mechanism of action of MDL-860 remain unknown. Here, we have characterized antipoliovirus activity of MDL-860 and identified host phosphatidylinositol-4 kinase III beta (PI4KB) as the target. MDL-860 treatment caused covalent modification and irreversible inactivation of PI4KB. A cysteine residue at amino acid 646 of PI4KB, which locates at the bottom of a surface pocket apart from the active site, was identified as the target site of MDL-860. This work reveals the mechanism of action of this class of PI4KB inhibitors and offers insights into novel allosteric regulation of PI4KB activity. KEYWORDS: picornavirus, enterovirus, MDL-860, PI4KB, infection, virus
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drug candidates known as enviroxime-like compounds (i.e., enviroxime, pachypodol [Ro 09-0179], oxoglaucine, GW5074, T-00127-HEV1, and BF-738735).12−18 OSBP family I was then identified as the target of a minor group of enviroxime-like compounds (i.e., AN-12-H5, T-00127-HEV2, 25-HC, and itraconazole) by a siRNA-sensitization screening.19,20 OSBP transfers cholesterol between endoplasmic reticulum and trans Golgi by a phosphatidylinositol 4-phosphate (PI4P)-dependent manner and contributes to homeostasis of cholesterol and lipid.21,22 A functional link between PI4KB and OSBP family I in PV replication was suggested by a conserved mutation in the 3A protein (Ala70Thr) that confers resistance to both PI4KB and OSBP inhibitors,16,23 by enhancing the processing of viral 3AB protein to 3A protein without hyperactivation of PI4KB.24 The current model of the role of the PI4KB/OSBP pathway in
oliovirus (PV) is a small nonenveloped virus with a positivesense single-stranded RNA genome of about 7500 nucleotides (nt) belonging to Enterovirus C species in the genus Enterovirus, the family Picornaviridae. In the global PV eradication program initiated in 1988, antivirals for PV are anticipated to have important roles in the endgame and in posteradication era.1 However, currently, there is no antiviral available for PV infection. Phosphatidylinositol-4 kinase III beta (PI4KB) is one of the four mammalian PI4 kinases (PI4K2A, PI4K2B, PI4KA, and PI4KB).2 PI4KB produces PI4P mainly at the Golgi3 and is involved in Ceramid transport,4 membrane trafficking from the Golgi,5 and normal function of lysosome.6 PI4KB is an essential gene in a human cell line.7 In Drosophila and Plasmodium falciparum, PI4KB is essential for spermatocyte cytokinesis and membrane ingression, respectively.8,9 The importance of PI4KB in the replication of enterovirus was first recognized by Hsu et al.,10 with a potent PI4KB inhibitor PIK93.11 Subsequently, PI4KB was identified as the target of a group of antipicornavirus © 2017 American Chemical Society
Received: April 18, 2017 Published: June 12, 2017 585
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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Figure 1. Anti-PV activity of MDL-860. (A) (Top) Structure of MDL-860. (Bottom) Inhibitory effect of MDL-860 on PV1pv infection and the viability of RD cells after a 2 day treatment. PV1pv infection at 7 h p.i. or the cell viability after a 2 day treatment in the absence of MDL-860 was taken as 100%. (B) Effect of MDL-860 on the infection of PV1pv mutants with drug resistance mutations. RD cells were infected with PV1pv wt or resistant mutants to GuHCl, BFA, or enviroxime. PV1pv infections at 7 h p.i. in the absence of MDL-860 were taken as 100%. n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Effect of MDL-860 on in vitro activities of PI4KB and PI4KA. T-00127-HEV1 was included as a positive control of PI4KB inhibition.
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RESULTS AND DISCUSSION MDL-860 Is an Atypical Enviroxime-Like Compound. We analyzed the specificity of the antiviral effect of MDL-860 (Figure 1A). MDL-860 showed moderate anti-PV activity (EC50 of 6.8 μM) with no apparent cytotoxicity in the concentrations examined (CC50 of >100 μM, and SI of >14). An enviroximeresistant type 1 PV pseudovirus (PV1pv) mutant showed resistance against MDL-860 (Figure 1B), suggesting that MDL-860 targets the PI4KB/OSBP pathway. However, MDL860 did not affect in vitro PI4KB activity (Figure 1C), nor did it enhance the localization of OSBP at the Golgi in vivo in contrast to the PI4KB/OSBP inhibitors (Figure S1).20,26,50 MDL-860 treatment reduced UC in the cells similar to treatment with the PI4KB/OSBP inhibitors (Figure S1). MDL-860 did not inhibit encephalomyocarditis virus (EMCV) replication (Figure S2), which depends on the PI4KA/OSBP pathway,51 suggesting that MDL-860 is an atypical enviroxime-like compound possibly targeting PI4KB activity. MDL-860 Affects in Vivo PI4KB Activity. First, we analyzed the effect of MDL-860 treatment on the enhanced localization of PI4KB at the Golgi, which could be caused by treatment with PI4KB inhibitors (Figure S3).52 Treatment of the cells with a PI4KB inhibitor T-00127-HEV1 or MDL-860 enhanced the localization of PI4KB at the Golgi but not of the PI4KB interactant RAB11A. The enhancement caused by MDL860 treatment showed a 1 to 2 h delay compared to that caused by T-00127-HEV1 treatment. We then analyzed the effect of MDL-860 treatment on the localization of OSBP on the virusinduced membrane structure in PV1pv-infected cells, which depends on the overproduced PI4P by PI4KB in the infected cells (Figure 2A).26 Treatment with T-00127-HEV1 caused prompt dissociation of OSBP from the membrane structure to the nucleus within 1 h (5 to 6 h p.i. treatment) as previously reported.26 MDL-860 treatment caused the dissociation of OSBP
enterovirus replication is as follows: viral proteins (2C, 2BC, 3AB, and 3D) activate PI4KB to produce PI4P for recruitment of OSBP to accumulate unesterified cholesterol (UC) on virusinduced membrane structure for the formation of a virus replication complex required for the synthesis of viral plus-strand RNA.13,20,23−27 A similar pathway is conserved in hepatitis C virus replication, where PI4KA and in part PI4KB act for the recruitment of OSBP.28−33 Currently, targets of substantially noncytotoxic compounds (i.e., selectivity index of >100) are confined to viral proteins (capsid proteins, 2C, 3C, and 3D)34−37 and host proteins PI4KB and OSBP family I.12,13,16,18,19,38−43 Retrospective analysis of uncharacterized antipicornavirus drug candidates underscores the importance of the PI4KB/OSBP pathway as the sole potential host-target of anti-PV drug in the viral RNA replication step.44 MDL-860 is an antipicornavirus compound identified in 1982 by a group of The Dow Chemical Company.45 MDL-860 showed a broad-spectrum antiviral activity against enteroviruses and rhinoviruses but was not active against other virus families, including positive-sense RNA viruses (feline calicivirus, coronavirus), negative-sense RNA viruses (Newcastle disease virus, vesicular stomatitis virus, and influenza A virus), and a DNA virus (herpes simplex virus type 1).45,46 Effectiveness of MDL-860 in vivo has been shown in mouse models of coxsackievirus infection,47,48 and its potent derivatives have also been synthesized.49 MDL-860 inhibits some early step of viral replication after uncoating,45,46 but its direct target and the mechanism of action remain unknown. In the present study, we have identified PI4KB as the target of MDL-860 for its anti-PV activity and characterized the mechanism of action. We provide insights into novel allosteric regulation of PI4KB activity by this class of antipicornavirus compounds. 586
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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Figure 2. Effect of MDL-860 on the amount of PI4P in PV1pv-infected cells. (A) Effect of MDL-860 on the localization of OSBP in PV1pv-infected OSBP-EGFP-HEK293 cells. GuHCl (2 mM), MDL-860 (20 μM), or T-00127-HEV1 (PI4KB inhibitor, 10 μM) were added to the OSBP-EGFPHEK293 cells infected with PV1pv at 3, 4, or 5 h p.i. The cells were fixed at 6 h p.i and then subjected to indirect immunofluorescence. Green, OSBPEGFP; magenta, 2B; blue, nucleus. Pearson’s correlation coefficient between 2B protein and OSBP-EGFP was shown in the OSBP-EGFP image. OSBPEGFP colocalized with 2B in the MDL-860-treated cells is indicated by an arrow. (B) Flow cytometry analysis of HEK293 cells infected with PV1pv(wt) (MOI = 2) after transient treatment (from 3, 4, or 5 to 6 h p.i.) with PI4KB inhibitor (T-00127-HEV1, 10 μM) or MDL-860 (20 μM). The cells were detected with anti-2B and PI4P antibodies. Ratios of geometric means of PI4P signals in the PV1pv-infected cells to noninfected cells are shown in the right graphs.
but after a 2 h treatment (4 to 6 h p.i. or 3 to 6 h p.i. treatment). We also measured the amount of PI4P in the infected cells after MDL-860 treatment (Figures 2B and S4). T-00127-HEV1 treatment reduced the amount of PI4P within 1 h of treatment, but MDL-860 treatment could cause a similar level of decrease of
PI4P only after 3 h of treatment. An apparent discrepancy in the effects of MDL-860 on the conventional in vitro PI4KB assay and in vivo PI4KB activity might suggest that some factor(s) of the in vivo condition might be required for the effect (e.g., temperature, incubation time, oxidation−reduction potential). These results 587
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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Figure 3. Effect of pretreatment with MDL-860 on PV1pv infection. (A) RD cells were pretreated with the indicated concentration of anti-PV compounds for 0 or 24 h at 37 °C before PV1pv infection. PV1pv infection in the absence of anti-PV compounds at 7 h p.i. was taken as 100%. (B) Evaluation of the effective period of the addition of MDL-860 for the anti-PV activity. MDL-860 (20 μM), T-00127-HEV1 (20 μM), or GuHCl (2 mM) was added to RD cells at the indicated time (−24 to 6 h p.i.). Time of drug addition, where significant suppression of PV1pv infection was observed, is indicated by the line. PV1pv infection in the absence of anti-PV compounds at 7 h p.i. was taken as 100%. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (C) Evaluation of the effective period after the removal of MDL-860 after pretreatment for its anti-PV activity. MDL-860 (20 μM), T-00127-HEV1 (20 μM), or GuHCl (2 mM) was added to RD cells at −24 h p.i. Supernatants containing the compounds were removed at 0 h p.i., and then, the cells were washed three times before PV1pv infection. PV1pv infection in the absence of anti-PV compounds at 7 h p.i. was taken as 100%. n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
suggest that MDL-860 targets PI4KB activity in vivo and requires pretreatment for its activity in contrast to the conventional PI4KB inhibitor. MDL-860 Confers an Irreversible Anti-PV Effect to the Cells and Activates the Nrf2-Keap1 Antioxidant Pathway. We analyzed the effect of pretreatment of the cells with MDL860 on the anti-PV activity (Figure 3). Pretreatment with a 2C inhibitor guanidine hydrochloride (GuHCl) or a PI4KB inhibitor had no effect on the anti-PV activities. In contrast, pretreatment with OSBP inhibitors (T-00127-HEV2 and itraconazole) enhanced their activities by 2- to 3-fold (Figure 3A). Pretreatment with MDL-860 drastically enhanced the anti-PV activity (about 7-fold enhancement, EC50 of 6.4 μM [no pretreatment] vs 0.97 μM [24 h pretreatment]). The anti-PV activity of MDL860 could be enhanced by 3 h of pretreatment (>95% inhibition) and more significantly by 24 h of pretreatment (>98% inhibition) (Figure 3B). After removal of MDL-860 after 24 h of pretreatment, significant anti-PV activity remained (86% inhibition), in contrast to a 2C inhibitor and PI4KB inhibitor (P < 0.0001) (Figure 3C). These results suggest that MDL-860 treatment causes an irreversible anti-PV effect to the cells.
To further analyze the effect of pretreatment with MDL-860, we performed microarray analysis of MDL-860-treated cells (Table S1). Gene ontology analysis of the regulated genes (fold change ≥2.0) indicated that MDL-860 treatment activates the Nrf2-Keap1 antioxidant pathway that is activated by electrophiles or oxidants by targeting Keap1 via covalent modification of the sensor cysteine residues (Table S2).53,54 A representative Nrf2 activator sulforaphane, however, did not show anti-PV activity in noncytotoxic concentrations, nor did it enhance the activity after pretreatment (Figure S5). These results suggest that MDL-860 could serve as an electrophile or oxidant to activate the Nrf2Keap1 pathway in the cells; however, the activation of this pathway is not essential for its anti-PV activity. Mechanism of Action of MDL-860. To analyze irreversible anti-PV effect of the cells caused by MDL-860 treatment, Nterminally FLAG-tagged PI4KB (N-FLAG-PI4KB) was expressed in HEK293 cells in the presence of MDL-860 or T00127-HEV1 or the absence of these compounds (mocktreatment) and then purified to analyze the activity (Figure 4). In vitro activity of N-FLAG-PI4KB purified from MDL-860-treated cells was significantly suppressed compared to those of N-FLAG588
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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Figure 4. Activity of PI4KB purified from MDL-860-treated cells. (A) Activity of FLAG-tagged PI4KB purified from HEK293 cells that are treated with T-00127-HEV1 (20 μM) or MDL-860 (20 μM) or with no compound (mock-treatment). Left panel: Image of SDS-PAGE analysis of purified FLAGtagged PI4KBs stained by Instant Bands. BSA (0.1 to 0.0063 μg/ μL) was used as control. Right panel: In vitro PI4KB activity. PI4KB activity of purified FLAG-tagged PI4KBs (diluted by 1-, 3-, or 10-fold) is shown. The activity of FLAG-tagged PI4KB purified from mock-treated cells (1/1 diluted) was taken as 100%. n = 3. *, P < 0.05; **, P < 0.01; ***, P < 0.001. (B) Intact mass measurement of purified FLAG-tagged PI4KB. Upper panel: Distribution of FLAG-tagged PI4KB in a chromatogram. The ratio of two molecular species of purified FLAG-tagged PI4KB is shown. Lower panel: Molecular mass distribution of purified FLAG-tagged PI4KB and a model of modification.
PI4KBs purified from mock-treated cells or T-00127-HEV1treated cells (4.7−9.8% activity of N-FLAG-PI4KB purified from mock-treated cells, P < 0.000015) (Figure 4A). This suggested that MDL-860 caused irreversible inactivation of PI4KB in vivo. To confirm the presence of irreversible modification caused by MDL-860 treatment, we measured intact molecular mass of
PI4KB purified from MDL-860-treated cells (Figure 4B). Purified N-FLAG-PI4KB (expected molecular mass of 90 801 Da) consisted of two molecular species; one is around 90 924 Da, and the other is around 95 184 Da (ratio of 1:1.85). The main peaks of each molecular species were surrounded by 4 to 5 molecular species (90 849−91 172 or 95 108−95 503 Da, 589
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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Figure 5. Target site of MDL-860 on PI4KB. (A) In vitro PI4KB activity of purified FLAG-tagged PI4KBs (wt and cysteine-to-serine mutants) from HEK293 cells treated with MDL-860 (20 μM) or with no compound (mock-treatment). The activity of FLAG-tagged PI4KB (wt) purified from mocktreated cells was taken as 100%. n = 3. ***, P < 0.001. (B) Location of C646 on a crystal structure of PI4KB (PDB: 4WAG) visualized by using Chimera. C646 and a PI4KB inhibitor (MI103) in the active site are colored by magenta and cyan, respectively. (C) Intact mass measurement of purified FLAGtagged PI4KBs (wt and C646S mutant).
respectively) with increased molecular mass of every 79.0 (standard deviation 0.47), possibly derived from phosphorylation.55,56 The identity of modification of 4260 Da (between 90 924 and 95 184 Da) remained unknown. N-FLAG-PI4KBs purified from mock-treated cells or T-00127-HEV1-treated cells showed a similar profile of molecular mass. However, N-FLAGPI4KB purified from MDL-860-treated cells showed increased molecular mass of 146.2 Da (standard deviation of 0.52) of the corresponding peaks, suggesting a covalent modification of PI4KB. The chemical structure and an electrophilic property of MDL-860 observed in vivo suggests its susceptibility to nucleophilic attack possibly by a cysteine residue at the foot of an electron-accepting group (Figure 4B), which could result in a net increase of 146.01 Da. To map the site of modification, we first performed proteomic analysis of N-FLAG-PI4KB purified from MDL-860-treated cells
by liquid chromatography-tandem mass spectrometry (LC/MS/ MS) analysis (Table S3). Detected peptides covered almost an entire region of PI4KB (798/801 amino acid residues); however, no amino acid residue had a 146 Da modification in its substantial population. Because current LC/MS/MS analysis has yet to attain robustness to detect all the peptides species with equal efficiency, we next performed mutagenesis analysis of the cysteine residues of PI4KB (total of 13 cysteine residues of PI4KB encoded by transcript variant 2; GenBank: NM_001198773; UniProt: Q9UBF8_2) to identify the target site of MDL-860. We measured the activity of PI4KB mutants that have a single substitution at corresponding cysteine residue to serine, purified from mock-treated or MDL-860-treated cells (Figure 5A). Among the mutations, only a C646S mutation conferred resistance to MDL-860 treatment (110% of the activity of PI4KB(C646S) purified from mock-treated cells). C646 590
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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(Takara Bio Inc.). Antibody against PV 2B raised in rabbits with peptides WLRKKACDVLEIPYVIKQ (amino acids 80 to 97 of PV 2B protein) and anti-PI4P antibody (mouse IgM antibody, Echelon Biosciences) were used for flow cytometry as previously described.44 A specific PI4KB inhibitor, T-00127-HEV1,16 was supplied by Pharmeks Ltd. (Moscow) (purity >99%, determined by liquid chromatography−mass spectrometry [LC-MS]). An OSBP inhibitor, T-00127-HEV2,19 was kindly provided from Hirotatsu Kojima (Drug Discovery Initiative, The University of Tokyo) (purity >99%, determined by LC-MS). Itraconazole was purchased from Tokyo Chemical Industry Co., Ltd. (purity ≥98%, determined by HPLC). MDL-860 was prepared by chemical synthesis (purity >99.5%, determined by NMR).49 In Vitro Kinase Assay. Inhibitory effect of MDL-860 at a concentration of 10, 20, and 40 μM or of T-00127-HEV1 at concentration of 1 and 10 μM on in vitro PI4KB and PI4KA activities was assessed by SelectScreen Kinase Profiling Service with ATP concentration of 10 μM (Invitrogen). In vitro activity of purified N-FLAG-PI4KB was evaluated by using an ADP-Glo Lipid Kinase Systems kit (Promega) as previously described.44 Eluents from mock-transfected HEK293 cells were used as negative control. In a total 5.5 μL reaction solution, PI4KB activity of 340, 110, or 34 ng of purified N-FLAG-PI4KB (1/1, 1/ 3, and 1/10 dilution, respectively; with a final concentration of 680, 230, and 68 nM) with lipid substrates (0.025 mg/mL of phosphatidylinositol and 0.075 mg/mL of phosphatidylserine) and 25 μM ATP was measured in the presence or the absence of PI4KB inhibitor (T-00127-HEV1, 10 μM). For the analysis of cysteine-to-serine PI4KB mutants, 110 ng of purified proteins was used for the assay. The net signals of PI4KB activity of the samples were determined as below:
locates at the bottom of a surface pocket in kinase C-lobe domain apart from the active site of PI4KB (Figure 5B).41,57,58 To further confirm that C646 of PI4KB is the direct target site of MDL-860, intact molecular masses of purified PI4KB(C646S) from mocktreated or from MDL-860-treated cells were determined (Figure 5C). The molecular mass of N-FLAG-PI4KB(C646S) (main peaks at 90 906 and 95 167 Da) from mock-treated cells was smaller than N-FLAG-PI4KB(wt) (main peaks at 90 922 and 95 183 Da) by 16 Da corresponding to the cysteine to serine substitution. After MDL-860 treatment, in contrast to PI4KB(wt) that had a 146 Da modification (main peaks at 91 069 and 95 329 Da), PI4KB(C646S) showed no shift of molecular mass (main peaks at 90 906 and 95 167 Da). These results indicate that MDL-860 regulates the activity of PI4KB by an allosteric manner via covalent modification of C646. To evaluate the importance of PI4KB as the target of MDL860 for its anti-PV activity, we attempted to rescue PV1pv infection in the presence of MDL-860 by ectopic expression of PI4KB (Figure S6). Expression of a kinase-dead mutant NFLAG-PI4KB (D656A) could not rescue PV1pv infection in the presence of MDL-860 as well as empty vector (1.0−1.1% PV1pv infection in 20 μM MDL-860). PV1pv infection was slightly restored by the expression of N-FLAG-PI4KB(wt) in the presence of MDL-860 (3.1% PV1pv infection in 20 μM MDL860) but showed dose dependent suppression by MDL-860 as well as in mock-transfected RD cells (1.1% PV1pv infection in 40 μM MDL-860). In contrast, expression of N-FLAG-PI4KB(C646S) substantially rescued PV1pv infection in the presence of MDL-860 (16% PV1pv infection in 20 μM MDL-860) and eliminated dose dependent suppression by MDL-860 (16% PV1pv infection in 40 μM MDL-860). Transfection efficiency was about 10% in the condition examined, suggesting that the expression of PI4KB(C646S) substantially rescued PV1pv infection in the transfected cells in the presence of MDL-860. These results indicate that PI4KB is the major target of MDL-860 for its anti-PV activity. In summary, we have identified PI4KB as the target of an antipicornavirus compound MDL-860 and mapped the target site at an allosteric site of PI4KB. Since the discovery of PI4KB as an essential and druggable host factor for the infection of pathogenetic microorganisms, drastic progress has been made in both structural study and the development of inhibitors. This report offers insights into the mechanism of action of this class of antipicornavirus compounds via novel allosteric regulation of PI4KB activity.
The net signals of PI4KB activity = (thesignalsinthe absenceof PI4KBinhibitor − thesignalsinthepresenceof PI4KBinhibitor)
The net signals of PI4KB activity of N-FLAG-PI4KB purified from mock-treated cells (1/1 diluted) were taken as 100% of PI4KB activity. Flow Cytometry. HEK293 cells (8.0 × 105 cells) were infected with PV1pv (parental or G5318A mutant) at multiplicities of infection (MOI) of 2 and then treated with T-00127HEV1 (10 μM) or MDL-860 (20 μM) from 5 to 6 h p.i., 4 to 6 h p.i., or 3 to 6 h p.i. The cells were collected at 6 h p.i. in 0.8 mL of 10%FCS-DMEM and then fixed with 3% paraformaldehyde for 10 min at room temperature and permeabilized with 20 μM digitonin in HBS for 5 min. The cells were incubated with primary antibodies for 30 min at 37 °C. Cells were washed 2 times by 0.5% BSA in HBS and then incubated with secondary antibodies conjugated with Alexa Fluor 647 and 488 dyes (Molecular Probes) for 20 min at 37 °C. The cells were suspended in 250 μL of HBS. About 5.0 × 104 cells were analyzed per sample with a BD FACSCanto II Flow Cytometer (BD Biosciences) and FlowJo software (FLOWJO, LLC). Relative intensity of the PI4P signals was determined as below:
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METHODS Cells, Viruses, Plasmids, Antibodies, and Compounds. RD cells (human rhabdomyosarcoma cell line) and HEK293 cells (human embryonic kidney cells) were cultured as monolayers in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS). PV1pv, which encapsidated luciferase-encoding PV replicon with capsid proteins derived from type 1 Mahoney strain, was used for evaluation of anti-PV activity of compounds.59 PV1pv mutants that have known drug resistance mutations, including G5318A (enviroxime and GW5074 resistance, 3A-Ala70Thr),23 U4614A (GuHCl resistance, 2C-Phe164Tyr),60 and G4361A and C5190U (brefeldin A [BFA] resistance, 2C-Val80Ile, and 3AAla27Val)),61 were used for characterization of anti-PV activity of MDL-860. Expression vectors for N-terminally FLAG-tagged PI4KB (GenBank: NM_001198773; UniProt: Q9UBF8_2) were constructed with pHEK293 Ultra Expression Vector I
Relative intensity of the PI4P signals ⎛ the PI4P signals in PV1pv ‐infected cells ⎞ =⎜ ⎟ ⎝ the PI4P signals innoninfected cells ⎠
Net amount of produced PI4P was determined as below: 591
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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ACKNOWLEDGMENTS We are grateful to Junko Wada and Yuzuru Aoi for excellent technical assistance, Hiroyuki Shimizu and Takaji Wakita for kind support, Tadaki Suzuki (Department of Pathology, National Institute of Infectious Diseases) and Yuichiro Nakatsu (Department of Virology III, National Institute of Infectious Diseases) for kindly providing EGFP-RAB11 expression vector, Ann Palmenberg (Institute for Molecular Virology, University of Wisconsin-Madison) for kindly providing mengovirus replicon, Hiroshi Sezaki (Agilent technologies Japan, Ltd.) for kind support of mass spectrometry, and Hirotatsu Kojima (Drug Discovery Initiative, The University of Tokyo) and Kentaro Hanada (Department of Biochemistry and Cell Biology, National Institute of Infectious Diseases) for insightful discussion on the chemical property of MDL-860. This study was in part supported by JSPS KAKENHI Grant Number 25460579 and Advanced Research & Development Programs for Medical Innovation (AMED-CREST) from Japan Agency for Medical Research and Development, AMED to M.A., and by Bulgarian Science Fund - project B02/11 12.12.2014 “Synthesis and anti-enterovirus activity of novel diaryl ethers and their complexes with cyclodextrins” to G.D. and A.S.G. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Net amount of produced PI4P = Relative intensity of the PI4P signals − 1
Protein Purification. HEK293 cells (8.0 × 106 cells) were transfected with N-FLAG-PI4KB expression vectors by using Lipofectamine 3000 (Invitrogen) in the presence or the absence of 20 μM of T-00127-HEV1 or MDL-860 and then were lysed with xTractor buffer (Clontech Laboratories, Inc.) at 24 h posttransfection (p.t.). N-FLAG-PI4KB was purified by using antiFLAG M2 magnetic beads (Sigma-Aldrich Co. LLC.) and was eluted with 3xFLAG peptide. Eluent from mock-transfected HEK293 cells was used as negative control. Intact Protein Mass Measurement. Intact mass of purified N-FLAG-PI4KB (expected molecular mass of 90 801 Da, 0.16 μg/ μL, 10 μL) was measured by using a LC-QTOF system with an Agilent 1260 Infinity HPLC system (AdvanceBio RP-mAb Diphenyl column, flow rate of 0.4 mL/min, 60 °C) and an Agilent LCMS 6530 QTOF LCMS system (positive ion mode, capillary voltage of 4000 V, fragmentor voltage of 250 V, drying gas flow of 8 L/min, 350 °C, sheath gas flow of 11 L/min, 400 °C) (Agilent technologies Japan, Ltd.). Statistical Analysis. The results of the experiments are shown as the averages with standard deviations. P values of less than 0.05 by a one-tailed t test were considered to have a significant difference and were indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).
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ABBREVIATIONS BFA, brefeldin A; GuHCl, guanidine hydrochloride; OSBP, oxysterol-binding protein; PI4KB, phosphatidylinositol-4 kinase III beta; PI4P, phosphatidylinositol 4-phosphate; PV, poliovirus; PV1pv, type 1 PV pseudovirus; UC, unesterified cholesterol
ASSOCIATED CONTENT
S Supporting Information *
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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsinfecdis.7b00053. Effects of MDL-860 on the localization of OSBP and the amount of unesterified cholesterol in the cells; specificity of the antiviral activity of MDL-860; effect of MDL-860 on the localization of PI4KB in the cells; quantitation of PI4P in PV1pv-infected cells; anti-PV activity of sulforaphane; rescue of PV1pv infection in the presence of MDL-860 by ectopic expression of PI4KB; methods (PDF) Genes regulated by MDL-860 treatment; upstream analysis of up-regulated genes by MDL-860 treatment by Ingenuity Pathway Analysis (Top 50 hits); peptide mapping of N-FLAG-PI4KB purified from MDL-860treated cells (XLSX)
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Article
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AUTHOR INFORMATION
Corresponding Author
*Phone:+81-42-561-0771. Fax:+81-42-561-4729. E-mail:
[email protected]. ORCID
Minetaro Arita: 0000-0002-3314-6626 Author Contributions
M.A. and A.S.G. conceived the project. M.A. designed the study. G.D., G.P., and A.S.G. performed chemical synthesis of MDL860; M.A. performed biological experiments with MDL-860. M.A. wrote the manuscript; all authors commented on the manuscript. Notes
The authors declare no competing financial interest. 592
DOI: 10.1021/acsinfecdis.7b00053 ACS Infect. Dis. 2017, 3, 585−594
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