Allosteric Regulation of Phosphatidylinositol 4-Kinase III Beta by an

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Allosteric regulation of phosphatidylinositol 4-kinase III beta by an anti-picornavirus compound MDL-860 Minetaro Arita, Georgi Dobrikov, Gerhard Pürstinger, and Angel Galabov ACS Infect. Dis., Just Accepted Manuscript • Publication Date (Web): 12 Jun 2017 Downloaded from http://pubs.acs.org on June 14, 2017

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ACS Infectious Diseases

Allosteric regulation of phosphatidylinositol 4-kinase III beta by an anti-picornavirus compound MDL-860

Minetaro Arita1*, Georgi Dobrikov 2, Gerhard Pürstinger3, and Angel S. Galabov4

1

Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan

2

Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Academician Georgi Bonchev Street, Bl. 9,1113 Sofia, Bulgaria

3

Institute of Pharmacy, University of Innsbruck, Innrain 80/82, A-6020 Innsbruck, Austria 4

The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, 26 Academician Georgi Bonchev Street, BG-1113, Sofia, Bulgaria

*To whom correspondence should be addressed: Department of Virology II, National Institute of Infectious Diseases, 4-7-1 Gakuen, Musashimurayama-shi, Tokyo 208-0011, Japan Phone:+81-42-561-0771; Fax:+81-42-561-4729 e-mail: [email protected]

1 ACS Paragon Plus Environment


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Abstract MDL-860 is a broad-spectrum anti-picornavirus 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 anti-poliovirus 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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Introduction Poliovirus (PV) is a small non-enveloped virus with a positive-sense 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 post-eradication era 1. However, currently there is no antiviral available for PV infection. PI4KB is one of the four mammalian PI4 kinases (PI4K2A, PI4K2B, PI4KA, and PI4KB) 2. PI4KB produces PI4P mainly at the Golgi 3, and involves 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. with a potent PI4KB inhibitor PIK93

10-11

. Subsequently,

PI4KB was identified as the target of a group of anti-picornavirus 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 phosphatidylinositol 4-phosphate (PI4P)-dependent manner, and contributes to homeostasis of cholesterol and lipid

21-22

. Functional link between PI4KB and OSBP

family I in PV replication was suggested by a conserved mutation in the 3A protein



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(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. Current model of the role of PI4KB/OSBP pathway in 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 virus-induced membrane structure for the formation of a virus replication complex required for the synthesis of viral plus-strand RNA

13, 20, 23-27

. 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 non-cytotoxic 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 anti-picornavirus drug candidates underscores the importance of PI4KB/OSBP pathway as the sole potential host target of anti-PV drug in viral RNA replication step 44. MDL-860 is an anti-picornavirus 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 DNA virus (herpes simplex virus type 1)

45-46

. Effectiveness of MDL-860 in vivo

has been shown in mouse models of coxsackievirus infection derivatives have also been synthesized

47 48

, and its potent

49

. MDL-860 inhibits some early step of viral

replication after uncoating 45-46, but its direct target and the mechanism of action remain


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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 anti-picornavirus compounds.



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Results and Discussion MDL-860 is an atypical enviroxime-like compound. We analyzed the specificity of antiviral effect of MDL-860 (Figure 1A). MDL-860 showed moderate anti-PV activity (EC50 of 6.8 µM) with apparent no cytotoxicity in the concentrations examined (CC50 of > 100 µM, and SI of > 14). An enviroxime-resistant type 1 PV pseudovirus (PV1pv) mutant showed resistance against MDL-860 (Figure 1B), suggesting that MDL-860 targets PI4KB/OSBP pathway. However, MDL-860 did not affect in vitro PI4KB activity (Figure 1C), nor enhanced 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)51, which depends on PI4KA/OSBP pathway, 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 a PI4KB interactant RAB11A. The enhancement caused by MDL-860 treatment showed 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 virus-induced membrane structure in PV1pv-infected cells, which depends on the overproduced PI4P by PI4KB in the infected cells (Figure 2A)26. Treatment with


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T-00127-HEV1 caused prompt dissociation of OSBP from the membrane structure to nucleus within 1 h (5 to 6 h p.i. treatment) as previously reported

26

. MDL-860

treatment caused the dissociation of OSBP, but after 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 (Figure 2B and S4). T-00127-HEV1 treatment reduced the amount of PI4P within 1 h treatment, but MDL-860 treatment could cause similar level of decrease of PI4P only after 3 h treatment. Apparent discrepancy in the effects of MDL-860 on conventional in vitro PI4KB assay and in vivo PI4KB activity might suggest that some factor(s) in vivo condition might be required for the effect (e.g. temperature, incubation time, oxidation-reduction potential). These results suggest that MDL-860 targets PI4KB activity in vivo, and requires pre-treatment for its activity in contrast to conventional PI4KB inhibitor.

MDL-860 confers an irreversible anti-PV effect to the cells and activates Nrf2-Keap1 antioxidant pathway. We analyzed the effect of pre-treatment of the cells with MDL-860 on the anti-PV activity (Figure 3). Pre-treatment with a 2C inhibitor guanidine hydrochloride (GuHCl) or a PI4KB inhibitor had no effect on the anti-PV activities. In contrast, pre-treatment with OSBP inhibitors (T-00127-HEV2 and itraconazole) enhanced their activities by 2 to 3-fold (Figure 3A). Pre-treatment with MDL-860 drastically enhanced the anti-PV activity (about 7-fold enhancement, EC50 of 6.4 µM [no pre-treatment] vs. 0.97 µM [24 h pre-treatment]). The anti-PV activity of MDL-860 could be enhanced by 3 h pre-treatment (> 95% inhibition), and more significantly by 24 h pre-treatment (> 98% inhibition) (Figure 3B). After removal of



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MDL-860 after 24 h pre-treatment, 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 pre-treatment 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 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 non-cytotoxic concentrations, nor enhanced the activity after pre-treatment (Figure S5). These results suggest that MDL-860 could serve as an electrophile or oxidant to activate Nrf2-Keap1 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, N-terminally FLAG-tagged PI4KB (N-FLAG-PI4KB) was expressed in HEK293 cells in the presence of MDL-860, T-00127-HEV1, or the absence of these compounds (mock-treatment), 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-FLAG-PI4KBs purified from mock-treated cells or T-00127-HEV1-treated cells (4.7 to 9.8% activity of N-FLAG-PI4KB purified from mock-treated cells, P < 0.000015) (Figure 4A). This


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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). Main peaks of each molecular species were surrounded by 4 to 5 molecular species (90,849 Da to 91,172 Da, or 95,108 Da to 95,503 Da, respectively) with increased molecular mass of every 79.0 (standard deviation 0.47), possibly derived from phosphorylation

55-56

. The identity of

modification of 4,260 Da (between 90,924 Da and 95,184 Da) remained unknown. N-FLAG-PI4KBs purified from mock-treated cells or T-00127-HEV1-treated cells showed similar profile of molecular mass. However, N-FLAG-PI4KB purified from MDL-860-treated cells showed increased molecular mass of 146.2 Da (standard deviation 0.52) of the corresponding peaks, suggesting a covalent modification of PI4KB. 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 entire region of PI4KB (798/801 amino acid residues); however, no amino acid residue had a 146 Da modification in its substantial population.



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Because current LC/MS/MS analysis has yet attained robustness to detect all the peptides species with equal efficiency, we next performed mutagenesis analysis of the cysteine residues of PI4KB (total 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 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 mock-treated or from MDL-860-treated cells were determined (Figure 5C). The molecular mass of N-FLAG-PI4KB(C646S) (main peaks at 90,906 Da and 95,167 Da) from mock-treated cells was smaller than N-FLAG-PI4KB(wt) (main peaks at 90,922 Da 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 Da and 95,329 Da), PI4KB(C646S) showed no shift of molecular mass (main peaks at 90,906 Da and 95,167 Da). These results indicate that MDL-860 regulates the activity of PI4KB by allosteric manner via covalent modification of C646. To evaluate the importance of PI4KB as the target of MDL-860 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 N-FLAG-PI4KB


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(D656A) could not rescue PV1pv infection in the presence of MDL-860 as well as empty vector (1.0 to 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 MDL-860), 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 anti-picornavirus 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 anti-picornavirus compounds via novel allosteric regulation of PI4KB activity.



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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, G4361A and C5190U (brefeldin A [BFA] resistance, 2C-Val80Ile and 3A-Ala27Val))

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 (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.


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In vitro kinase assay. Inhibitory effect of MDL-860 at concentration of 10, 20, and 40 µM or of T-00127-HEV1 at concentration of 1 and 10 µM on in vitro PI4KB ad 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, 1/10 dilution, respectively. Final concentration of 680, 230, 68 nM) with lipid substrates (0.025 mg/mL of phosphatidylinositol and 0.075 mg/mL or 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 were used for the assay. The net signals of PI4KB activity of the samples were determined as below: The net signals of PI4KB activity = ℎ ℎ !" #$ %&4'( ℎ!#) − ℎ ℎ +)" #$ %&4'( ℎ!#) , 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) infected with PV1pv (parental or G5318A mutant) at multiplicities of infection (MOI) of 2, and then treated with



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T-00127-HEV1 (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 FACSCantoTM II Flow Cytometer (BD Biosciences) and FlowJo software (FLOWJO, LLC). Relative intensity of the PI4P signals was determined as below: Relative intensity of the PI4P signals the PI4P signals in PV1pv − infected cells =. 3 the PI4P signals in non − infected cells Net amount of produced PI4P was determined as below: 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 post-transfection (p.t.). N-FLAG-PI4KB was purified by using anti-FLAG M2 magnetic beads (Sigma-Aldrich Co. LLC.), and was eluted with 3xFLAG peptide. Eluent from mock-transfected HEK293 cells was used as negative control.


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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 0.4 mL/min, 60°C) and an Agilent LCMS 6530 QTOF LCMS system (positive ion mode, capillary voltage 4000 V, fragmentor voltage 250 V, drying gas flow 8L/min, 350°C, sheath gas flow 11L/min, 400°C) (Agilent technologies Japan, Ltd.).

Statistical analysis. The results of experiments are shown as the averages with standard deviations. P values of less than 0.05 by one-tailed t test were considered significant difference, and were indicated by asterisks (*P < 0.05, **P < 0.01, ***P < 0.001).



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Supporting information Figures S1, S2, S3, S4, S5, and S6, Tables S1, S2, and S3; methods, reference

Figure S1: Effects of MDL-860 on the localization of OSBP and the amount of unesterified cholesterol in the cells Figure S2: Specificity of the anti-viral activity of MDL-860 Figure S3: Effect of MDL-860 on the localization of PI4KB in the cells Figure S4: Quantitation of PI4P in PV1pv-infected cells Figure S5: Anti-PV activity of sulforaphane Figure S6: Rescue of PV1pv infection in the presence of MDL-860 by ectopic expression of PI4KB Table S1: Genes regulated by MDL-860 treatment Table S2: Upstream analysis of up-regulated genes by MDL-860 treatment by Ingenuity Pathway Analysis (Top 50 hits) Table S3: Peptide mapping of N-FLAG-PI4KB purified from MDL-860-treated cells Methods: Methods for Supporting information Reference: Reference for Supporting information

Author Information Corresponding Author *Phone:+81-42-561-0771. Fax:+81-42-561-4729. E-mail: [email protected]

ORCID


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Minetaro Arita: 0000-0002-3314-6626

Author Contributions M.A. and A.G. conceived the project. M.A. designed the study. G.D., G.P., and A.G. performed chemical synthesis of MDL-860, M.A. performed biological experiments with MDL-860. M.A. wrote the manuscript, all authors commented on the manuscript.

Funding 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.

Notes The authors declare no competing financial interest.

Acknowledgements We are grateful to Junko Wada and Yuzuru Aoi for excellent technical assistance, and Hiroyuki Shimizu and Takaji Wakita for kind supports, Tadaki Suzuki



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(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, and Hiroshi Sezaki (Agilent technologies Japan, Ltd.) for kind support of mass spectrometry, 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. 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


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References 1. Committee on Development of a Polio Antiviral and Its Potential Role in Global Poliomyelitis Eradication, N. R. C., Exploring the role of antiviral drugs in the eradication of polio: Workshop Report. THE NATIONAL ACADEMIES PRESS, Washington, D.C. 2006. DOI: https://doi.org/10.17226/11599. 2. Balla, A.; Balla, T., Phosphatidylinositol 4-kinases: old enzymes with emerging functions. Trends Cell Biol. 2006, 16 (7), 351-361. DOI: 10.1016/j.tcb.2006.05.003. 3. Godi, A.; Pertile, P.; Meyers, R.; Marra, P.; Di Tullio, G.; Iurisci, C.; Luini, A.; Corda, D.; De Matteis, M. A., ARF mediates recruitment of PtdIns-4-OH kinase-beta and stimulates synthesis of PtdIns(4,5)P2 on the Golgi complex. Nat. Cell Biol. 1999, 1 (5), 280-287. DOI: 10.1038/12993. 4. Toth, B.; Balla, A.; Ma, H.; Knight, Z. A.; Shokat, K. M.; Balla, T., Phosphatidylinositol 4-kinase IIIbeta regulates the transport of ceramide between the endoplasmic reticulum and Golgi. J. Biol. Chem. 2006, 281 (47), 36369-36377. DOI: 10.1074/jbc.M604935200. 5. Jovic, M.; Kean, M. J.; Szentpetery, Z.; Polevoy, G.; Gingras, A. C.; Brill, J. A.; Balla, T., Two phosphatidylinositol 4-kinases control lysosomal delivery of the Gaucher disease enzyme, beta-glucocerebrosidase. Mol. Biol. Cell 2012, 23 (8), 1533-1545. DOI: 10.1091/mbc.E11-06-0553. 6. Sridhar, S.; Patel, B.; Aphkhazava, D.; Macian, F.; Santambrogio, L.; Shields, D.; Cuervo, A. M., The lipid kinase PI4KIIIbeta preserves lysosomal identity. EMBO J. 2013, 32 (3), 324-339. DOI: 10.1038/emboj.2012.341. 7. Wang, T.; Birsoy, K.; Hughes, N. W.; Krupczak, K. M.; Post, Y.; Wei, J. J.; Lander, E. S.; Sabatini, D. M., Identification and characterization of essential genes in the human genome. Science 2015, 350 (6264), 1096-1101. DOI: 10.1126/science.aac7041. 8. Polevoy, G.; Wei, H. C.; Wong, R.; Szentpetery, Z.; Kim, Y. J.; Goldbach, P.; Steinbach, S. K.; Balla, T.; Brill, J. A., Dual roles for the Drosophila PI 4-kinase four wheel drive in localizing Rab11 during cytokinesis. J. Cell Biol. 2009, 187 (6), 847-858. DOI: 10.1083/jcb.200908107. 9. McNamara, C. W.; Lee, M. C.; Lim, C. S.; Lim, S. H.; Roland, J.; Nagle, A.; Simon, O.; Yeung, B. K.; Chatterjee, A. K.; McCormack, S. L.; Manary, M. J.; Zeeman, A. M.; Dechering, K. J.; Kumar, T. R.; Henrich, P. P.; Gagaring, K.; Ibanez, M.; Kato, N.; Kuhen, K. L.; Fischli, C.; Rottmann, M.; Plouffe, D. M.; Bursulaya, B.; Meister, S.; Rameh, L.; Trappe, J.; Haasen, D.; Timmerman, M.; Sauerwein, R. W.; Suwanarusk, R.; Russell, B.; Renia, L.; Nosten, F.; Tully, D. C.; Kocken, C. H.; Glynne, R. J.; Bodenreider, C.; Fidock, D. A.; Diagana, T. T.; Winzeler, E. A., Targeting Plasmodium PI(4)K to eliminate malaria. Nature 2013, 504 (7479), 248-253. DOI: 10.1038/nature12782. 10. Knight, Z. A.; Gonzalez, B.; Feldman, M. E.; Zunder, E. R.; Goldenberg, D. D.; Williams, O.; Loewith, R.; Stokoe, D.; Balla, A.; Toth, B.; Balla, T.; Weiss, W. A.; Williams, R. L.; Shokat, K. M., A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell 2006, 125 (4), 733-747. DOI: 10.1016/j.cell.2006.03.035. 11. Hsu, N. Y.; Ilnytska, O.; Belov, G.; Santiana, M.; Chen, Y. H.; Takvorian, P.



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

M.; Pau, C.; van der Schaar, H.; Kaushik-Basu, N.; Balla, T.; Cameron, C. E.; Ehrenfeld, E.; van Kuppeveld, F. J.; Altan-Bonnet, N., Viral reorganization of the secretory pathway generates distinct organelles for RNA replication. Cell 2010, 141 (5), 799-811. DOI: 10.1016/j.cell.2010.03.050. 12. Wikel, J. H.; Paget, C. J.; DeLong, D. C.; Nelson, J. D.; Wu, C. Y.; Paschal, J. W.; Dinner, A.; Templeton, R. J.; Chaney, M. O.; Jones, N. D.; Chamberlin, J. W., Synthesis of syn and anti isomers of 6-[[(hydroxyimino)phenyl]methyl]-1-[(1-methylethyl)sulfonyl]-1H-benzimidaz ol-2-amine. Inhibitors of rhinovirus multiplication. J. Med. Chem. 1980, 23 (4), 368-372. DOI: 10.1021/jm00178a004. 13. Ishitsuka, H.; Ohsawa, C.; Ohiwa, T.; Umeda, I.; Suhara, Y., Antipicornavirus flavone Ro 09-0179. Antimicrob. Agents Chemother. 1982, 22 (4), 611-616. DOI: 10.1128/AAC.22.4.611. 14. Galabov, A. S.; Nikolaeva, L.; Philipov, S., Aporphinoid alkaloid glaucinone: a selective inhibitor of poliovirus replication. Antiviral. Res. 1995, 26 (suppl I), A347. 15. Arita, M.; Wakita, T.; Shimizu, H., Characterization of pharmacologically active compounds that inhibit poliovirus and enterovirus 71 infectivity. J. Gen. Virol. 2008, 89 (Pt 10), 2518-2530. DOI: 10.1099/vir.0.2008/002915-0. 16. Arita, M.; Kojima, H.; Nagano, T.; Okabe, T.; Wakita, T.; Shimizu, H., Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity. J. Virol. 2011, 85 (5), 2364-2372. DOI: 10.1128/JVI.02249-10. 17. Delang, L.; Paeshuyse, J.; Neyts, J., The role of phosphatidylinositol 4-kinases and phosphatidylinositol 4-phosphate during viral replication. Biochem. Pharmacol. 2012, 84 (11), 1400-1408. DOI: 10.1016/j.bcp.2012.07.034. 18. MacLeod, A. M.; Mitchell, D. R.; Palmer, N. J.; Van de Poel, H.; Conrath, K.; Andrews, M.; Leyssen, P.; Neyts, J., Identification of a series of compounds with potent antiviral activity for the treatment of enterovirus infections. ACS Med. Chem. Lett. 2013, 4 (7), 585-589. DOI: 10.1021/ml400095m. 19. Arita, M.; Kojima, H.; Nagano, T.; Okabe, T.; Wakita, T.; Shimizu, H., Oxysterol-binding protein family I is the target of minor enviroxime-like compounds. J. Virol. 2013, 87 (8), 4252-4260. DOI: 10.1128/JVI.03546-12. 20. Strating, J. R.; van der Linden, L.; Albulescu, L.; Bigay, J.; Arita, M.; Delang, L.; Leyssen, P.; van der Schaar, H. M.; Lanke, K. H.; Thibaut, H. J.; Ulferts, R.; Drin, G.; Schlinck, N.; Wubbolts, R. W.; Sever, N.; Head, S. A.; Liu, J. O.; Beachy, P. A.; De Matteis, M. A.; Shair, M. D.; Olkkonen, V. M.; Neyts, J.; van Kuppeveld, F. J., Itraconazole Inhibits Enterovirus Replication by Targeting the Oxysterol-Binding Protein. Cell Rep. 2015. DOI: 10.1016/j.celrep.2014.12.054. 21. Mesmin, B.; Bigay, J.; Moser von Filseck, J.; Lacas-Gervais, S.; Drin, G.; Antonny, B., A Four-Step Cycle Driven by PI(4)P Hydrolysis Directs Sterol/PI(4)P Exchange by the ER-Golgi Tether OSBP. Cell 2013, 155 (4), 830-843. DOI: 10.1016/j.cell.2013.09.056. 22. Olkkonen, V. M.; Li, S., Oxysterol-binding proteins: sterol and phosphoinositide sensors coordinating transport, signaling and metabolism. Prog. Lipid Res. 2013, 52 (4), 529-538. DOI: 10.1016/j.plipres.2013.06.004. 23. Heinz, B. A.; Vance, L. M., The antiviral compound enviroxime targets the 3A coding region of rhinovirus and poliovirus. J. Virol. 1995, 69 (7), 4189-4197.


Page 20 of 34

Page 21 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


24. Arita, M., Mechanism of Poliovirus Resistance to Host Phosphatidylinositol-4 Kinase III β Inhibitor. ACS Infectious Diseases 2016, 2 (2), 140-148. DOI: 10.1021/acsinfecdis.5b00122. 25. Rodriguez, P. L.; Carrasco, L., Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol. J. Virol. 1992, 66 (4), 1971-1976. 26. Arita, M., Phosphatidylinositol-4 kinase III beta and oxysterol-binding protein accumulate unesterified cholesterol on poliovirus-induced membrane structure. Microbiol. Immunol. 2014, 58 (4), 239-256. DOI: 10.1111/1348-0421.12144. 27. Roulin, P. S.; Lotzerich, M.; Torta, F.; Tanner, L. B.; van Kuppeveld, F. J.; Wenk, M. R.; Greber, U. F., Rhinovirus Uses a Phosphatidylinositol 4-Phosphate/Cholesterol Counter-Current for the Formation of Replication Compartments at the ER-Golgi Interface. Cell Host Microbe 2014, 16 (5), 677-690. DOI: 10.1016/j.chom.2014.10.003. 28. Sagan, S. M.; Rouleau, Y.; Leggiadro, C.; Supekova, L.; Schultz, P. G.; Su, A. I.; Pezacki, J. P., The influence of cholesterol and lipid metabolism on host cell structure and hepatitis C virus replication. Biochem. Cell Biol. 2006, 84 (1), 67-79. DOI: 10.1139/o05-149. 29. Bianco, A.; Reghellin, V.; Donnici, L.; Fenu, S.; Alvarez, R.; Baruffa, C.; Peri, F.; Pagani, M.; Abrignani, S.; Neddermann, P.; De Francesco, R., Metabolism of phosphatidylinositol 4-kinase IIIalpha-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity. PLoS Pathog. 2012, 8 (3), e1002576. DOI: 10.1371/journal.ppat.1002576. 30. Wang, H.; Perry, J. W.; Lauring, A. S.; Neddermann, P.; De Francesco, R.; Tai, A. W., Oxysterol-binding protein is a phosphatidylinositol 4-kinase effector required for HCV replication membrane integrity and cholesterol trafficking. Gastroenterology 2014, 146 (5), 1373-1385 e1-11. DOI: 10.1053/j.gastro.2014.02.002. 31. Esser-Nobis, K.; Harak, C.; Schult, P.; Kusov, Y.; Lohmann, V., Novel perspectives for hepatitis A virus therapy revealed by comparative analysis of hepatitis C virus and hepatitis A virus RNA replication. Hepatology 2015, 62 (2), 397-408. DOI: 10.1002/hep.27847. 32. Harak, C.; Meyrath, M.; Romero-Brey, I.; Schenk, C.; Gondeau, C.; Schult, P.; Esser-Nobis, K.; Saeed, M.; Neddermann, P.; Schnitzler, P.; Gotthardt, D.; Perez-Del-Pulgar, S.; Neumann-Haefelin, C.; Thimme, R.; Meuleman, P.; Vondran, F. W.; Francesco, R.; Rice, C. M.; Bartenschlager, R.; Lohmann, V., Tuning a cellular lipid kinase activity adapts hepatitis C virus to replication in cell culture. Nat. Microbiol. 2016, 2, 16247. DOI: 10.1038/nmicrobiol.2016.247. 33. Wang, H.; Tai, A. W., Continuous de novo generation of spatially segregated hepatitis C virus replication organelles revealed by pulse-chase imaging. J. Hepatol. 2017, 66 (1), 55-66. DOI: 10.1016/j.jhep.2016.08.018. 34. Otto, M. J.; Fox, M. P.; Fancher, M. J.; Kuhrt, M. F.; Diana, G. D.; McKinlay, M. A., In vitro activity of WIN 51711, a new broad-spectrum antipicornavirus drug. Antimicrob. Agents Chemother. 1985, 27 (6), 883-886. 35. Patick, A. K.; Binford, S. L.; Brothers, M. A.; Jackson, R. L.; Ford, C. E.; Diem, M. D.; Maldonado, F.; Dragovich, P. S.; Zhou, R.; Prins, T. J.; Fuhrman, S. A.; Meador, J. W.; Zalman, L. S.; Matthews, D. A.; Worland, S. T., In vitro antiviral



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

activity of AG7088, a potent inhibitor of human rhinovirus 3C protease. Antimicrob. Agents Chemother. 1999, 43 (10), 2444-2450. 36. Shimizu, H.; Agoh, M.; Agoh, Y.; Yoshida, H.; Yoshii, K.; Yoneyama, T.; Hagiwara, A.; Miyamura, T., Mutations in the 2C region of poliovirus responsible for altered sensitivity to benzimidazole derivatives. J. Virol. 2000, 74 (9), 4146-4154. DOI: 10.1128/JVI.74.9.4146-4154.2000. 37. Furuta, Y.; Takahashi, K.; Shiraki, K.; Sakamoto, K.; Smee, D. F.; Barnard, D. L.; Gowen, B. B.; Julander, J. G.; Morrey, J. D., T-705 (favipiravir) and related compounds: Novel broad-spectrum inhibitors of RNA viral infections. Antiviral Res. 2009, 82 (3), 95-102. DOI: 10.1016/j.antiviral.2009.02.198. 38. De Palma, A. M.; Thibaut, H. J.; van der Linden, L.; Lanke, K.; Heggermont, W.; Ireland, S.; Andrews, R.; Arimilli, M.; Altel, T.; De Clercq, E.; van Kuppeveld, F.; Neyts, J., Mutations in the non-structural protein 3A confer resistance to the novel enterovirus replication inhibitor TTP-8307. Antimicrob. Agents Chemother. 2009, 53 (5), 1850-1857. DOI: 10.1128/AAC.00934-08. 39. Lamarche, M. J.; Borawski, J.; Bose, A.; Capacci-Daniel, C.; Colvin, R.; Dennehy, M.; Ding, J.; Dobler, M.; Drumm, J.; Gaither, L. A.; Gao, J.; Jiang, X.; Lin, K.; McKeever, U.; Puyang, X.; Raman, P.; Thohan, S.; Tommasi, R.; Wagner, K.; Xiong, X.; Zabawa, T.; Zhu, S.; Wiedmann, B., Anti-hepatitis C virus activity and toxicity of type III phosphatidylinositol-4-kinase beta inhibitors. Antimicrob. Agents Chemother. 2012, 56 (10), 5149-5156. DOI: 10.1128/AAC.00946-12. 40. Spickler, C.; Lippens, J.; Laberge, M. K.; Desmeules, S.; Bellavance, E.; Garneau, M.; Guo, T.; Hucke, O.; Leyssen, P.; Neyts, J.; Vaillancourt, F. H.; Decor, A.; O'Meara, J.; Franti, M.; Gauthier, A., Phosphatidylinositol 4-Kinase III Beta Is Essential for Replication of Human Rhinovirus and Its Inhibition Causes a Lethal Phenotype In Vivo. Antimicrob. Agents Chemother. 2013, 57 (7), 3358-3368. DOI: 10.1128/AAC.00303-13. 41. Mejdrova, I.; Chalupska, D.; Kogler, M.; Sala, M.; Plackova, P.; Baumlova, A.; Hrebabecky, H.; Prochazkova, E.; Dejmek, M.; Guillon, R.; Strunin, D.; Weber, J.; Lee, G.; Birkus, G.; Mertlikova-Kaiserova, H.; Boura, E.; Nencka, R., Highly Selective Phosphatidylinositol 4-Kinase IIIbeta Inhibitors and Structural Insight into Their Mode of Action. J. Med. Chem. 2015, 58 (9), 3767-93. DOI: 10.1021/acs.jmedchem.5b00499. 42. Rutaganira, F. U.; Fowler, M. L.; McPhail, J. A.; Gelman, M. A.; Nguyen, K.; Xiong, A.; Dornan, G. L.; Tavshanjian, B.; Glenn, J. S.; Shokat, K. M.; Burke, J. E., Design and Structural Characterization of Potent and Selective Inhibitors of Phosphatidylinositol 4 Kinase IIIbeta. J. Med. Chem. 2016, 59 (5), 1830-1839. DOI: 10.1021/acs.jmedchem.5b01311. 43. Albulescu, L.; Bigay, J.; Biswas, B.; Weber-Boyvat, M.; Dorobantu, C. M.; Delang, L.; van der Schaar, H. M.; Jung, Y. S.; Neyts, J.; Olkkonen, V. M.; van Kuppeveld, F. J.; Strating, J. R., Uncovering oxysterol-binding protein (OSBP) as a target of the anti-enteroviral compound TTP-8307. Antiviral Res. 2017, 140, 37-44. DOI: 10.1016/j.antiviral.2017.01.008. 44. Arita, M.; Philipov, S.; Galabov, A. S., Phosphatidylinositol 4-kinase III beta is the target of oxoglaucine and pachypodol (Ro 09-0179) for their anti-poliovirus activities, and is located at upstream of the target step of brefeldin A. Microbiol. Immunol. 2015, 59 (6), 338-347. DOI: 10.1111/1348-0421.12261.


Page 22 of 34

Page 23 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


45. Torney, H. L.; Dulworth, J. K.; Steward, D. L., Antiviral activity and mechanism of action of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860). Antimicrobial agents and chemotherapy 1982, 22 (4), 635-638. DOI: 10.1128/AAC.22.4.635. 46. Powers, R. D.; Gwaltney, J. M., Jr.; Hayden, F. G., Activity of 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile (MDL-860) against picornaviruses in vitro. Antimicrobial agents and chemotherapy 1982, 22 (4), 639-642. DOI: 10.1128/AAC.22.4.639. 47. Padalko, E.; Verbeken, E.; De Clercq, E.; Neyts, J., Inhibition of coxsackie B3 virus induced myocarditis in mice by 2-(3,4-dichlorophenoxy)-5-nitrobenzonitrile. J. Med. Virol. 2004, 72 (2), 263-267. DOI: 10.1002/jmv.10570. 48. Stoyanova, A.; Nikolova, I.; Purstinger, G.; Dobrikov, G.; Dimitrov, V.; Philipov, S.; Galabov, A. S., Anti-enteroviral triple combination of viral replication inhibitors: activity against coxsackievirus B1 neuroinfection in mice. Antivir. Chem. Chemother. 2016, 24 (5-6), 136-137. DOI: 10.1177/2040206616671571. 49. Purstinger, G.; De Palma, A. M.; Zimmerhofer, G.; Huber, S.; Ladurner, S.; Neyts, J., Synthesis and anti-CVB 3 evaluation of substituted 5-nitro-2-phenoxybenzonitriles. Bioorg. Med. Chem. Lett. 2008, 18 (18), 5123-5125. DOI: 10.1016/j.bmcl.2008.07.099. 50. Burgett, A. W.; Poulsen, T. B.; Wangkanont, K.; Anderson, D. R.; Kikuchi, C.; Shimada, K.; Okubo, S.; Fortner, K. C.; Mimaki, Y.; Kuroda, M.; Murphy, J. P.; Schwalb, D. J.; Petrella, E. C.; Cornella-Taracido, I.; Schirle, M.; Tallarico, J. A.; Shair, M. D., Natural products reveal cancer cell dependence on oxysterol-binding proteins. Nat. Chem. Biol. 2011, 7 (9), 639-647. DOI: 10.1038/nchembio.625. 51. Dorobantu, C. M.; Albulescu, L.; Harak, C.; Feng, Q.; van Kampen, M.; Strating, J. R.; Gorbalenya, A. E.; Lohmann, V.; van der Schaar, H. M.; van Kuppeveld, F. J., Modulation of the Host Lipid Landscape to Promote RNA Virus Replication: The Picornavirus Encephalomyocarditis Virus Converges on the Pathway Used by Hepatitis C Virus. PLoS Pathog. 2015, 11 (9), e1005185. DOI: 10.1371/journal.ppat.1005185. 52. van der Schaar, H. M.; van der Linden, L.; Lanke, K. H.; Strating, J. R.; Purstinger, G.; de Vries, E.; de Haan, C. A.; Neyts, J.; van Kuppeveld, F. J., Coxsackievirus mutants that can bypass host factor PI4KIIIbeta and the need for high levels of PI4P lipids for replication. Cell Res. 2012, 22 (11), 1576-1592. DOI: 10.1038/cr.2012.129. 53. Itoh, K.; Wakabayashi, N.; Katoh, Y.; Ishii, T.; Igarashi, K.; Engel, J. D.; Yamamoto, M., Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev. 1999, 13 (1), 76-86. DOI: 10.1101/gad.13.1.76. 54. Hayes, J. D.; McMahon, M., NRF2 and KEAP1 mutations: permanent activation of an adaptive response in cancer. Trends Biochem. Sci. 2009, 34 (4), 176-188. DOI: 10.1016/j.tibs.2008.12.008. 55. Zhao, X. H.; Bondeva, T.; Balla, T., Characterization of recombinant phosphatidylinositol 4-kinase beta reveals auto- and heterophosphorylation of the enzyme. J. Biol. Chem. 2000, 275 (19), 14642-14648. DOI: 10.1074/jbc.275.19.14642. 56. Suer, S.; Sickmann, A.; Meyer, H. E.; Herberg, F. W.; Heilmeyer, L. M., Jr., Human phosphatidylinositol 4-kinase isoform PI4K92. Expression of the recombinant



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

enzyme and determination of multiple phosphorylation sites. Eur. J. Biochem. 2001, 268 (7), 2099-2106. DOI: 10.1046/j.1432-1327.2001.02089.x. 57. Burke, J. E.; Inglis, A. J.; Perisic, O.; Masson, G. R.; McLaughlin, S. H.; Rutaganira, F.; Shokat, K. M.; Williams, R. L., Structures of PI4KIIIbeta complexes show simultaneous recruitment of Rab11 and its effectors. Science 2014, 344 (6187), 1035-8. DOI: 10.1126/science.1253397. 58. Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E. C.; Ferrin, T. E., UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 2004, 25 (13), 1605-1612. DOI: 10.1002/jcc.20084. 59. Arita, M.; Nagata, N.; Sata, T.; Miyamura, T.; Shimizu, H., Quantitative analysis of poliomyelitis-like paralysis in mice induced by a poliovirus replicon. J. Gen. Virol. 2006, 87 (Pt 11), 3317-3327. DOI: 10.1099/vir.0.82172-0. 60. Baltera, R. F., Jr.; Tershak, D. R., Guanidine-resistant mutants of poliovirus have distinct mutations in peptide 2C. J. Virol. 1989, 63 (10), 4441-4444. 61. Crotty, S.; Saleh, M. C.; Gitlin, L.; Beske, O.; Andino, R., The poliovirus replication machinery can escape inhibition by an antiviral drug that targets a host cell protein. J. Virol. 2004, 78 (7), 3378-3386. DOI: 10.1128/JVI.78.7.3378-3386.2004.


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Figures and legends

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 2-days treatment. PV1pv infection at 7 h p.i. or the cell viability after 2 days-treatment in the absence of MDL-860 were 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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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-EGFP-HEK293 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, OSBP-EGFP; magenta, 2B, blue, nucleus. Pearson’s correlation coefficient between 2B protein and OSBP-EGFP was shown in the OSBP-EGFP image. OSBP-EGFP co-localized with 2B in the MDL-860-treated cells is indicated by 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 non-infected cells are shown in the right graphs.


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Figure 3. Effect of pre-treatment with MDL-860 on PV1pv infection. (A) RD cells were pre-treated with indicated concentration of anti-PV compounds for 0 or 24 h at 37C 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 indicated time (-24 to 6 h p.i.). Time of drug addition, where significant suppression of PV1pv infection was observed, is indicated by 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 pre-treatment 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.


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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 FLAG-tagged 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 chromatogram. 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.


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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 mock-treated 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 FLAG-tagged PI4KBs (wt and C646S mutant).


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For Table of Contents Use Only Title: Allosteric regulation of phosphatidylinositol 4-kinase III beta by an anti-picornavirus compound MDL-860

Author: Minetaro Arita, Georgi Dobrikov, Gerhard Pürstinger, and Angel S. Galabov


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Allosteric Regulation of Phosphatidylinositol 4-Kinase III Beta by an

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