Characterization of SPP inhibitors suppressingpropagation of HCV and protozoaJunki Hiranoa, Toru Okamotoa,1, Yukari Sugiyamaa, Tatsuya Suzukia, Shinji Kusakabea, Makoto Tokunagaa,Takasuke Fukuharaa, Miwa Sasaib, Takahiro Touganc, Yasue Matsunagad, Kazuo Yamashitae, Yusuke Sakaif,Masahiro Yamamotob, Toshihiro Horiic, Daron M. Standleyg, Kohji Moriishih, Kyoji Moriyai, Kazuhiko Koikei,and Yoshiharu Matsuuraa,1

aDepartment of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; bDepartment ofImmunoparasitology, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; cDepartment of Molecular Protozoology, ResearchInstitute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; dPlanning and Promotion Office for University-Industry Collaboration, OsakaUniversity, Osaka 565-0871, Japan; eKOTAI Biotechnologies, Inc., Osaka, Japan; fDepartment of Veterinary Pathology, Yamaguchi University, Yamaguchi753-0841, Japan; gDepartment of Genome Informatics, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; hDepartment ofMicrobiology, Faculty of Medicine, University of Yamanashi, Yamanashi 400-8510, Japan; and iDepartment of Gastroenterology, Graduate School ofMedicine, The University of Tokyo, Tokyo 113-8654, Japan

Edited by Peter Palese, Icahn School of Medicine at Mount Sinai, New York, NY, and approved November 3, 2017 (received for review July 17, 2017)

Signal peptide peptidase (SPP) is an intramembrane aspartic proteaseinvolved in the maturation of the core protein of hepatitis C virus(HCV). The processing of HCV core protein by SPP has been reportedto be critical for the propagation and pathogenesis of HCV. Here weexamined the inhibitory activity of inhibitors for γ-secretase, anotherintramembrane cleaving protease, against SPP, and our findingsrevealed that the dibenzoazepine-type structure in the γ-secretaseinhibitors is critical for the inhibition of SPP. The spatial distributionshowed that the γ-secretase inhibitor compound YO-01027 with thedibenzoazepine structure exhibits potent inhibiting activity againstSPP in vitro and in vivo through the interaction of Val223 in SPP.Treatment with this SPP inhibitor suppressed the maturation of coreproteins of all HCV genotypes without the emergence of drug-resistant viruses, in contrast to the treatment with direct-acting an-tivirals. YO-01027 also efficiently inhibited the propagation ofprotozoa such as Plasmodium falciparum and Toxoplasma gondii.These data suggest that SPP is an ideal target for the developmentof therapeutics not only against chronic hepatitis C but also againstprotozoiasis.

SPP | HCV | pathogenesis | Protozoa | propagation

Hepatitis C virus (HCV) is a major causative agent of chronicliver diseases, including steatosis, cirrhosis, and hepatocel-

lular carcinoma (1). HCV infection is also epidemiologicallycorrelated with extrahepatic manifestations such as type 2 di-abetes, mixed cryoglobulinemia, and non-Hodgkin lymphoma(2). Combination therapy using pegylated IFN plus ribavirinachieved an ∼50% sustained virological response (SVR) in pa-tients infected with genotype (GT) 1b with high viral loads (3).With the introduction of direct-acting antivirals (DAAs) target-ing viral protease and polymerase (4), over 90% of chronichepatitis C patients achieved SVR (5, 6). However, HCV that isresistant to DAAs has already been reported (7), and thus thereis need for novel therapeutics with a low frequency of emergenceof breakthrough viruses.HCV, which belongs to the family Flaviviridae, possesses a

positive-sense and single-stranded RNA genome and has sevenmajor genotypes (GT1–GT7) (8). The viral RNA is translated intoa large single polyprotein (∼3,000 amino acids) and is processedinto 10 viral proteins through cleavage by host and viral-encodedproteases. The core protein of HCV is a component of viral capsids,and E1 and E2 are envelope glycoproteins. The p7 protein acts as aproton pump for the efficient release of the virus. Nonstructural(NS) 2 and 3 proteins possess protease activities. NS4 is thought tobe a scaffold for the viral replication complex. NS5A interacts withvarious host factors and regulates viral replication. NS5B has RNA-dependent RNA polymerase activity (9, 10). HCV is trapped byglycosaminoglycans such as heparin and heparan sulfate on the cell

surface and then is transferred to protein receptors. HCV entersthe cells via endocytosis, replicates on the endoplasmic reticulum(ER) membrane, and buds into the ER lumen.The core protein of HCV is a multifunctional protein present in

many cellular components, such as the nucleus, ER, lipid droplets,lipid rafts, and mitochondria. The core protein participates inapoptosis, autophagy, cell cycles, and oncogenesis (11–13). Al-though the precise molecular mechanisms of HCV-inducedpathogenesis remain unknown (14), liver-specific HCV coretransgenic (CoreTg) mice have been reported to exhibit insulinresistance, steatosis, and hepatocellular carcinoma (15, 16). Sterolregulatory element binding protein 1c (SREBP-1c), which posi-tively regulates the production of saturated and monounsaturatedfatty acids and triglycerides, is enhanced in the CoreTg mouseliver (17), suggesting that the core protein participates in the de-velopment of liver diseases and extrahepatitis manifestations.HCV core protein is cleaved off from the polyprotein at amino

acid position 191/192 by the host signal peptidase, and the signalsequence in the C-terminal region is further cleaved by theintramembrane aspartic protease known as “signal peptide

Significance

Signal peptide peptidase (SPP) is an essential host factor forpropagation of hepatitis C virus (HCV). Here, we show thatdibenzoazepine-type γ-secretase inhibitors suppressed the mat-uration of all genotypes of HCV core proteins through a specificinteraction with Val223 in SPP, and no drug-resistant virusemerged after several passages of HCV in the presence of the SPPinhibitors. In addition, SPP encoded by Plasmodium falciparumwas functionally similar to human SPP, and treatment with theSPP inhibitors suppressed the propagation of protozoa, includingP. falciparum and Toxoplasma gondii. Structural analysis in silicorevealed that Phe258 of SPP participates in binding to the inhib-itors. Compounds possessing a high affinity to Val223/Phe258 inSPP might be novel therapeutics for chronic hepatitis C andprotozoiasis.

Author contributions: T.O. and Y. Matsuura designed research; J.H., T.O., Y. Sugiyama,T.S., S.K., M.T., T.F., M.S., T.T., and Y. Sakai performed research; M.S., T.T., Y. Matsunaga,K.Y., M.Y., T.H., D.M.S., K. Moriishi, K. Moriya, and K.K. contributed new reagents/analytictools; K. Moriishi analyzed data; and J.H., T.O., and Y. Matsuura wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.1To whom correspondence may be addressed. Email: toru@biken.osaka-u.ac.jp or matsuura@biken.osaka-u.ac.jp.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1712484114/-/DCSupplemental.

E10782–E10791 | PNAS | Published online November 29, 2017 www.pnas.org/cgi/doi/10.1073/pnas.1712484114

peptidase” (SPP) to form a mature core protein (18). We pre-viously demonstrated that the maturation of HCV core proteinby cleavage with SPP is essential for the stable expression of thecore protein and the induction of steatosis in CoreTg mice, andwe observed that immature core protein unprocessed by SPP wasdegraded by proteasomes after ubiquitination by TRC8, anE3 ubiquitin ligase (19). Those findings suggested that SPPwould be an ideal target for the development of therapeuticsagainst chronic hepatitis C.SPP has nine transmembrane domains that are members of

the family of GxGD-type intramembrane cleaving proteases,including γ-secretase (20). The SPP gene is also encoded inprotozoa such as Plasmodium falciparum and was suggested tobe essential for their survival (21). SPP was also reported tocleave cellular substrates such as the histocompatibility antigenHLA-E (22), the unsplicing variant of X-box binding protein 1(XBP1μ) (23), heme oxygenase-1 (HO-1) (24), and viral pro-teins (25–29). Although some of the inhibitors for γ-secretaseinhibit SPP (19), the molecular mechanisms underlying theability of the compounds to inhibit γ-secretase and SPP re-main unclear.In our present investigation of the structure–activity relationship of

the γ-secretase inhibitors, we determined that the dibenzoazepine-type structure of γ-secretase inhibitors plays a crucial role in theirinhibition of SPP, and we identified a compound, YO-01027, thatexhibits potent inhibition of SPP in vitro and in vivo. Our findingsalso demonstrate that human SPP (hsSPP) and P. falciparum SPP(pfSPP) are functionally similar and that YO-01027 suppresses the

propagation of protozoa, including P. falciparum and Toxoplasmagondii. In addition, the treatment of CoreTg mice with YO-01027suppressed core-induced steatosis. We also show that treatment withYO-01027 suppressed the production of infectious particles of HCVby inhibiting the maturation of HCV core proteins and that no drug-resistant virus emerged even after 15 passages in the presence of theinhibitor. In a subsequent analysis using an in silico structural-basedapproach, we demonstrate that Val223 of SPP interacts with thedibenzoazepine structure of YO-01027 and that Phe258 of SPPparticipates in binding to inhibitors for SPP and γ-secretase. Thesedata provide clues for the development of novel SPP-targetingtherapeutics for chronic hepatitis C and protozoiasis.

Materials and MethodsCell Lines.Mouse embryonic fibroblasts (MEFs), a human embryonic kidney cellline (HEK293T), and two human hepatocellular carcinoma cell lines (Huh7 andHuh7.5.1) were cultured in DMEM supplemented with 10% FBS, 100 U/mLpenicillin, and 100 μg/mL streptomycin. GT1 (Con1 strain) (30) and GT2(JFH1 strain) (31) replicon cells were cultured in DMEM supplemented with10% FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 1 mg/mL of G418.

Mice. Mice were maintained under a 12-h light/dark cycle (lights on at 08:00AM) at room temperature (23 °C ± 2 °C). All animal experiments conformedto the Guidelines for the Care and Use of Laboratory Animals (32) and wereapproved by the Institutional Committee of Laboratory Animal Experimen-tation of the Research Institute for Microbial Diseases, Osaka University. Allefforts were made to minimize animal suffering and to reduce the numberof animals used in the experiments.

Fig. 1. The dibenzoazepine-type structure of γ-secretase inhibitors participates in the inhibitory activity of SPP. (A) Schematic of the maturation of HCV coreprotein and its inhibition by SPP. HCV core protein is cleaved off from the precursor polyprotein by signal peptidase and then matures through the processingby SPP. The immature core protein that was not processed by SPP due to the inhibition of SPP was degraded by the proteasome pathway through ubiq-uitination by the E3 ligase TRC8. (B) Structure of a dicistronic lentiviral vector encoding the FLAG-tagged core protein under the ubiquitin promoter and GFPunder the encephalomyocarditis virus (EMCV) IRES. (C) SPP inhibitory activity of the γ-secretase inhibitors. Huh7 cells expressing FLAG-tagged core proteinwere treated with the compounds at concentrations of 0.1, 0.3, 1.0, or 3.0 μM for 2 d, and the mature core protein was detected by immunoblotting. Data arerepresentative of three independent experiments.

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Preparation of HCV and T. gondii. HCV derived from the GT2a JFH-1 strainmutated in E2, p7, and NS2 as shown (33) was prepared by serial passages inHuh7.5.1 cells. Briefly, 1.5 × 106 Huh7.5.1 cells seeded on a 10-cm dish wereincubated for 1 d and were inoculated with HCV at a multiplicity of infection(MOI) of 1.0, and the culture medium was changed to fresh medium at 2 hpostinfection. Culture supernatants were collected at 4 d postinfection, andinfectious titers were determined. J6/JFH1 and Con1/JFH1 were prepared aspreviously described (34, 35).

Antibodies and Reagents. The following antibodies were obtained from thesources indicated: anti-HCV NS5A monoclonal antibody (5A27) (36), anti-HCVcore mouse monoclonal antibody (Fujirebio), anti-actin mouse monoclonalantibody (A228; Sigma), anti-HA rat monoclonal antibody (3F10; Roche),anti-GFP mouse monoclonal antibody (JL-8; Clontech), Alexa Fluor (AF) 488-conjugated anti-rabbit IgG antibodies (Life Technologies), and horseradishperoxidase-conjugated anti-FLAG mouse monoclonal antibody (M2; Sigma).YO-01027 was obtained from SYNthesis med chem, and LY-411575 wasobtained from Sigma. RO-4929097, avagacestat, LY-450139, DAPT, MK-0752,telaprevir (VX-950), and daclatasvir (BMS-790052) were purchased fromSelleck Chemicals. Compound E and compound 34 were purchased fromSanta Cruz Biotechnology.

Plasmids. A lentiviral vector, FUGW (#14883) was obtained from Addgene.FUGW was inserted into the puromycin N-acetyl-transferase gene and the GFPgene under the internal ribosomal entry site (IRES) sequence, and the resultingplasmids were designated “FUIPW” and “FUIGW,” respectively (19). Theplasmid pCMV-VSV-G (#8454) was obtained from Addgene. The plasmidspMDLg/pRRE and pRSV-Rev were obtained from David Huang, The Walter &Eliza Hall Institute, Parkville, Australia. The cDNAs of GT1 (TN strain), GT1(H77C strain), GT2 (J6 strain), GT2 (J8 strain), GT2 (JFH1 strain), GT3(S52 strain), GT4 (ED43 strain), GT5 (SA13 strain), GT6 (HK6a strain), and GT7

(QC69 strain) were synthesized by Integrated DNA Technologies (IDT). ThesecDNAs were amplified by PCR and cloned into FUIGW together with FLAG tagsequences. The SPP gene encoded in P. falciparum was synthesized by IDT,amplified by PCR, and cloned into FUIPW together with an ER retrieval se-quence fused with HA tag sequences (19). SPP mutants were generated by theoverlap extension method and were cloned into FUIPW. All cloning proce-dures were performed using the In-Fusion HD cloning kit (Clontech), and thesequences were confirmed by using an ABI Prism 3130 genetic analyzer.

Generation of Lentiviruses. HEK293T cells (2 × 106) were seeded on a 10-cmdish and incubated at 37 °C for 1 d. The lentiviral transfer vector FUIPW(1.5 μg), 2 μg of pMDLg/pRRE, 2 μg of pRSV-Rev, and 1 μg of pCMV-VSV-Gwere mixed with 500 μL of Opti-MEM and 40 μL of polyethylenimine andwere incubated for 15 min. The DNA complex was inoculated into HEK293Tcells, and the culture medium was changed at 4 h posttransfection. Theculture supernatants were collected at 3 d posttransfection. For the infectionof lentivirus, 2 × 105 cells (2 mL) were seeded on six-well plates and wereincubated for 1 d. The virus-containing culture supernatants (2 mL) and 8 μLof Polybrene (4 mg/mL; Sigma) were inoculated into cells and centrifuged at1,220 × g for 45 min at 32 °C. Stable cell lines were selected by puromycin at2 d postinfection.

qRT-PCR. qRT-PCR for HCV RNA was performed using a TaqMan RNA-to-Ct1-Step Kit and a ViiA7 real-time PCR system (Life Technologies). The followingprimers were used: HCV, 5′-GAGTGTCGTGCAGCCTCCA-3′ and 5′-CACTCGC-AAGCACCCTATCA-3′; GAPDH, 5′-TGTAGTTGAGGTCAATGAAGGG-3′ and 5′-ACATCGCTCAGACACCATG-3′. The following probes were used: HCV, 5′-6-FAM/CTGCGGAAC/ZEN/CGGTGAGTACAC/-3′IABkFQ; GAPDH, 5′-6-FAM/AAGGTCGG-A/ZEN/GTCAACGGATTTGGTC/-3′IABkFQ. HCV RNA was determined by the ΔΔCtmethod using GAPDH as an internal control.

Fig. 2. Classification of γ-secretase inhibitors. (Upper) All the γ-secretase inhibitors exhibiting inhibition of SPP have a dibenzoazepine structure (green circle).(Lower) The other γ-secretase inhibitors exhibiting no inhibitory activity toward SPP lack the dibenzoazepine-type structure. Notably, the only differencebetween YO-01027 and compound E is the dibenzoazepine structure (purple circle).

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HCV Titration. Huh7.5.1 cells were seeded on 24-well plates (3 × 104 cells perwell) and were incubated for 1 d. The culture supernatants serially diluted bymedium were inoculated and incubated for 2 h. Then the culture super-natants were removed, 1% methylcellulose containing DMEM supplementedwith 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin was added,and the culture was further incubated for 2 d. The supernatants were re-

moved, washed once with PBS, and then incubated with 4% para-formaldehyde (PFA) in PBS for 2 h. The cells were washed with PBS threetimes and were permeabilized by incubation with 0.2% Triton X-100 con-taining PBS for 15 min. After washing with PBS three times, the cells wereincubated for 1 h with anti-NS5A antibody (1/2,000) diluted by 2% FBS/PBSat room temperature. After washing with PBS three times, the cells were

Fig. 3. YO-01027 is a potent inhibitor of the production of infectious particles of HCV. (A) Huh7 cells infected with HCV JFH1 strain at an MOI of 5 were treatedwith LY-411575 or YO-01027 for 4 d. Infectious titers in the culture supernatants were determined by a focus-forming assay. The P values were determined by aStudent’s t test. The IC99 values were calculated by using GraphPad Prism software. FFU, focus-forming units. (B) Huh7 cells infected with HCV JFH1 strains at anMOI of 5 were treated with the SPP inhibitors LY-411575 (0, 300, 1,000, or 3,000 nM) or YO-01027 (0, 30, 100, or 300 nM) and the DAAs telaprevir (TPV) ordaclatasvir (DCV) for 4 d. Infectious titers in the culture supernatants were determined by a focus-forming assay. (C) Fractional IC99 (FC) values were calculated asthe IC99 of an inhibitor combination divided by the IC99 of an inhibitor alone. The effect of synergy was investigated by isobologram analysis. (D) SPPKOHuh7 cellswere lentivirally transduced with hsSPP and pfSPP. SPPKOHuh7 cells stably expressing either hsSPP (Hs) or pfSPP (Pf) were selected with puromycin, and theexpression of hsSPP and pfSPP was confirmed byWestern blotting. (E) Huh7 and SPPKOHuh7 cells stably expressing hsSPP (Hs) and pfSPP (Pf) were infected with alentivirus expressing FLAG-tagged HCV core protein. The mature core protein was detected by Western blotting. (F) SPPKOHuh7 cells stably expressing hsSPP (Hs)and pfSPP (Pf) were infected with HCV at an MOI of 5. Infectious titers in the culture supernatants were determined by a focus-forming assay at 4 d postinfection.The P values were determined by a Student’s t test. (G) SPPKOHuh7 cells stably expressing pfSPP were infected with lentivirus vector expressing HCV core proteinand were treated with YO-01027 at a concentration of 0.1, 0.3, 1.0, or 3.0 μM. The matured core protein was detected by immunoblotting. (H) Treatment with YO-01027 at a concentration of 1, 10, or 20 μM for 4 d suppressed the production of infectious particles of HCV in SPPKOHuh7 cells expressing pfSPP upon infectionwith HCV at an MOI of 5. The P values were determined by a Student’s t test. (I) The growth rate of P. falciparum treated with YO-01027 or LY-411575 wasmeasured. The P values were determined by a Student’s t test. (J) MEFs were infected with the T. gondii strain ME-49, which possesses a luciferase gene, at anMOIof 1 and then were incubated with the inhibitors for 1 d. The luminescent signal was measured. The P values were determined by a Student’s t test. (K) MEFs, SPP-KO MEFs (SPPKOMEFs), Huh7 cells, and SPPKOHuh7 cells were infected with T. gondii at an MOI of 1. The luminescent signal was measured at 1 d postinfection.The P values were determined by a Student’s t test. ns, not significant. (L) BALB/c mice i.p. infected with T. gondii (RH strain: 1 × 102 parasites) were i.p. treatedwith 5 μmol/kg or 10 μmol/kg of YO-01027 (n = 6 mice for each treatment) or vehicle (n = 6 mice) at 24 h postinfection. Bioluminescent analysis was performed atthe indicated time points to visualize parasite growth. (M) The survival of mice was monitored. The P values were determined by a log-rank text. Data are themean ± SD of two independent experiments performed with cell lines (A, B, F, and H–K) and are representative of three (G) or two (D, E, L, and M) independentexperiments. Significant differences are indicated by double asterisks (**P < 0.01) or a single asterisk (*P < 0.05).

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incubated for 1 h with AF488-conjugated anti-rabbit antibody (1/2,000) di-luted by 2% FBS/PBS at room temperature. After the cells were washedthree final times with PBS, the viral proteins expressing foci were countedunder an immunofluorescent microscope (Olympus).

Immunoblotting. Cell lysates were prepared by adding lysis buffer consistingof 20 mM Tris·HCl (pH 7.4), 135 mM NaCl, 1% Triton X-100, 1% glycerol, andprotease inhibitor mixture tablets (Roche), incubation for 30 min at 4 °C, andcentrifugation at 14,000 × g for 15 min at 4 °C. The supernatants were in-cubated at 95 °C for 5 min. Proteins were resolved by SDS/PAGE (Novex gels;Invitrogen) and were transferred onto nitrocellulose membranes. Themembranes were then blocked with Tris-buffered saline containing 20 mMTris·HCl (pH 7.4), 135 mM NaCl, 0.05% Tween 20, and 5% skim milk, in-cubated with the primary antibody at room temperature for 1 h, and thenincubated with the HRP-conjugated secondary antibody at room tempera-ture for 1 h. The immune complexes were visualized with SuperSignal WestFemto substrate (Pierce) and were detected by an LAS-3000 image analyzersystem (Fujifilm).

Determination of IC99 in the Combination of SPP Inhibitors and DAAs. Huh7 cellswere seeded on a 24-well plate (3 × 104 cells per well) and incubated for 24 h.The cells were then infected with HCV (JFH1 strain) at an MOI of 5 and wereincubated for 2 h. The supernatants were replaced with fresh medium con-taining compounds. At 4 d postinfection, the infectious titers in the culturesupernatants were determined by using a focus-forming assay. IC99 curves weregenerated using GraphPad Prism 7 software (GraphPad).

Dosing. Dosing of YO-01027 was performed as previously described (37).Briefly, YO-01027 was formulated in 1-mM solutions containing 0.5%(hydroxypropyl)methyl cellulose (Sigma) and 0.1% Tween-80 in H2O. CoreTg

mice were dosed i.p. once a day for 14 d with vehicle or with 5.0 μmol/kg YO-01027. BALB/c mice were dosed i.p. once a day with vehicle or with 5.0 μmol/kgor 10 μmol/kg YO-01027 at 24 h postinfection.

Live Imaging Analysis. The 8-wk-old BALB/c mice were i.p. infected with 1 ×102 parasites (RH strains) expressing luciferase in 100 mL PBS, and bio-luminescence was assessed on the indicated days after infection. For thedetection of bioluminescence emission, mice were i.p. injected with 3 mg ofD-luciferin (Promega) in 200 mL PBS, maintained for 5 min, and then anes-thetized with isoflurane (Dainippon Sumitomo Pharma). Abdominal photonemission was assessed during a 3-s exposure by an in vivo imaging system(IVIS 100; Xenogen) and was analyzed as described previously (38).

Hematoxylin and Oil Red O Staining. Formalin-fixed livers were impregnatedwith 30% sucrose and frozen with Tissue-Tek OCT compound (Sakura Fine-tek). Ten-micrometer-thick frozen sections were stained with hematoxylin(H&E). To visualize lipids, frozen sections were stained with Oil Red O. Thefrozen sections were washed with running tap water for 5 min, rinsed with60% isopropanol for 1 min, and then stained with freshly prepared Oil RedO solution for 15 min. The sections were then rinsed with 60% isopropanol,followed by light staining of the nuclei with HE for 15 s. The sections wereobserved under a microscope (Olympus).

Homology Modeling and Docking Simulation of SPP. Structural information ofa presenilin/SPP homolog (PSH) from Methanocellus marisnigri JR1 wasobtained from the Protein Data Bank (PDB)(PDB ID code: 4HYG.A; https://pdbj.org/mine/resources/4hyg) as a template to model SPP. Jr1 SPP se-quences were aligned by HHPred (https://toolkit.tuebingen.mpg.de/#/tools/hhpred), and a structural model of SPP was built using the program Spanner(https://sysimm.ifrec.osaka-u.ac.jp/spanner/). Poorly aligned and flexible re-gions of modeled SPP were selected manually and resampled by Spanner.The 3D information of YO-01027 was obtained from ZINC (https://zinc.docking.org/substance/22056928), and docking simulation of YO-01027 withSPP was conducted using the AutoDock Vina docking program (39). Struc-tural alignment of modeled SPP and presenilin-1 (PDB ID code: 5A63.B;https://pdbj.org/mine/summary/5a63) was conducted by the SALIGN server(https://modbase.compbio.ucsf.edu/salign/) (40). The electrostatic potentialwas calculated using an Adaptive Poisson–Boltzmann Solver (APBS) tool (41–43). To render the structure images (and make mutants of SPP in silico), theopen-source PyMOL Molecular Graphics System 1.8.4.0 was used.

Cell Viability Assay. Huh7 cells (2 × 104 cells in 50 μL of culture medium) wereadded to black 96-well plates (Corning) and then serially diluted compounds(50 μL) were added to each well. Cell viability was measured by using aCellTiter-Glo Luminescent Cell Viability Assay (Promega) according to themanufacturer’s protocol.

Parasite Culture and Drug Treatment. P. falciparum strain 3D7 was cultured inRPMI 1640 medium (Nacalai Tesque) supplemented with 25 μg/mL L-glutamine,Hepes, NaHCO3, 50 μg/mL hypoxanthin, gentamicin, 10% human serum, andRBCs at 3% hematocrit in an atmosphere of 5% CO2, 5% O2, and 90% N2 at37 °C as previously described (44). Parasites were synchronized to ring stageby three 5% sorbitol treatments (45) and then were treated with the in-dicated drugs for 48 h. Parasitemia and morphological changes were ob-served under a microscope. The ME49 and RH strains of T. gondii weremaintained in Vero cells in RPMI 1640 supplemented with 2% FBS, 100 U/mLpenicillin, and 0.1 mg/mL streptomycin.

Statistical Analysis. Statistical analyses were performed using GraphPad Prismsoftware (GraphPad). Significant differences were determined using Stu-dent’s t test and are indicated with asterisks (*P < 0.05) and double asterisks(**P < 0.01) in each figure. Significant differences in the in vivo survival datawere determined using a log-rank test and are indicated with asterisks (*P <0.05) and double asterisks (**P < 0.01) in each figure.

ResultsThe Dibenzoazepine-Type Structure in γ-Secretase Inhibitors Participatesin the Inhibitory Activity of SPP. Our prior study demonstrated thatthe cleavage of HCV core protein by SPP is essential for maturationof the core protein and that an immature core protein generatedby the inhibition of SPP activity was degraded by proteasomesafter ubiquitination by TRC8 (Fig. 1A) (19). In the present study,to assess the inhibitory activity of γ-secretase inhibitors for SPP, we

Fig. 4. Treatment of YO-01027 ameliorates liver steatosis in CoreTg mice.(A) CoreTg male mice (24 wk old) were i.p. administered YO-01027 or vehicleone time/d for 14 d. (Upper) Total RNA was obtained from the livers ofCoreTg mice (n = 5), and core mRNA was determined by qPCR. Data are therelative expression levels after standardization by actin expression and areshown as the means ± SE. (Lower) Body weights of mice before and afterYO-01027 administration (n = 5). (B) The livers of CoreTg mice treated withYO-01027 or vehicle for 2 wk or left untreated (WT) were subjected to im-munoblotting. (C) Liver sections of mice after treatment with YO-01027 werestained with Oil Red O (Upper) and H&E (Lower). (Scale bars, 100 μm.) (D)The expression of mRNA of SREBP-1c (Left), SREBP-1a (Center), and SREBP-2(Right) in the livers was determined by qPCR (n = 5). Data are the mean ± SDfrom two independent measurements in five mice per compound treatment.A significant reduction (*P < 0.05) of SREBP-1c expression was observed inCoreTg mice in response to YO-01027 treatment. Images in B and C arerepresentative of at least three independent mice per compound treatment.The P values were determined by a Student’s t test.

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constructed a dicistronic lentiviral vector to express the FLAG-tagged core protein and GFP (Fig. 1B). Among the γ-secretaseinhibitors we examined, YO-01027 exhibited more potent sup-pression of the maturation of HCV core protein than LY-411575 orRO-0492907, as we reported previously (19).In contrast, other compounds such as compound E, compound

34, LY-450139, DAPT, avagacestat, and MK-0752 showed noeffect on the processing of HCV core protein (Fig. 1C). Wetherefore classified these γ-secretase inhibitors into two groupsbased on whether they inhibited SPP activity (Fig. 2) and foundthat the γ-secretase inhibitors capable of inhibiting SPP activityhave a conserved dibenzoazepine-type structure. YO-01027 andcompound E have the same structure except for dibenzoazepine,and YO-01027 but not compound E inhibited SPP activity,suggesting that dibenzoazepine-type γ-secretase inhibitors caninhibit SPP.

YO-01027 Is a Potent Inhibitor of the Propagation of HCV and Protozoa.To examine the toxicity of the γ-secretase inhibitors, we de-termined the viability of Huh7 cells treated with the inhibitors ata concentration of 3 μM. Although treatment with LY-411575,YO-01027, and LY-450139 exhibited no significant cell toxicity,avagacestat showed cell toxicity at 4 d posttreatment (Fig. S1A).Next, to examine the effect of YO-01027 and LY-411575 on thereplication of HCV, we treated GT2 and GT1 replicon cells with

YO-01027 and LY-411575 for 3 d. Treatment with YO-01027 andLY-411575 exhibited no effect on the replication of HCV RNA(Fig. S1B).To examine the effects of the γ-secretase inhibitors on the

production of infectious HCV particles, Huh7 cells infected withHCV were treated with various concentrations of the inhibitors.The IC99 values were 230 nM for YO-01027 and 76.7 μΜ for LY-411575 (Fig. 3A), suggesting that YO-01027 exhibited a morepotent inhibitory effect on the production of infectious particlesin the culture supernatants (Fig. 3A). We did not see any effectsby using LY-450139 and avagacestat (Fig. S1C). Next, we ex-amined the effects of LY411575 and YO-01027 on the propa-gation of two chimeric HCVs, J6/JFH1 and Con1/JFH1, thatpossessed the structural proteins of GT2 and GT1, respectively.Huh7 cells infected with the chimeric viruses were incubatedwith LY-411575 or YO-01027. Propagation of both J6/JFH1 andCon1/JFH1 was significantly suppressed by the treatment withLY-411575 and YO-01027. In particular, treatment with YO-01027 impaired the propagation of J6/JFH1 to an undetectablelevel (Fig. S1D). To examine the synergistic effects of SPP in-hibitors and DAAs on the propagation of HCV, we first de-termined the IC99 values of telaprevir and daclatasvir on HCVpropagation; these values were 114 nM and 5.28 nM, respectively(Fig. S1E). Next, Huh7 cells infected with HCV were treated

Fig. 5. SPP inhibitors are broad-spectrum antivirals against HCV of all genotypes with a low risk of emergence of drug-resistant breakthrough viruses.(A) Inhibitory effects of YO-01027 against the processing of core proteins of various genotypes of HCV. FLAG-tagged core proteins of GT1 (TN strain), GT1(H77C strain), GT2 (J6 strain), GT2 (J8 strain), GT2 (JFH1 strain), GT3 (S52 strain), GT4 (ED43 strain), GT5 (SA13 strain), GT6 (HK6a strain), and GT7 (QC69 strain)were expressed in Huh7 cells and were treated with YO-01027 at a concentration of 0.1, 0.3, 1.0, or 3.0 μM. HCV core protein was detected by immuno-blotting. Images are representative of at least three independent experiments. (B) Culture supernatant of Huh7.5.1 cells infected with HCV in the presence ofLY-411575 (1.0 μM), YO-01027 (0.1 μM), or telaprevir (TPV, 0.1 μM) was passaged in naive cells in the presence of these reagents. After five or 15 passages, thesupernatants were collected, and the inhibitory effects of the reagents were determined. (C) After five passages in the presence or absence of telaprevir,culture supernatants were inoculated into Huh7.5.1 cells in the presence of various concentrations of telaprevir, and the infectious titers were determined 4 dpostinfection. (D) After 15 passages in the presence or absence of LY-411575 or YO-01027, the culture supernatants were inoculated into Huh7.5.1 cells in thepresence of various concentrations of the inhibitors, and infectious titers were determined 4 d postinfection. Data in C and D are the mean ± SD of twoindependent experiments. **P < 0.01. P values were determined by a Student’s t test.

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with SPP inhibitors and DAAs, and the IC99 values against eachcombination were determined (Fig. 3B). All IC99 values wereplotted on an isobologram based on the Loewe combinationindex (46), and the combination of SPP inhibitors and DAAs wasfound to exhibit synergistic effects on the propagation of HCV(Fig. 3C). Collectively, these results suggest that YO-01027 is apotent inhibitor of the production of infectious HCV particles.A previous study demonstrated that the SPP gene is encoded

in protozoa such as P. falciparum and is likely to be essential fortheir survival (21). To examine the role of P. falciparum-encodedSPP (pfSPP) on the maturation of HCV core protein, we de-termined the expression of HCV core protein in SPP-knockoutHuh7 (SPPKOHuh7) cells by assessing the expression of pfSPP(Fig. 3D). The expression of core protein in SPPKOHuh7 wasrestored by the exogenous expression of either hsSPP or pfSPP(Fig. 3E), suggesting that pfSPP can compensate for the functionof hsSPP in cleaving HCV core protein in SPPKOHuh7 cells.Next, to examine the effect of pfSPP on the propagation of

HCV, we determined the intracellular viral RNA and infectioustiters in the supernatants of SPPKOHuh7 cells expressing eitherhsSPP or pfSPP upon infection with HCV. HCV propagation inSPPKOHuh7 cells was restored by the expression of either pfSPPor hsSPP (Fig. 3F and Fig. S1F). In addition, the maturation of coreprotein (Fig. 3G) and HCV propagation (Fig. 3H) were impaired inSPPKOHuh7 cells expressing pfSPP by treatment with YO-01027,suggesting that pfSPP was functionally similar to hsSPP.For investigation of the effects of the γ-secretase inhibitors on

the propagation of P. falciparum, synchronized P. falciparum wasinoculated with hematocrit and γ-secretase inhibitors. Treatmentwith YO-01027 exhibited 10-fold higher inhibition of the propa-gation of P. falciparum compared with treatment with LY-411575(Fig. 3I). In addition, propagation of the ME49 strain of T. gondii,which possesses a luciferase reporter gene, was also significantlymore inhibited by treatment with YO-01027 than by treatmentwith LY-411575 (Fig. 3J). Treatment with LY-411575 or YO-01027 at concentrations up to 30 μM exhibited no significant celltoxicity in SPPKOHuh7 cells expressing pfSPP (Fig. S1G).Next, to examine the effect of host SPP on the propagation of

the ME49 strain of T. gondii, we inoculated parental and SPP-knockout MEFs and Huh7 cells with the ME49 strain. Thepropagation of T. gondii was similar among the cell lines (Fig.

3K), suggesting that a parasite but not the host SPP participatesin the propagation of T. gondii. Finally, we examined the effect ofYO-01027 on T. gondii in vivo. BALB/c mice i.p. infected withthe RH strain possessing a luciferase reporter gene were treatedwith 5 μmol/kg or 10 μmol/kg of YO-01027 i.p. at 24 h post-infection, and the growth of parasites was determined using anIVIS imaging system. The growth of parasites was significantlysuppressed by treatment with YO-01027 (Fig. 3L), and the sur-vival times of infected mice were slightly extended by the YO-01027 treatment (Fig. 3M). Collectively, these results suggestthat YO-01027 is capable of inhibiting the propagation of HCVand protozoa.

YO-01027 Ameliorates Liver Steatosis in CoreTg Mice. To determinethe effect of YO-01027 on the liver pathogenesis in CoreTgmice, we administered YO-01027 to the mice for 2 wk. Althoughthe mRNA expression of the HCV core protein in the livers of

Fig. 6. In silico profiling of the structure of SPP. (A) Homology modeling ofhsSPP was deduced based on a PSH derived from M. marisnigri JR1 (PDB IDcode: 4HYG). The enzymatic active site of SPP was identified in Asp219 andAsp265, which are located on TM6 and TM7, respectively. (B) The structureof SPP is shown with an electrostatic surface representation. As in previousstudies of PSH and presenilin-1 (47, 48), the modeled structure of SPPexhibited a cavity-like structure and a large hole.

Fig. 7. Val223 of SPP interacts with the dibenzoazepine-type structure ofYO-01027. (A) The docking simulation of YO-01027 revealed three putativedrug-binding sites in SPP (sites 1–3). (B) SPPKOHuh7 cells were lentivirallytransduced with wild-type or mutant SPP in which Val223 was replaced withIle (V223I), Phe192 was replaced with Try (F192W), or Ala259 was replacedwith Leu (A259L), and SPP expression was confirmed by immunoblottingafter selection with puromycin. (C) Huh7 cells and SPPKOHuh7 cells stablyexpressing wild-type or mutant SPP were infected with a lentivirusexpressing the FLAG-tagged HCV core protein. The mature core protein wasdetected by immunoblotting. (D) SPPKOHuh7 cells expressing wild-type ormutant SPP were transduced with a lentiviral vector encoding the FLAG-tagged HCV core protein and were treated with YO-01027 at a concentra-tion of 0.1, 0.3, 1.0, or 3.0 μM for 2 d. The mature core protein was detectedby immunoblotting. (E) Close-up views of the interaction of SPP and YO-01027. The dibenzoazepine structure of YO-01027 interacted with SPPVal223. Asp219 of the enzymatic active site of SPP was also close to YO-01027. The data are representative of two (B and C) or three (D) in-dependent experiments.

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CoreTg mice and the body weights of the mice were not changedby the treatment with YO-01027 (Fig. 4A), the expression of thecore protein was significantly reduced by the treatment (Fig. 4B).Oil Red O staining of the liver sections revealed that the lipidaccumulation in the livers of the CoreTg mice was also amelio-rated by 2-wk treatment with YO-01027 when the mice were24 wk old (Fig. 4C).The synthesis of liver fatty acids is tightly regulated by SREBP-

1c, a family of steroid regulatory element-binding proteins. Here,the mRNA expression of SREBP-1c, but not that of SREBP-1aor SREBP-2, was reduced in the livers of CoreTg mice by the YO-01027 treatment (Fig. 4D). These results suggest that treatment withYO-01027 ameliorates liver steatosis in CoreTg mice by suppressingthe expression of HCV core protein.

SPP Inhibitors Are Broad-Spectrum Antivirals Against HCV of All Genotypeswith a Low Risk of the Emergence of Drug-Resistant Breakthrough Viruses.Next, to assess the effects of YO-01027 on the maturation of HCVcore proteins derived from various genotypes of HCV, we constructedexpression vectors for HCV core proteins derived from GT1 to GT7.The treatment of Huh7 cells expressing these core proteins with YO-01027 efficiently suppressed the maturation of core proteins derivedfrom all HCV genotypes (Fig. 5A). For a further examination of theemergence of drug-resistant viruses by treatment with the inhibitors,culture supernatants from Huh7.5.1 cells infected with HCV andtreated with 1 μΜ of LY-411575, 0.1 μΜ of YO-01027, or 0.1 μM oftelaprevir for 4 d were inoculated into naive Huh7.5.1 cells treatedwith same compounds (Fig. 5B). After five rounds of passages, HCVin the culture supernatants exhibited resistance to the treatment

with telaprevir (Fig. 5C), but HCV was sensitive to the treatmentwith LY-411575 and YO-01027 even after 15 passages (Fig. 5D),suggesting that the SPP inhibitors are broad-spectrum antiviralsagainst all genotypes of HCV with a low risk of the emergence ofdrug-resistant breakthrough viruses.

Val223 of SPP Interacts with the Dibenzoazepine-Type Structure ofYO-01027. We next investigated the molecular interactions be-tween SPP and γ-secretase inhibitors. Because the crystal struc-ture of a presenilin/SPP homolog (PSH) from M. marisnigriJR1 has been resolved (47), we constructed the 3D structure ofhuman SPP in silico based on the structure of PSH (Fig. 6A). Theenzymatic active site of SPP had been identified as Asp219 andAsp265 (20), and the structures of PSH and presenilin-1 show acavity-like structure in an enzymatic active site and a large holesurrounded by TM2, TM3, TM5, and TM7 (47, 48). The mod-eled structure of SPP showed the same cavity-like structure and alarge hole (Fig. 6B).To examine the interaction of SPP with γ-secretase inhibitors,

we performed a docking simulation by using YO-01027. This insilico simulation indicated that the dibenzoazepine structure ofYO-01027 interacts with Val223, Phe192, or Ala259 of SPP (Fig.7A). To determine the amino acid residue responsible for theinteraction with YO-01027, we established SPPKOHuh7 celllines stably expressing mutant SPP with Val223, Phe192, andAla259 replaced by Ile, Trp, and Leu, respectively.The results showed that the expression of SPP was comparable

in the wild type and mutants (Fig. 7B) and that the SPP mutantsretained the ability to process HCV core proteins (Fig. 7C).

Fig. 8. Phe258 in SPP determines the accessibility of the γ-secretase inhibitors. (A) Close-up views of the interaction of SPP Phe258 with YO-01027. (B) Thededuced structure of SPP (blue) was compared with that of human presenilin-1 (PDB ID code: 5A63; yellow). The structure of presenilin-1 corresponding to SPPPhe258 was not resolved. (C) Amino acid comparison between SPP and presenilin-1. (Upper) The amino acid residue of presenilin-1 corresponding to SPPPhe258 is Gly. (Lower) A hypothetical model of the interaction of SPP with compound E. (Left) In wild-type SPP, Phe258 is an obstacle to the incorporation ofcompound E. (Right) A small side chain such as Gly258 accepts the incorporation of compound E into the P258G SPP mutant. (D) Docking simulation ofcompound E with the P258G SPP mutant revealed an interaction similar to that seen between YO-01027 and wild-type SPP (Fig. 6E). (E) SPPKOHuh7 cellsexpressing the P258G SPP mutant were transduced with a lentiviral vector encoding the FLAG-tagged HCV core protein and were treated with compound E(Upper) or YO-01027 (Lower) at a concentration of 0.1, 0.3, 1.0, or 3.0 μM for 2 d. The mature core protein was detected by immunoblotting. The data arerepresentative of three independent experiments.

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Although the mutant SPPs in which Phe192 was replaced by Trpand Ala259 was replaced by Leu were still sensitive to the YO-01027 treatment, the substitution of Ile for Val223 resulted inresistance to the YO-01027 treatment (Fig. 7D). In addition, theSPP mutant in which Val223 was replaced by Ile also showedresistance to other dibenzoazepine-type γ-secretase inhibitors,i.e., LY-411575 and RO-0492907 (Fig. S2). These results suggestthat YO-01027 interacts directly with enzymatic active sites(Asp219 and Asp265) of SPP and that the dibenzoazepinestructure of YO-01027 interacts with Val223 of SPP (Fig. 7E).

Phe258 in SPP Determines the Accessibility of the γ-Secretase Inhibitors.To further elucidate the structure–activity relationship betweenγ-secretase inhibitors and SPP, we compared the docking simula-tion of YO-01027 to SPP with that to presenilin-1 (Fig. 8A). Wefocused on Phe258 of SPP because this amino acid is close to thedibenzoazepine structure in YO-01027. Although the 3D structureof presenilin-1 around SPP Phe258 has not yet been determined(Fig. 8B), the amino acid residue corresponding to SPP Phe258 inpresenilin-1 is Gly378 (Fig. 8C). We therefore hypothesized that thebenzyl group of SPP Phe258 prevents compound E from interactingwith SPP (Fig. 8C), because the only difference between YO-01027 and compound E is the dibenzoazepine structure.To test our hypothesis, we constructed the in silico structure of

a mutant SPP in which Phe258 was replaced with Gly, and weperformed a docking simulation of both the wild-type SPP andmutant SPP with compound E. Although the wild-type SPPexhibited no interaction with compound E in our docking simu-lation, the Phe258Gly mutant of SPP interacted with compound Emuch as the wild-type SPP interacted with YO-01027 (Fig. 8D).To further confirm our in silico data in vitro, we established

SPPKOHuh7 cells expressing the Phe258Gly mutant of SPP. Theexpression of SPP was comparable in the wild type and thePhe258Gly mutant (Fig. S3A), and the SPP mutant still retainedthe ability to process HCV core proteins (Fig. S3B). SPPKOHuh7cells expressing the Phe258Gly mutant of SPP were treated witheither compound E or YO-0127 upon the expression of HCV coreprotein. Treatment not only with YO-01027 but also with com-pound E resulted in significant inhibition of the core protein ex-pression in SPPKOHuh7 cells expressing the SPP Phe258Glymutant (Fig. 8E), whereas other compounds such as semagacestat,avagacestat, compound 34, DAPT, and MK-0752 showed no effecton the processing of HCV core protein (Fig. S3C). Collectively,these results suggest that SPP Phe258 determines the accessibilityof the γ-secretase inhibitors to an active site of SPP.

DiscussionOur previous research revealed that SPP plays crucial roles in thepropagation of HCV and the development of core-inducedpathogenesis and that SPP is therefore a promising target forthe development of novel therapeutics against chronic hepatitisC (19). In addition, SPP encoded in protozoa participates in theirpropagation (21), suggesting that SPP inhibitors would be ap-plicable as not only antivirals but also as antiprotozoals. How-ever, the molecular mechanisms underlying the suppression ofSPP by the γ-secretase inhibitors have been largely unknown.Therefore in the present study we attempted to determine theinteraction between the γ-secretase inhibitors and SPP.Our findings showed that some γ-secretase inhibitors are capable

of inhibiting SPP activity. Although YO-01027 shares the samestructure as compound E, except for the dibenzoazepine structure,YO-01027 inhibited SPP in our experiments, but compound E didnot, suggesting that the dibenzoazepine-type structure plays a crucialrole in the inhibition of SPP. Our in silico docking simulation ofhsSPP with YO-01027 suggested that three binding sites and themutation of Val223 in SPP abolished the inhibitory activity of YO-01027, indicating that SPP Val223 interacts with the dibenzoazepine-type structure in YO-01027.

Our results also showed that Phe258 is a spatial obstacle to theincorporation of compound E into SPP. Because the inhibitoryactivity of YO-01027 was still higher than that of compound E inthe Phe258Gly mutant of SPP, we speculate that the interactionbetween SPP and the dibenzoazepine-type structure of YO-01027 might be responsible for the potent inhibitory activity ofYO-01027. Our comparison of the amino acid sequences ofmembers of the SPP family revealed that the amino acid residueVal223 in SPP is conserved not only in presenilin-1 but also in SPP-like 2A (SPPL2A), SPPL2B, and SPPL3. Therefore, dibenzoazepine-type γ-secretase inhibitors might be able to inhibit other SPPfamily enzymes. In contrast, Phe258 in SPP is conserved in SPPL3but not in presenilin-1, SPPL2A, or SPPL2B. Thus, enhancing theinteraction of a compound containing SPP Val223 and Phe258might improve SPP-specific inhibition.We also observed that compound E and compound 34 have a

benzodiazepine structure, but compound 34 could not inhibit theactivity of the Phe258Gly SPP mutant (Fig. S3C), suggesting thatstructures other than the dibenzoazepine structure participate indetermining specificity for SPP inhibition. These data couldprovide clues for the development of novel inhibitors that arespecific for SPP.Although treatment with DAAs targeting viral proteins in-

cluding NS3, NS5A, and NS5B have achieved a 90% SVR rate (5,6), the emergence of viruses resistant to DAAs is an importantissue that must be considered (7). The inhibition of host proteinsthat are crucial for efficient viral propagation is a suitable methodof suppressing the emergence of drug-resistant breakthroughviruses (49, 50). HCV has various genotypes and a highly diverseamino acid sequence due to the low fidelity of RNA-dependentRNA polymerase, and thus it is difficult for antivirals targetingviral proteins to inhibit all genotypes of HCV. In this study, wedemonstrated that the inhibition of SPP suppressed both thematuration of HCV core proteins derived from all the HCVgenotypes examined and the propagation of HCV without theemergence of drug-resistant viruses. Moreover, combined treat-ment with SPP inhibitors and DAAs showed synergistic effects,suggesting that combination therapy with SPP inhibitors and DAAsmight be an effective treatment for patients with chronic hepatitisC, with a low frequency of emergence of drug-resistant viruses.HCV belongs to the family Flaviviridae, which contains four

genera: Flavivirus, Pestivirus,Hepacivirus, and Pegivirus (51, 52). TheC-terminal signal sequences of the core proteins of Hepacivirus andPestivirus are processed by SPP (53), and those of Flavivirus arecleaved by the viral protease NS3 (54). Therefore, cleavage of thesignal sequence of the core protein is essential for the maturation ofthe core protein in viruses of the family Flaviviridae.We also explored the possibility of the application of SPP in-

hibitors in other infectious diseases. Some protozoa encode SPP intheir genomes, and SPP has been suggested to play a crucial rolein the life cycle of these protozoa (21). Here we observed that SPPinhibitors suppressed the propagation of P. falciparum andT. gondii by inhibiting the SPP encoded in their genomes, not by theinhibiting SPP in the host cell genomes. LY-411575, NITD679, andNITD731 were previously shown to inhibit the activity of pfSPP(21), and all three of these compounds contain the dibenzoazepinestructure, which is consistent with our data (Fig. 2).Collectively, these results suggest that SPP is an ideal drug

target for the development of novel therapeutics for chronichepatitis C and protozoiasis. However, the development of SPP-specific inhibitors is still required, because all the compoundsused in this study were developed as γ-secretase inhibitors. Thephase III clinical trials of semagacestat (LY-450139) resulted inno improvement of Alzheimer’s disease and unfortunately en-hanced tumorigenesis of skin and other cancers (55). In thisstudy, we showed that LY-450139 had no inhibitory activity onSPP (Fig. S1C). Therefore, specific inhibitors for SPP whichexert no inhibitory effects on γ-secretase are needed for the

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treatment of chronic hepatitis C. Toward this goal, further in-vestigations will be necessary to elucidate the 3D structure ofSPP and the structural interaction between SPP and YO-01027.

ACKNOWLEDGMENTS. We thank M. Tomiyama and J. Higuchi for secretarialwork; S. Haga and M. Ishibashi for technical assistance; and Bartenschlager,

F. Chisari, and T. Wakita for providing experimental materials. This work wassupported by Grants 17fk0210206h0002, 17fk0210305h0003, 17fk0210210h0002,17fk0210209h0502, and 17fk0210304h0003 from the Program for Basic andClinical Research on Hepatitis of the Japan Agency for Medical Research andDevelopment and by Grants 16H06432, 16H06429, 16K21723, 15H04736,and 16K19139 from the Ministry of Education, Culture, Sports, Science, andTechnology of Japan.

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Hirano et al. PNAS | Published online November 29, 2017 | E10791

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