HBx-derived constrained peptides inhibit the secretion of hepatitis B

Subscriber access provided by Kaohsiung Medical University

Article

HBx-derived constrained peptides inhibit the secretion of hepatitis B virus antigens Xiaodan Cai, Weihao Zheng, Xiaodong Shi, Longjian Chen, Zhihong Liu, and Zigang Li Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.8b00807 • Publication Date (Web): 30 Oct 2018 Downloaded from http://pubs.acs.org on November 4, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 8 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

Molecular Pharmaceutics

HBx-derived constrained peptides inhibit the secretion of hepatitis B virus antigens Xiaodan Cai,‡,# Weihao Zheng,‡,# Xiaodong Shi,‡,# Longjian Chen,# Zhihong Liu, # Zigang Li#,* #State

Key Laboratory of Chemical Oncogenomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, Shenzhen 518055, China. ‡These authors contributed equally to this work. ABSTRACT: Hepatitis B virus (HBV) infection is the primary cause of cirrhosis and liver cancer. Protein-protein interactions (PPIs) between HBV x protein (HBx) and its host targets including Bcl-2 are important for cell death and viral replication. No modulators targeting these PPIs have been reported yet. Here, we developed HBx-derived constrained peptides generated by a facile macrocyclization method by joining two methionine side chains of unprotected peptides with chemoselective alkylating linkers. The resulting constrained peptides with improved cell permeability and binding affinity were effective anti-HBV modulators by blocking secretion of viral antigens. This study clearly demonstrated HBx as a potentially important PPI target and the potential application of this efficient peptide macrocyclization strategy for targeting key PPIs. KEYWORDS：Hepatitis B virus, HBx, Bcl-2, HBsAg, HBeAg, peptide, protein-protein interaction, inhibitor

INTRODUCTION Peptide stapling strategies have been developed to improve the cell membrane permeability and metabolic stability of peptides, which are important approaches toward protein-protein interaction (PPI) modulator discovery.1-7 PPIs are essential for cellular physiology and pathology processes, representing an important target space for drug development.8, 9 PPIs with large or flat interface are generally difficult for small molecule development, but easily targetable by conformationally constrained peptides.10-14 Various chemical methods have been developed for peptide macrocyclization, including disulfide/thioether bond formation,2, 15-21 ring-closing olefin/alkyne metathesis,4, 22, 23 lactam ring formation,3 “click” chemistry,24 addition of perfluoroarenes,25, 26 acetone-linked peptides,27 and vinyl-sulfide formation.6 And some of these methods have been utilized to tackle human diseases by modulating various PPIs, including p53/MDM2, estrogen receptor/coactivator, and Ras/effector, to name a few.7, 28-32 Notably, stapled peptides NYAD-1, SAH-gp41(626−662) and hydrogen-bondsurrogate-derived α helices were reported to inhibit HIV1 (human immunodeficiency virus type 1) infection via effectively targeting the viral capsid or the viral fusion apparatus.33-36 Hepatitis B virus (HBV) infection is a global public health issue and considered as the primary cause of cirrhosis and liver cancer.37 HBV encodes a regulatory HBx protein, which is crucial for viral replication and transcription of HBV cccDNA (covalently closed circular DNA),38, 39 and is also considered to contribute to HBV oncogenicity.40 Interaction between HBx and Bcl-2 is important for viral replication, and could induce elevation of cytosolic calcium, and cell death.41-43 Besides, HBx interacts with DDB1 (DNA-damage-binding protein 1)containing E3 ubiquitin ligase to promote degradation of

the Smc5/6 (structural maintenance of chromosomes 5/6) complex to enhance HBV replication.44-47 Thus, although there is limited knowledge about the effects of HBxmediated PPIs on the secretion of HBV antigens, the roles of these PPIs in HBV replication imply that HBx could be a promising PPI target for HBV therapeutics. However, the effect of targeting HBx associated PPIs in cells remains to be explored. It is worth noting that, functional cure is defined as sustained loss of HBsAg (hepatitis B surface antigen), which is regarded as the objective of new HBV therapies.48, 49 However, currently available treatments including interferon and nucleos(t)ide analogues for chronic hepatitis B (CHB) are related to low rates of functional cure.50 Evidences suggested that high amounts of circulating HBsAg may contribute to T cell exhaustion in chronic hepatitis B.51-54

Scheme 1. A peptide macrocyclization strategy was used to generate constrained peptides that target the HBx/Bcl-2 interaction. Constrained peptides with enhanced cell permeability

ACS Paragon Plus Environment

Molecular Pharmaceutics 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

and binding affinity showed inhibitory effects on HBV antigens secretion.

Notably, several studies demonstrate HBsAg negatively altering HBV-specific immune responses by impairing the functions of natural killer (NK) cells, monocytes, and dendritic cells (DCs).55-58 Thus, the inhibitors of HBsAg secretion may be combined with interferon, nucleos(t)ide analogues or therapeutic vaccines including preS VLP (virus-like particle) that was recently developed by our group,59, 60 for treating HBV infection. We previously developed a novel facile method for peptide macrocyclization by joining side chains of methionine from unprotected peptides with chemoselective alkylating linkers (Figure 1A), which allowed us to generate a series of constrained peptides with different stapling positions and sequence lengths. And the bis-alkylated peptides showed increased proteolysis resistance and negligible cytotoxicity.61 However, the biological applications of peptides constrained with this method are yet to be explored. Here, we employed this strategy in the design of HBx-derived constrained peptides to generate potential anti-HBV candidates, and studied how linkers affect the peptides’ anti-HBV activities through sequence optimization (Scheme 1).

EXPERIMENTAL SECTION Reagents DMEM and FBS were purchased from Thermo Fisher Scientific. Amino acids and resins were purchased from TopPeptide Biotechnology (Shanghai). Adefovir (PMEA) and entecavir (ETV) were purchased from MedchemExpress. Primers were synthesized by Sangon (Shanghai). Peptide Synthesis and Characterization Linear acetylated or FAM labelled peptides were purchased from Top-peptide Biotechnology. Constrained peptide was synthesized according to our previous reports. Generally, linear peptide was dissolved in CH3CN/H2O (1:2 in volume) to reach a concentration of ~10 mM, followed by added with 1% (in volume) formic acid to adjust the pH of the solution to about 3. The alkylation reagent (1.2 equiv.) were dissolved by a small amount of DMF and mixed with the peptide solution, followed by stirring for ~ 48 h at room temperature. During the course of experiment, turbid solution would become pellucid. Then the solution was diluted and purified by reversed phase high-performance liquid chromatography (HPLC) immediately. Peptide Screening Assay To investigate the anti-HBV activities of the peptides, HepG2.2.15 cells (2 × 104 cells/well) were seeded into 96-well plates and incubated for 12 h at 37°C in DMEM supplemented with 10% FBS, and then treated with various concentrations (2.5, 5, 10, 20, 40, 80, 160, and 320 μM) of the peptides. The culture medium was changed with new peptide-containing medium every 3 days. On day 7, cell culture supernatant was collected for HBV antigens and DNA detection. And HepG2.2.15 cells were used for MTT assay to measure the cell viability. ELISA

HBV antigens, such as HBsAg and HBeAg, were measured using 50 μL of HepG2.2.15 cell supernatant with commercial ELISA Kits (Wantai Pharmacy, Beijing, China) according to manufacturer’s procedures. qRT-PCR Analysis HBV DNA were extracted and purified using 250 μL HepG2.2.15 cell supernatant with commercial tissue DNA Kits (Omega Bio-tek, Inc) following manufacturer’s instructions. 5 μL of each DNA sample was used for qRTPCR (quantitative real-time polymerase chain reaction) analysis. Primers HBV2270-F and HBV2392-R used for quantifying copy numbers of HBV DNA were listed in Table S1. qRT-PCR was conducted as described previously.62 HBV DNA copy numbers were quantified based on a standard curve generated from pHBV1.3 plasmid with known nucleic acid quantities. For investigating the effects of peptides 2a and 2b on the transcription of p53 and its downstream genes, HepG2.2.15 cells (2 × 105 cells/well) were seeded into 12-well plates and incubated for 12 h at 37°C. Peptides at different concentrations (25 μM, 100 μM) were added to the wells every three days. On day 7, cells were washed by PBS, and total RNA was isolated using Trizol (Takara) as described by the manufacturer. Then 1 μg of total RNA was reverse transcribed into cDNA using the PrimeScript RT Reagent Kit with gDNA Eraser (Takara). The primers used for transcripts of p53 and its downstream genes were listed in Table S1, including primers for an endogenous control β-actin gene. qRT-PCR was performed using SYBR green (Takara) as described previously.62 Cell Viability After HepG2.2.15 cells treated with serial dilution of peptides for six days, MTT reagent (5 mg/mL, 10 μL) was added to each well and incubated at 37°C for 4 h. After aspirating the MTT reagent, 150 μL of DMSO was added, and then shaken for 10 min. The absorbance of each well was detected by a microplate reader (BioTek Synergy H1) at 490 nm. The cell viability was analyzed using OriginPro 8. Flow Cytometry Analysis HepG2.2.15 cells (2 × 105 cells/well) were seeded into 24well plates and grown for 12 h at 37°C. The cells were then incubated with 40 μM FAM-labelled peptides overnight at 37°C. After removing the peptide solution, the cells were washed with phosphate buffered saline (PBS), and digested with 0.25% trypsin. The cells were then treated with 0.05% trypan blue for 5 min and re-suspended in PBS. Cellular mean fluorescence intensities were detected using a FACS Calibur flow cytometer (BD). Fluorescence Polarization Assay Bcl-2 protein was expressed and purified as described previously.62, 63 Briefly, DNA encoding Bcl-2 (Accession Number NM_000633) was cloned into pCold I plasmid and overexpressed in E. coli (BL21 (DE3) pLysS). Cultures were grown in Luria-Bertani medium at 37°C. The cells were induced with 0.5 mM isopropyl b-D-1-thiogalactopyranoside at 16°C for 20 h till OD600 = 0.6–0.8. Bacteria were collected and purified using an HisTrap™ HP column and a SuperdexTM 200 10/300 GL column with desalting buffer (20 mM TrisHCl, 150 mM NaCl, pH 7.5). SDS-PAGE analysis was used to verify the fractions of the Bcl-2 peak, which were further concentrated to ~4 mg/mL.


Page 2 of 8

Page 3 of 8 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

Molecular Pharmaceutics Fluorescence polarization (FP) experiments were conducted in 96-well black plates. Various concentrations of purified Bcl-2 protein (40 μL) and FAM-labelled peptides (10 nM, 40 μL) in FP assay buffer (20 mM Tris-HCl, 150 mM NaCl, pH 7.5) were mixed by shaking for 5 min and incubated for 24 h at room temperature in the dark. The anisotropy values were measured on a plate reader (PerkinElmer, 2104 EnVision® Multilabel Reader) with excitation at 485 nm and emission at 520 nm. The binding affinity (Kd) values were calculated by nonlinear regression analysis using Hill equation in OriginPro 8.0 (OriginLab). Kd was determined as the concentration of the Bcl-2 protein at which 50% of the peptide is bound. Confocal Microscopy Imaging HepG2.2.15 cells were grown in 24-well plates on coverslips for 12 h at 37°C. Then 40 μM FAM-labelled peptides were added into each well, following incubation at 37°C for 12 h. After fixing with 4% paraformaldehyde at room temperature for 20 min, cells were permeabilized with Triton X-100 (0.25%) for 25 min. All cells were blocked with PBS containing 5% BSA for 1 h, followed by incubation with rabbit anti-Bcl-2 antibody (Cell Signaling Technology) for 12 h at 4°C. After washing, cells were incubated with Alexa Fluor® 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (Jackson ImmunoResearch Laboratories, Inc.) at 37°C for 1 h. Cells were washed, and stained with DAPI for 10 min, followed by mounting onto microscope slides. Confocal slices were acquired with a 100× objective, using a Zeiss 510 confocal microscope with random sampling.

Figure 1. Constrained peptides were prepared by methionine bisalkylation. A) Methionine bis-alkylation of peptide 2 or 3 with different linkers as described previously.61 Peptides were dissolved in a solution of 1% HCOOH in MeCN/H2O (30 : 70, v/v), followed by stirring at room temperature for 48 h. B) Sequences and conversion rate of peptides in this work.

residues that locate on the solvent surface and do not contribute to direct PPI were selected for methionine substitutions. Peptide 1 derived from the BH3-like motif of HBx43 was used as a control peptide, peptides 2 and 3 were designed by substituting specific residues of peptide 1 with two methionine residues for cyclization, and peptide 4 is the truncate form of peptide 1. Besides, peptide 6 was derived from the central fragment of HBx,44 peptides 7 and 8 were designed the same as peptides 2 and 3 (Figure S1A). As shown in Figure 1A, we utilized alkylating linker b to react with methionine-containing peptides to generate corresponding bis-alkylated cyclic peptides. The antiviral activities of the designed peptides in HBV-replicating HepG2.2.15 cells were examined by measuring the levels of HBV markers, such as secreted HBsAg, HBeAg (hepatitis B e antigen) and HBV DNA, after six days of compound treatment as described in EXPERIMENTAL SECTION (Figure S1B). Among peptides tested, peptides 1, 2b and 3b derived from the

RESULTS AND DISCUSSION During the sequence optimization process, we tested a panel of peptides designed based on the interacting interface of HBx/Bcl-2 and HBx/DDB1 interactions (Figure S1A).43, 44 To minimize the impacts of amino acid substitutions on binding affinities of constrained peptides,



analogues adefovir (PMEA) and entecavir (ETV) only reduced the amount of extracellular HBV DNA (EC50 < 0.08 µM or 1.56 nM, respectively), without affecting the secretion of HBV antigens as previously reported64 (Figure 2A-C and S2A-C). Although some peptides exhibited inhibitory effects on extracellular level of HBV DNA, they did not show dose-dependent manners (Figure 2D). Subsequently, an MTT assay was performed to measure cell viability. There is no significant difference in cytotoxicity for almost all the peptides even under the highest concentration tested (320 μM), except that peptide 3a showed cytotoxicity (cell viability: 47.0%) at the highest concentration (Figure S2D-F). Taken together, these results demonstrate that constrained peptides 2a and 2b are the most effective inhibitors that block the secretion of HBV antigens. The bioactivity of peptides requires promising cell permeability and target-binding affinity. We first investigated the target-binding features of the peptides. Peptides 1, 2a, 2b, 3a and 3b were labelled with 5carboxyfluorescein (FAM). We investigated the binding affinity of peptides to Bcl-2 by fluorescence polarization assay. Constrained peptides FAM-2a (14.1±1.1 µM), FAM-2b (5.3±0.8 µM), FAM-3a (23.4±3.8 µM) and FAM-3b (11.7±0.2 µM) showed enhanced binding affinity with Bcl-2 compared to linear peptide FAM-1 (>45.6 µM) (Figure 3 and S3).

Figure 2. Antiviral activities of peptides in HepG2.2.15 cells. AB) Data shown are mean dose-response curves of HBsAg and HBeAg inhibition. The data represent the mean ± SD of three independent experiments. C-D) Relative level of extracellular HBV DNA after compound treatment. PMEA, adefovir; EC50: 50% effective concentration.

interacting interface of HBx/Bcl-2 interaction were found to have anti-HBV potentials (Figure S1A). Thus, using these peptides as templates, different linkers were tested to investigate the reaction efficiencies and their effects on the peptides’ biological functions. As shown in Figure 1B, all linkers reacted cleanly with unprotected peptides. We then evaluated the antiviral activities of peptides from Figure 1B. Compared to linear peptide 1 with an EC50 value of 157.2 µM, constrained peptides 2a, 2c, 2d and 3a reduced the extracellular HBsAg in a dosedependent way with EC50 values of 53.6, 196.4, 110.8 and 126.3 µM, respectively (Figure 2A). Besides, in a dosedependent way, constrained peptides 2a, 2b, 2c and 2d potently decreased the levels of secreted HBeAg with EC50 values of 45.4, 30.0, 154.5 and 140.0 µM, respectively (Figure 2B). Notably, different linking positions and different linkers could affect the biological activity of these constrained peptides, suggesting that the constrained conformation of peptides may influence the bioactivity of peptides. Interestingly, the nucleos(t)ide

Figure 3. Binding affinities of FAM-labelled peptides to Bcl-2. Binding affinity (Kd) was analysed by fluorescence polarization (FP) assay. The data are presented as mean ± standard deviation from at least three independent experiments.

We next set out to explore cell permeability of the designed peptides. The cell-penetrating peptide FITCTAT was used as a positive control. HepG2.2.15 cells incubated with 40 µM peptides overnight were analysed by flow cytometry. As shown in Figure 4, peptides FAM1, FAM-2a, FAM-2b, FAM-3a and FAM-3b showed mean fluorescence intensity (MFI) values of 126, 134, 226, 67 and 70, respectively. Thus, constrained peptide FAM-2b showed significant improved cell permeability than linear peptide FAM-1, while constrained peptides FAM-2a, FAM-3a and FAM-3b exhibited similar or even poor membrane permeability compared to linear peptide FAM-1. These results were further confirmed by confocal microscopy. Constrained peptides FAM-2a and FAM-2b showed significant higher cellular uptake than peptides FAM-1, FAM-3a and FAM-3b (Figure 5). Thus, high cell permeability of constrained peptides FAM-2a and FAM-2b may contribute to their more effective antiviral activities.


Page 4 of 8

Page 5 of 8 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

Molecular Pharmaceutics Furthermore, co-localization of the peptides with Bcl-2 in HepG2.2.15 cells was quantified using Pearson’s correlation coefficient (r) on confocal microscopy images. Constrained peptides FAM-2a (r = 0.67 ± 0.026) and FAM-2b (r = 0.69 ± 0.027) displayed significant colocalization with Bcl-2. On the contrary, peptides FAM-1 (r = 0.036 ± 0.029), FAM-3a (r = 0.016 ± 0.009) and FAM-3b (r = 0.018 ± 0.009) showed negligible colocalization with Bcl-2 (Figure 5), which may be hampered by the poor membrane permeability or lower binding affinity of these peptides. Collectively, these results demonstrate that constrained peptides FAM-2a and FAM-2b bind to Bcl-2 in cells, implying that these peptides may block the interactions between HBx and Bcl-2, which contributes to their antiviral activities.

Figure 4. Cell permeability of peptides. A) Flow cytometry analysis of HepG2.2.15 cells incubated with peptides FAM1, FAM-2a, FAM-2b, FAM-3a and FAM-3b (40 µM) at 37℃ for 12 hours, respectively. After incubation with 0.05% trypan blue for 5 min, the cells were subjected to flow cytometry analysis. B) The mean fluorescence intensity of each peptide was analysed. The data represent the mean ± SD of three independent experiments.

Figure 5. Co-localization of FAM-labelled peptides and Bcl-2. HepG2.2.15 cells were incubated with peptides FAM-1, FAM-2a, FAM-2b, FAM-3a and FAM-3b (40 µM) at 37°C for 12 hours, respectively. Cells were incubated with rabbit anti-Bcl-2 antibody and Alexa Fluor® 594-conjugated Goat Anti-Rabbit secondary antibody. After washing, cells were analyzed by confocal microscopy. The mean Pearson’s correlation coefficient (r) was calculated from 3 groups of images by ImageJ. The data are presented as mean ± SD.

CONCLUSION In conclusion, HBx-derived constrained peptides were developed by our macrocyclization strategy, which involved methionine bis-alkylation chemistry. This is the first time to investigate the biological profiles of the constrained peptides generated by this method. The collective data showed that constrained peptides 2a and 2b with greatly enhanced cell permeability and binding affinity are the most effective antiHBV inhibitors that block HBV antigens secretion. As a potent inhibitor of HBsAg secretion, peptide 2a could potentially be used to combine with nucleos(t)ide analogues, interferon or therapeutic vaccines for treating HBV infection. Furthermore, our findings revealed that different linking positions and different linkers may affect the cell permeability and binding affinity of the constrained peptides, which may further influence the biological profiles of these peptides, demonstrating the value of tunable property of the strategy. Importantly, as a proof of concept, we demonstrated that HBx is a potential PPI target, laying the foundations for further developing new inhibitors of HBx-mediated PPIs as alternative strategies to combat HBV infection. As a multifunctional protein, HBx is able to interplay with other host targets (e.g., p53) through the region involved in HBx/Bcl-2 interaction.65, 66 Our preliminary results showed that peptides 2a and 2b didn’t significantly alter the transcription of p53 and its downstream genes (Figure S4), implying that these peptides



may not influence the p53 signaling pathway. Further investigation will be performed to fully illustrate the detailed mechanisms of these peptides’ inhibition on the secretion of HBV antigens.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: # Supplementary Figure S1-S4; Table S1; HPLC traces and MS spectra of synthetic peptides.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected].

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This work is supported by the National Natural Science Foundation of China (Grant 21778009, 81701818 and 81572198), the Shenzhen Science and Technology Innovation Committee (JCYJ20170412150609690, KQJSCX20170728101942700 and JCYJ20170807144449135).

REFERENCES 1. Hu, K.; Geng, H.; Zhang, Q.; Liu, Q.; Xie, M.; Sun, C.; Li, W.; Lin, H.; Jiang, F.; Wang, T.; Wu, Y. D.; Li, Z. An In-tether Chiral Center Modulates the Helicity, Cell Permeability, and Target Binding Affinity of a Peptide. Angew. Chem. Int. Ed. Engl. 2016, 55, 8013-8017. 2. Jackson, D. Y.; King, D. S.; Chmielewski, J.; Singh, S.; Schultz, P. G. General approach to the synthesis of short .alpha.helical peptides. J. Am. Chem. Soc. 1991, 113, 9391-9392. 3. Osapay, G.; Taylor, J. W. Multicyclic polypeptide model compounds. 1. Synthesis of a tricyclic amphiphilic .alpha.-helical peptide using an oxime resin, segment-condensation approach. J. Am. Chem. Soc. 1990, 112, 6046-6051. 4. Schafmeister, C. E.; Po, J.; Verdine, G. L. An AllHydrocarbon Cross-Linking System for Enhancing the Helicity and Metabolic Stability of Peptides. J. Am. Chem. Soc. 2000, 122, 5891-5892. 5. Shepherd, N. E.; Hoang, H. N.; Abbenante, G.; Fairlie, D. P. Single turn peptide alpha helices with exceptional stability in water. J. Am. Chem. Soc. 2005, 127, 2974-2983. 6. Tian, Y.; Li, J.; Zhao, H.; Zeng, X.; Wang, D.; Liu, Q.; Niu, X.; Huang, X.; Xu, N.; Li, Z. Stapling of unprotected helical peptides via photo-induced intramolecular thiol-yne hydrothiolation. Chem. Sci. 2016, 7, 3325-3330. 7. Zhao, H.; Liu, Q. S.; Geng, H.; Tian, Y.; Cheng, M.; Jiang, Y. H.; Xie, M. S.; Niu, X. G.; Jiang, F.; Zhang, Y. O.; Lao, Y. Z.; Wu, Y. D.; Xu, N. H.; Li, Z. G. Crosslinked Aspartic Acids as Helix-Nucleating Templates. Angew. Chem. Int. Ed. Engl. 2016, 55, 12088-12093. 8. Jin, L.; Wang, W.; Fang, G. Targeting protein-protein interaction by small molecules. Annu. Rev. Pharmacol. Toxicol. 2014, 54, 435-456. 9. Sawyer, N.; Watkins, A. M.; Arora, P. S. Protein Domain Mimics as Modulators of Protein-Protein Interactions. Acc. Chem. Res. 2017, 50, 1313-1322. 10. Dado, G. P.; Gellman, S. H. Redox control of secondary structure in a designed peptide. J. Am. Chem. Soc. 1993, 115, 12609-12610.

11. Douat, C.; Aisenbrey, C.; Antunes, S.; Decossas, M.; Lambert, O.; Bechinger, B.; Kichler, A.; Guichard, G. A Cell‐Penetrating Foldamer with a Bioreducible Linkage for Intracellular Delivery of DNA. Angew. Chem. Int. Ed. Engl. 2015, 54, 11133-11137. 12. Fremaux, J.; Mauran, L.; Pulka‐Ziach, K.; Kauffmann, B.; Odaert, B.; Guichard, G. α‐Peptide–Oligourea Chimeras: Stabilization of Short α‐Helices by Non‐Peptide Helical Foldamers. Angew. Chem. Int. Ed. Engl. 2015, 54, 9816-9820. 13. Milroy, L.-G.; Grossmann, T. N.; Hennig, S.; Brunsveld, L.; Ottmann, C. Modulators of Protein–Protein Interactions. Chem. Rev. 2014, 114, 4695-4748. 14. PetersonKaufman, K. J.; Haase, H. S.; Boersma, M. D.; Lee, E. F.; Fairlie, W. D.; Gellman, S. H. Residue-Based Preorganization of BH3-Derived α/β-Peptides: Modulating Affinity, Selectivity and Proteolytic Susceptibility in α-Helix Mimics. ACS Chem. Biol. 2015, 10, 1667-1675. 15. Zhang, F.; Sadovski, O.; Xin, S. J.; Woolley, G. A. Stabilization of folded peptide and protein structures via distance matching with a long, rigid cross-linker. J. Am. Chem. Soc. 2007, 129, 14154-14155. 16. Heinis, C.; Rutherford, T.; Freund, S.; Winter, G. Phageencoded combinatorial chemical libraries based on bicyclic peptides. Nat. Chem. Biol. 2009, 5, 502-507. 17. Jo, H.; Meinhardt, N.; Wu, Y.; Kulkarni, S.; Hu, X.; Low, K. E.; Davies, P. L.; DeGrado, W. F.; Greenbaum, D. C. Development of alpha-helical calpain probes by mimicking a natural protein-protein interaction. J. Am. Chem. Soc. 2012, 134, 17704-17713. 18. Cui, H. K.; Guo, Y.; He, Y.; Wang, F. L.; Chang, H. N.; Wang, Y. J.; Wu, F. M.; Tian, C. L.; Liu, L. Diaminodiacid‐Based Solid‐Phase Synthesis of Peptide Disulfide Bond Mimics. Angew. Chem. Int. Ed. Engl. 2013, 52, 9558-9562. 19. Wang, Y.; Chou, D. H. A Thiol-Ene Coupling Approach to Native Peptide Stapling and Macrocyclization. Angew. Chem. Int. Ed. Engl. 2015, 54, 10931-10934. 20. Guo, Y.; Sun, D. M.; Wang, F. L.; He, Y.; Liu, L.; Tian, C. L. Diaminodiacid Bridges to Improve Folding and Tune the Bioactivity of Disulfide-Rich Peptides. Angew. Chem. Int. Ed. Engl. 2016, 54, 14276-14281. 21. Shi, Y.; Challa, S.; Sang, P.; She, F.; Li, C.; Gray, G. M.; Nimmagadda, A.; Teng, P.; Odom, T.; Wang, Y.; van der Vaart, A.; Li, Q.; Cai, J. One-Bead-Two-Compound Thioether Bridged Macrocyclic gamma-AApeptide Screening Library against EphA2. J. Med. Chem. 2017, 60, 9290-9298. 22. Cromm, P. M.; Schaubach, S.; Spiegel, J.; Furstner, A.; Grossmann, T. N.; Waldmann, H. Orthogonal ring-closing alkyne and olefin metathesis for the synthesis of small GTPase-targeting bicyclic peptides. Nat. Commun. 2016, 7, 11300. 23. Dietrich, L.; Rathmer, B.; Ewan, K.; Bange, T.; Heinrichs, S.; Dale, T. C.; Schade, D.; Grossmann, T. N. Cell Permeable Stapled Peptide Inhibitor of Wnt Signaling that Targets β-Catenin Protein-Protein Interactions. Cell Chem. Biol. 2017, 24, 958-968. 24. Yu, H. L.; Andrade, P. D.; Quah, S. T.; Rossmann, M.; Laraia, L.; Sköld, N.; Sum, T. J.; Rowling, P. J. E.; Joseph, T. L.; Verma, C. Functionalised staple linkages for modulating the cellular activity of stapled peptides. Chem. Sci. 2014, 5, 18041809. 25. Lautrette, G.; Touti, F.; Hong, G. L.; Peng, D.; Pentelute, B. L. Nitrogen Arylation for Macrocyclization of Unprotected Peptides. J. Am. Chem. Soc. 2016, 138, 8340-8343. 26. Spokoyny, A. M.; Zou, Y.; Ling, J. J.; Yu, H.; Lin, Y. S.; Pentelute, B. L. A Perfluoroaryl-Cysteine SNAr Chemistry Approach to Unprotected Peptide Stapling. J. Am. Chem. Soc. 2013, 135, 5946-5949. 27. Assem, N.; Ferreira, D. J.; Wolan, D. W.; Dawson, P. E. Acetone-Linked Peptides: A Convergent Approach for Peptide


Page 6 of 8

Page 7 of 8 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

Molecular Pharmaceutics Macrocyclization and Labeling. Angew. Chem. Int. Ed. Engl. 2015, 54, 8665-8668. 28. Chang, Y. S.; Graves, B.; Guerlavais, V.; Tovar, C.; Packman, K.; To, K. H.; Olson, K. A.; Kesavan, K.; Gangurde, P.; Mukherjee, A.; Baker, T.; Darlak, K.; Elkin, C.; Filipovic, Z.; Qureshi, F. Z.; Cai, H.; Berry, P.; Feyfant, E.; Shi, X. E.; Horstick, J.; Annis, D. A.; Manning, A. M.; Fotouhi, N.; Nash, H.; Vassilev, L. T.; Sawyer, T. K. Stapled alpha-helical peptide drug development: a potent dual inhibitor of MDM2 and MDMX for p53-dependent cancer therapy. Proc. Natl. Acad. Sci. USA 2013, 110, E3445-54. 29. Leshchiner, E. S.; Parkhitko, A.; Bird, G. H.; Luccarelli, J.; Bellairs, J. A.; Escudero, S.; Opoku-Nsiah, K.; Godes, M.; Perrimon, N.; Walensky, L. D. Direct inhibition of oncogenic KRAS by hydrocarbon-stapled SOS1 helices. Proc. Natl. Acad. Sci. USA 2015, 112, 1761-1766. 30. Speltz, T. E.; Fanning, S. W.; Mayne, C. G.; Fowler, C.; Tajkhorshid, E.; Greene, G. L.; Moore, T. W. Stapled Peptides with gamma-Methylated Hydrocarbon Chains for the Estrogen Receptor/Coactivator Interaction. Angew. Chem. Int. Ed. Engl. 2016, 55, 4252-4255. 31. Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. The MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis sensitizer. Nat. Chem. Biol. 2010, 6, 595-601. 32. Upadhyaya, P.; Qian, Z.; Selner, N. G.; Clippinger, S. R.; Wu, Z.; Briesewitz, R.; Pei, D. Inhibition of Ras signaling by blocking Ras-effector interactions with cyclic peptides. Angew. Chem. Int. Ed. Engl. 2015, 54, 7602-7606. 33. Bhattacharya, S.; Zhang, H.; Debnath, A. K.; Cowburn, D. Solution structure of a hydrocarbon stapled peptide inhibitor in complex with monomeric C-terminal domain of HIV-1 capsid. J. Biol. Chem. 2008, 283, 16274-16278. 34. Bird, G. H.; Madani, N.; Perry, A. F.; Princiotto, A. M.; Supko, J. G.; He, X.; Gavathiotis, E.; Sodroski, J. G.; Walensky, L. D. Hydrocarbon double-stapling remedies the proteolytic instability of a lengthy peptide therapeutic. Proc. Natl. Acad. Sci. USA 2010, 107, 14093-14098. 35. Zhang, H.; Zhao, Q.; Bhattacharya, S.; Waheed, A. A.; Tong, X.; Hong, A.; Heck, S.; Curreli, F.; Goger, M.; Cowburn, D.; Freed, E. O.; Debnath, A. K. A cell-penetrating helical peptide as a potential HIV-1 inhibitor. J. Mol. Biol. 2008, 378, 565-580. 36. Wang, D.; Lu, M.; Arora, P. S. Inhibition of HIV-1 fusion by hydrogen-bond-surrogate-based alpha helices. Angew. Chem. Int. Ed. Engl. 2008, 47, 1879-1882. 37. Ganem, D.; Prince, A. M. Hepatitis B virus infection--natural history and clinical consequences. N. Engl. J. Med. 2004, 350, 1118-1129. 38. Belloni, L.; Pollicino, T.; De Nicola, F.; Guerrieri, F.; Raffa, G.; Fanciulli, M.; Raimondo, G.; Levrero, M. Nuclear HBx binds the HBV minichromosome and modifies the epigenetic regulation of cccDNA function. Proc. Natl. Acad. Sci. USA 2009, 106, 19975-19979. 39. Lucifora, J.; Arzberger, S.; Durantel, D.; Belloni, L.; Strubin, M.; Levrero, M.; Zoulim, F.; Hantz, O.; Protzer, U. Hepatitis B virus X protein is essential to initiate and maintain virus replication after infection. J. Hepatol. 2011, 55, 996-1003. 40. Levrero, M.; Zucman-Rossi, J. Mechanisms of HBV-induced hepatocellular carcinoma. J. Hepatol. 2016, 64, S84-S101. 41. Geng, X.; Harry, B. L.; Zhou, Q.; Skeen-Gaar, R. R.; Ge, X.; Lee, E. S.; Mitani, S.; Xue, D. Hepatitis B virus X protein targets the Bcl-2 protein CED-9 to induce intracellular Ca2+ increase and cell death in Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 2012, 109, 18465-18470. 42. Geng, X.; Huang, C.; Qin, Y.; McCombs, J. E.; Yuan, Q.; Harry, B. L.; Palmer, A. E.; Xia, N. S.; Xue, D. Hepatitis B virus X protein targets Bcl-2 proteins to increase intracellular calcium,

required for virus replication and cell death induction. Proc. Natl. Acad. Sci. USA 2012, 109, 18471-18476. 43. Jiang, T.; Liu, M.; Wu, J.; Shi, Y. Structural and biochemical analysis of Bcl-2 interaction with the hepatitis B virus protein HBx. Proc. Natl. Acad. Sci. USA 2016, 113, 2074-2079. 44. Li, T.; Robert, E. I.; van Breugel, P. C.; Strubin, M.; Zheng, N. A promiscuous alpha-helical motif anchors viral hijackers and substrate receptors to the CUL4-DDB1 ubiquitin ligase machinery. Nat. Struct. Mol. Biol. 2010, 17, 105-111. 45. Decorsiere, A.; Mueller, H.; van Breugel, P. C.; Abdul, F.; Gerossier, L.; Beran, R. K.; Livingston, C. M.; Niu, C.; Fletcher, S. P.; Hantz, O.; Strubin, M. Hepatitis B virus X protein identifies the Smc5/6 complex as a host restriction factor. Nature 2016, 531, 386-389. 46. Hodgson, A. J.; Hyser, J. M.; Keasler, V. V.; Cang, Y.; Slagle, B. L. Hepatitis B virus regulatory HBx protein binding to DDB1 is required but is not sufficient for maximal HBV replication. Virology 2012, 426, 73-82. 47. Murphy, C. M.; Xu, Y.; Li, F.; Nio, K.; Reszka-Blanco, N.; Li, X.; Wu, Y.; Yu, Y.; Xiong, Y.; Su, L. Hepatitis B Virus X Protein Promotes Degradation of SMC5/6 to Enhance HBV Replication. Cell Rep. 2016, 16, 2846-2854. 48. Lok, A. S.; Zoulim, F.; Dusheiko, G.; Ghany, M. G. Hepatitis B cure: From discovery to regulatory approval. J. Hepatol. 2017, 67, 847-861. 49. Hu, P.; Shang, J.; Zhang, W.; Gong, G.; Li, Y.; Chen, X.; Jiang, J.; Xie, Q.; Dou, X.; Sun, Y.; Liu, Y.; Liu, G.; Mao, D.; Chi, X.; Tang, H.; Li, X.; Xie, Y.; Zhao, P.; Hou, J.; Gao, Z.; Fan, H.; Ding, J.; Zhang, D.; Ren, H. HBsAg Loss with Peg-interferon Alfa-2a in Hepatitis B Patients with Partial Response to Nucleos(t)ide Analog: New Switch Study. J. Clin. Transl. Hepatol. 2018, 6, 25-34. 50. Kwon, H.; Lok, A. S. Hepatitis B therapy. Nat. Rev. Gastroenterol. Hepatol. 2011, 8, 275-284. 51. Frebel, H.; Richter, K.; Oxenius, A. How chronic viral infections impact on antigen-specific T-cell responses. Eur. J. Immunol. 2010, 40, 654-663. 52. Isogawa, M.; Chung, J.; Murata, Y.; Kakimi, K.; Chisari, F. V. CD40 activation rescues antiviral CD8(+) T cells from PD-1mediated exhaustion. PLoS Pathog. 2013, 9, e1003490. 53. Ochel, A.; Cebula, M.; Riehn, M.; Hillebrand, U.; Lipps, C.; Schirmbeck, R.; Hauser, H.; Wirth, D. Effective intrahepatic CD8+ T-cell immune responses are induced by low but not high numbers of antigen-expressing hepatocytes. Cell. Mol. Immunol. 2016, 13, 805-815. 54. Zhu, D.; Liu, L.; Yang, D.; Fu, S.; Bian, Y.; Sun, Z.; He, J.; Su, L.; Zhang, L.; Peng, H.; Fu, Y. X. Clearing Persistent Extracellular Antigen of Hepatitis B Virus: An Immunomodulatory Strategy To Reverse Tolerance for an Effective Therapeutic Vaccination. J. Immunol. 2016, 196, 30793087. 55. Chen, Y.; Wei, H.; Sun, R.; Tian, Z. Impaired function of hepatic natural killer cells from murine chronic HBsAg carriers. Int. Immunopharmacol. 2005, 5, 1839-1852. 56. Op den Brouw, M. L.; Binda, R. S.; van Roosmalen, M. H.; Protzer, U.; Janssen, H. L.; van der Molen, R. G.; Woltman, A. M. Hepatitis B virus surface antigen impairs myeloid dendritic cell function: a possible immune escape mechanism of hepatitis B virus. Immunology 2009, 126, 280-289. 57. Woltman, A. M.; Op den Brouw, M. L.; Biesta, P. J.; Shi, C. C.; Janssen, H. L. Hepatitis B virus lacks immune activating capacity, but actively inhibits plasmacytoid dendritic cell function. PLoS One 2011, 6, e15324. 58. Kondo, Y.; Ninomiya, M.; Kakazu, E.; Kimura, O.; Shimosegawa, T. Hepatitis B surface antigen could contribute to the immunopathogenesis of hepatitis B virus infection. ISRN Gastroenterol. 2013, 2013, 935295.



59. Autran, B.; Carcelain, G.; Combadiere, B.; Debre, P. Therapeutic vaccines for chronic infections. Science 2004, 305, 205-208. 60. Cai, X.; Zheng, W.; Pan, S.; Zhang, S.; Xie, Y.; Guo, H.; Wang, G.; Li, Z.; Luo, M. A virus-like particle of the hepatitis B virus preS antigen elicits robust neutralizing antibodies and T cell responses in mice. Antiviral. Res. 2018, 149, 48-57. 61. Shi, X.; Zhao, R.; Jiang, Y.; Zhao, H.; Tian, Y.; Jiang, Y.; Li, J.; Qin, W.; Yin, F.; Li, Z. Reversible stapling of unprotected peptides via chemoselective methionine bisalkylation/dealkylation. Chem. Sci. 2018, 9, 3227-3232. 62. Zheng, W.; Cai, X.; Xie, M.; Liang, Y.; Wang, T.; Li, Z. Structure-Based Identification of a Potent Inhibitor Targeting Stp1-Mediated Virulence Regulation in Staphylococcus aureus. Cell Chem Biol 2016, 23, 1002-13. 63. Zheng, W.; Liang, Y.; Zhao, H.; Zhang, J.; Li, Z. 5,5'Methylenedisalicylic Acid (MDSA) Modulates SarA/MgrA Phosphorylation by Targeting Ser/Thr Phosphatase Stp1. Chembiochem 2015, 16, 1035-40. 64. Mueller, H.; Wildum, S.; Luangsay, S.; Walther, J.; Lopez, A.; Tropberger, P.; Ottaviani, G.; Lu, W.; Parrott, N. J.; Zhang, J. D.; Schmucki, R.; Racek, T.; Hoflack, J. C.; Kueng, E.; Point, F.;

Zhou, X.; Steiner, G.; Lutgehetmann, M.; Rapp, G.; Volz, T.; Dandri, M.; Yang, S.; Young, J. A. T.; Javanbakht, H. A novel orally available small molecule that inhibits hepatitis B virus expression. J. Hepatol. 2018, 68, 412-420. 65. Lin, Y.; Nomura, T.; Yamashita, T.; Dorjsuren, D.; Tang, H.; Murakami, S. The transactivation and p53-interacting functions of hepatitis B virus X protein are mutually interfering but distinct. Cancer Res. 1997, 57, 5137-5142. 66. Stohwasser, R.; Holzhutter, H. G.; Lehmann, U.; Henklein, P.; Kloetzel, P. M. Hepatitis B virus HBx peptide 116-138 and proteasome activator PA28 compete for binding to the proteasome alpha4/MC6 subunit. Biol. Chem. 2003, 384, 39-49.

Table of Contents/Abstract Graphic

HBx-derived constrained peptides with improved cell permeability and binding affinity act as effective anti-HBV modulators by blocking secretion of viral antigens.


Page 8 of 8

HBx-derived constrained peptides inhibit the secretion of hepatitis B

Recommend Documents