Tat47-57 binding analogue based on decafluorobiphenyl as well as preparation method and application of Tat47-57 binding analogue

Abstract

The invention discloses a decafluorobiphenyl-based Tat47-57 binding peptide, the amino acid sequence of the binding peptide is shown as SEQ ID NO.1 or SEQ ID NO.2, the fourth amino acid and the eighth amino acid of the SEQ ID NO.1 are bound through decafluorobiphenyl, and the fifth amino acid and the twelfth amino acid of the SEQ ID NO.2 are bound through decafluorobiphenyl. In particular, the decafluorobiphenyl-based Tat47-57 binding peptides (P3 and P6) are efficient CPPs, capable of delivering a variety of cargo molecules into the cytoplasm of mammalian cells. Meanwhile, decafluorobiphenyl cyclization plays a key role in cellular uptake and endosome escape. The two peptides P3 and P6 show remarkable cell permeability, and are speculated to enter cells through gridding protein dependent endocytosis. They also show efficient endosome escape ability, and have successfully applied P3 and P6 as CPPs to deliver phospholipid peptides and avidin.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a Tat47-57 binding peptide based on decafluorobiphenyl, a preparation method thereof, and an application thereof. Background Art

[0002] The cell membrane, composed of a lipid bilayer, membrane-embedded proteins, carbohydrates, and their conjugates, is a barrier that controls the transport of substances. Many biomacromolecules such as proteins, peptides, and nucleic acids have high and specific biological activities in vitro, but due to their poor cell permeability, they cannot be used as therapeutic agents or research tools. To overcome this barrier, a series of strategies have been developed, such as viral vectors, nanomaterial-based delivery systems, and direct injection, but their efficiency and safety still need to be improved. Since the discovery of the highly positively charged HIV Tat peptide 47-57 YGRKKRRQRRR, cytoplasmic delivery strategies based on "cell-penetrating peptides" (CPPs) have been developed in biomedical research. CPPs have been used to deliver small molecule drugs, peptides, proteins, and nucleic acids into cultured mammalian cells and living organisms. Generally, CPPs deliver biologics into mammalian cells through the endocytic pathway, which requires efficient endosomal escape efficiency to avoid degradation in lysosomes. Unfortunately, the endosomal membrane has been shown to be an important barrier to the cytoplasmic delivery of these CPPs. Therefore, developing CPPs with efficient escape has become an ideal solution. Due to the similarity between the cell membrane and the endosomal membrane, it is difficult to design peptides that can recognize specific lipids. In recent years, new methods for improving the efficiency of CPPs have been studied, especially strategies for designing peptides that can improve their endosomal escape efficiency.For example, a series of small amphiphilic cyclic peptides have shown efficient penetration effects and high cytoplasmic delivery efficiencies (Qian, Z.; Liu, T.; Liu, Y.Y.; Briesewitz, R.; Barrios, A.M.; Jhiang, S.M.; Pei, D., Efficient delivery of cyclic peptides into mammalian cells with short sequence motifs. ACS chemical biology 2013, 8(2), 423-31; Qian, Z.; LaRochelle, J.R.; Jiang, B.; Lian, W.; Hard, R.L.; Selner, N.G.; Luechapanichkul, R.; Barrios, A.M.; Pei, D., Early endosomal escape of a cyclic cell-penetrating peptide allows effective cytosolic cargo delivery. Biochemistry 2014, 53(24), 4034-46.20. Oba, M.; Kunitake, M.; Kato, T.; Ueda, A.; Tanaka, M., Enhanced and Prolonged Cell-Penetrating Abilities of Arginine-Rich Peptides by Introducing Cyclic α,α-Disubstituted α-Amino Acids with Stapling. Bioconjugate chemistry 2017, 28(7), 1801-1806; Saha, A.; Mandal, S.; Arafiles, J.V.V.; Gómez-González, J.; Hackenberger, C.P.R.; Brik, A., Structure-Uptake Relationship Study of DABCYL Derivatives Linked to Cyclic Cell-Penetrating Peptides for Live-Cell Delivery of Synthetic Proteins. Angew Chem Int Ed Engl 2022, 61(47), e202207551.).Hydrocarbon stapled Tat47-57 analogs with an α-helical structure exhibit high endosomal escape efficiency. Cell-penetrating peptide-bismuth bicycles show efficient cellular uptake at concentrations as low as 10 nM (Voss, S.; Adair, L.D.; Achazi, K.; Kim, H.; Bergemann, S.; Bartenschlager, R.; New, E.J.; Rademann, J.; Nitsche, C., Cell-Penetrating Peptide-Bismuth Bicycles. Angew Chem Int Ed Engl 2024, 63(10), e202318615.). Despite these remarkable advances, the design and discovery of efficient CPPs still require further exploration.

[0003] Fluorine chemistry has developed for more than 200 years, and fluorinated organic compounds are widely used in the fields of pharmaceuticals, agrochemicals, materials, etc. When a fluorine atom is introduced into a molecule, due to its highest electronegativity and second smallest size, the molecule will exhibit unique properties. The introduction of fluorine endows the molecule with various properties, such as molecular conformation, dipole moment, acidity and basicity, which will affect toxicity, selectivity, potency, as well as pharmacodynamic and pharmacokinetic properties. Bradley L.P. et al. reported a method that allows the macrocyclization of unprotected peptides using perfluoroaryl linkers between two cysteine residues through S~N~Ar chemistry (Zou, Y.; Spokoyny, A.M.; Zhang, C.; Simon, M.D.; Yu, H.; Lin, Y.S.; Pentelute, B.L., Convergent diversity-oriented side-chain macrocyclization scan for unprotected polypeptides. Organic & biomolecular chemistry 2014, 12(4), 566 - 73). They demonstrated that compared with their linear counterparts, these perfluoroarene-based peptide macrocycles have increased cellular uptake and improved ability of the peptides to cross the blood-brain barrier (Fadzen, C.M.; Wolfe, J.M.; Cho, C.F.; Chiocca, E.A.; Lawler, S.E.; Pentelute, B.L., Perfluoroarene-Based Peptide Macrocycles to Enhance Penetration Across the Blood-Brain Barrier. J Am Chem Soc 2017, 139(44), 15628 - 15631; Ngambenjawong, C.; Pineda, J.M.; Pun, S.H., Engineering an Affinity-Enhanced Peptide through Optimization of Cyclization Chemistry. Bioconjugate chemistry 2016, 27(12), 2854 - 2862). Summary of the Invention

[0004] Objective of the Invention: Aiming at the deficiencies of the prior art, the present invention provides a Tat47-57 binding peptide based on decafluorobiphenyl for enhanced in vivo escape.

[0005] To solve the above technical problems, the present invention discloses a Tat47-57 binding peptide based on decafluorobiphenyl, wherein the amino acid sequence of the binding peptide is shown in SEQ ID NO.1 or SEQ ID NO.2. Among them, the 4th amino acid and the 8th amino acid of SEQ ID NO.1 are bound by decafluorobiphenyl to obtain the binding peptide P3, and the 5th amino acid and the 12th amino acid of SEQ ID NO.2 are bound by decafluorobiphenyl to obtain the binding peptide P6, as shown in A in the structural formula diagram Figure 1 shown in

[0006] The present application further provides a preparation method of the above-mentioned Tat47-57 binding peptide. The Tat47-57 peptide analog is synthesized by the Fmoc solid-phase peptide synthesis method, wherein the amino acid sequence of the Tat47-57 peptide analog is shown in SEQ ID NO.1 or SEQ ID NO.2, and then the obtained Tat47-57 peptide analog, decafluorobiphenyl and cyclizing agent are obtained through a cyclization reaction.

[0007] Among them, the cyclizing agent is Tris.

[0008] Preferably, the molar ratio of Tat47-57 peptide analog: decafluorobiphenyl: cyclizing agent is 1:2-6:30-50.

[0009] Among them, the reaction solvent in the cyclization reaction is DMF.

[0010] Preferably, the reaction conditions are stirring at room temperature for 4-5 h, then removing the reaction solution by rotary evaporation, and the crude product is purified by RP-HPLC to obtain the cyclized product.

[0011] The present invention further provides an application of the above-mentioned Tat47-57 binding peptide in the cytoplasmic delivery of chemical probes.

[0012] Among them, the chemical probe is a negatively charged phospholipid peptide.

[0013] The present invention also provides an application of the above-mentioned Tat47-57 binding peptide in the cytoplasmic delivery of biological macromolecules.

[0014] In one embodiment, the biological macromolecule is avidin

[0015] Beneficial effects: The present application first applies the decafluorobiphenyl stapling strategy to study enhanced endosomal escape. In the present application, fluorinated stapling bridges were introduced into peptide molecules, and their effects on cell-penetrating activity were studied. The results showed that decafluorobiphenyl-based Tat47-57 stapling analogs (P3 and P6) are efficient CPPs that can deliver a variety of cargo molecules into the cytoplasm of mammalian cells. We also found that decafluorobiphenyl cyclization plays a key role in cell uptake and endosomal escape. The two peptides P3 and P6 showed significant cell permeability and were presumably internalized into cells via clathrin-dependent endocytosis. They also showed endosomal escape ability, and P3 and P6 have been successfully applied as CPPs to deliver phospholipid peptides (sequence: GpYEEI) and avidin. Description of the Drawings

[0016] Figure 1 In it, A is a Tat47-57 analog designed by decafluorobiphenyl-based peptide stapling, B is conventional Fmoc solid-phase peptide synthesis (SPPS), and C is the synthesis of decafluorobiphenyl-based stapled peptides;

[0017] Figure 2 CD spectra of the newly designed peptides in (A) H 2 O and (B) TFE / H 2 O (1:1, v / v);

[0018] Figure 3 Cell uptake results for evaluating decafluorobiphenyl stapled peptides as CPP candidates. All results are mean ± SEM (n = 4). ***p < 0.001, 0.001 ≤ **p < 0.01, 0.01 ≤ *p < 0.05 vs Tat;

[0019] Figure 4 Cell uptake of Tat, P3, and P6 (2 μM, 2 h) in Hela cells in the presence of trypan blue or trypan blue and Triton-X100. The results are mean ± SEM (n = 4);

[0020] Figure 5 Effect of heparinase III treatment on the uptake of Tat47-57 and its analogs;

[0021] Figure 6is the endosomal escape efficiency of decafluorobiphenyl-conjugated peptides. Among them, A-C are the leakage results of calcein dye in liposomes mimicking endosomal membranes in PBS buffer at pH 5.5. The results are mean ± SEM (n = 4); D is a confocal micrograph of HeLa cells incubated with 5 μM NF-labeled peptides for 2 hours. Scale bar = 20 μm; E is the uptake of these NF-labeled peptides detected by flow cytometry (FACS). The results are mean ± SEM (n = 4); F and G are the endosomal escape pathways of Tat47-57, P3, and P6. Scale bar is 20 mm. ***p < 0.001, 0.001 ≤ **p < 0.01, 0.01 ≤ *p < 0.05 vs Tat;

[0022] Figure 7 shows that at 4°C, Filipin, EIPA, and CPZ inhibit the cellular internalization of P3 (A) and P6 (C), respectively. In Hela cells, the cellular uptake of FITC-labeled P3 (B) and P6 (D) (5 μM, 1 h) after incubation with endocytosis inhibitors or at 4°C for 1 hour. The results are mean ± standard error (n = 4); compared with the group without inhibitor, ***p < 0.001, 0.001 ≤ **p < 0.01, 0.01 ≤ *p < 0.05. The nuclear stain Hoechst 33342 (blue) is used to counterstain the cell nuclei. Scale bar represents 20 μm;

[0023] Figure 8 is for cytotoxicity and hemolysis analysis. Among them, A is the cytotoxicity of TAT47-57, P3, and P6 in Hela, NIH2T3, and CHO-k1 cells. Cells were treated with DMSO (control) or Tat47-57, P3, and P6 at 10, 20, and 50 μM for 24 hours. The results are mean ± SEM (n = 4); B is the LDH assay data (2 h) of TAT47-57, P3, and P6 in Hela, NIH2T3, and CHO-k1 cells. The results are mean ± SEM (n = 4); C is the hemolysis (2 h) of TAT47-57, P3, and P6 in mouse red blood cells. The results are mean ± SEM (n = 4);

[0024] Figure 9Results of delivering phospholipid peptides and avidin into living HeLa cells. Among them, A is the live cell confocal microscopy images of HeLa cells treated with GpTEEI, Tat-GpTEEI conjugate, P3-GpTEEI conjugate and P6-GpTEEI conjugate (5 μM, 2 h); B is the cellular uptake of GpTEEI, Tat-GpTEEI conjugate, P3-GpTEEI conjugate and P6-GpTEEI conjugate (5 μM, 2 h) in HeLa cells, ***p < 0.001, 0.001 ≤ **p < 0.01, 0.01 ≤ *p < 0.05 vs Tat&GpTEEI, the results are mean ± SEM (n = 4); C is the co-localization of FITC-labeled Tat-GpTEEI conjugate, P3-GpTEEI conjugate and P6-GpTEEI conjugate (5 μM, 2 h) with lysosomes in HeLa cells; D is the live cell confocal microscopy images of HeLa cells treated with avidin, TatD-avidin conjugate, P3D-avidin conjugate and P6D-avidin conjugate (5 μM, 2 h); E is the cellular uptake of avidin, TatD-avidin conjugate, P3D-avidin conjugate and P6D-avidin conjugate (5 μM, 2 h) in HeLa cells, ***p < 0.001, 0.001 ≤ **p < 0.01, 0.01 ≤ *p < 0.05 vs TatD&avidin. The results are mean ± SEM (n = 4); F is the co-localization of FITC-labeled TatD-avidin conjugate, P3D-avidin conjugate and P6D-avidin conjugate (5 μM, 2 h) with lysosomes in HeLa cells. The images were obtained by live cell confocal microscopy, scale bar = 20 μm;

[0025] Figure 10 HPLC and mass spectrometry spectra of FITC-labeled P3;

[0026] Figure 11 HPLC and mass spectrometry spectra of FITC-labeled P6. Detailed implementation manners

[0027] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners, and the above and / or other advantages of the present invention will become clearer.

[0028] The detection methods involved in the following examples are as follows:

[0029] RP-HPLC: Unless otherwise stated, all RP-HPLC separations were performed using a mobile phase consisting of 0.1% (v / v) TFA in water (solvent A) and 0.1% (v / v) TFA in MeCN (solvent B). UV detection of all peptides was recorded at a wavelength of 220 nm in both analytical HPLC and preparative HPLC.

[0030] Chromatographic separation for analytical HPLC was carried out using an Agilent Technologies 1260 Infinity LC system equipped with an Exsil Pure 300 C18 column (5.0 μm, 4.6 × 150 mm) at a flow rate of 1 mL / min. Preparative HPLC separation was performed using a Hanbang Technology NP7005C solvent delivery system and a Hanbang Technology NU3010C UV detector, with an Exsil Pure 300 C18 column (10 μm, 20 × 250 mm), and the flow rate was set at 8 mL / min.

[0031] Circular dichroism: Peptide samples were prepared at a concentration of 40 μM in H 2 O. Circular dichroism spectra were measured at room temperature. Spectral data were acquired using an Olis DSM 1000 CD (USA) instrument at a scanning speed of 1 nm / s in the wavelength range of 190 to 260 nm. The structure of the peptides was determined using the K2D2 tool in the wavelength range of 190 - 240 nm. The obtained CD spectra were then converted to mean residue ellipticity using the following formula:

[0032] θ M =(θ obs × 1000) / (c × l × n)

[0033] where θ M is the residue ellipticity (deg·cm 2 ·dmol -1 ), θ obs is the observed ellipticity (mdeg) corrected for the buffer at a specific wavelength, c is the peptide concentration (μM), l is the path length (mm), and n is the number of amino acids.

[0034] Cell culture: Human cervical cancer cell lines HeLa, NIHT3, and CHO-K1 cells were cultured in a medium containing DMEM, 10% fetal bovine serum, and 1% penicillin / streptomycin at 37 °C and 5% CO 2 conditions.

[0035] Cell viability and hemolysis assays: The cytocompatibility of various analogs was detected in HeLa cells, NIH3T3, and CHO-K1 cells by MTT and LDH assays. For the MTT assay, cells were seeded in 96-well plates (5,000 cells per well) and cultured at 37 °C for 24 h, then replaced with fresh medium containing each peptide segment and incubated for another 24 h. Subsequently, 10 μL of MTT solution (2 mg / mL) was added to each well, and after incubation for 4 h, the supernatant was discarded. Then, 150 μL of DMSO was added and incubated at 37 °C for 10 min, and the detection was carried out using a microplate reader. In the LDH assay, cells were cultured in 96-well plates (15,000 cells per well) at 37 °C for 24 h. After culture, different concentrations of peptides were added and incubated for another 2 h, and then detected using the CytoTox-ONE TM Homogeneous Membrane Integrity Detection Kit (Promega), and the results were measured by a microplate reader.

[0036] The hemolytic activity of the peptides was determined using fresh mouse blood. Briefly, fresh mouse blood was collected into a heparin-containing centrifuge tube and centrifuged at 800 rpm for 10 min to obtain red blood cells (RBCs), which were then washed three times with PBS and diluted to 4-fold volume. 4.0×10 8 cells / mL were used in the hemolysis assay. A series of concentrations of peptides were incubated with red blood cells at 37 °C for 2 h. After incubation, the samples were centrifuged at 1,000 rpm for 15 min, and the absorbance of the supernatant of each sample was measured at 490 nm. 0.2% Triton X-100 was used as the maximum hemolysis control (100%). The data represent the mean ± standard deviation of three independent experiments.

[0037] Flow cytometry: HeLa cells were cultured in 24-well plates (1×10^5 cells per well) overnight. The old medium was removed, and fresh DMEM containing 2 μM fluorescein isothiocyanate (FITC)-labeled peptide was added. Alternatively, 5 μM naphthalene fluorescein (NF)-labeled peptide was used. Subsequently, the cells were incubated at 37 °C for 2 h. After washing the cells 3 times with PBS, the cells were collected by treating with 0.25% trypsin for 15 min. After adding medium containing 10% FBS, the cells were centrifuged at 800 rpm for 5 min. The cell pellet was resuspended in PBS, centrifuged again at 800 rpm for 5 min, and finally resuspended in PBS. The fluorescence intensity was analyzed using a BD FACS LSR II flow cytometer. For the naphthalene fluorescein-labeled peptide, its fluorescence was measured and analyzed according to the previously reported method. The data were analyzed using Flowjo software.

[0038] Confocal microscopy: 1 mL of HeLa cell suspension (about 2×10^4 cells) was seeded in a Fluorodish and incubated at 37 °C, 5% CO2 Cultivate for 24 hours under the conditions. After the cells were incubated with DMEM medium containing 2 μM FITC or 5 μM NF-labeled compound for 2 hours, they were washed 3 times with PBS and then DMEM medium was added. The cells were observed using ZEN 880 (Carl Zeiss AG, Germany). Before imaging, the nuclei were stained with Hoechst 33342 for 5 minutes, and after labeling late endosomes / lysosomes with LysoTracker Red for 30 minutes, the intracellular distribution was observed using ZEN 880. The images were further processed using Photoshop.

[0039] Use trypan blue for extracellular fluorescence quenching

[0040] Flow cytometry analysis was performed in the presence of trypan blue to ensure extracellular fluorescence quenching. After the cells were treated with the peptide under the above conditions, the first flow cytometry signal was collected. Subsequently, the cells were incubated with trypan blue (0.05% w / v in PBS) for 3 minutes and then the second signal was collected. Finally, the same cells were permeabilized with 0.05% (v / v) triton-X100 and the fluorescence was measured by cytometry for the last time.

[0041] Heparinase III inhibits cell uptake: HeLa cells were pre-treated with 3 mIU / ml heparinase III at 37 °C for 1 hour and then treated with 2 μM FITC-labeled peptide at 4 °C for 2 hours. The cell uptake of 2 μM FITC-labeled peptide was used as a positive control and was determined by flow cytometry and set as 100%. The data represent the mean ± standard deviation of three independent experiments.

[0042] Isothermal titration calorimetry (ITC): The binding of the peptide to heparin (HS) was measured at 25 °C using an ITC200 microcalorimeter. The experiment was carried out using the method reported previously (Li, S.; Zhang, X.; Guo, C.; Peng, Y.; Liu, X.; Wang, B.; Zhuang, R.; Chang, M.; Wang, R., Hydrocarbon staple constructing highly efficient α-helix cell-penetrating peptides for intracellular cargo delivery. Chem Commun (Camb) 2020, 56(100), 15655-15658.). Briefly, heparin and the peptide were dissolved in HBSS respectively. After degassing all the solutions, the peptide solution (40 μM) was filled into the sample cell, and 2 μL of the heparin solution (30 μM) was added dropwise into the sample cell each time for 19 titrations. The data were analyzed by ITC200 Microcal software.

[0043] Study on the endocytosis mechanism: To identify the possible internalization mechanisms of P3 and P6, HeLa cells were pre-incubated at 4 °C or treated with specific inhibitors at 37 °C for 1 h, and then re-treated with 5 μM FITC-labeled peptide at 37 °C at 4 °C or in the presence of inhibitors. The concentrations of the inhibitors used were: chlorpromazine 10 μg / mL (clathrin-mediated pathway inhibitor), amiloride 0.5 mM (macropinocytosis inhibitor), methyl-β-cyclodextrin 2.5 mM (M-β-CD, lipid raft inhibitor), and heparin 50 units / mL (heparan sulfate proteoglycan inhibitor). The cellular uptake of the FITC-labeled peptide was used as a positive control. The results were measured by flow cytometry and set as 100%. The data represent the mean ± standard deviation of three independent experiments.

[0044] Calcein release assay: Large unilamellar vesicles (LUVs) composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG, POPC:POPG = 3:1) were used to mimic the endosomal membrane. Briefly, the lipids were dried from chloroform under vacuum overnight to obtain a lipid film. The lipid film was rehydrated in PBS (pH 5.4) containing 70 mM calcein to a lipid concentration of 7 mM. After hydration in a 37 °C water bath for 1 h, the liposome suspension was frozen and thawed five cycles and extruded 20 times through a 0.1 μm nucleopore polycarbonate filter using a LiposoFast extrusion system (Avestin). It was dialyzed for 1 day through a dialysis membrane (molecular weight cut-off, 10,000 Da) at room temperature to remove free calcein. The prepared liposomes were stored at 4 °C and used for the calcein release experiment within 2 - 3 days. The diameter of the unilamellar vesicles was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern Instruments, USA). Using the calcein dye leakage assay, the experiment was carried out on a 96-well plate and monitored by a Thermo Varioskan Flash microplate reader with 490 nm laser excitation and a 520 emission filter. The assay was performed in triplicate. After mixing 10 μL of peptide solutions at different concentrations (5 and 10 μM) with 90 μL of the liposome suspension, it was incubated at 37 °C for 30 min. The leakage percentage (%) was calculated as follows:

[0045] Dye leakage percentage (%) = 100 × [(Fobs - F0) / (Ft - F0)]

[0046] where Fobs is the intensity measured at a given peptide concentration. F0 is the background intensity of the liposome sample, and Ft is the maximum fluorescence intensity after adding Triton X-100 (0.1% (v / v)).

[0047] RFP-RAB5 and RFP-RAB7 translocation analysis and co-localization with P3 and P6: Approximately 2×10^4 HeLa cells were transfected with RFP-RAB5 or RFP-RAB7 plasmids and cultured at 37 °C for 24 h. Subsequently, the cells were incubated with DMEM medium containing 5 μM FITC-labeled compound for 1 or 2 h. After washing the cells three times with PBS, DMEM medium was added. The cells were observed using ZEN 880 (Carl Zeiss, Germany).

[0048] Delivery of cargo into living cells: Phospholipid peptide synthesis and its cellular uptake. The FITC-labeled phospholipid peptide (FITC-Acp-Gly-pThr-Glu-Glu-Ile-OH) was synthesized according to the above solid-phase peptide synthesis (SPPS) method. The FITC-labeled phospholipid peptide (20 μM) was pre-incubated with the analog (5 μM) at 37 °C for 30 minutes to form a complex, and then this mixture was added to the cells and allowed to act for 2 hours. The uptake of the phosphorylated peptide by the cells was detected by flow cytometry, and its intracellular distribution was observed using a Zeiss 880 microscope.

[0049] Delivery of avidin into living cells. FITC-labeled avidin (1 equivalent) was pre-incubated with biotinylated TatD, P3D, or P6D peptide (1 equivalent) at 37 °C for 30 minutes to form a peptide-avidin complex through biotin-avidin interaction. Subsequently, the mixture was added to a culture dish or a 24-well plate and cultured for 2 hours. After the cells were washed 3 times with PBS, they were observed using a Zeiss 880 microscope. The cellular uptake of FITC-labeled streptavidin was detected by BD FACSAria TM III flow cytometer (BD Biosciences, San Jose, California, USA). The cellular uptake of the fluorescently labeled TatD-avidin conjugate was used as a positive control, and its uptake was measured by flow cytometry and set to 100%.

[0050] Statistical analysis was performed using SPSS 16.0. Statistical values are expressed as mean ± SEM. A p-value < 0.05 was considered significantly different.

[0051] Preparation of a series of Tat47-57 binding peptides in Example 1.

[0052] Materials: Rink Amide AM resin with a loading of 0.45 mmol / g was purchased from Tianjin Nankai Hecheng Technology Co., Ltd., China. Fluorescein isothiocyanate (FITC), ethyl cyanoacetaldehyde oxime (Oxyma), N,N'-diisopropylcarbodiimide (DIC), and Fmoc-protected amino acids were purchased from Shanghai Gil Biochemical Co., Ltd. Naphthofluorescein succinimidyl ester was purchased from Sigma-Aldrich. Trifluoroacetic acid (TFA) and N,N-diisopropylethylamine (DIEA) were purchased from Aladdin Reagent Co., Ltd. Decafluorobiphenyl (DFBP) and 4,4'-bis(bromomethyl)biphenyl were purchased from Shanghai Energy Chemical Co., Ltd. Hoechst 33342 and Lysosome Red Tracker were purchased from Shanghai Beyotime Biotechnology Co., Ltd. Chlorpromazine (CPZ), methyl-β-cyclodextrin (Me-β-CD), 5-(N-ethyl-N-isopropyl)-amiloride (EIPA), and Filipin were provided by Sigma Aldrich. The HeLa cell line was purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences. Cell culture medium, fetal bovine serum, and penicillin-streptomycin solution were purchased from other biological reagent suppliers. 25% Trypsin-EDTA (Gibco) was purchased from Thermo Fisher Scientific. All animal experiments were conducted in accordance with the guidelines for the care and use of laboratory animals at Lanzhou University and were approved by the Animal Ethics Committee of Lanzhou University (approval number: EAF2020022).

[0053] (I) Solid-phase peptide synthesis

[0054] Peptides were synthesized on Rink Amide AM resin using the Fmoc solid-phase peptide synthesis method. In each coupling reaction, Fmoc-protected amino acids (3 equivalents based on the initial resin loading) were used. The coupling reagent (Oxyma / DIC) (3 equivalents) was dissolved in DMF and reacted with shaking at room temperature for 60 minutes. The removal of the Fmoc group was completed using a 20% (v / v) piperidine / DMF solution. For active fluorescent labeling (fluorescein isothiocyanate), the resin was incubated overnight in the dark with a mixture of the active fluorescent labeling reagent (3 equivalents) and DIEA (6 equivalents) dissolved in DMF. After synthesis, the resin was dried under vacuum and then suspended in a mixture of TFA / H 2 2O / EDT / TIS (94:2.5:2.5:1) for cleavage to obtain the crude peptide.

[0055] (II) Decafluorobiphenyl-based peptide cyclization procedure

[0056] Dissolve the crude Tat47-57 peptide or its analog in DMF. Add Tris (30 mM) to a DMF solution containing the crude Tat47-57 peptide or its analog (1 mM) and decafluorobiphenyl, 4,4'-bis(bromomethyl)biphenyl, or hexafluorobenzene (5 mM). Stir the reaction solution at room temperature for 4 hours. Then remove the reaction solution by rotary evaporation. Purify the crude product by RP-HPLC using a linear gradient of 10 to 60% acetonitrile over 50 minutes. The fractions collected during the preparation were confirmed by ESI-TOF-MS (ESI-Q-TOFmaXis-4G, Bruker Daltonics, Germany),

[0057] A decafluorobiphenyl stapled peptide was designed based on the previously reported natural short-chain cell-penetrating peptide Tat47-57, which is derived from the trans-activating transcriptional activator of human immunodeficiency virus type 1. A series of Tat47-57 analogs were synthesized using conventional Fmoc solid-phase peptide synthesis (SPPS) and subsequently stapled by decafluorobiphenyl-cysteine SNAr chemistry (as Figure 1 shown in A). In this library, analogs of the model peptide Tat47-57 containing two cysteine residues were prepared in the i, i + 4 or i, i + 7 configurations, respectively (Table 1). Cysteine substitutions were made instead of insertions in the sequence to maintain the spatial relationship between the other amino acids in the peptide, except for P6. We systematically varied four parameters of the decafluorobiphenyl stapling: (1) the position of decafluorobiphenyl stapling (P1 - P6); (2) the biphenyl bridge, fluorine-free (P3b and P6b); (3) the hexafluorobenzene bridge, reducing the number of fluorines and benzenes (P3a); (4) the disulfide bridge (P3c and P6c). We manipulated the fluorine groups of the stapling bridge by replacing decafluorobiphenyl with 4,4'-bis(bromomethyl)biphenyl. By varying the stapling position and type, 13 conformationally restricted analogs were constructed. All peptides were purified by reverse-phase high-performance liquid chromatography (RP-HPLC) with a purity > 95% (Table 1). Among them, the HPLC and NMR spectra of FITC-labeled P3 and P6 are shown in Figure 10 and Figure 11 respectively.

[0058] Table 1 Peptides studied, their purity, and molecular weight

[0059]

[0060] The secondary structure of the newly designed peptides described above was characterized by circular dichroism spectroscopy (CD). Previous studies have shown that changes in the environment affect the biological activity of cell-penetrating peptides by altering their conformation. The secondary structure of the stapled peptides was measured in a simulated water membrane environment (50% trifluoroethanol TFE) to explore the relationship between conformation and cell uptake of the stapled peptides. The CD data showed that neither the parent peptide Tat47-57 nor the stapled peptide analogs adopted a stable secondary structure in water or TFE / H2 O(1:1, v / v Figure 2 ) did not show a specific secondary structure.

[0061] These results suggest that decafluorobiphenyl stapling may not induce the formation of a new secondary structure. Considering secondary structure and related cellular uptake together, it was found that a defined secondary structure is not essential for the stapled analogs to achieve efficient cellular uptake or endosomal escape.

[0062] Characterization of the cellular penetration efficiency of stapled peptide analogs in Example 2.

[0063] To visualize and quantify the cellular penetration efficiency of these newly designed stapled analogs, we labeled their N-terminus with fluorescein isothiocyanate (FITC). To avoid techniques that require cell fixation, as fixation often produces false positive results, we combined laser confocal microscopy and flow cytometry, both of which use live cells to detect the cellular penetration ability of our newly designed stapled peptides. Flow cytometry showed that the mean fluorescence intensity (MFI) of all analogs except P5 was higher than that of the positive control group using Tat47-57 ( Figure 3 ). Among all the analogs, P3 and P6 had the highest fluorescence intensity, with an efficiency 3.5 - 4 times higher than that of Tat47-57, while the uptake of P5 was lower ( Figure 3 B)

[0064] Using confocal microscopy, we confirmed the effect of these analogs on HeLa cells ( Figure 3 A). To confirm that the fluorescence in flow cytometry was intracellular rather than cell surface-associated, the uptake experiments of Tat47-57, P3, and P9 were repeated in the presence of trypan blue to quench extracellular fluorescence ( Figure 4 ).

[0065] No major loss of fluorescence was observed for P3 and P6 in the presence of trypan blue, indicating that the observed fluorescence was indeed intracellular. In addition, to evaluate the effect of fluorine and the biphenyl linker, the cellular uptake of uP3, P3a, P3b, P3c, uP6, P6b, and P6c was measured using flow cytometry. Interestingly, when the decafluorobiphenyl linker was replaced with a biphenyl linker (P3 vs P3a), the MFI of P3a decreased significantly ( Figure 3D and 3F), indicating that the introduction of F atoms into the peptide is crucial for the activity of CPPs. P3b and P6b are stapled with hexafluorobenzene bridges. Their cellular uptake is significantly lower than that of P3 or P6, indicating that both F atoms and the biphenyl bridge greatly affect the activity of CPPs. P3c and P6c with disulfide bridges are less active than their model peptide Tat47-57, indicating that the aromatic bridge and F atoms endow them with cell permeability. In addition, the activities of the unstapled peptides (uP3 and uP6) are lower than those of their stapled peptide counterparts (P3 and P6). Confocal microscopy imaging subsequently confirmed these effects on HeLa cells ( Figure 3 C and 3E). These results clearly show that F atoms and aromatic bridge stapling are important factors determining cell penetration efficiency. Negatively charged cell surface glycans have been shown to interact with cationic cell-penetrating peptides and affect the uptake of cell-penetrating peptides (Stanzl, E.G.; Trantow, B.M.; Vargas, J.R.; Wender, P.A., Fifteen years of cell-penetrating, guanidinium-rich molecular transporters: basic science, research tools, and clinical applications. Acc Chem Res 2013, 46(12), 2944-54; Li, M.; Schlesiger, S.; Knauer, S.K.; Schmuck, C., A tailor-made specific anion-binding motif in the side chain transforms a tetrapeptide into an efficient vector for gene delivery. Angew Chem Int Ed Engl 2015, 54(10), 2941-4). As Figure 5 shown, the cellular uptake of the analogs was found to be related to their binding to heparin (an analog of heparan sulfate). Notably, for P4 and P5, the cellular uptake was not affected by cell surface heparan sulfate, indicating the possible existence of other negatively charged cell surface glycans. Therefore, we determined the binding affinity of the examined cell-penetrating peptides to heparin by isothermal titration calorimetry (Table 2 and Figure 5)。These experiments revealed a high binding affinity of heparin to P3 (KD = 0.054 ± 0.005 μM) and P6 (KD = 0.041 ± 0.005 μM). The binding of heparin to P3 and P6 was 3 - 4 times higher than that of Tat47 - 57 (KD = 0.160 ± 0.015 μM). The results indicated that the binding affinity of these CPPs to heparin was closely related to their uptake efficiency.

[0066] Table 2 Results of ITC binding studies of HS and analogues

[0067]

[0068] To investigate the endosomal membrane lysis behavior of the control peptide Tat47 - 57 and the decafluorobiphenyl - stapled analogues, liposomes were used to mimic endosomes to evaluate the leakage of calcein dye. The calcein dye was encapsulated in large unilamellar vesicles (LUVs), and the release of the calcein dye was examined to evaluate the disruption of the liposomes. As Figure 6 shown, at concentrations of 5 μM or 10 μM, all peptides produced a leakage effect on the liposomes of the simulated endosomal membrane after 30 - minute incubation (<10% leakage). The leakage effect of all analogues increased in a dose - dependent manner. Notably, Tat47 - 57 had the lowest leakage of LUVs, while the decafluorobiphenyl - stapled analogues (P1 - P6) showed a significantly increased leakage effect ( Figure 6 A). Compared with P3a, P3b, P3c, and uP3, we observed that P3 had a higher leakage effect ( Figure 6 B). Similarly, for P6 and its analogues, results consistent with those shown in Figure 6 C were obtained. The results of the calcein dye leakage experiments indicated that the decafluorobiphenyl - stapled analogues had a potent effect on disrupting the endosomal membrane. The F atoms played a key role in selectively disrupting the endosomal membrane.

[0069] To further evaluate the endosomal escape behavior of the analogues, quantitative measurements of endosomal escape were performed using naphthyl - fluorescein - labeled CPPs, a pH - sensitive fluorophore that has been reported to quantitatively measure the endosomal escape of CPPs (Qian, Z.; Dougherty, P.G.; Pei, D., Monitoring the cytosolic entry of cell - penetrating peptides using a pH - sensitive fluorophore. Chem Commun (Camb) 2015, 51(11), 2162 - 5.). As Figure 6As shown in Figures D and 6E, the results of flow cytometry showed that P3 and P6 had strong endosomal escape abilities (9-fold and 6-fold higher than Tat47-57), while P3b and P6b were slightly better than Tat47-57 (3.4-fold and 2.8-fold). Compared with P3 and P6, the MFI of P3b and P6b was significantly reduced, indicating that the F atoms of the decafluorobiphenyl bridge were crucial for selectively disrupting the endosomal membrane. These data were consistent with the results confirmed by confocal microscopy in live HeLa cells. Intracellular localization was achieved by using the late endosome / lysosome stain LysoTracker Red (red) in live HeLa cells by confocal microscopy. As Figure 6 shown in Figure G, we observed that the green fluorescence of FITC-P3 and FITC-P6 was mostly located in the cytoplasm, indicating that the endosomal escape efficiency of P3 and P6 was better than that of Tat47-57. In summary, these results indicated that the decafluorobiphenyl-conjugated analogs P3 and P6 had high cytoplasmic delivery efficiency.

[0070] Rab5 is a major regulator of endosome biogenesis and recruits additional cytokines required for maintaining, fusing, and maturing vesicles. The resulting early endocytic compartments fuse with other Rab5+ vesicles and deliver cargo to other cellular locations by budding of transport vesicles. Some cargo is delivered to late endosomes, marked by Rab7, for degradation in lysosomes. To characterize the intracellular pathways taken by Tat47-57, P3, and P6, we looked for the overlap of these molecules with endocytic uptake markers and RFP-tagged Rab proteins. HeLa cells were transfected with GFP-Rab5 and treated with 5 μM Tat47-57, P3, and P6 for 1 hour or 2 hours ( Figure 6 Figure F). When these cells were examined by confocal microscopy, the major FITC signal overlapped with the signal of RFP-Rab5, confirming the presence of Tat47-57, P3, and P6 in Rab5+ vesicles. Since Rab5 vesicles subsequently deliver their cargo to downstream vesicles, we also evaluated the co-localization of Tat47-57, P3, and P6 with RFP-Rab7. HeLa cells transfected with RFP-Rab7 were treated with Tat47-57, P3, and P6 for 2 hours, and the results showed that a small fraction of the FITC signal was located in the RFP-Rab7 compartments, confirming that P3 and P6 entered early (Rab5+) and escaped from late (Rab7+) endosomes or entered lysosomes ( Figure 6 Figure F). HeLa cells treated with Tat47-57, P3, and P6 for 2 hours were stained with LysoTracker Red (red, Figure 6 Figure G) for late endosomes / lysosomes, and a part of the FITC signal was located in the late endosome / lysosome compartments, indicating that Tat47-57, P3, and P6 escaped from late (Rab7+) endosomes.

[0071] To explore the possible mechanisms of P3 and P6, we incubated HeLa cells in the presence of different inhibitors or at different temperatures to comprehensively inhibit endocytosis. The cells were treated with various inhibitors, including ethylisopropylamiloride (EIPA, a macropinocytosis inhibitor) (Commisso, C.; Davidson, S.M.; Soydaner-Azeloglu, R.G.; Parker, S.J.; Kamphorst, J.J.; Hackett, S.; Grabocka, E.; Nofal, M.; Drebin, J.A.; Thompson, C.B.; Rabinowitz, J.D.; Metallo, C.M.; Vander Heiden, M.G.; Bar-Sagi, D., Macropinocytosis of protein is an amino acid supply route in Ras-transformed cells. Nature 2013, 497(7451), 633-7.), chlorpromazine (CPZ, a clathrin-mediated endocytosis pathway inhibitor) (Wang, L.H.; Rothberg, K.G.; Anderson, R.G., Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation. The Journal of cell biology 1993, 123(5), 1107-17.), filipin (a caveolae-mediated endocytosis inhibitor) (Schnitzer, J.E.; Oh, P.; Pinney, E.; Allard, J., Filipin-sensitive caveolae-mediated transport in endothelium: reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. The Journal of cell biology 1994, 127(5), 1217-32) and methyl-β-cyclodextrin (M-β-CD, a lipid raft-mediated endocytosis inhibitor) (Ruan, H.; Chen, X.; Xie, C.; Li, B.; Ying, M.; Liu, Y.; Zhang, M.; Zhang, X.; Zhan, C.; Lu, W.; Lu, W.,Stapled RGDPeptide Enables Glioma-Targeted Drug Delivery by Overcoming MultipleBarriers.ACS applied materials&interfaces 2017,9(21),17745-17756.).Subsequent cell permeability assessment showed that, on the one hand, the cellular uptake of FITC-labeled P3 or P6 almost dropped to the basal level at 4 °C. On the other hand, treatment with CPZ led to a reduction in peptide uptake of P3 and P6 by approximately 41% and 45%, respectively. In contrast, treatment of cells with EIPA, filipin, or M-β-CD did not significantly affect the cellular uptake of P3 or P6 (. Figure 7 ). These results together indicate that the cellular uptake of P3 or P6 is energy-dependent and may mainly occur through the clathrin-mediated endocytic pathway. Cytotoxicity and hemolytic properties are key indicators for evaluating drug safety. The cytotoxicity of Tat47-57 and the newly designed stapled analogs in HeLa, NIH3T3, and CHO-K1 cells was evaluated using the CCK-8 assay. The analysis showed that none of the cyclic analogs (P3 and P6) exhibited obvious toxicity, and even at the highest tested concentration (50 μM, Figure 8 A), their cell viability remained above 95%. In addition, the results of the lactate dehydrogenase (LDH) release assay confirmed that neither P3 nor P6 peptide caused obvious cell membrane damage ( Figure 8 B). Then, to further understand drug safety, the hemolytic toxicity of Tat47-57 and the newly designed stapled analogs was evaluated using mouse red blood cells. The results showed that even at the highest tested concentration (100 μM, Figure 8 C), Tat47-57 and the analogs (P3 and P6) were safe for cells, with hemolysis values less than 5%. Since the analogs P3 and P6 had better cellular uptake efficiency and safety, they were further evaluated.

[0072] Negatively charged phospholipopeptides are not easily able to cross cell membranes, so studying these peptides in a cellular system is challenging. Phospholipopeptides mimic the interactions between the negatively charged phosphate groups of phosphoproteins and the positively charged amino acids in various protein-binding pockets, and these peptides are used as probes to explore phosphoprotein-protein interactions. We evaluated the cellular delivery of negatively charged phospholipopeptides using Tat47-57, P3, and P6 peptides. The intracellular localization of the phospholipopeptides was monitored by confocal microscopy. By treating HeLa cells with a mixture of Tat47-57, P3, or P6 and the phospholipopeptide, both P3 and P6 were able to enhance the cellular uptake of the cargo phospholipopeptide. However, Tat47-57 was hardly able to deliver the phospholipopeptide into the cells ( Figure 9A and 9B). Individually labeled phospholipopeptides did not show any cellular uptake. The images confirmed a significant increase in the uptake of GpYEEI in the presence of the stapled analog. Additionally, after staining late endosomes / lysosomes with LysoTracker Red (red, Figure 9 C), the intracellular localization of the phospholipopeptide / decabromobiphenyl stapled analog complex was examined by confocal microscopy. As Figure 9 shown in C, the punctate signals of the phospholipopeptide / Tat47-57 complex were distributed in the cytoplasm and overlapped with the lysosome tracker. In contrast, for the phospholipopeptide / decabromobiphenyl cyclic analog complex, a diffused FITC signal was observed in the cells, indicating that the decabromobiphenyl cyclic analog might have undergone endosomal escape. Taken together, the decabromobiphenyl cyclic analog led to an increase in the intracellular delivery of negatively charged phospholipopeptides.

[0073] Based on the above experiments, we further evaluated the cellular delivery effect of using the decabromobiphenyl stapled analog to deliver a biomacromolecule (avidin). Biotinylated P3 and P6 (Table S1) were treated with FITC-labeled avidin to form peptide-avidin complexes through biotin-avidin interaction. Then, by treating HeLa cells with these complexes, the results of flow cytometry showed that P3 and P6 delivered avidin into the cells with efficiencies of 319% and 327%, respectively, compared to Tat47-57 ( Figure 9 E). These results were confirmed by live-cell confocal microscopy, as Figure 9 shown in D. To confirm the endosomal escape ability of the decabromobiphenyl cyclic analog-avidin complex, after staining late endosomes / lysosomes with LysoTracker Red (red, Figure 9 F), the intracellular localization of the decabromobiphenyl cyclic analog-avidin complex was examined by confocal microscopy. For the Tat47-57-avidin complex, some punctate signals were distributed in the cytoplasm and overlapped with the lysosome tracker, with a small amount of cytoplasmic diffused signals. But when we evaluated the decabromobiphenyl cyclic analog-avidin complex, some cytoplasmic diffused signals were observed, and only a small number of punctate signals were distributed in the cytoplasm and overlapped with the lysosomes. These results indicate that the decabromobiphenyl cyclic analog exhibits potential intracellular delivery ability for biomacromolecules.

[0074] In summary, the present application created a decafluorobiphenyl-conjugated peptide library with different conjugation positions, conjugation types, and fluorine atoms. A detailed study of cellular uptake and endosomal escape activities revealed several insightful pieces of information. Among these analogs, P3 and P6 were found to have several advantages, such as superior cellular uptake efficiency and the ability to escape from late (Rab7+) endosomes. The results of calcein dye leakage and pH-sensitive fluorophores indicated that P3 and P6 had significant endosomal escape efficiency. Our study also showed that the decafluorobiphenyl bridge had a significant impact on endosomal escape. Combining its high endosomal escape efficiency and ease of preparation, these conjugated CPPs provide a general membrane transport strategy for cytoplasmic delivery of chemical probes and proteins.

[0075] There are many methods and ways to specifically implement this technical solution. The above is only the preferred embodiment of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention. Each component not clearly defined in this embodiment can be implemented by existing technologies.

Claims

1. A Tat47-57 stapled peptide based on decafluorobiphenyl, characterized in that: The amino acid sequence of the stapled peptide is shown in SEQ ID NO.1 or SEQ ID NO.2, wherein the 4th amino acid and the 8th amino acid of SEQ ID NO.1 are stapled by decafluorobiphenyl, and the 5th amino acid and the 12th amino acid of SEQ ID NO.2 are stapled by decafluorobiphenyl.

2. The method for preparing the Tat47-57 stapled peptide according to claim 1, characterized in that: The Tat47-57 peptide analog is synthesized by Fmoc solid phase peptide synthesis, wherein the amino acid sequence of the Tat47-57 peptide analog is shown in SEQ ID NO.1 or SEQ ID NO.2, and then the obtained Tat47-57 peptide analog, decafluorobiphenyl and a cyclizing agent are subjected to cyclization reaction to obtain the obtained peptide.

3. The preparation method according to claim 2, characterized in that: The cyclizing agent is Tris.

4. The preparation method according to claim 2, characterized in that: The molar ratio of Tat47-57 peptide analog: decafluorobiphenyl: cyclizing agent is 1:2~6:30~50.

5. The preparation method according to claim 2, characterized in that: The solvent used in the cyclization process was DMF.

6. The method according to claim 2, characterized in that The reaction conditions are stirring at room temperature for 4-5 hours, then removing the reaction solution by rotary evaporation, and the crude product is purified by RP-HPLC to obtain the cyclized product.

7. Use of the Tat47-57 stapled peptide according to claim 1 in cytoplasmic delivery of chemical probes.

8. The use according to claim 6, characterized in that: The chemical probe is a negatively charged phospholipid peptide.

9. Use of the Tat47-57 stapled peptide according to claim 1 in the cytoplasmic delivery of biological macromolecules.

10. The use according to claim 9, characterized in that: The biomacromolecule is avidin.

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