A polypeptide type protac molecule targeting degradation of sting protein and application thereof in treatment of dry eye

Abstract

The application provides a polypeptide type PROTAC molecule targeting degradation of a STING protein and application thereof in treatment of dry eye, and belongs to the technical field of biotechnology.The polypeptide type PROTAC molecule provided by the application comprises a STING targeting unit capable of specific binding with the STING protein, a connecting arm and an E3 ubiquitin ligase ligand, and can specifically degrade the STING protein.The application further provides application of the polypeptide type PROTAC molecule in preparation of a medicine for treating and / or preventing dry eye and a medicine for preventing and / or treating dry eye, has significant anti-inflammatory and tissue protection effects, can improve corneal epithelial damage, inflammatory reaction and goblet cell loss, and has excellent biocompatibility and safety, and is suitable for long-term or repeated administration on an ocular surface.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a polypeptide PROTAC molecule that targets and degrades STING protein and its application in the treatment of dry eye syndrome. Background Technology

[0002] Dry eye syndrome is one of the most common ocular surface diseases in clinical practice, characterized by dryness of the ocular surface, foreign body sensation, burning sensation, fluctuating vision, and ocular surface inflammation. In severe cases, it can lead to corneal epithelial damage, ulceration, and even vision loss, significantly reducing patients' quality of life. In recent years, with the increasing aging population, excessive use of electronic products, changes in environmental factors, and the increased prevalence of eye surgery, the incidence of dry eye syndrome has continued to rise, making clinical treatment increasingly urgent.

[0003] The core pathological mechanism of dry eye syndrome has been confirmed to be closely related to non-infectious inflammation of the ocular surface, with abnormal activation of innate immune inflammation being a key driver of disease occurrence and progression. Related studies have found that in dry eye conditions, various external factors such as a hyperosmolar environment on the ocular surface and dryness can lead to mitochondrial dysfunction in corneal epithelial cells, causing the opening of the mitochondrial membrane permeability transition pore (mPTP). This allows mitochondrial DNA (mtDNA) to leak into the cytoplasm, thereby activating the cyclic guanosine monophosphate-adenosine monophosphate synthase-interferon gene stimulating factor (cGAS-STING) signaling pathway, ultimately mediating the occurrence and progression of ocular surface inflammation. Interferon gene stimulating factor (STING), as a core hub protein of the cGAS-STING signaling pathway, is widely expressed in ocular surface cells such as corneal epithelial cells and immune cells. Its normal physiological function is to participate in the body's innate immune response against viruses and pathogens. However, in the pathological state of dry eye, STING protein is abnormally highly expressed and overactivated, which initiates downstream TBK1-IRF3 and NF-κB signaling pathways, promoting the massive release of pro-inflammatory cytokines such as interleukins and tumor necrosis factor. This exacerbates damage to ocular surface epithelial cells and loss of conjunctival goblet cells, leading to reduced tear secretion and increased tear osmotic pressure, forming a vicious cycle of "inflammatory damage - worsening dry eye." Clinical studies have confirmed that the level of STING protein in the tears of dry eye patients is significantly elevated, and its expression level is positively correlated with corneal fluorescein staining scores and negatively correlated with aqueous tear secretion, suggesting that STING protein is a potential key target for the treatment of dry eye.

[0004] Currently, clinical treatment for dry eye syndrome mainly focuses on symptomatic and supportive care, including artificial tear replacement therapy, anti-inflammatory treatment with corticosteroid eye drops, immunosuppressant therapy (such as cyclosporine), and punctal plugging. However, these treatments all have significant limitations: artificial tears only temporarily relieve dry eye symptoms and cannot inhibit the progression of inflammation at its root; long-term use may also lead to ocular surface flora imbalance. While corticosteroid eye drops can quickly reduce inflammation, long-term use can easily cause serious adverse reactions such as high intraocular pressure, cataracts, and ocular surface infections. Immunosuppressants have a slow onset of action and suffer from significant individual variability in efficacy and poor tolerance in some patients. Physical therapies such as punctal plugging are invasive procedures that may cause complications such as dacryocystitis and cannot address the core inflammatory problem mediated by abnormal activation of the STING protein. Therefore, developing novel therapeutic drugs that can precisely target the STING protein and block the inflammatory pathway at its root has become a key direction for breaking through the current bottleneck in dry eye syndrome treatment. Summary of the Invention

[0005] To address the problems existing in the prior art, the primary objective of this invention is to provide a polypeptide-type PROTAC molecule that targets and degrades STING protein, and a method for preparing the same.

[0006] A second object of the present invention is to provide the use of the above-mentioned polypeptide PROTAC molecule in the preparation of medicaments for the treatment and / or prevention of dry eye syndrome.

[0007] A third objective of this invention is to provide a medicament for the prevention and / or treatment of dry eye syndrome.

[0008] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a polypeptide PROTAC molecule for targeted degradation of STING protein. The polypeptide PROTAC molecule includes a STING targeting unit, a linker arm, and an E3 ubiquitin ligase ligand. The STING targeting unit includes a chemical group capable of specifically binding to the STING protein. The E3 ubiquitin ligase ligand includes a VHL ligand.

[0009] Preferably, the connecting arm comprises any one or a combination of several of the following: amide bond, ether bond, alkyl chain, and PEG unit.

[0010] Preferably, the structure of the STING targeting unit is as shown in formula (I): .

[0011] Preferably, the structure of the VHL ligand is shown in formula (II): .

[0012] Preferably, the structure of the polypeptide PROTAC molecule is shown in formula (Ⅲ): .

[0013] This invention also provides a method for preparing the above-mentioned polypeptide-type PROTAC molecule, comprising the following steps: (1) VHL ligands were prepared by solid-phase polypeptide synthesis. (2) The STING targeting unit is linked to the N-terminus of the VHL ligand via amidation reaction to obtain a polypeptide PROTAC molecule.

[0014] The present invention also provides the use of the above-mentioned polypeptide PROTAC molecule in the preparation of medicaments for the treatment and / or prevention of dry eye syndrome.

[0015] Preferably, the dry eye syndrome includes chemically induced dry eye syndrome, hyperosmolar environment-induced dry eye syndrome, inflammation-related dry eye syndrome, and dry eye syndrome accompanied by corneal epithelial damage and goblet cell reduction.

[0016] The present invention also provides a medicament for the prevention and / or treatment of dry eye syndrome, the medicament comprising the above-described polypeptide PROTAC molecule.

[0017] Preferably, the drug comprises any one or a combination of several of ophthalmic eye drops, ophthalmic gels, ophthalmic sustained-release formulations, and ophthalmic nanodelivery systems.

[0018] Compared with the prior art, the beneficial effects of the technical solution of the present invention are as follows: The polypeptide-type PROTAC molecule provided by this invention can specifically degrade STING protein, rather than just inhibit its activity. It can introduce STING protein into the ubiquitin-proteasome system through the PROTAC mechanism to achieve continuous and cyclical protein degradation, thus avoiding the problems of traditional STING inhibitors that rely solely on high occupancy rates and short duration of action.

[0019] The peptide-type PROTAC molecule provided by this invention exhibits significant anti-inflammatory and tissue-protective effects in dry eye-related inflammatory models. In a hyperosmolar corneal epithelial cell model, it effectively inhibits the STING / TBK1 / IRF3 signaling pathway. In a chemically induced dry eye animal model, it significantly improves corneal epithelial damage, inflammatory response, and goblet cell loss. Furthermore, it possesses excellent biocompatibility and safety, making it suitable for long-term or repeated topical administration to the ocular surface. Compared to traditional STING small molecule inhibitors, it significantly reduces cytotoxicity and hemolytic risk, and long-term ocular instillation has not caused significant toxic side effects on the cornea, conjunctiva, or systemic organs. Attached Figure Description

[0020] Figure 1 Synthetic route of the polypeptide PROTAC molecule SPP; Figure 2 High-resolution mass spectrum of the polypeptide PROTAC molecule SPP; Figure 3 The degradation effect of the polypeptide-type PROTAC molecule SPP on human cell STING protein; Figure 4 The application effect of peptide-type PROTAC molecule SPP in a hyperosmolar stress-induced corneal epithelial cell injury model; Figure 5 The therapeutic effect of the polypeptide-type PROTAC molecule SPP in an in vivo model of dry eye disease; Figure 6 Synthetic route of the small molecule-based PROTAC drug molecule SPB; Figure 7 High-resolution mass spectrum of the small molecule PROTAC drug molecule SPB; Figure 8 : The 1H NMR spectrum of the small molecule PROTAC drug molecule SPB; Figure 9 : Carbon NMR spectrum of the small molecule PROTAC drug molecule SPB; Figure 10 Comparison of STING degradation capabilities between SPP and SPB; Figure 11 Comparison of biosafety between SPP and SPB. Detailed Implementation

[0021] This invention provides a polypeptide PROTAC molecule that targets and degrades STING protein. The polypeptide PROTAC molecule includes a STING targeting unit, a linker arm, and an E3 ubiquitin ligase ligand.

[0022] The STING targeting unit (warhead) of this invention includes a chemical group capable of specifically binding to the STING protein; the targeting unit can be in a covalent or non-covalent binding form, and may be a derivative group derived from a known STING inhibitor structure. Preferably, the structure of the STING targeting unit is shown in formula (I): .

[0023] The E3 ubiquitin ligand of the present invention can recruit ligands for E3 ubiquitin ligands; the ligand can be a polypeptide or a polypeptide mimic. Preferably, it is a VHL (von Hippel-Lindau) protein ligand; the VHL ligand is extended to a main chain (SEQ ID No. 1: PIYPALASGSG) by sequentially linking proline (Pro, P), isoleucine (Ile, I), tyrosine (Tyr, Y), proline (Pro, P), alanine (Ala, A), leucine (Leu, L), alanine (Ala, A), serine (Ser, S), glycine (Gly, G), serine (Ser, S), and glycine (Gly, G), and the structure of the VHL ligand is shown in Formula (II): .

[0024] The linker arm of this invention connects the STING targeting unit to the E3 ubiquitin ligase ligand. The linker arm can be a flexible or semi-flexible structure; preferably, its length ranges from 3 to 30 atoms, more preferably from 3 to 20 atoms. The linker arm of this invention includes any one or a combination of several of the following: amide bond (-CONH-), ether bond (-O-), alkyl chain (-CH2-…), and PEG unit (-O-CH2-CH2-O-…). For example, the linker arm of the polypeptide-type PROTAC molecule (SPP) shown in formula (III) of this invention is a linker fragment containing a succinyl backbone, comprising a -CO-CH2-CH2-CO- structure, and is connected to the STING targeting unit and the VHL ligand respectively via amide bonds.

[0025] The peptide-type PROTAC molecule for targeted degradation of STING protein described in this invention can be represented by the following general formula: STING targeting unit - linker arm - E3 ubiquitin ligand ligand (VHL ligand). This invention utilizes a peptide-type E3 ubiquitin ligand (VHL ligand) to increase molecular polarity and reduce nonspecific membrane damage, resulting in better biosafety and a wider therapeutic window. This invention embeds an inhibitory unit with STING binding capacity into the PROTAC structure, achieving a "repression-degradation" functional conversion. By targeting and degrading STING, it inhibits the cascade amplification of multiple inflammatory factors upstream of the signaling pathway, achieving continuous and cyclical protein degradation, avoiding the problems of traditional STING inhibitors that rely solely on high occupancy rates and short durations of action.

[0026] Preferably, the structure of the polypeptide-type PROTAC molecule of the present invention is shown in formula (Ⅲ): .

[0027] In equation (Ⅲ), the linker between the STING targeting unit and the VHL ligand is a linker segment containing -CO-CH2-CH2-CO-.

[0028] This invention also provides a method for preparing the above-mentioned polypeptide PROTAC molecule (the synthetic route of the polypeptide PROTAC molecule is as follows). Figure 1 As shown in the figure, the final target PROTAC molecule obtained (denoted as SPP) includes the following steps: (1) VHL ligands were prepared by solid-phase polypeptide synthesis. (2) The STING targeting unit is linked to the N-terminus of the VHL ligand via amidation reaction to obtain a polypeptide PROTAC molecule.

[0029] As an optional implementation, the method for preparing the polypeptide PROTAC molecule of formula (III) of the present invention includes: 1. Peptides were assembled on 2-chloro resin by the standard Fmoc-SPPS method, and side-chain protected amino acids were introduced in sequence, in the order PIYPALASGSG (SEQ ID No. 1): (1) Take 2-chloro resin, put it into a reaction tube, add an appropriate amount of DCM (dichloromethane), shake on a shaker, and blow out the DCM in the tube. (2) Take the first amino acid (P), dissolve it in DCM, add condensing agent DIEA, make it completely dissolved, drain the liquid in the reverse phase tube, and then transfer the dissolved amino acid with a pipette to continue the reaction. After the reaction, drain the solution in the tube and wash with DCM. Then, block the solution with blocking solution, and after completion, wash with DCM and DMF respectively. (3) Remove the Fmoc protecting group: use 20% piperidine solution for cutting, and wash with DMF after the reaction. (4) Take the next amino acid (I), add condensing agent HBTU and DIEA, then add it to DMF, dissolve it completely, add it to the reactor, blow out the solution after the reaction, and wash with DMF. (5) Repeat steps (3) and (4) above, adding each amino acid in turn (starting from the fifth amino acid, the reaction time is appropriately extended to improve the yield of the final product) until the last amino acid.

[0030] 2. After the last amino acid is ligated, the mixture is washed sequentially with DMF and DCM. Then, the purified, dried, and identified STING target protein ligand is added to a solid-phase reaction tube and coupled using the same method as the amino acid. Afterward, the mixture is washed with DMF and DCM, and then the resin is cut.

[0031] 3. Resin cutting: Prepare the cutting solution (19 mL of 95% TFA + 500 μL of 2.5% H2O + 500 μL of 2.5% TIS), add it to the reaction tube, and collect the cutting solution into a glass bottle. Then wash with DCM and collect the liquid.

[0032] 4. Connect the collected liquid to a rotary evaporator to begin concentration. When it is nearly dry, add DCM to assist in washing, and repeat the process until an oily substance is obtained in the bottle.

[0033] 5. Add anhydrous diethyl ether to the evaporated substance, let stand, and a solid will precipitate. Wash with diethyl ether, dry the ether, and collect the solid to obtain the crude product (denoted as SPP). Analyze the product structure and purity using LC-MS. If the purity is not up to standard, purify it further using HPLC.

[0034] This invention also provides the use of the above-mentioned polypeptide PROTAC molecule in the preparation of medicaments for the treatment and / or prevention of dry eye syndrome. Preferably, the dry eye syndrome includes chemically induced dry eye syndrome, hyperosmolar environment-induced dry eye syndrome, inflammation-related dry eye syndrome, and dry eye syndrome accompanied by corneal epithelial damage and goblet cell reduction.

[0035] The present invention also provides a medicament for the prevention and / or treatment of dry eye syndrome, the medicament comprising the aforementioned polypeptide PROTAC molecule. The medicament of the present invention is administered topically to the ocular surface; preferably, the medicament comprises any one or a combination of several of ophthalmic eye drops, ophthalmic gels, ophthalmic sustained-release formulations, and ophthalmic nanodelivery systems.

[0036] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0037] Unless otherwise specified, the following embodiments are all conventional methods.

[0038] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0039] Example 1 Preparation steps of peptide-type PROTAC molecule SPP (synthetic route of peptide-type PROTAC molecule SPP as follows) Figure 1 (as shown) Peptides were assembled on 2-chloro resin using the standard Fmoc-SPPS method, with side-chain protected amino acids introduced in sequence, in the order PIYPALASGSG (SEQ ID No. 1).

[0040] (1) Accurately weigh 0.516 g of dichloromethane resin and place it in a reaction tube. Then, add an appropriate amount of DCM (dichloromethane) and place it on a shaker for 10 min. Then, blow out the DCM from the tube using a syringe.

[0041] (2) Weigh 1 mmol of the first amino acid (P), dissolve it in DCM, and add 330 μL of condensing agent DIEA to completely dissolve it. Use a syringe rubber bulb to drain the liquid from the reversed-phase tube, and then use a pipette to transfer the dissolved amino acid. Continue the reaction for 2 hours. After the reaction is complete, drain the solution from the tube and wash it 5 times with DCM. Then, use blocking solution (composed of 17 mL DCM, 2 mL MeOH and 1 mL DIEA) for blocking treatment for 15 min. After the reaction is complete, wash it 5 times with DCM and DMF, 1 min each time.

[0042] (3) Removal of the Fmoc protecting group: The Fmoc protecting group was removed using a 20% piperidine solution (piperidine:DMF = 1:4) for 30 min. After the reaction, the Fmoc protecting group was washed 5 times with DMF for 1 min each time.

[0043] (4) Weigh 1 mmol of the next amino acid (I), add 1 mmol of condensing agent HBTU and 330 μL of DIEA (ratio 1:1:2), then add to 10-15 mL of DMF. After complete dissolution, add to the reactor and continue the reaction for 2 h. Then blow out the solution with a syringe and wash with DMF 5 times.

[0044] (5) Repeat steps (3) and (4) above, adding each amino acid in turn (starting from the fifth amino acid, the reaction time is appropriately extended to improve the yield of the final product) until the last amino acid.

[0045] (6) After the last amino acid is ligated, wash with DMF and DCM five times each, for 1 min each time. Then, add the purified, dried and identified STING target protein ligand to the solid-phase reaction tube and perform the coupling reaction for 10 hours in the same manner as the amino acid. After the reaction, wash with DMF and DCM five times each, and then cut the resin.

[0046] (7) Resin cutting: Prepare the cutting solution (19 mL of 95% TFA + 500 μL of 2.5% H2O + 500 μL of 2.5% TIS), add it to the reaction tube, and after reacting for 45 min, collect the cutting solution into a glass bottle. Then wash once with DCM and collect the liquid.

[0047] (8) Connect the collected liquid to a rotary evaporator to begin concentration. When it is close to drying, add DCM to assist washing. Repeat the operation 4 times until an oily substance is obtained in the bottle.

[0048] (9) Add anhydrous diethyl ether to the evaporated substance and let stand for 3-5 min. A solid will precipitate out. Wash with diethyl ether 4 times, and then remove the diethyl ether by vacuum filtration (using a water pump, plug the interface with paper towels, and pump for 15 min). Collect the solid to obtain the product (SPP).

[0049] The prepared SPP was subjected to high-resolution mass spectrometry using a liquid chromatography-quadrupole time-of-flight mass spectrometer. The high-resolution mass spectrum is shown below. Figure 2 As shown. Combining the high-resolution mass spectrum, the final structure of the SPP was determined, as shown in equation (Ⅲ): .

[0050] The prepared SPP was used for subsequent experiments.

[0051] Example 2 The human cell line expressing STING protein (THP-1 cells) was selected as an experimental model to verify the degradation effect of SPP in vitro.

[0052] (1) Concentration-dependent degradation of STING protein by SPP The cells were seeded in culture plates, and after the cells stabilized, different concentrations of SPP (0, 1, 5, 10, 20, and 40 μmol / L) were added and incubated at 37°C with 5% CO2 for a predetermined time (12 h). After treatment, the cells were collected and total protein was extracted. The expression level of STING protein was detected by Western blot, with β-Tubulin used as an internal control protein.

[0053] (2) Time-dependent degradation of STING protein by SPP The cells were seeded in culture plates and, after stabilization, treated with a fixed concentration of SPP (10 μmol / L) at 37°C and 5% CO2 for 0, 6, 12, 24, and 48 hours, respectively. After treatment, cells were collected and total protein was extracted. The expression level of STING protein was detected by Western blot, with β-Tubulin used as an internal control.

[0054] (3) Effect of proteasome inhibitors on SPP degradation To confirm whether the SPP-induced decrease in STING protein depends on the proteasome pathway, a control experiment was conducted using the proteasome inhibitor MG132, building upon the previous experiments. The specific method was as follows: cells were seeded in culture plates, and after stabilization, they were pretreated with MG132 (10 μM) at 37°C and 5% CO2 for 2 h, followed by the addition of SPP (10 μmol / L) and cultured for another 24 h. After treatment, cells were collected and total protein was extracted. The expression level of STING protein was detected using Western blot, with β-Tubulin used as an internal control protein.

[0055] The results of the WB experiment are as follows: Figure 3 As shown in the figure, A is a schematic diagram of the changes in STING protein expression levels after cells were treated with different concentrations of SPP; B is a schematic diagram of the changes in STING protein expression levels after cells were treated with a fixed concentration of SPP for different time periods; C is a schematic diagram of the effect of proteasome inhibitors on the degradation of STING protein by SPP.

[0056] The results showed that with increasing SPP concentration, the expression level of STING protein in cells gradually decreased, exhibiting a clear concentration-dependent trend; while the expression level of the internal reference protein β-Tubulin remained relatively stable. This indicates that the SPP molecule described in this invention can effectively induce the degradation of STING protein, and this degradation is concentration-dependent (A). After SPP treatment, the STING protein level gradually decreased with prolonged treatment time, showing a clear time-dependent characteristic, while the β-Tubulin expression level did not change significantly. This indicates that the SPP molecule of this invention can exert a sustained effect in cells, effectively reducing the expression level of STING protein (B). In the absence of MG132, SPP treatment significantly reduced the STING protein level; however, in the presence of MG132, the reducing effect of SPP on STING protein was significantly weakened or partially blocked. This indicates that the process by which the SPP molecule of this invention induces the reduction of STING protein levels depends on the intracellular proteasome-mediated degradation pathway (C).

[0057] In summary, the results of this embodiment demonstrate that the SPP provided by this invention can effectively induce the degradation of STING protein in cells. This degradation is concentration- and time-dependent, and relies on a proteasome-mediated protein degradation mechanism. These results prove the feasibility and effectiveness of the SPP molecule as a targeted degrader of STING.

[0058] Example 3 Human corneal epithelial cells (HCE-T) were used to establish an in vitro cell damage model induced by hyperosmotic stress (HS), and the effects of SPP on cell viability and the expression levels of STING / TBK1 / IRF3 signaling-related proteins were investigated.

[0059] (1) Establishment of hyperosmolar stress model and effect of SPP on cell viability HCE-T cells were seeded in culture plates and cultured to the logarithmic growth phase. A hyperosmolar stress model was established by stimulating the cells with a hyperosmolar culture condition of 500 mOsm: NaCl was added to DMEM medium (containing 10% Gibco serum), and the concentration was quantified to 500 mOsm using an osmometer. Subsequently, different concentrations of SPP (1, 5, 10, 20, 40, and 60 μM) were added under hyperosmolar stimulation. Cell viability was assessed after treatment. A normal control group (Ctrl) without hyperosmolar stimulation and a model group treated only with hyperosmolar stress (500 mOsm) were also included.

[0060] (2) Effect of SPP on the STING / TBK1 / IRF3 signal axis under high osmotic stress conditions To further evaluate the role of SPP in relation to the STING signaling pathway, cells from the normal control group (Ctrl), model group (500 mOsm), and SPP (1, 5, 10, 20 μM) treatment groups in step (1) were collected and total protein was extracted. The expression levels of STING and its downstream signal-related proteins TBK1, IRF3 and their phosphorylated forms (p-TBK1, p-IRF3) were detected by Western blot, with GAPDH used as an internal reference protein.

[0061] Experimental results are as follows Figure 4 As shown, a is a schematic diagram of the effect of different concentrations of SPP treatment on the viability of human corneal epithelial cells (HCE-T) under hyperosmolar stress (HS) conditions; b is a schematic diagram of the effect of SPP treatment on the expression levels of STING / TBK1 / IRF3 signaling-related proteins under hyperosmolar stress conditions.

[0062] The results showed that cell viability decreased under hyperosmolar stress conditions; after the addition of SPP, cell viability improved with increasing SPP concentration, indicating that SPP has a certain protective effect against hyperosmolar stress-induced corneal epithelial cell damage (a). Hyperosmolar stress can cause changes in the phosphorylation levels of STING protein and its downstream signaling proteins; after SPP treatment, the expression / phosphorylation levels of the above-mentioned STING and downstream signaling proteins decreased or were inhibited with changes in SPP dosage, while the expression of the internal reference protein remained stable. This indicates that the SPP described in this invention can regulate the STING-related signaling pathway under hyperosmolar stress conditions (b).

[0063] In summary, the results of this embodiment demonstrate that the SPP provided by the present invention can improve cell viability in a hyperosmolar stress-induced corneal epithelial cell injury model and inhibit the expression / activation levels of STING and its downstream TBK1 / IRF3 signaling axis-related proteins, thus demonstrating its application potential under dry eye-related pathological stimuli.

[0064] Example 4 A benzalkonium chloride (BAC)-induced mouse dry eye model was used to verify the therapeutic effect of SPP in an in vivo dry eye model and its ameliorative effect on ocular surface structure and inflammatory response.

[0065] (1) Establishment of dry eye animal model and drug administration regimen Healthy mice (C57BL / 6 mice, 6-8 weeks old) were randomly divided into groups. Except for the normal control group, all other groups were treated with 0.2% BAC eye drops to establish a dry eye model (benzalkonium chloride was repeatedly applied to the eyes at a concentration of 0.2%, twice daily, 5 μL each time, for 7 consecutive days). After model establishment, different treatments were administered: Group G1 was the blank control group for healthy mice; Group G2 was the BAC modeling group; Group G3 was the BAC modeling group treated with 100 μM SPP; Group G4 was the BAC modeling group treated with 200 μM SPP; and Group G5 was the BAC modeling group treated with 100 μM C-176 (a small molecule STING protein inhibitor). For Groups G3-G5, the drug was dissolved in PBS solution and administered topically as eye drops. Animals were observed and tested at predetermined time points (days 0, 1, 3, and 5) during the treatment period.

[0066] (2) Assessment of ocular surface damage by corneal fluorescein staining Before administration (day 0) and at different time points after administration (day 1, day 3, and day 5), corneal fluorescein staining was performed on mice in each group, and the corneal staining was observed using a slit-lamp microscope.

[0067] (3) Histopathological observation of corneal tissue Eye tissues from mice in each group were collected, paraffin sections were prepared, and hematoxylin-eosin (H&E) staining was performed.

[0068] (4) PAS staining analysis of changes in goblet cells To assess changes in conjunctival goblet cells, conjunctival tissue was stained with PAS.

[0069] (5) Immunohistochemical detection of inflammatory factor expression Immunohistochemistry was used to detect the expression of inflammatory factors TNF-α and IL-1β in corneal and conjunctival tissues.

[0070] Experimental results are as follows Figure 5 As shown in the figure, A represents the grouping of each experimental group, G1 is the blank control group, G2 is the BAC model group, G3 is the BAC+SPP (100 μmol / L) treatment group, G4 is the BAC+SPP (200 μmol / L) treatment group, and G5 is the BAC+C-176 (100 μmol / L) treatment group; B represents the corneal fluorescein staining images of different experimental groups at different time points before and after drug administration, used to assess corneal epithelial damage and repair; C represents the hematoxylin-eosin (H&E) staining results of corneal tissue in each experimental group; D represents the PAS staining results of conjunctival tissue in each experimental group, used to observe the distribution and number changes of conjunctival goblet cells; E represents the immunohistochemical staining results of TNF-α in corneal or conjunctival tissue in each experimental group; and F represents the immunohistochemical staining results of IL-1β in corneal or conjunctival tissue in each experimental group.

[0071] The results showed that the corneas of mice in the BAC treatment group exhibited significant fluorescent staining, indicating impaired corneal epithelial integrity. In the SPP treatment group, the area and intensity of corneal fluorescent staining gradually decreased with prolonged administration, with the higher concentration SPP treatment group showing more significant improvement, approaching the state of the normal control group (B). The corneal epithelium in the BAC model group was thinned and accompanied by tissue structural disorder; while the corneal structure in the SPP treatment group was more intact, with varying degrees of improvement in epithelial thickness and tissue arrangement (C). BAC treatment led to a decrease in the number of conjunctival goblet cells; after SPP treatment, the number of goblet cells recovered somewhat, suggesting that SPP can improve the damage to mucin secretion-related structures in the dry eye model (D). The expression of the aforementioned inflammatory factors was enhanced in the BAC model group; while in the SPP treatment group, their expression levels were significantly reduced, indicating that SPP can inhibit local inflammatory responses in the dry eye model (E-F).

[0072] In summary, the results of this embodiment demonstrate that the SPP described in this invention can improve corneal epithelial damage, promote goblet cell recovery, and reduce the expression of inflammatory factors in a BAC-induced dry eye animal model, showing a good therapeutic effect on dry eye.

[0073] Example 5 Synthetic small molecule-based STING PROTAC drug molecule SPB (synthetic route as follows) Figure 6 As shown in the figure, the final target PROTAC molecule obtained is denoted as SPB. The steps are as follows: (1) 5-Nitro-2-furanic acid (314 mg, 2 mmol) was dissolved in 10 mL of DMF. HATU (836 mg, 2.2 mmol) and DIPEA (695 μL, 4 mmol) were added, and the mixture was stirred for 15 minutes. Then 4-(tert-Butoxycarbonylamino)aniline (416 mg, 2 mmol) was added, and the reaction was allowed to proceed at room temperature for 6 hours. The reaction was terminated with water (30 mL), and the product was extracted with ethyl acetate (3 × 30 mL). The combined organic layers were washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, concentrated under vacuum, and purified by rapid column chromatography to give the desired amide (compound 1) (583 mg, 84%).

[0074] (2) At room temperature, the intermediate (compound 1) (347 mg, 1 mmol) was suspended in hydrochloric acid solution (2 mL, 4 M, soluble in dioxane) and stirred overnight. Then, the solvent was removed under reduced pressure, and the residue was prepared with saturated sodium bicarbonate solution (8 mL). The precipitate was filtered, washed with water, and dried to give the crude product (compound 2).

[0075] (3) The crude product (compound 2), succinic anhydride (1.1 mmol), and DIPEA (1.2 mmol) were dissolved in anhydrous tetrahydrofuran (5 mL) and stirred under reflux for 4 hours. The solvent was removed under vacuum, and the residue was poured into water (5 mL). Then, dilute hydrochloric acid was added to adjust the pH to 3-4, the precipitate was filtered, and washed with water to obtain the product (compound 3) (226 mg, 65%).

[0076] (4) The intermediate (compound 3) (191 mg, 0.55 mmol), HATU (209 mg, 0.55 mmol), and DIPEA (17 μL, 1 mmol) were dissolved in DMF (1 mL), and the mixture was stirred at room temperature for 15 minutes. Subsequently, the thalidomide derivative (172 mg, 0.50 mmol) was added and stirred for 10 hours. Afterward, the reaction mixture was poured into water (10 mL) and extracted with EtOAc (3 × 10 mL). The combined organic phases were washed with physiological saline, dried over anhydrous sodium sulfate, and the solvent was concentrated under reduced pressure. The residues were purified by column chromatography (DCM / MeOH = 50:1) to give the final product (SPB).

[0077] The prepared SPB was subjected to high-resolution mass spectrometry using a liquid chromatography-quadrupole time-of-flight mass spectrometer. The high-resolution mass spectrum is shown below. Figure 7 As shown; nuclear magnetic resonance (NMR) identification was performed using a nuclear magnetic resonance (NMR) spectrometer, and the hydrogen NMR spectrum is shown below. Figure 8 As shown, the carbon NMR spectrum is as follows: Figure 9 As shown in the figure. Simultaneous use of 1H NMR, 1C NMR, and high-resolution mass spectrometry confirmed the successful synthesis of the small molecule-based SPB. The prepared SPB was used in subsequent experiments.

[0078] 1. Comparison of the STING degradation capabilities of SPB and SPP THP-1 cells were used as an experimental model and treated with different concentrations of SPP (0, 1, 5, 10, 20, 40 μM) or SPB (0, 1, 5, 10, 20, 40 μM) at 37℃ and 5% CO2 for 24 h. Cells were collected and total protein was extracted under the same treatment time conditions. The expression level of STING protein was detected by Western blotting and normalized analysis was performed using the internal reference protein (β-Tubulin).

[0079] The results show that... Figure 10 As shown, the intracellular STING protein expression level gradually decreased with increasing SPP or SPB treatment concentration. Further dose-response analysis of the relative expression level of STING protein revealed that both SPP and SPB effectively reduced STING protein levels within the micromolar concentration range, with corresponding half-maximal degradation concentrations (DCs) of [missing information]. 50 The values ​​were in a similar range. The results indicate that the small molecule-based PROTAC drug SPB and the peptide-based PROTAC drug SPP both exhibit comparable degradation abilities against STING protein under in vitro conditions.

[0080] 2. Comparison of biosafety between SPB and SPP The SPP and SPB were compared and evaluated from two aspects: hemolytic reaction and cell compatibility with various normal cells.

[0081] (1) Comparison of hemolysis experiments Fresh red blood cells were collected, washed with physiological saline, and prepared into a red blood cell suspension (2% v / v). This suspension was then co-incubated with different concentrations of SPP (1, 6.25, 12.5, 25, 50, 100, 200 μM) or SPB (1, 6.25, 12.5, 25, 50, 100, 200 μM) in PBS solution at 37°C and 5% CO2 for 6 h. Deionized water was used as a positive control, and physiological saline as a negative control. After incubation, hemolysis was assessed by visual observation and spectrophotometry.

[0082] (2) Comparison of cell compatibility Normally derived human corneal epithelial cells (HCE-T) were treated with different concentrations of SPP (0, 5, 10, 20, 50, 100, 200 μM) or SPB (0, 5, 10, 20, 50, 100, 200 μM). Normally derived mouse renal tubular epithelial cells (TCMK-1) and mouse cardiomyocytes (HL-1) were also treated with different concentrations of SPP (0, 1, 5, 10, 20, 50, 100 μM) or SPB (0, 1, 5, 10, 20, 50, 100 μM). All treatments were carried out at 37℃ with 5% CO2 for 24 h. Cell viability was assessed after treatment.

[0083] The results are as follows Figure 11 As shown in the figure, A is a schematic diagram of the hemolysis experiment results after co-incubation of different concentrations of SPP with red blood cells and the corresponding hemolysis rate analysis; B is a schematic diagram of the hemolysis experiment results after co-incubation of different concentrations of SPB with red blood cells and the corresponding hemolysis rate analysis; C shows the changes in cell viability after SPP and SPB acted on human corneal epithelial cells, respectively; D shows the changes in cell viability after SPP and SPB acted on mouse renal tubular epithelial cells, respectively; and E shows the changes in cell viability after SPP and SPB acted on mouse cardiomyocytes, respectively.

[0084] The results showed that within the tested concentration range, the SPP-treated group exhibited clear erythrocyte sedimentation and a clear supernatant, with no obvious hemolysis observed. However, under the same or higher concentrations, the SPB-treated group showed varying degrees of supernatant discoloration, indicating a hemolytic reaction. Quantitative analysis revealed that the hemolysis rate significantly increased at higher SPB concentrations, while the hemolysis rate remained low within the same concentration range with SPP. Within the tested concentration range, the survival rate of various cell types remained high after SPP treatment; in contrast, the SPB-treated group showed a decreasing trend in cell viability under medium-to-high concentrations, and different cell types exhibited varying degrees of tolerance to SPB.

[0085] In summary, the results of this embodiment demonstrate that, while maintaining similar STING protein degradation capabilities, SPP exhibits significantly better performance than SPB in terms of hemolytic reaction and cell compatibility with various normal cells, thus possessing superior biosafety characteristics.

[0086] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A polypeptide-type PROTAC molecule that targets and degrades STING protein, characterized in that, The polypeptide PROTAC molecule includes a STING targeting unit, a linker arm, and an E3 ubiquitin ligase ligand. The STING targeting unit includes chemical groups capable of specifically binding to the STING protein; The E3 ubiquitin ligand includes the VHL ligand.

2. The polypeptide-type PROTAC molecule according to claim 1, characterized in that, The connecting arm includes any one or a combination of several of the following: amide bond, ether bond, alkyl chain, and PEG unit.

3. The polypeptide-type PROTAC molecule according to claim 1, characterized in that, The structure of the STING targeting unit is shown in equation (Ⅰ): 。 4. The polypeptide-type PROTAC molecule according to claim 1, characterized in that, The structure of the VHL ligand is shown in formula (II): 。 5. The polypeptide-type PROTAC molecule according to claim 1, characterized in that, The structure of the polypeptide PROTAC molecule is shown in formula (Ⅲ): 。 6. The method for preparing the polypeptide-type PROTAC molecule according to claim 5, characterized in that, Includes the following steps: (1) VHL ligands were prepared by solid-phase polypeptide synthesis. (2) The STING targeting unit is linked to the N-terminus of the VHL ligand via amidation reaction to obtain a polypeptide PROTAC molecule.

7. The use of the polypeptide PROTAC molecule according to any one of claims 1 to 5 in the preparation of a medicament for the treatment and / or prevention of dry eye syndrome.

8. The application according to claim 7, characterized in that, The dry eye syndromes mentioned include chemically induced dry eye syndrome, hyperosmolar environment-induced dry eye syndrome, inflammation-related dry eye syndrome, and dry eye syndrome accompanied by corneal epithelial damage and goblet cell reduction.

9. A medicine for the prevention and / or treatment of dry eye syndrome, characterized in that, The drug comprises the polypeptide PROTAC molecule as described in any one of claims 1 to 5.

10. The medicament according to claim 9, characterized in that, The drug includes any one or a combination of ophthalmic eye drops, ophthalmic gels, ophthalmic sustained-release formulations, and ophthalmic nanodelivery systems.

Images