Polypeptide compound, medicine for preventing and treating eye diseases and application

Abstract

The invention relates to the technical field of biology, in particular to a polypeptide compound, a medicine for preventing and treating eye diseases and application. According to the present invention, a mitochondrial targeting module (triphenylphosphine) and a superoxide-initiated persulfide donor (SOPD) are integrated into a specific polypeptide to obtain the ROS-responsive mitochondrial targeting polypeptide compound; experiments show that the polypeptide compound has remarkable oxidative stress resistance, can effectively relieve RGC damage caused by intraocular hypertension, has a good prevention and treatment effect on glaucoma optic neuropathy, has the prevention and treatment effect obviously improved compared with that of other polypeptide compounds with similar structures, and has good biocompatibility and biological safety.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a polypeptide compound, a medicine for preventing and treating eye diseases and applications thereof. Background Art

[0002] Glaucoma is a degenerative optic nerve disease characterized by progressive optic atrophy and visual field loss, with elevated intraocular pressure as the main risk factor. Its exact pathogenesis has not yet been fully elucidated. Among the many pathological factors related to the pathogenesis of glaucomatous optic neuropathy, mitochondrial dysfunction and oxidative stress damage are closely related. Mitochondrial dysfunction and the accompanying oxidative stress, mainly manifested in excessive reactive oxygen species (ROS), are important links in the pathogenesis of glaucoma. Research on optic nerve protection strategies targeting mitochondria, relieving oxidative stress and improving respiratory chain function is of great significance and potential translational application value for the diagnosis and treatment of glaucomatous optic neuropathy. Hydrogen sulfide (H2S), as an important regulatory signal molecule in cells, has been shown to play an important regulatory role in maintaining the redox homeostasis of biological cells and the function of the mitochondrial electron transport respiratory chain. In recent years, the academic community has reported on the research results on the protective effect of exogenous H2S on the glaucomatous optic nerve. However, to date, research on the specific signal regulation role of H2S as a signal molecule in the pathogenesis of glaucomatous optic neuropathy and its neuroprotective mechanism is still relatively blank.

[0003] A major bottleneck factor that limits the further development of H2S research in the field of glaucoma is the lack of H2S donors that are safe, long-lasting, and have controllable release characteristics. Currently, the commonly used H2S donor is NaHS. The H2S component produced by this inorganic salt when dissolved in water is produced in a transient burst and evaporates within a few minutes. The actual concentration of the reagent and the target concentration in the intraocular tissue are difficult to accurately grasp. Recent studies on the content of H2S in the body under physiological conditions have shown that the actual content of H2S in mammals in the form of pure molecules (sub-μM level) is much lower than the previously reported level. As a signal molecule in the body, H2S plays a biological regulatory role in different scenarios in the form of different sulfur-containing compounds (sulfane sulfur "metabolic pool"), showing an active and unstable morphological switching mode, including hydrosulfide ions (HS-), sulfide ions (S2-), thiol compounds (RSH), persulfide (RSSH) and polysulfide [polysulfide, RS(S)nH], etc. Among them, persulfide can be converted to release H2S under physiological conditions, but it itself has more significant nucleophilicity and reducing ability than H2S molecules, showing a stronger ability to remove ROS and maintain intracellular redox homeostasis. Persulfide is a key intermediate and the main effector molecule in the H2S signal regulation mechanism. The cysteine ​​residues of specific proteins are modified by transsulfurization to achieve the regulation of the conformation and activity of the protein. Therefore, using persulfide donors instead of traditional H2S donors in the study of H2S signal regulation mechanism in the context of glaucomatous optic neuropathy can eliminate the interference introduced by the instability of the H2S molecule itself. At the same time, the stronger redox homeostasis maintenance ability and more efficient protein persulfurization modification of persulfide are expected to bring breakthroughs in the research field of optic nerve protection strategies for glaucomatous optic nerves. Summary of the invention

[0004] In view of this, the present invention provides a polypeptide compound, a drug for preventing and treating eye diseases and its application.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The polypeptide compound is a polypeptide modified with a triphenylphosphine group and a persulfide donor group; the polypeptide has an amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80% homology thereto.

[0007] Among them, the sequence shown in SEQ ID NO: 1 is: CFFEE.

[0008] In the present invention, the persulfide donor group has a structure shown in Formula I:

[0009]

[0010] Wherein, R is hydrogen, substituted or unsubstituted straight chain or branched C1-C14 alkyl;

[0011] The substituted substituents are selected from hydroxy, cyano, nitro, oxo, thioxo or halogen.

[0012] In some embodiments, the N-terminal cysteine ​​of the polypeptide compound is modified with a triphenylphosphine group and a persulfide donor group shown in Formula I.

[0013] In some embodiments, the persulfide donor group of Formula I forms a disulfide bond with the cysteine ​​of the polypeptide; and the carboxyl group of the triphenylphosphine group forms an amide bond with the amino group of the cysteine.

[0014] In some specific embodiments, the polypeptide compound has a structure shown in Formula II, or a stereoisomer thereof, or a pharmaceutically acceptable hydrate, solvate or salt thereof:

[0015]

[0016] The present invention adds a mitochondrial targeting module triphenylphosphine and a specific water-soluble group to a polypeptide persulfide sustained-release precursor SOPD-pep that responds to ROS (especially superoxide ions therein), and obtains a reactive oxygen responsive mitochondrial targeted delivery polypeptide persulfide precursor, which is named RPDP-Mito (ROS responsive persulfide donating peptides, targeting mitochondria) in the present invention. Experiments show that RPDP-Mito has high biocompatibility and utilization. Its innovative design realizes the focus of H2S signal regulation mechanism research on mitochondria at the organelle level, maximally excludes possible interference from other organelles, and uses ROS as a trigger switch, which not only caters to the characteristics of glaucomatous optic neuropathy with oxidative stress as an important pathological factor, but also avoids the toxic side effects caused by excessive release of persulfide or H2S, creating optimal conditions for the specific regulation mechanism research of H2S on the redox and mitochondrial homeostasis of retinal ganglion cells (RGCs) in the pathological process of glaucoma and neuroprotection.

[0017] The present invention also provides a method for preparing the polypeptide compound, comprising:

[0018] synthesizing a peptide segment shown in SEQ ID NO: 1 modified with TPP;

[0019] The peptide segment is reacted with a persulfide donor shown in formula I to obtain the polypeptide compound.

[0020] In some embodiments, the peptide CFFEE is prepared by solid phase synthesis. In some specific embodiments, the solid phase synthesis method is Fmoc solid phase synthesis. The present invention has no special restrictions on the synthesis strategy of Fmoc solid phase synthesis, which can be synthesized one by one or in sections after splitting. The present invention has no special restrictions on the specific synthesis steps, and the synthesis can be performed according to the common steps and parameters in the art.

[0021] The present invention also provides the use of the polypeptide compound in preparing medicines for preventing and treating eye diseases.

[0022] The eye disease includes at least one of glaucoma, diabetic retinopathy, ischemic optic neuropathy, etc.

[0023] The glaucoma includes high-tension glaucoma, normal-tension glaucoma or secondary glaucoma.

[0024] In the present invention, the prevention and treatment includes: improving the ability to resist oxidative stress and / or protecting retinal ganglion cells.

[0025] In the present invention, the improvement of the anti-oxidative stress ability includes inhibiting at least one of the ROS level, the hydrogen peroxide level, and the glutathione level, and / or improving the anti-oxidative ability.

[0026] In the present invention, the protection of retinal ganglion cells includes: improving the survival rate of retinal ganglion cells and / or reducing retinal ganglion cell damage, improving or alleviating visual function impairment.

[0027] The present invention also provides medicines for preventing and treating eye diseases, comprising the polypeptide compound of the present invention.

[0028] The medicine of the present invention also includes pharmaceutically acceptable excipients.

[0029] The drug of the present invention has dosage forms including eye drops, eye drops, eye ointments, eye gels, injections, oral preparations and implants.

[0030] The present invention also provides a method for preventing and treating eye diseases, comprising: administering the polypeptide compound or the medicine of the present invention.

[0031] The drug of the present invention can be used alone or in combination with other drugs. The combined use includes: using with other drugs together or separately, and the use method is not limited.

[0032] The present invention integrates a mitochondrial targeting module (i.e., triphenylphosphine) and a superoxide-triggered persulfide donor (SOPD) into a specific polypeptide to obtain a ROS-responsive mitochondrial-targeted polypeptide compound. Experiments show that the polypeptide compound has significant antioxidant stress resistance, can effectively reduce RGC damage caused by high intraocular pressure, has a good preventive and therapeutic effect on glaucomatous optic neuropathy, and has significantly improved preventive and therapeutic effects compared to other polypeptide compounds with similar structures, and has good biocompatibility and biosafety. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Figure 2 shows the characterization, physicochemical properties and persulfide release rate of RPDP-Mito; A is the molecular structure of RPDP-Mito. B is the molecular structure of RPDP-Cont. C is the mass spectrum of RPDP-Mito; D is the mass spectrum of RPDP-Cont; E is the analytical HPLC trace of RPDP-Mito; F is the analytical HPLC trace of RPDP-Cont; G is the UV-visible spectra of RPDP-Mito and RPDP-Cont at 20 μM; H is the release of H2S by RPDP-Mito within 55 minutes at 37°C;

[0034] Figure 2 Figures show the biosafety, superoxide reactivity, and mitochondria-targeted delivery results of RPDP-Mito. (A) CCK-8 assay evaluates the viability of R28 cells treated with different concentrations of RPDP-Mito. (B) Live / dead assay confirms that RPDP-Mito is not cytotoxic to R28 cells. (C, D) EdU detection and quantitative analysis show that RPDP-Mito does not significantly inhibit R28 cell proliferation. (EG) Confocal microscopy imaging of retinal cryosections after retrograde labeling with fluorescent gold to evaluate retinal layer thickness and RGCs counts, indicating that intravitreal injection of RPDP-Mito does not damage retinal structure or RGCs survival. (H) Immunofluorescence staining of mitochondria and H2S after treatment of R28 cells with BSO and various H2S donors illustrates the ROS reactivity and mitochondrial targeting ability of RPDP-Mito. The experiment was repeated three times, and the data are expressed as mean ± SEM (n = 3). Error bars represent mean ± SD (n = 3). (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001). Scale bars: 50 μm in B and E, 200 μm in C, 10 μm in H.

[0035] Figure 3The antioxidant capacity of RPDP-Mito was shown in Figure 2. A is the immunofluorescence staining of each group after BSO pretreatment. B is the detection result of H2O2 level of each group after BSO pretreatment. C is the quantification of GSH level of each group after BSO pretreatment. D is the measurement of T-AOC level of each group after BSO pretreatment. E is the ROS level of COH rats after treatment. F is the H2O2 level of COH rats after treatment. G is the GSH level of COH rats after treatment. H is the T-AOC level of COH rats after treatment. Data are expressed as mean ± SEM (n = 3). Error bars represent mean ± SD (n = 3). One-way ANOVA and Tukey post hoc test were used to determine statistical significance (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001); Scale bar: 20 μm in A.

[0036] Figure 4 The protective effect of RPDP-Mito on the optic nerve is shown; A is sham surgery + DMSO (average 10.52±2.20 mmHg), sham surgery + RPDP-Mito (average 10.17±2.01 mmHg), COH 2 weeks + DMSO (average 35.16±3.20 mmHg), COH Changes in intraocular pressure of rats in each group after 2 weeks of COH (average 34.90±2.99 mmHg); B and C are the results of RGC fluorescent gold staining and quantitative analysis, respectively; D is the F-VEP detection waveform of rats that received 2 weeks of COH and treated with DMSO or RPDP-Mito; E and F are the quantitative analysis results of P1 latency and amplitude of rats that received 2 weeks of COH and treated with DMSO or RPDP-Mito, respectively; data are expressed as mean±SEM (n=3); error bars represent mean±SD (n=3); one-way ANOVA and Tukey post hoc test were used to determine statistical significance (*p<0.05; **p<0.01; ***p<0.001; ****p<0.0001); scale bar: 100 μm in (B). DETAILED DESCRIPTION

[0037] The present invention provides a polypeptide compound, a drug for preventing and treating eye diseases, and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0038] The test materials used in the present invention are all common commercial products and can be purchased in the market.

[0039] In order to improve the defects of the current H2S donors, such as insufficient water solubility and poor cellular internalization efficiency, a reliable donor is provided for the mechanism study of H2S-related glaucomatous optic neuropathy. The applicant has previously designed a polypeptide-type persulfide sustained-release precursor SOPD-pep (PMID: 32592216) that responds to ROS (especially superoxide ions therein). The polypeptide-type material can be self-assembled into a supramolecular nanostructure, showing good biocompatibility and utilization. It triggers a self-cleavage reaction under ROS stress to release persulfide, and can play the role of maintaining redox homeostasis and protein persulfurization modification of persulfide while consuming superoxide ions as a response switch. In this polypeptide structure, the present invention further adds a mitochondrial targeting module triphenylphosphine and a water-soluble group, thereby obtaining a reactive oxygen-responsive mitochondrial targeted delivery polypeptide-type persulfide precursor RPDP-Mito (ROS responsive persulfide donating peptides, targeting mitochondria). RPDP-Mito has high biocompatibility and availability. Its innovative design allows us to focus the study of H2S signal regulation mechanisms on mitochondria at the organelle level, excluding possible interference from other organelles to the greatest extent, and using ROS as a trigger switch. It not only caters to the characteristics of glaucomatous optic neuropathy with oxidative stress as an important pathological factor, but also avoids the toxic side effects caused by excessive release of persulfide or H2S, creating optimal conditions for studying the specific regulatory mechanisms of H2S on the redox and mitochondrial homeostasis of retinal ganglion cells (RGCs) in the pathological process of glaucoma and its neuroprotective effects.

[0040] The present invention will be further described below in conjunction with embodiments:

[0041] Example 1 Characterization, physicochemical properties and persulfide release rate of RPDP-Mito

[0042] To achieve controlled release of subcellular persulfide / H2S, we developed RPDP-Mito by incorporating a mitochondrial targeting module (i.e., triphenylphosphine) and a superoxide-triggered persulfide donor (SOPD) into the peptide.

[0043] The specific preparation process is as follows:

[0044] Preparation of the polypeptide RPDP-Mito of the present invention: TPP-CFFEE (TPP = triphenylphosphine group) was synthesized by Fmoc solid-phase peptide synthesis (SPPS) method. After SOPD was synthesized according to the reported scheme (PMID: 32592216), TPP-CFFEE was treated with disulfide-functionalized SOPD to obtain the target peptide conjugate RPDP-Mito ( Figure 1 A).

[0045] Preparation of the control peptide RPDP-Cont: The peptide of CFFEE conjugated to a diphenylphosphonate group was synthesized by Fmoc solid-phase peptide synthesis (SPPS) method. After SOPD was synthesized according to the reported protocol, TPP-CFFEE (PMID: 32592216) was treated with disulfide functionalized SOPD, and the other peptide, referred to as RPDP-Cont, was able to scavenge superoxide anions but lacked the ability to release persulfide / H2S ( Figure 1 B).

[0046] The two peptides prepared above were purified by reverse phase HPLC. Then, they were purified by mass spectrometry ( Figure 1 C, D) and analytical HPLC ( Figure 1 E, F) Confirmation of purity and expected molecular weight.

[0047] In addition, we verified the presence of TPP in RPDP-Mito and control groups by UV-visible spectrophotometry. The characteristic absorption peak of TPP between 260 and 280 nm was observed in both groups, verifying that TPP had been successfully modified onto both peptides and was able to target mitochondria ( Figure 1 G) Based on SOPD 2·- To investigate the responsiveness of RPDP-Mito, we mixed 500 μM RPDP-Mito with O at 37 °C. 2·- The release of H2S was detected by incubating with RPDP-Mito and monitoring the fluorescence increment of the H2S selective fluorescence probe WSP-5 every 5 minutes at 502 / 525 nm. The results showed that RPDP-Mito could achieve controlled and sustainable release of H2S, reaching a peak release at around 55 minutes ( Figure 1 H).

[0048] Example 2 Biosafety, ROS responsiveness and mitochondrial targeted delivery performance of RPDP-Mito

[0049] To evaluate the biocompatibility and biosafety of RPDP-Mito, we performed CCK-8, EdU, and live / dead cell assays on R28 cells. Figure 2 As shown in Figure 2, RPDP-Mito showed almost no cytotoxicity at concentrations less than 1000 μM, and only had significant cytotoxic effects at concentrations of 2000 μM or higher ( Figure 2 A). We selected 200 μM as the effective concentration for further EdU and live / dead cell assays, confirming that there was no significant toxicity after 24 h of incubation ( Figure 2 BD). For in vivo biosafety assessment, we injected RPDP-Mito into the vitreous cavity of SD rats. One week later, retinal tissues were collected for histological analysis. Examination of retinal structure (including layer thickness and ganglion cell counts) showed that there was no significant difference in the thickness of retinal layers and RGCs counts between the vehicle solution group (DMSO) and the RPDP-Mito-treated group compared with the control group ( Figure 2 EG), indicating that intravitreal injection of RPDP-Mito does not damage the retinal structure or RGCs survival rate. The results show that at a specific concentration, RPDP-Mito is harmless to the rat retina.

[0050] Based on this biosafety, we further evaluated the ROS responsiveness and mitochondrial targeting ability of RPDP-Mito. R28 cells were subjected to oxidative stress treatment using BSO to increase ROS levels, and then treated with RPDP-Mito and traditional H2S donors (NaHS and GYY4137) and monitored H2S release. The results showed that compared with the control + RPDP-Mito group, the H2S release in the BSO + RPDP-Mito group was significantly increased, accompanied by obvious H2S staining (green fluorescence) and mitochondrial colocalization. In contrast, the BSO + GYY4137 group and the BSO + NaHS group released less H2S, and no mitochondrial colocalization was observed ( Figure 2 These findings suggest that RPDP-Mito effectively exhibits ROS-responsive H2S release and can target mitochondria.

[0051] Example 3 Antioxidative stress ability and RGC protective effect of RPDP-Mito

[0052] To evaluate the antioxidant capacity of RPDP-Mito, we treated BSO-pretreated R28 cells with RPDP-Mito, other common H2S donors (NaHS and GYY4137). We evaluated intracellular ROS, hydrogen peroxide (H2O2) levels, glutathione (GSH) levels, and total cellular antioxidant capacity (T-AOC). Immunofluorescence staining results showed that ROS levels were significantly increased in BSO-treated cells compared with the control group. RPDP-Mito significantly inhibited the BSO-induced increase in ROS, and the effect was the most obvious, followed by NaHS and GYY4137 ( Figure 3 (A). H2O2 assay showed that H2O2 levels were significantly increased after BSO treatment, while RPDP-Mito significantly reduced H2O2 levels, while GYY4137 and NaHS had no significant effect on H2O2 levels ( Figure 3 Middle B). RPDP-Mito and GYY4137 restored the GSH level significantly reduced by BSO, while NaHS had no effect ( Figure 3 In addition, total antioxidant capacity (T-AOC) analysis showed that T-AOC increased slightly after BSO treatment, but not significantly, indicating partial activation of the antioxidant response. Compared with NaHS, RPDP-Mito and GYY4137 significantly enhanced T-AOC ( Figure 3 (middle D).

[0053] In chronic ocular hypertension (COH) rats, the effect of RPDP-Mito on retinal antioxidant capacity was evaluated by intravitreal injection. Two weeks after COH modeling, the level of ROS in the retina of rats increased, and RPDP-Mito significantly inhibited the increase in the level. GYY4137 also reduced ROS, while NaHS had no significant effect on this, which means that continuous H2S release is essential for better ROS inhibition ( Figure 3 This trend is consistent with the H2O2 detection results ( Figure 3 Middle F). Retinal GSH levels were reduced by elevated intraocular pressure, which was restored by RPDP-Mito, partially restored by GYY4137, and ineffective by NaHS ( Figure 3 Middle G). Retinal T-AOC decreased two weeks after COH modeling. Unlike the results of cells, the antioxidant capacity of the retina increased in the early stage of modeling and decreased with the extension of oxidative damage time. In addition, RPDP-Mito significantly increased retinal T-AOC, and GYY4137 injection showed some improvement, but the effect was poor. NaHS did not enhance T-AOC ( Figure 3 Middle H).

[0054] We further investigated the protective effect of RPDP-Mito on retinal ganglion cells (RGCs) in the COH rat model. Two weeks later, the survival rate of retinal ganglion cells (RGCs) was assessed by retrograde fluorescent gold labeling in the superior colliculus. Compared with the sham-operated group, COH rats showed a significant decrease in RGCs, while the RGC survival rate increased after RPDP-Mito administration ( Figure 4 B, C), indicating that it can effectively reduce RGC damage caused by high intraocular pressure. In addition, we evaluated the visual function of COH rats. F-VEP analysis showed that the P1 wave latency was prolonged and the amplitude was reduced in COH rats, indicating impaired visual function. Compared with the DMSO group, RPDP-Mito treatment reduced the P1 wave latency and increased the P1 wave amplitude, indicating that the pressure-induced visual function impairment was alleviated ( Figure 4 D in F).

[0055] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A polypeptide compound, characterized in that: The invention is a polypeptide modified with a triphenylphosphine group and a persulfide donor group; the polypeptide has an amino acid sequence shown in SEQ ID NO: 1 or a sequence having at least 80% homology thereto.

2. The polypeptide compound according to claim 1, characterized in that The persulfide donor group has the structure shown in Formula I: Wherein, R is hydrogen, substituted or unsubstituted straight chain or branched C1-C14 alkyl; The substituted substituents are selected from hydroxy, cyano, nitro, oxo, thioxo or halogen.

3. The polypeptide compound according to claim 1 or 2, characterized in that: The cysteine ​​at the N-terminus is modified with a triphenylphosphine group and a persulfide donor group shown in formula I.

4. The polypeptide according to any one of claims 1 to 3, characterized in that The persulfide donor group shown in formula I forms a disulfide bond with the cysteine ​​of the polypeptide; the carboxyl group of the triphenylphosphine group forms an amide bond with the amino group of the cysteine.

5. The polypeptide compound according to any one of claims 1 to 4, characterized in that: It has the structure shown in Formula II, or its stereoisomers, or its pharmaceutically acceptable hydrates, solvates or salts:

6. The method for preparing a polypeptide compound according to any one of claims 1 to 5, characterized in that: include: synthesizing a peptide segment shown in SEQ ID NO: 1 modified with TPP; The peptide segment is reacted with a persulfide donor shown in formula I to obtain the polypeptide compound.

7. Use of the polypeptide compound according to any one of claims 1 to 5 in the preparation of drugs for preventing and treating eye diseases.

8. The use according to claim 7, characterized in that: The eye disease includes at least one of glaucoma, diabetic retinopathy, and ischemic optic neuropathy; the glaucoma includes high-tension glaucoma, normal-tension glaucoma, or secondary glaucoma.

9. The use according to claim 7, characterized in that: The prevention and treatment include: improving the ability to resist oxidative stress and / or protecting retinal ganglion cells; The improving the anti-oxidative stress ability includes inhibiting at least one of the ROS level, the hydrogen peroxide level, and the glutathione level, and / or improving the anti-oxidative ability; The protecting of retinal ganglion cells includes: increasing the survival rate of retinal ganglion cells and / or reducing the damage of retinal ganglion cells, and improving or alleviating the impairment of visual function.

10. A drug for preventing and treating eye diseases, characterized in that: It includes the polypeptide compound according to any one of claims 1 to 5.

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