An enzyme-responsive peptide-conjugated drug targeting ACE2, its preparation method and application

Abstract

This invention belongs to the field of biomedical technology and relates to an enzyme-responsive peptide-conjugated drug targeting ACE2, its preparation method, and its application. The peptide-conjugated drug consists of dexamethasone and an ACE2-targeting peptide covalently linked by an enzyme-responsive linker; wherein the enzyme-responsive linker contains any one or more of carboxylic acid ester bonds, thioester bonds, or phosphate ester bonds; the amino acid sequence of the ACE2-targeting peptide is YKYRYL. The peptide-conjugated drug provided by this invention not only forms nanoparticles, avoiding rapid degradation, but also ensures specific binding to ACE2, thereby actively accumulating at lesions such as inflammatory tissues with high ACE2 expression. After accumulation, it specifically releases active dexamethasone under enzymatic action, significantly reducing the systemic side effects of dexamethasone while maintaining anti-inflammatory efficacy, providing a safe, effective, and universal platform for the precision treatment of inflammatory diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to an enzyme-responsive polypeptide-conjugated drug targeting angiotensin-converting enzyme 2 (ACE2), its preparation method, and its application. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Dexamethasone, a classic glucocorticoid, is widely used as an adjunct therapy for various inflammatory diseases, autoimmune diseases, and some cancers due to its potent anti-inflammatory and immunosuppressive effects. However, long-term or high-dose use can cause serious systemic side effects, including glucose metabolism disorders, bone metabolism abnormalities, immunosuppression, sodium and water retention, and hypothalamic-pituitary-adrenal axis suppression, significantly impacting patient treatment adherence and quality of life. To reduce its systemic toxicity, researchers have explored targeted delivery strategies such as liposomes, nanoparticle encapsulation, or conjugation with antibodies and peptides. While these strategies have achieved some degree of tissue enrichment of the drug, they still cannot prevent premature release or systemic exposure of the drug before reaching the target tissue. Therefore, developing a precise delivery system that can both actively enrich the drug at the lesion site and specifically release the active drug at the lesion site is of significant clinical importance.

[0004] Angiotensin-converting enzyme 2 (ACE2) is a transmembrane receptor protein widely distributed on the surface of cells in tissues such as the lungs, intestines, kidneys, and heart. Under physiological conditions, it exerts anti-inflammatory, antioxidant, and vasodilatory effects by degrading angiotensin II. Under pathological conditions, it is a major receptor for pathogens such as the novel coronavirus to invade cells. Studies have shown that ACE2 expression levels are significantly upregulated in inflamed tissues such as the lungs of patients with pneumonia caused by the novel coronavirus and the intestinal mucosa of patients with ulcerative colitis, making it an ideal tissue-specific target for inflammation. Conjugating ACE2-targeting peptides with dexamethasone can construct peptide-conjugated drugs with both active targeting and site-directed release functions, potentially significantly reducing the systemic side effects of dexamethasone while maintaining anti-inflammatory efficacy. However, peptides are easily degraded by proteases in vivo. Simply conjugating ACE2-targeting peptides with dexamethasone will result in the peptide being degraded in vivo, leading to a lack of targeting function and making it difficult to reduce the systemic side effects of dexamethasone. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an enzyme-responsive peptide-conjugated drug targeting ACE2, its preparation method, and its application. The peptide-conjugated drug provided by the present invention can not only form nanoparticles to avoid rapid degradation, but also ensure specific binding with ACE2, thereby actively accumulating in inflamed tissues with high ACE2 expression. After accumulation, it can specifically release active dexamethasone under the action of enzymes, significantly reducing the systemic side effects of dexamethasone while maintaining anti-inflammatory efficacy.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows: In the first aspect, there is an enzyme-responsive peptide-conjugated drug targeting ACE2, consisting of dexamethasone and an ACE2-targeting peptide covalently linked by an enzyme-responsive linker. The enzyme-responsive linker contains one or more of carboxylic acid ester bonds, thioester bonds, or phosphate ester bonds; the amino acid sequence of the ACE2 targeting peptide is Tyr-Lys-Tyr-Arg-Tyr-Leu (YKYRYL), as shown in SEQ ID NO: 1.

[0007] This invention utilizes YKYRYL as an ACE2-targeting peptide to conjugate dexamethasone, forming a peptide-conjugated drug. This peptide-conjugated drug can self-assemble into nanoparticles with a particle size of 30-200 nm. Firstly, these nanoparticles consist of a hydrophilic shell and a hydrophobic core, encapsulating dexamethasone within the core. The small particle size (30-200 nm) significantly reduces contact with degrading factors such as proteases and hydrolases in body fluids, preventing degradation and improving the stability of the peptide-conjugated drug. Secondly, the nanoparticles maintain their ACE2-targeting specificity, allowing the peptide-conjugated drug to increase its residence time at the lesion site through specific binding and accumulate there. This increases the contact of esterases, thioesterases, or phosphatases at the lesion site, leading to the breakage of carboxylic acid ester bonds, thioester bonds, or phosphate ester bonds, and transforming the nanoparticles into nanofiber structures. This enables the specific release of dexamethasone at the lesion site, achieving anti-inflammatory efficacy and avoiding systemic side effects.

[0008] In a second aspect, a method for preparing the polypeptide-conjugated drug described in the first aspect of the present invention includes the following steps: The compound represented by Formula II was obtained by acylation of dexamethasone with succinic anhydride. ; ACE2-targeting peptides were prepared using a solid-phase Fmoc synthesis method. The compound shown in Formula II was subjected to an amidation reaction with the ACE2-targeting peptide.

[0009] Thirdly, a pharmaceutical composition comprising the polypeptide-conjugated drug and pharmaceutical excipients described in the first aspect of the present invention.

[0010] Since the therapeutic effect of peptide-conjugated drugs mainly depends on dexamethasone, which not only has the effect of treating inflammation but also has the effect of adjuvant treatment of tumors, and tumor tissue also has high expression of ACE2, that is, it can also accumulate and release dexamethasone in tumor tissue, thereby achieving the effect of adjuvant treatment of tumors, the fourth aspect of the present invention provides the use of the peptide-conjugated drug of the first aspect of the present invention or the pharmaceutical composition of the third aspect of the present invention in the preparation of drugs for treating inflammatory diseases, treating viral diseases, or anti-tumor.

[0011] The beneficial effects of this invention are as follows: The present invention utilizes short amino acid peptides with high biocompatibility to construct an enzyme-responsive peptide-conjugated drug targeting ACE2. The preparation process is simple, with good responsiveness and high biosafety. The enzyme-responsive peptide-conjugated drug targeting ACE2 provided by the present invention can be hydrolyzed in response to highly expressed carboxylesterase at the lesion site to release the active drug dexamethasone at a specific site. The hydrated nanoparticles with a diameter of 30-200 nm are transformed into nanofiber structures with a diameter of 10-100 nm.

[0012] The enzyme-responsive peptide-conjugated drug targeting ACE2 provided by this invention releases active dexamethasone on demand via carboxylesterase-responsive bond breaking, while retaining the active navigation capability of the ACE2-targeting peptide. It has significant advantages in precisely enriching itself in inflammatory or tumor lesions and reducing the systemic toxic side effects of hormones, thereby achieving the therapeutic goals of anti-inflammatory, antiviral and synergistic anti-tumor treatment. It provides a novel drug design strategy for the precision treatment of inflammatory diseases, COVID-19 infection and tumors with high carboxylesterase expression. Attached Figure Description

[0013] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0014] Figure 1 The 1H NMR spectrum of DS-pep, an enzyme-responsive peptide-conjugated drug targeting ACE2 prepared in this embodiment of the invention; Figure 2 Electrospray mass spectra of DS-pep, an enzyme-responsive peptide-conjugated drug targeting ACE2 prepared in an embodiment of the present invention; Figure 3 Electron transmission microscope image of the enzyme-responsive peptide-conjugated drug DS-pep targeting ACE2, prepared for an embodiment of the present invention, self-assembling into nanoparticles. Figure 4The particle size distribution of DS-pep, an enzyme-responsive peptide-conjugated drug targeting ACE2 prepared in an embodiment of the present invention, is shown in the figure. Figure 5 The images are electron transmission microscopy images of different concentrations of the enzyme-responsive peptide-conjugated drug DS-pep targeting ACE2 after incubation with carboxylesterase for 12 h, (a) 5 μM, (b) 200 μM. Figure 6 The release curves of dexamethasone drug prepared by the present invention, which is an enzyme-responsive peptide-conjugated drug targeting ACE2, DS-pep, after incubation with esterase at different pH for 72 h. Figure 7 The binding affinity between the enzyme-responsive peptide-conjugated drug DS-pep, which targets ACE2 and is prepared according to an embodiment of the present invention, and recombinant human ACE2 protein. Detailed Implementation

[0015] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0016] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0017] Given that long-term or high-dose use of dexamethasone can cause serious systemic side effects, this invention proposes an enzyme-responsive peptide-conjugated drug targeting ACE2, its preparation method, and its application.

[0018] In one typical embodiment of the present invention, an enzyme-responsive peptide-conjugated drug targeting ACE2 is provided, wherein dexamethasone and an ACE2-targeting peptide are covalently linked by an enzyme-responsive linker. The enzyme-responsive linker contains one or more of carboxylic acid ester bonds, thioester bonds, or phosphate ester bonds; the amino acid sequence of the ACE2 targeting peptide is YKYRYL, as shown in SEQ ID NO: 1.

[0019] The enzyme-responsive peptide-conjugated drug targeting ACE2 can be hydrolyzed in response to esterases, thioesterases, and phosphoesterases highly expressed at the lesion site, releasing active dexamethasone and achieving targeted drug release. The conjugated drug remains stable in the bloodstream and is actively enriched in inflammatory or tumor tissues with high ACE2 expression via the YKYRYL hexapeptide, thereby reducing the systemic side effects of dexamethasone.

[0020] In some embodiments, the enzyme-responsive linker contains a carboxylic acid ester bond. Carboxylesterases (CES) are hydrolases widely present in mammalian tissues, and their expression levels are significantly elevated in various inflammatory diseases (such as ulcerative colitis) and some solid tumors (such as head and neck squamous cell carcinoma and colorectal cancer). When the enzyme-responsive linker contains a carboxylic acid ester bond, the highly expressed CES at the lesion site can specifically hydrolyze the carboxylic acid ester bond, thereby facilitating the release of dexamethasone from the peptide-conjugated drug at the lesion site. Specifically, the enzyme-responsive linker is formed from a dicarboxylic acid or a dicarboxylic acid derivative, with one end connected to the hydroxyl group of dexamethasone by forming an ester group, and the other end connected to the amino group of the ACE2 targeting peptide by forming a peptide bond (or amide bond). The dicarboxylic acid can be oxalic acid, malonic acid, succinic acid, glutaric acid, etc. The dicarboxylic acid derivative can be an anhydride of a dicarboxylic acid. More specifically, the enzyme-responsive linker is formed from succinic acid or a succinic acid derivative. The succinic acid derivative can be succinic anhydride.

[0021] In some embodiments, the enzyme-responsive peptide-conjugated drug targeting ACE2 has the structure shown in Formula I: .

[0022] Another embodiment of the present invention provides a method for preparing the above-mentioned polypeptide-conjugated drug, comprising the following steps: The compound represented by Formula II was obtained by acylation of dexamethasone with succinic anhydride. ; ACE2-targeting peptides were prepared using a solid-phase Fmoc synthesis method. The compound shown in Formula II was subjected to an amidation reaction with the ACE2-targeting peptide.

[0023] In some embodiments, the specific steps are as follows: Step 1: Dexamethasone is dissolved in the first solvent and then contacted with succinic anhydride and an acylation catalyst. The reaction is carried out at a certain temperature to obtain the compound shown in Formula II: Step 2: ACE2-targeting peptides were prepared using the standard solid-phase Fmoc synthesis method; wherein, the compound shown in Formula II was covalently linked as the last compound, and after separation and purification, the compound shown in Formula I was obtained; Step 3: Dissolve the compound shown in Formula I under room temperature ultrasound, and after dissolving it, allow it to stand under ultrasound to obtain the enzyme-responsive peptide-conjugated drug targeting ACE2.

[0024] Specifically, in step one, the first solvent is one or more of pyridine, acetone, acetonitrile, methanol, and ethanol, preferably a solvent containing only pyridine; the contact temperature is 20~45℃, for example 20℃, 25℃, 28℃, 30℃, 35℃, 38℃, 40℃, or 45℃; the contact time is 18~38 h, for example 18 h, 20 h, 25 h, 28 h, 30 h, 35 h, or 38 h. h; the acylation catalyst is 4-dimethylaminopyridine; the molar ratio of the active drug dexamethasone to succinic anhydride is 1:1, 1:1.1, 1:1.25, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4; the molar ratio of the active drug dexamethasone to the acylation catalyst is 1:0.1, 1:0.15, 1:0.2, 1:0.25, 1:0.3, 1:0.35, 1:0.4, 1:0.45, 1:0.5; when the active drug dexamethasone is in contact with succinic anhydride and the acylation catalyst, an organic base solvent is used to provide an alkaline environment, such as triethylamine or N,N-diisopropylethylamine; Specifically, in step two, the standard solid-phase Fmoc synthesis method is as follows: the resin swelling solvent used is ultra-dry dichloromethane (DCM), the reaction contact solvent is ultra-dry N,N-dimethylformamide (DMF), the Fmoc deprotection solvent is 20% piperidine-DMF solution, the resin cutting solvent is trifluoroacetic acid:water = 95:5, and the crude purification precipitation solvent is ice-pre-cooled anhydrous diethyl ether. Specifically, in the standard solid-phase Fmoc synthesis method, the coupling time for each amino acid, aromatic group, or hydrophobic protecting group is 30-120 min, and the coupling temperature is 20-35℃; the Fmoc deprotection time is 20-45 min; the temperature is 20-35℃; the resin cutting time is 1.5-2.5 h; before and after coupling each amino acid, aromatic group, or hydrophobic protecting group, the resin needs to be washed 3-5 times with ultra-dry DMF solvent, with each wash using a solvent volume of 5-10 mL. Before and after coupling, TLC (thin-layer chromatography) sampling is required to confirm that the resin has been completely cleaned. Specifically, in step two, the standard solid-phase Fmoc synthesis method is as follows: the amino acid sequence of the ACE2 targeting peptide is synthesized as Tyr-Lys-Tyr-Arg-Tyr-Leu (YKYRYL). The carboxyl terminus of the Leu amino acid protected by Fmoc or other side chain protecting groups is coupled in the following order: Leu-Tyr-Arg-Tyr-Lys-Tyr. Finally, the compound shown in Formula II is covalently linked as the last compound. After separation and purification, the final product DS-pep shown in Formula I is obtained. Specifically, in step three, the concentration of the aqueous solution of the compound represented by Formula I is 1~2000 µM, preferably 20~500 µM; the solution to which the compound is dissolved is one of aqueous solution, PBS solution and physiological saline solution; the sonication time is 5~30 min; The reaction formula for synthesizing DS-pep molecules using 4-dimethylaminopyridine (4-DMAP) as an acylation catalyst, succinic anhydride as a condensing agent, and N,N-diisopropylethylamine (DIPEA) as an organic base is shown below: ; Specifically, in step three, the concentration of the aqueous solution of the compound represented by formula I is 1~2000 µM; The solution to be dissolved is one of the following: aqueous solution, PBS solution, and physiological saline solution, with aqueous solution being the most preferred; the sonication time is 5-30 min.

[0025] A third embodiment of the present invention provides a pharmaceutical composition comprising the above-mentioned polypeptide-conjugated drug and pharmaceutical excipients.

[0026] In some embodiments, the pharmaceutical excipient is selected from one or more of diluents, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants. The pharmaceutical excipient may constitute 1-98% by mass in the pharmaceutical composition, typically approximately 80%.

[0027] In some embodiments, the dosage form is tablet, capsule, granule, powder for injection, oral liquid or injection, etc.

[0028] A fourth embodiment of the present invention provides the use of the above-mentioned polypeptide-conjugated drug or pharmaceutical composition in the preparation of a drug for treating inflammatory diseases, treating viral diseases, or fighting tumors.

[0029] In some embodiments, the drug is administered orally, intravenously, via nasal drops, or via spray.

[0030] In some embodiments, the inflammatory disease is a lung inflammatory disease, an intestinal inflammatory disease, a cardiovascular disease, or a kidney inflammatory disease, etc.

[0031] In some embodiments, viral diseases are respiratory diseases such as pneumonia caused by the novel coronavirus.

[0032] In some embodiments, the tumor is a solid tumor that specifically highly expresses carboxylesterase, including small cell lung cancer, pancreatic cancer, liver cancer, colorectal cancer, or nasopharyngeal carcinoma.

[0033] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0034] Example 1 Synthesis of Dex-SA: Dexamethasone (785 mg, 2 mmol) was dissolved in 30 mL of pyridine, and succinic anhydride (300 mg, 3 mmol), 4-DMAP (24.4 mg, 0.2 mmol), and DIPEA (2 mL) were added. The mixture was stirred at 40 °C for 24 h. After the reaction was completed, the solid product was collected by filtration to obtain the compound Dex-SA represented by Formula II.

[0035] The Dex-SA NMR information is as follows: 1 H NMR (400 MHz, DMSO-d6) δ 12.23 (s, 1H), 7.29 (d, J = 10.1 Hz, 1H), 6.23 (dd, J = 10.1, 1.9 Hz, 1H), 6.01 (d, J = 1.6 Hz,1H), 5.41 (dd, J = 4.9, 1.5 Hz, 1H), 5.16 (s, 1H), 5.05 (d, J = 17.6 Hz, 1H), 4.80 (d, J = 17.6 Hz, 1H), 4.18 – 4.11 (m, 1H), 2.88 (ddd, J = 11.3, 7.4, 4.2Hz, 1H), 2.68 – 2.55 (m, 3H), 2.34 (d, J = 5.4 Hz, 1H), 2.33 – 2.26 (m, 1H), 2.21 – 2.06 (m, 2H), 1.81 – 1.73 (m, 1H), 1.70 – 1.60 (m, 1H), 1.60 – 1.52(m, 1H), 1.49 (s, 3H), 1.35 (qd, J = 12.9, 5.1 Hz, 1H), 1.07 (ddd, J = 12.2,8.2, 4.2 Hz, 1H), 0.88 (s, 3H), 0.79 (d, J = 7.2 Hz, 3H). Example 2 Synthesis of DS-pep: Tyr-Lys-Tyr-Arg-Tyr-Leu (YKYRYL) was synthesized from the C-terminus using a standard solid-phase peptide synthesis method. An appropriate amount of 2-chloro-triphenylmethyl chloride resin was weighed into a peptide solid-phase synthesis tube, and anhydrous DCM was added for activation. The resin was bubbled and stirred thoroughly twice under nitrogen atmosphere, and then washed clean with anhydrous DMF. Subsequently, an appropriate amount of Fmoc-L-Leu-OH was weighed into a centrifuge tube, dissolved in a small amount of anhydrous DMF, and then DIPEA was added. The dissolved Fmoc-L-Leu-OH solution was transferred to the synthesis tube, and the reaction was carried out under nitrogen atmosphere for 30 min. After the reaction, unattached amino acids were washed away with anhydrous DMF. A mixed solvent of DCM:MeOH:DIPEA = 16:3:1 was added, and the reaction was carried out twice under nitrogen atmosphere for 10 min each time. The resin was then washed clean with anhydrous DMF. Add 10 mL of 20% piperidine-anhydrous DMF solution to the synthesis tube. After reacting for 30 min under nitrogen stirring, wash away the removed Fmoc protecting group with anhydrous DMF solution. Then, weigh appropriate amounts of Fmoc-L-Tyr(tBu)-OH and HBTU into a centrifuge tube, dissolve them in a small amount of anhydrous DMF, add DIPEA, transfer the dissolved Fmoc-L-Tyr(tBu)-OH solution to the synthesis tube, react under nitrogen stirring for 30 min, wash the resin with anhydrous DMF, and check with a TLC plate until clean. Repeat the above Fmoc removal steps. Other amino acids are coupled to the resin successively according to the same procedure. Finally, Dex-SA is covalently coupled to the target peptide as the last amino acid. Add TFA solution to the synthesis tube, react for 2 h under nitrogen stirring, dry with a vacuum pump, and wash the resin successively with small amounts of TFA / MeOH until the extracted liquid is colorless. The collected product was blown to near dryness with nitrogen, precipitated in a large amount of ice-cold diethyl ether, filtered through two layers of filter paper on a sintered core funnel, the product was collected and separated and purified by high performance liquid chromatography (HPLC) to obtain the final product DS-pep shown in Formula I.

[0036] The DS-PEP NMR information is as follows: 1H NMR (400 MHz, DMSO-d6) δ 9.99 (s, 2H), 9.22 (s,2H), 8.37 (s, 3H), 8.12 – 7.93 (m, 11H), 7.79 (d, J = 8.2 Hz, 3H), 7.31 (d, J= 10.1 Hz, 4H), 7.17 (d, J = 5.9 Hz, 3H), 6.99 (d, J = 8.4 Hz, 6H), 6.88 (dd,J = 13.0, 8.3 Hz, 8H), 6.61 (ddd, J = 14.1, 8.5, 4.2 Hz, 11H), 6.23 (dd, J =10.1, 1.9 Hz, 4H), 6.02 (s, 3H), 5.43 (s, 2H), 5.17 (s, 2H), 5.06 (d, J =17.6 Hz, 4H), 4.78 (d, J = 17.6 Hz, 4H), 4.49 – 4.35 (m, 8H), 4.23 (dd, J =15.7, 9.8 Hz, 6H), 4.15 (d, J = 10.9 Hz, 3H), 3.98 (q, J = 7.8 Hz, 4H), 3.91(q, J = 5.3 Hz, 4H), 3.52 (s, 2H), 3.03 (s, 2H), 3.00 (d, J = 5.7 Hz, 4H),2.90 – 2.81 (m, 1.80 (s,2H), 1.77 (d, J = 6.3 Hz, 3H), 1.65 (t, J = 11.8 Hz, 5H), 1.55 (d, J = 14.0Hz, 5H), 1.49 (s, 8H), 1.36 (t, J = 7.4 Hz, 8H), 1.24 (s, 3H), 1.17 – 1.02(m, 8H), 0.91 – 0.76 (m, 21H). (e.g.) Figure 1 As shown.

[0037] The mass spectrometry information is as follows: MS(ESI)(m / z): C71H95N10O17calcd.1378.69; found1379.6971[M+1] + ,like Figure 2 As shown.

[0038] Example 3 Preparation procedure for the self-assembly of DS-pep, an enzyme-responsive peptide-conjugated drug targeting ACE2, into nanoparticles: A 50 µM DS-pep solution was obtained by dissolving the ACE2-targeting enzyme-responsive peptide conjugate DS-pep prepared in Example 2 in water three times. This solution was then sonicated and allowed to stand overnight at room temperature to obtain DS-pep nanoparticles.

[0039] Figure 3 The electron microscopy morphology of DS-pep, an enzyme-responsive peptide-coupled drug targeting ACE2, after self-assembly, is shown as nanoparticles with a morphology of 30–200 nm. Figure 4 The results show that when the DS-pep solution concentration is 50 µM, the hydrated particle size of the self-assembled nanoparticles is approximately 77.4 nm.

[0040] Example 4 Morphological changes of the same concentration of ACE2-targeting enzyme-responsive peptide conjugate DS-pep after 12 h of incubation with carboxylesterase: The morphological transformation of DS-pep nanoparticles prepared according to Example 3 under the action of carboxylesterase was investigated. DS-pep was prepared into 5 µM and 200 µM aqueous solutions, respectively, and porcine liver esterase (5 U / mL) was added and incubated at 37 °C for 12 h. The incubated samples were dropped onto a copper grid, and the morphology was observed by transmission electron microscopy after negative staining.

[0041] Figure 5 The morphological changes of different concentrations of enzyme-responsive peptide-conjugated drugs targeting ACE2 after incubation with carboxylesterase for 12 h are shown. According to the transmission electron microscopy results, the DS-pep nanoparticles prepared in Example 3 of this invention transform from nanoparticles to nanofiber structures under the action of esterase, and the fiber network formed in the high concentration group (200 µM) is more dense.

[0042] Example 5 The cumulative in vitro release rate of dexamethasone from DS-pep, an enzyme-responsive peptide conjugate targeting ACE2, was determined by high-performance liquid chromatography (HPLC) under different pH conditions (7.4, 6.5, 5.0) and in the presence of carboxylesterase (CES) to verify the enzyme-responsive bond-breaking release characteristics of DS-pep. The specific steps included: A1: Sample Preparation pH 7.4: 0.1 M phosphate-buffered saline (PBS); pH 6.5: 0.1 M MES buffer; pH 5.0: 0.1 M acetate-sodium acetate buffer; Carboxylesterase: derived from pig liver, enzyme activity ≥ 15 U / mg.

[0043] A2: Dissolve the DS-pep solution in each of the three buffer solutions mentioned above to prepare solutions with a final concentration of 50 µM. Divide each sample into two portions: one portion contains carboxylesterase (final concentration 5 U / mL), and the other portion contains an equal volume of buffer as an enzyme-free control. Incubate the samples in a 37°C water bath in the dark for 72 h. At time points of 0, 2, 4, 8, 12, 24, 48, and 72 h, take 100 µL of the incubation solution, add 200 µL of glacial acetonitrile (containing 0.1% formic acid) to terminate the reaction and precipitate the protein. Vortex for 1 min, centrifuge at 12,000 rpm for 10 min at 4°C, and filter the supernatant through a 0.22 μm filter membrane for HPLC analysis.

[0044] Figure 6 The HPLC release curves of dexamethasone after 72 h of incubation with DS-pep solution under different pH and esterase conditions are shown. The results indicate that DS-pep remains highly stable in the absence of esterase (pH 5.0–7.4), with a dexamethasone release rate of less than 5%. However, in the presence of carboxylesterase, 38%–82% of dexamethasone can be released within 72 h, with the highest release efficiency observed in an acidic environment. These results fully demonstrate that DS-pep possesses excellent esterase-responsive drug release characteristics, aligning with the "on-demand release" precision treatment design principle, and providing strong experimental evidence for its targeted drug release at lesion sites (inflammatory or tumor tissues, which are often weakly acidic and have high CES expression).

[0045] Example 6 The binding affinity between the enzyme-responsive peptide-conjugated drug DS-pep targeting ACE2 and recombinant human ACE2 protein was determined by isothermal titration microcalorimetry (ITC), verifying the specific binding ability of the YKYRYL targeting peptide in DS-pep to the ACE2 receptor.

[0046] Figure 7 The study demonstrated a strong and specific binding between DS-pep nanoparticles and recombinant human ACE2 protein (KD value of 19.8 nM), with a stoichiometric ratio close to 1:1, indicating that one DS-pep molecule binds to one ACE2 protein molecule. The binding process was enthalpy-driven (ΔH < 0), suggesting that interactions such as hydrogen bonding and van der Waals forces dominate the binding. These results fully demonstrate that the enzyme-responsive peptide-conjugated drug targeting ACE2 described in this invention possesses the ability to actively target the ACE2 receptor.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An enzyme-responsive peptide-conjugated drug targeting ACE2, characterized in that, Dexamethasone and the ACE2-targeting peptide are covalently linked by an enzyme-responsive linker; The enzyme-responsive linker contains one or more of carboxylic acid ester bonds, thioester bonds, or phosphate ester bonds; the amino acid sequence of the ACE2 targeting peptide is shown in SEQ ID NO:

1.

2. The polypeptide-conjugated drug as described in claim 1, characterized in that, The enzyme-responsive linker is formed from a dicarboxylic acid or a dicarboxylic acid derivative, with one end connected to the hydroxyl group of dexamethasone by forming an ester group, and the other end connected to the amino group of the ACE2-targeting peptide by forming a peptide bond.

3. The polypeptide-conjugated drug as described in claim 2, characterized in that, The enzyme-responsive linker is formed from succinic acid or a succinic acid derivative.

4. The polypeptide-conjugated drug as described in claim 1, characterized in that, The enzyme-responsive peptide-conjugated drug targeting ACE2 has the structure shown in Formula I: 。 5. A method for preparing the polypeptide-conjugated drug according to claim 4, characterized in that, Includes the following steps: The compound represented by Formula II was obtained by acylation of dexamethasone with succinic anhydride. ； ACE2-targeting peptides were prepared using a solid-phase Fmoc synthesis method. The compound shown in Formula II was subjected to an amidation reaction with the ACE2-targeting peptide.

6. A pharmaceutical composition, characterized in that, This includes the polypeptide-conjugated drug and pharmaceutical excipients as described in any one of claims 1 to 4.

7. The pharmaceutical composition of claim 6, characterized in that, The pharmaceutical excipients are selected from one or more of the following: diluents, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorbents, and lubricants. Alternatively, its dosage form may be tablets, capsules, granules, powder for injection, oral liquid, or injection.

8. The use of a polypeptide-conjugated drug according to any one of claims 1 to 4 or a pharmaceutical composition according to claim 6 or 7 in the preparation of a medicament for treating inflammatory diseases, treating viral diseases, or for antitumor purposes.

9. The application as described in claim 8, characterized in that, The drug can be administered orally, intravenously, via nasal drops, or via spray.

10. The application as described in claim 8, characterized in that, Inflammatory diseases include inflammatory diseases of the lungs, intestines, cardiovascular diseases, or kidney inflammation; Alternatively, the viral disease could be pneumonia caused by the novel coronavirus; Alternatively, the tumor may be small cell lung cancer, pancreatic cancer, liver cancer, colorectal cancer, or nasopharyngeal carcinoma.

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