Kidney-targeting polypeptide specifically binding to kim-1 and use thereof in the preparation of a medicament for treating kidney disease

Abstract

The application provides a KIM-1 specific binding kidney targeting polypeptide and application thereof in preparation of a medicine for treating kidney diseases, and an amino acid sequence of the KIM-1 specific binding kidney targeting polypeptide is shown as SEQ ID NO. 1. In the application, the targeting polypeptide can be specifically combined with an extracellular Ig V domain of kidney injury molecule-1 (KIM-1 / HAVCR1), so that kidney targeting and treatment effect on the damaged kidney can be realized after the occurrence of kidney diseases such as kidney injury.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of KIM-1-specifically binding kidney-targeting peptides in the preparation of drugs for treating kidney diseases. Background Technology

[0002] Acute kidney injury (AKI) has a high morbidity and mortality rate, making it a major global public health problem. AKI is a clinical syndrome characterized by a rapid decline in kidney function over a short period of time due to damage to the kidney's structure or function; its pathogenesis is complex. Elderly individuals, patients with chronic kidney disease, cardiovascular disease, diabetes, and hypertension are more susceptible to AKI. Globally, approximately 13.3 million people suffer from AKI each year, and 1.7 million die from it.

[0003] Kidney Injury Molecule-1 (KIM-1 / HAVCR1) is a single-pass transmembrane protein encoded by the HAVCR1 gene. Initially discovered to be highly expressed during kidney injury, it is considered a biomarker for kidney damage. The extracellular structure of KIM-1 protein contains an immunoglobulin V (IgV) domain and a mucin domain; the IgV domain participates in the interaction of the extracellular matrix and other proteins. KIM-1 is expressed only in trace amounts or not at all in normal kidney tissue, but it is rapidly and abundantly expressed after acute kidney injury (AKI), producing high levels of KIM-1 protein at the apical membrane of dedifferentiated proximal tubule cells. Multiple studies have shown that KIM-1 can not only serve as a biomarker for early detection of acute kidney injury, but its expression level can also reflect the severity of AKI damage and prognostic indicators.

[0004] Accurate early diagnosis and timely intervention in acute kidney injury (AKI) play a crucial role in the prognosis of AKI patients. Currently, there is no unified diagnostic standard for AKI, and clinical diagnosis relies excessively on serum creatinine levels. However, serum creatinine levels are influenced by various factors, such as age, sex, muscle mass, and diet. Furthermore, elevated serum creatinine often remains undetectable for some time after kidney injury, potentially delaying early diagnosis and missing the optimal window for early intervention. Although some drugs and treatment strategies are used to treat AKI, due to the diversity of etiologies and disease courses, a definitive and effective treatment remains lacking. Therefore, discovering timely diagnostic methods and effective treatments is key to managing acute kidney injury.

[0005] The kidneys are one of the body's main metabolic organs, playing a crucial role in drug metabolism and excretion. Due to the reabsorption of drugs within the renal tubules and their excretion in urine, drug concentrations in the kidneys are often insufficient to reach therapeutic levels. Furthermore, drug distribution in the kidneys is influenced by renal structure and function. The kidney's unique tissue structure includes different parts such as the glomeruli, tubules, and interstitium, each with varying effects on drug distribution and metabolism. Drugs must pass through the tubular epithelial cells and tubulointerstitium to enter the tubular lumen, and the permeability of the tubular epithelial cells, the presence of drug transport proteins, and the biochemical environment of the tubulointerstitial space all affect drug distribution and metabolism. Therefore, drug distribution and metabolism in the kidneys are influenced by multiple factors, making it difficult to accumulate drugs to therapeutic concentrations.

[0006] As shown above, discovering a harmless target that enables drug accumulation in the kidneys is key to the effective treatment of acute kidney injury. Therefore, it is necessary to develop a targeted peptide for kidney-targeted application in acute kidney injury. Summary of the Invention

[0007] This invention addresses the technical challenges of insufficient binding affinity of peptides to kidney-specific receptors and insufficient accumulation in damaged kidneys. It provides a targeted peptide for kidney-targeted application in acute kidney injury, composed of natural amino acid residues. This targeted peptide has the ability to specifically bind to the extracellular IgV domain of KIM-1, which is highly expressed in damaged kidneys. Following kidney injury, it rapidly accumulates in the damaged kidney, with a peptide ratio in the kidney to the liver reaching 3.43, and the peptide accumulation in the damaged kidney exceeding that in the undamaged kidney by more than 3.5 times. This innovation solves the technical problem of the inability of existing technologies to achieve targeted drug delivery to the kidney.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect of the invention, a KIM-1-specifically binding kidney-targeting polypeptide is provided, characterized in that the amino acid sequence of the KIM-1-specifically binding kidney-targeting polypeptide is as shown in SEQ ID NO.1.

[0010] Furthermore, the KIM-1-specifically bound kidney-targeting polypeptide also includes:

[0011] The polypeptide modification performed on the amino acid sequence shown in SEQ ID NO.1 includes N-terminal acetylation or fatty acidation, glycosylation or phosphorylation of intermediate residues;

[0012] Alternatively, it may have an amino acid sequence that has at least 90% homology with the amino acid sequence shown in SEQ ID NO.1 and can specifically bind to the extracellular Ig V domain of the kidney injury molecule: KIM-1 / HAVCR1.

[0013] The peptide modification includes N-terminal modification (acetylation, fatty acidation, etc.) and intermediate residue modification (glycosylation modification that binds to Ser-, Tyr-, Asn-, Thr-; phosphorylation modification that binds to Ser-, Tyr-, Thr-, etc.).

[0014] Furthermore, the KIM-1-specifically binding kidney-targeting polypeptide also includes polypeptides with the same function obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence shown in SEQ ID NO.1.

[0015] In a second aspect of the invention, a nucleic acid aptamer for a KIM-1-specifically binding kidney-targeting polypeptide is provided, the nucleic acid aptamer being capable of expressing the KIM-1-specifically binding kidney-targeting polypeptide.

[0016] Furthermore, the nucleic acid aptamer is selected from expression cassettes or vectors.

[0017] In a third aspect of the invention, a host cell containing the nucleic acid molecule or the nucleic acid aptamer is provided.

[0018] Furthermore, the host cells include prokaryotic host cells and eukaryotic host cells.

[0019] In a fourth aspect of the invention, the use of the KIM-1-specifically binding kidney-targeting polypeptide, the nucleic acid aptamer, or the host cell is provided in the preparation of a medicament for treating kidney diseases.

[0020] Furthermore, the kidney disease includes at least one of kidney injury and diabetic nephropathy.

[0021] In a fifth aspect of the invention, a pharmaceutical composition for treating kidney injury is provided, comprising the said polypeptide and a pharmaceutically acceptable excipient or a pharmaceutically acceptable targeted delivery carrier.

[0022] In a sixth aspect of the invention, a reagent for detecting kidney disease is provided, the reagent package containing a kidney-targeting polypeptide that specifically binds to KIM-1, and an imaging agent conjugated to the targeting polypeptide.

[0023] Furthermore, the imaging agent is a contrast agent or a fluorescent label. The kidney disease includes at least one of acute kidney injury and diabetic nephropathy.

[0024] One or more technical solutions in the embodiments of the present invention have at least the following technical effects or advantages:

[0025] (1) This invention utilizes artificial intelligence (AI) protein interaction model prediction technology to simulate and analyze the protein binding pocket of the KIM-1 Ig V region, and designs a series of peptides that can target the Ig V domain. Simultaneously, considering the peptide length and water solubility, it was found that the amino acid sequence shown in SEQ ID NO: 1 can bind to the human and mouse-derived KIM-1 Ig V domain with a low Gibbs free energy, while also possessing good water solubility, thus making it a more suitable candidate for the development of diagnostic and therapeutic agents for acute kidney injury.

[0026] (2) This invention demonstrates that the artificially synthesized KIM-1 Ig V domain-targeting peptide exhibits excellent targeting of damaged kidneys in acute kidney injury. We found that the amino acid sequence shown (SEQ ID NO: 1) can co-localize with human KIM-1 on the damaged kidney cell membrane. In cisplatin-induced acute kidney injury mice, this amino acid sequence was found to accumulate significantly in the damaged mouse kidneys.

[0027] (3) This invention investigated the effect of KIM-1 gene knockout on peptide targeting efficiency. The study found that in cisplatin-induced KIM-1 gene knockout HK-2 cells, the amino acid sequence shown in SEQ ID NO: 1 lost its co-localization with KIM-1 on the HK-2 cell membrane. In cisplatin-induced acute kidney injury KIM-1 gene knockout mice, the amino acid sequence shown in SEQ ID NO: 1 lost its high enrichment in the kidney, indicating that this amino acid sequence is a peptide that specifically targets KIM-1.

[0028] Furthermore, none of the synthetically produced peptides containing natural amino acid residues exhibited cytotoxicity. Therefore, the amino acid sequence shown in SEQ ID NO: 1 lays the foundation for the preparation of diagnostic and therapeutic drugs for acute kidney injury, and provides new ideas for developing more timely diagnostic agents and targeted therapies. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention, the accompanying drawings used in the description of the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a sequence diagram of the targeted polypeptide of the present invention;

[0031] Figure 2 This is a schematic diagram of the binding pattern of the targeted peptide of the present invention to the human KIM-1Ig V region;

[0032] Figure 3 This is a schematic diagram of the binding pattern of the targeted peptide of the present invention to the mouse KIM-1Ig V region;

[0033] Figure 4 This invention relates to the effect of the targeted peptide on HK-2 activity and its protective effect on HK-2 under cisplatin stimulation.

[0034] Figure 5 This is the co-localization of the targeted peptide of the present invention with HK-2 and KIM-1 in KIM-1 knockout under cisplatin stimulation;

[0035] Figure 6 This is a graph showing the enrichment intensity of the targeted peptide of the present invention in the kidneys of healthy or cisplatin-induced kidney-damaged mice.

[0036] Figure 7 This is a kidney enrichment intensity map of the KIM-1 targeting peptide of the present invention in mice with ischemia-reperfusion induced unilateral kidney injury; the right kidney is the control.

[0037] Figure 8 This is a graph showing the kidney enrichment intensity of the KIM-1 targeting peptide of this invention in KIM-1KO mice with cisplatin-induced acute kidney injury. Detailed Implementation

[0038] The present invention will be described in detail below with reference to specific embodiments and examples, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific embodiments and examples are for illustrative purposes only and are not intended to limit the present invention.

[0039] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, 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. In the event of any conflict, this specification shall prevail.

[0040] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be obtained by purchasing them from the market or by existing methods.

[0041] The technical solution of this application embodiment is to solve the above-mentioned technical problems, and the general idea is as follows:

[0042] A targeting polypeptide of the present invention contains an amino acid sequence from the N-terminus to the C-terminus as shown in SEQ ID NO: 1;

[0043] Alternatively, the targeted polypeptide may contain an amino acid sequence from the N-terminus to the C-terminus represented by SEQ ID NO: 1, which has been substituted, deleted, or added one or more amino acids and has the activity of sequence SEQ ID NO: 1.

[0044] The targeted peptide can specifically bind to the KIM-1Ig V domain, achieving high concentration enrichment in kidneys with kidney damage.

[0045] The targeted polypeptide structure in this invention is as follows: Figure 1 As shown.

[0046] In this invention, amino acids refer to naturally occurring amino acids. Unless otherwise specified, any amino acid generally or specifically indicated by name includes D and L stereoisomers if its structure allows for such stereoisomeric forms. Natural amino acids include alanine (Ala), aspartic acid (Asp), asparagine (Asn), arginine (Arg), cysteine ​​(Cys), glutamic acid (Glu), glutamine (Gln), glycine (Gly), histidine (His), leucine (Leu), isoleucine (Ile), lysine (Lys), methionine (Met), phenylalanine (Phe), proline (Pro), serine (Ser), threonine (Thr), tryptophan (Trp), tyrosine (Tyr), and valine (Val).

[0047] The targeting peptides of this invention have a strong affinity for the KIM-1Ig V region. The Gibbs free energy of their interaction with human KIM-1Ig V is -4.1 kcal / mol, and that of mouse KIM-1Ig V is -12.9 kcal / mol. They are highly enriched in kidneys that have been acutely injured and have a high expression of KIM-1. The sequences of each peptide are shown in Table 1.

[0048] Table 1. Amino acid sequences of targeted peptides

[0049]

[0050] The C57BL / 6 mice used in this embodiment were purchased from the Experimental Animal Research Center of Hubei Provincial Center for Disease Control and Prevention. The polypeptides of this invention were synthesized according to standard solid-phase polypeptide synthesis procedures. The polypeptides were purified using RP-HPLC with a C18 column. The direction of polypeptide synthesis was from C-terminus to N-terminus. First, the resin was swollen using chloropolymer resin. Then, the first amino acid was attached to the resin. The attachment of the first amino acid to the resin required double coupling, i.e., the amino acid reacted with the resin twice. Subsequently, the Fmoc group at the amino terminus of the amino acid was removed (deprotection). Then, the peptide chain elongation process was carried out. After repeated condensation, washing, deprotection, washing, and condensation steps, amino acids were attached one by one from the C-terminus to the N-terminus according to the polypeptide sequence until the desired peptide fragment was synthesized. After the resin was deswollen and dried, the peptide fragment was excised, and the crude peptide was purified, TKP 4 in Table 1 was synthesized.

[0051] In other embodiments, the polypeptides of the present invention can also be obtained by constructing expression vectors using conventional methods for expression and purification.

[0052] The kidney-targeting polypeptide TKP4, which specifically binds to KIM-1 in this invention, can accumulate in large quantities in the kidneys of mice with high KIM-1 expression in cisplatin-induced acute kidney injury.

[0053] The application of the KIM-1-specifically bound kidney-targeting polypeptide of this application in the preparation of drugs for treating kidney diseases will be described in detail below with reference to examples and experimental data.

[0054] Example 1

[0055] This invention relates to virtual screening of peptides.

[0056] Specific procedures: Proteins that interact with KIM-1 are subjected to interaction structure prediction with the KIM-1 Ig V region. The interaction segments are determined based on the docking results, and hotspot amino acid residues are identified. Targeted peptide sequences designed based on the main hotspot amino acid residues are then subjected to protein-peptide interaction structure prediction again with the human and mouse KIM-1 Ig V regions. The peptide TKP4, which requires target validation, is obtained by screening according to Gibbs free energy from low to high.

[0057] The binding patterns of peptide TKP4 to the human and mouse Kim-1 IgV regions are shown in the diagram below. Figure 2 , 3 As shown.

[0058] Example 2

[0059] The TKP 4 of this invention has a significant protective effect on cisplatin-stimulated cells and has no toxic side effects.

[0060] Specific procedures: HK-2 cells were seeded at a density of 7000 cells per well in 96-well plates. After cell attachment, the cells were stimulated with 5 μg / ml cisplatin alone or mixed with 1 μmol / L peptide and added to the culture medium, and cultured for another 24 hours. After the cell culture period, 10 μl of MTT solution (5 mg / ml, i.e., 0.5% MTT) was added to each well, and the cells were cultured for another 6 hours. The supernatant was then aspirated, and 150 μl of DMSO was added. The cells were shaken at low speed for 10 minutes to fully dissolve the crystals. Finally, the absorbance of each well was measured at 570 nm using an ELISA reader.

[0061] The results are as follows Figure 4 As shown, TKP 4 does not affect the cell viability of HK-2 cells, but it does have a certain protective effect on HK-2 cells stimulated by cisplatin.

[0062] Example 3

[0063] The TKP4 of this invention has significant co-localization with KIM-1 expressed in cisplatin-induced HK-2 cells, but no co-localization with cisplatin-induced HK-2 cells after KIM-1 knockout.

[0064] Specific procedures: Accurately weigh 5,6-FAM mg and 1 mg of peptide, add 800 μl of 0.1 M NaHCO3 solution, and incubate overnight at 4°C by rotation. After the reaction, transfer the mixture to a 1 kD dialysis bag and dialyze with PBS until the dialysate is colorless. Then, seed HK-2 cells (7000 cells / well) into 24-well plates with coverslips. Next, stimulate with 5 μg / ml cisplatin for 24 hours. After stimulation, administer 1 μmol / L of labeled fluorescent peptide, and aspirate the culture medium within 2 hours, washing twice with PBS. Next, fix cells with 4% paraformaldehyde for 15 min, wash with PBS, and then permeabilize with 0.1% Triton X-100-1% BSA for 10 min. Then, add KIM-1 antibody and incubate overnight at 4°C. After incubation, recover the antibody, add fluorescent secondary antibody, and incubate at room temperature for 4.5 hours. After antibody recovery, the samples were washed three times with PBS and stained with DAPI at room temperature for 10 min. After washing three times with PBS, the samples were mounted with anti-fluorescence quenching mounting medium and then incubated overnight at 4°C. Finally, the samples were observed using a fluorescence microscope.

[0065] The results are as follows Figure 5 As shown, in cisplatin-induced HK-2 cells expressing endogenous Kim-1, TKP4 and KIM-1 exhibit significant co-localization. However, in cells without cisplatin induction and without KIM-1 KO, the co-localization of TKP4 and KIM-1 was not observed.

[0066] Example 4

[0067] The TKP4 of this invention accumulates at high concentrations in the kidneys of mice with cisplatin-induced kidney injury.

[0068] Specific procedures: Accurately weigh 7 mg of Cy and 1 mg of the peptide, add 800 μl of 0.1 M NaHCO3 solution, and incubate overnight at 4°C by rotation. Transfer the reaction solution to a 1 kD dialysis bag and dialyze with PBS until the dialysate is colorless to obtain the labeled peptide. Then, 8-week-old male C57 mice were intraperitoneally injected with 10 mg / kg of cisplatin, followed by administration of the labeled peptide at 3 μmol / kg 24 hours later. Mice were sacrificed 2 and 4 hours after peptide administration, and organs such as brain, heart, lungs, liver, spleen, and kidneys were removed. Changes in near-infrared fluorescence intensity of each organ were observed using a small animal in vivo imaging instrument.

[0069] The results are as follows Figure 6 As shown, TKP4 can significantly accumulate in the damaged kidneys of mice with cisplatin-induced kidney injury, achieving a significant kidney-targeting effect.

[0070] Example 5

[0071] The TKP4 of the present invention accumulates at high concentrations in the damaged kidneys of mice with ischemia-reperfusion-induced unilateral kidney injury.

[0072] Specific procedures: Accurately weigh 7 mg Cy and 1 mg peptide, add 800 μL of 0.1 M NaHCO3 solution, and incubate overnight at 4°C by rotation. Transfer the reaction solution to a 1 kD dialysis bag and dialyze with PBS until the dialysate is colorless. Then, 8-week-old male C57 mice were anesthetized with isoflurane. The mice were shaved and their skin was disinfected. The skin and muscles were cut at the lower edge of the left rib on the back of the spine to expose the left kidney. The renal artery of the left kidney was carefully isolated and quickly clamped with an arterial clamp. After 45 minutes of ischemia, the arterial clamp was released to restore blood flow, and the kidney recovery was observed. 24 hours later, 3 μmol / kg of labeled peptide dissolved in PBS was administered (the labeled peptide is obtained by reacting the targeted peptide with the amino acid sequence shown in SEQ ID NO.1 in Example 1 with a fluorescent label, as detailed in Example 4). The mice were sacrificed 2 hours and 4 hours after peptide administration, and organs such as the brain, heart, lungs, liver, spleen, and kidneys were removed. The changes in near-infrared fluorescence intensity of each organ were observed using a small animal in vivo imaging instrument.

[0073] The results are as follows Figure 7 As shown, with the undamaged right kidney as a control, TKP 4 significantly accumulated in the damaged kidney (left kidney) in mice with ischemia-reperfusion-induced unilateral kidney injury, achieving a significant kidney-targeting effect.

[0074] Example 6

[0075] The TKP4 of this invention accumulates at high concentrations in the kidneys of cisplatin-induced Kim-1 KO kidney injury mice.

[0076] Specific procedures: Sulfo-Cyanine7 NHS ester (Cy 7) and TKP 4 were accurately weighed at a molar ratio of 7:1, and 800 μL of 0.1 M NaHCO3 solution was added. The reaction was carried out overnight at 4°C by rotation. The reaction solution was transferred to a 1 kD dialysis bag and dialyzed with PBS until the dialysate was colorless. After freeze-drying, TKP 5-Cy 7 was obtained. 8–12 week old male C57 Kim-1 KO mice (KO mice were constructed by our research team, and the results were published in Yang C, et al. Nat Commun. 2023) were intraperitoneally injected with cisplatin 30 mg / kg. 24 hours later, they were given 3 μmol / kg of a labeled peptide dissolved in PBS (the labeled peptide was obtained by reacting the targeting peptide with the amino acid sequence shown in SEQ ID NO.1 in Example 1 with a fluorescent label, as detailed in Example 4). Two hours after administration of the peptide, mice were sacrificed, and organs such as the brain, heart, lungs, liver, spleen, and kidneys were removed. Changes in the near-infrared fluorescence intensity of each organ were observed using a small animal in vivo imaging instrument.

[0077] The results are as follows Figure 8 As shown, TKP4 cannot achieve kidney-targeting effects in cisplatin-induced Kim-1 KO kidney injury model mice.

[0078] Finally, it should be noted that the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0079] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0080] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The use of a polypeptide with the amino acid sequence shown in SEQ ID NO.1 in the preparation of a medicament for the treatment of acute kidney injury.

2. The application according to claim 1, characterized in that, The acute kidney injury mentioned is ischemia-reperfusion induced kidney injury.

3. Use of the nucleic acid molecule encoding the polypeptide of claim 1 in the preparation of a medicament for treating acute kidney injury.

4. Use of a recombinant expression vector comprising a nucleic acid molecule encoding the polypeptide of claim 1 in the preparation of a medicament for treating acute kidney injury.

5. Use of host cells comprising the recombinant expression vector of claim 4 in the preparation of a medicament for treating acute kidney injury.

6. Use of a pharmaceutical composition comprising the polypeptide of claim 1 and a pharmaceutically acceptable targeted delivery carrier in the preparation of a medicament for treating acute kidney injury.

Images