Peptide inhibitors of human mitochondrial fission protein 1 and their methods of use

Abstract

The present disclosure provides mitochondrial fission protein 1 (FIS1) inhibitory peptides, polynucleotides and vectors encoding the peptides, and methods of using the peptides in the treatment of diseases such as arterial disease and vascular dysfunction associated with type 2 diabetes.

Description

[Technical Field]

[0001] [Cross-Reference to Related Application] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 110,457, filed November 6, 2020, the entire content of which is incorporated herein by reference.

[0002] [Statement of Federally Sponsored Research] This invention was made with government support under Grant No. R01 HL128240 and Grant No. R01-GM067180 awarded by the National Institutes of Health. The United States Federal Government has certain rights in this invention.

[0003] [Sequence Listing] The Sequence Listing attached to the present application is submitted as an ASCII text sequence listing file named "650053_00834_ST25.txt" created on November 5, 2021 with a file size of 11.4 KB. The Sequence Listing is electronically submitted with the present application via EFS-Web, and the entire content thereof is incorporated herein by reference.

[0004] The field of the present invention relates to fission protein 1 peptides, fusion peptides, and methods for treating diseases such as vascular disease and type 2 diabetes. [Background Art]

[0005] In patients with type 2 diabetes mellitus (T2DM), vascular endothelial dysfunction is observed before the onset of macrovascular and microvascular diseases. Emerging data suggest that abnormalities in mitochondrial morphology and function are involved in the pathogenesis of human vascular endothelial dysfunction derived from subjects affected with T2DM 1,2 . Mitochondria in endothelial cells derived from T2DM-affected subjects produce excessive superoxide. Such overproduction of superoxide is caused, in part, by polarization of the inner mitochondrial membrane 2. Excessive production of mitochondrial reactive oxygen species (mtROS) in endothelial cells causes severe epigenetic changes and activates cell signaling pathways, which leads to endothelial inflammation and vascular dysfunction 3 . As is clear from previous studies, partial depolarization of the inner mitochondrial membrane achieved by administering mitochondria-targeted antioxidants and pharmacologically active agents to human resistance arterioles derived from subjects affected by T2DM can improve endothelial-dependent vasodilatory dysfunction 2,4 .

[0006] Unfortunately, phase III clinical trials of antioxidant therapy for the prevention and treatment of vascular diseases have failed to verify the positive effects observed in separate small-scale physiological and / or non-randomized trials, and existing active agents that exert pharmacological effects targeting the inner mitochondrial membrane have toxicity profiles that impede their clinical use 7 .

[0007] Accordingly, there is a need for alternative therapies targeting the treatment of vascular dysfunction in, for example, patients with type 2 diabetes. Summary of the Invention Means for Solving the Problem

[0008] The present disclosure provides mitochondrial fission 1 protein (Fis1) activity-inhibiting peptides and methods of using the same.

[0009] One aspect of the present disclosure provides a mitochondrial fission protein 1 (Fis1) activity inhibitory peptide characterized by (a) comprising the amino acid sequence of SEQ ID NO: 38 (XLPYPZ), or a sequence having at least 80% sequence identity with SEQ ID NO: 38, wherein X and Z may be peptides consisting of 0 to 30 amino acids or optionally 1 to 20 amino acids. In some aspects, the inhibitory peptide is characterized by comprising an amino acid sequence selected from SEQ ID NOs: 33 to 37, or a sequence having at least 80% sequence identity with SEQ ID NOs: 33 to 37, and being a peptide with a length of 5 to 50 amino acids or optionally 5 to 30 amino acids.

[0010] Another aspect of the present disclosure provides a Fis1 inhibitory peptide comprising (a) the amino acid sequence of SEQ ID NO: 1 (SHKHDPLPYPHFLL) or a sequence having at least 90% sequence identity with SEQ ID NO: 1. Another aspect of the present disclosure provides a Fis1 inhibitory peptide comprising (b) an amino acid sequence encoding a carrier peptide, tag peptide, or cell-binding peptide, or a sequence linked to a carrier comprising (a) the amino acid sequence of SEQ ID NO: 1 (SHKHDPLPYPHFLL) or a sequence having at least 90% sequence identity with SEQ ID NO: 1.

[0011] Another aspect of this disclosure provides a mitochondrial fission protein 1 (Fis1) activity inhibitory peptide comprising (a) the amino acid sequence of any of SEQ ID NOs: 1, 16-21, 26, or 29, or a sequence having at least 80% sequence similarity, preferably at least 90%, to SEQ ID NOs: 1, 16-21, 26, or 29. In some aspects, the inhibitory peptide comprising (a) is linked to (b) an amino acid sequence or carrier encoding a carrier peptide, tag peptide, or cell-binding peptide. In some aspects, the inhibitory peptide is linked to or attached to a carrier peptide which is a cell-permeable peptide sequence, or optionally to TAT (SEQ ID NO: 2) or a sequence having at least 80% sequence similarity, preferably at least 90% sequence identity, to SEQ ID NO: 2. In some aspects, (a) and (b) are peptides linked by a linker amino acid sequence. In some aspects, the SEQ ID NO of the linker sequence is 4, 11, 12, 13, 14, or 15.

[0012] Another aspect of this disclosure provides a Fis1 inhibitory peptide comprising the amino acid sequence of SEQ ID NO: 1 (SHKHDPLPYPHFLL), or a sequence having at least 80%, preferably at least 90%, sequence identity with SEQ ID NO: 1. Another aspect provides a Fis1 inhibitory peptide that is SEQ ID NO: 3 (YGRKKRRQRRRGSGSGSSHKHDPLPYPHFLL), or a peptide having at least 90% sequence identity with SEQ ID NO: 3.

[0013] In another embodiment, the inhibitory peptide is a peptide having at least 80% sequence similarity, preferably about 90%, to SEQ ID NO: 3, SEQ ID NO: 31, or SEQ ID NO: 32, or to SEQ ID NO: 3, SEQ ID NO: 31, or SEQ ID NO: 32.

[0014] In yet another embodiment, this disclosure provides a method for treating vascular complications associated with type 2 diabetes, which includes administering an effective amount of the Fis1 inhibitor peptide according to this disclosure for the purpose of treating vascular complications.

[0015] Another aspect of this disclosure provides a method for improving vasodilatory dysfunction in a subject requiring treatment for vasodilatory dysfunction, comprising administering an effective amount of the Fis1 inhibitory peptide according to this disclosure for the purpose of restoring vasodilatory function in the subject. In one aspect, the subject is a patient with type 2 diabetes.

[0016] Another aspect of this disclosure provides a method for improving the bioavailability of nitric oxide (NO) in human microvascular endothelial cells, which includes administering an effective amount of the Fis1 inhibitory peptide according to this disclosure for the purpose of improving the bioavailability of NO in human endothelial cells. [Brief explanation of the drawing]

[0017] [Figure 1] This figure shows that transfection with Fis1 increases the response to acetylcholine in both high-glucose and low-glucose conditions, improving vasodilation compared to the control (siRNA). A. Effect of molecular inhibition of Fis1 expression on endothelial vasodilation in high-glucose conditions. Vasodilation impairment caused by high glucose conditions was improved under Fis1 knockdown conditions (overall P=0.0002, *P≦0.0002 between control siRNA and Fis1 siRNA at appropriate Ach doses, N=6). This improvement in vasodilation under Fis1 knockdown conditions may be overridden by L-NAME (P<0.0001 between Fis1 siRNA and siFis1+L-NAME, P=0.002 between control siRNA and siRNA+L-NAME). B. Effect of molecular knockdown of Fis1 expression on endothelial vasodilation in low-glucose conditions. Vasodilation impairment caused by hypoglucose was improved under Fis1 knockdown conditions (overall P=0.0008, *P≦0.0003 between control siRNA and Fis1 siRNA at Ach-appropriate doses, N=6). This improvement in vasodilation under Fis1 knockdown conditions may be overridden by L-NAME (P=0.0002 between Fis1 siRNA and Fis1 siRNA + L-NAME). Ach…Acetylcholine. [Figure 2]This figure shows that the bioavailability of NO in human arterioles increases with decreasing Fis1 concentration under high and low glucose conditions. A. Low glucose (LG): (n=8, overall P=0.01, P=0.03 between control siRNA and Fis1 siRNA, P=0.003 between control siRNA+L-NAME and Fis1 siRNA, P=0.03 between Fis1 siRNA and Fis1 siRNA+L-NAME). In the figure, each element of the rectangle represents the 25th and 75th percentiles. The horizontal line represents the median. B. High glucose: (n=9, overall P=0.04, P=0.01 between scrambled siRNA and Fis1 siRNA, P=0.03 between Fis1 siRNA and control siRNA+L-NAME, P=0.047 between Fis1 siRNA and Fis1 siRNA+L-NAME). [Figure 3] The figure shows that suppression of Fis1 expression in diabetic (DM) arterioles improves endothelial cell-dependent vasodilation impairment (n=6, P=0.002), and this effect is inhibited by L-NAME (P<0.0004 between Fis1 siRNA and Fis1 siRNA + L-NAME). *P<0.0005 compared to cases where other drugs were administered at Ach-appropriate doses. Ach...Acetylcholine. [Figure 4] This figure shows that molecular inhibition of Fis1 under high glucose (33 mM) and low glucose (2.5 mM) conditions improves the steady-state junction stability between monolayer endothelial cells. The figure compares electrocellular matrix impedance sensing (ECIS) measurements between high glucose and normal glucose conditions (A) and between normal glucose and low glucose conditions (B) in human microvascular endothelial cells (HMEC-1) transfected with Fis1 siRNA and control siRNA. In the figure, each element of the rectangle represents the 25th to 75th percentile. The horizontal line represents the median. Tukey's multiple comparison test (n=4 in each treatment) was performed following analysis of variance (ANOVA) (overall P<0.001 in both high glucose and low glucose tests); *-P<0.05, **P<0.01, ***P<0.001, ****P<0.0001). HG…High glucose, LG…Low glucose, NG…Normal glucose level. [Figure 5] This figure shows that molecular inhibition of Fis1 under high glucose (33 mM) and low glucose (2.5 mM) conditions does not alter the state of mitochondrial bioenergy synthesis compared to normal glucose (5 mM) conditions. The figure also shows the measured values ​​of extracellular acidification rate (ECAR, n=5) (A) and oxygen consumption rate (OCR, n=5) (B) in human microvascular endothelial cells (HMEC-1) that were previously cultured under high glucose (6 hours), normal glucose (5 mM, 2 hours), and low glucose (2 hours), and then transfected with Fis1 siRNA and control siRNA. Wild-type (WT) cells refer to HMEC-1 cells grown under normal glucose conditions without transfection. ECAR and OCR were measured under basal conditions, followed by the sequential addition of oligomycin (2.5 μM), FCCP (1 μM), and then rotenone (1 μM) and antimycin A (1 μM). No statistically significant differences were observed between the treatment methods. [Figure 6] Figures showing the binding of the novel peptide pep213 to Fis1. (A) Figure showing the overlap of 1H-15N HSQC spectra of 50 μM 15N-Fis1 samples titrated with gradually increasing amounts of unlabeled p8470 (0-2000 μM). (B) Figure showing that the affinity of pep213 binding to Fis1 was determined based on the NMR data in (A) by applying trend analysis to the entire spectrum of the titration series and fitting the normalized principal component 1 value (PC1) to a ligand depletion model to obtain the apparent KD = 7 ± 2 μM. (C) Figure showing affinity measurement by tryptophan-specific fluorescence analysis. Fis1 containing a single tryptophan was titrated with gradually increasing amounts of pep213 without tryptophan (0-1000 μM). By determining the average emission wavelength and fitting it, the apparent Kd = 3.3 ± 0.1 μM was obtained. The matching residues in Panel B and Panel C are shown above each panel. [Figure 7]Figure showing that Pep213-tat improves endothelial cell-dependent vasodilation impairment in healthy human blood vessels exposed to high glucose and in human blood vessels derived from T2DM patients. (A) High glucose-induced attenuation of acetylcholine-induced endothelial cell-dependent vasodilation was improved by treatment with tat sequences to facilitate cell entry by attaching 1-10 μM of pep213 (n=5, P<0.0001), but this improvement obtained by pep213-tat treatment was overturned by L-NAME (P<0.0001 between pep213 and pep-213+L-NAME, and P<0.0001 between pep213 at the appropriate concentration for *Ach and all other exposures). (B) In blood vessels taken from T2DM subjects, the attenuation of endothelial cell-dependent vasodilation by Ach was improved by pep213 (overall P<0.001, *P<0.05 between pep213 at Ach appropriate concentrations and all other exposures). Ach...acetylcholine, T2DM...type 2 diabetes mellitus. [Figure 8] Figures showing that pep213-tat improves the attenuation of endothelial cell-dependent vasodilation in healthy human blood vessels exposed to high glucose and in human blood vessels derived from T2DM patients, compared to a scrambled peptide control. (A) Figure showing that treatment with 1 μM pep213-tat improves the acetylcholine-induced endothelial cell-dependent vasodilation state after 6 hours of prior exposure to high glucose (33 mM) compared to a scrambled peptide containing the same amino acids as pep213 attached to the tat sequence (n=5, overall P<0.001, *Ach appropriate concentration P<0.05). (B) Figure showing similar results in blood vessels derived from T2DM subjects (n=4, overall P<0.001, *Ach appropriate concentration P<0.05). [Figure 9] This figure shows the efficiency of Fis1 knockdown in human arterioles by siRNA treatment. Human arterioles transfected with siRNA Fis1 showed a significantly lower Fis1 concentration than arterioles transfected with scrambled control siRNA (n=4, p<0.05). [Figure 10]Transfection with Drp1 siRNA can prevent hyperglucose-induced endothelial cell-dependent vasodilation impairment (overall P<0.0001, *P≦0.0001 between control siRNA and Ach-adapted doses of Drp1 siRNA, n=6), but the figure shows that the improvement in vasodilation under Drp1 knockdown conditions is overridden by L-NAME (P<0.0001 between Drp1 siRNA and Drp1 siRNA+L-NAME). [Figure 11] This figure shows that knockdown of Drp1 expression using Drp1 siRNA can prevent a decrease in nitric oxide (NO) bioavailability in healthy human arterioles exposed to high or low glucose conditions. (A) High glucose (HG, 33 mM, 6-hour exposure): n=9, P=0.02 overall, P<0.05 between Drp1 siRNA and all other exposures). (B) Low glucose (LG, 2.5 mM, 2-hour exposure): n=5, P=0.003 overall, P=≤0.03 between Drp1 siRNA and all other exposures). [Figure 12] This figure shows that in human arterioles derived from patients with T2DM, suppression of Drp1 expression using Drp1 siRNA tended to improve endothelial cell-dependent vasodilation impairment (n=4, P=0.076). [Figure 13] This figure shows the efficiency of Fis1 knockdown in HMEC-1 cells by siRNA treatment. In HMEC-1 cells transfected with Fis1 siRNA, Fis1 concentration was significantly lower compared to cells transfected with scrambled control siRNA (P=0.0002) and untransfected HMEC-1 cells (P=0.0001) (n=3, overall P<0.0001). [Figure 14]Figures showing that in HMEC-1 cells, phosphorylation of eNOS at Ser1177 and NO production increased after activation by the Ca+2 ionophore A23187. (A) Figures showing representative Western blots of p-eNOS-(Ser1177) and β-actin with and without A23187. (B) Figures showing quantitative measurements of p-eNOS-(Ser1177) with and without A23187 (P=0.03, n=6). (C) Figures showing that NO production, measured using DAF 2-DA (5uM), increased with the addition of A23187 (n=4, P=0.02). [Figure 15] This figure shows that Fis1 knockdown improved NO production in immortalized cultured human microvascular endothelial cells (HMEC-1) transfected with Fis1 siRNA (n=7, overall P<0.0001, P<0.0001 between siFis1 basal state and siFis1 stimulated state, P=0.0003 between siRNA basal state and siFis1 stimulated state, P=0.02 between siFis1 stimulated state and siRNA stimulated state). Cells exposed to L-NAME were cultured in L-NAME for 2 hours, then cultured in DAF2-DA (5uM) for 15 minutes, and then fluorescence intensity was measured. [Figure 16]This figure shows that molecular inhibition of Fis1 under high glucose (33 mM) and low glucose (2.5 mM) conditions did not alter the expression of other mitochondrial proteins. Immortalized HMEC-1 cells transfected or scrambled with siRNA Fis1 were pre-cultured under different glucose conditions: high glucose (HG, 33 mM, 6 hours), normal glucose (NG, 5 mM, 2 hours), and low glucose (LG, 2.5 mM, 2 hours). The expression of a predetermined mitochondrial protein, arbitrarily selected from these immortalized HMEC-1 cells, was measured. The expression of each protein was normalized to obtain the total protein amount between each sample (number of samples for each protein n=4-10). When compared after exposure to different glucose concentrations, differences were observed in the expression of some mitochondrial proteins, but knockdown of Fis1 expression with siRNA did not affect the expression of mitochondrial proteins other than Fis1. Statistical significance is indicated by "*" if p<0.05, "**" if p<0.01, "***" if p<0.001, and "****" if p<0.0001. [Figure 17] This figure shows increased bioavailability of nitric oxide (NO) in human microvascular endothelial cells (HMVECs). HMVEC cells were treated with 1 μM pep213-tat at 37°C for 1 hour, and NO was measured by diaminofluorescein-2 diacetate (DAF2-DA) staining (N=3, *P=0.04). [Figure 18] This figure shows that endothelial cell-dependent vasodilation impairment in arterioles associated with Fis1 overexpression is eNOS-dependent. Overexpression of Fis1 in healthy human resistance arterioles (cultured for 48 hours and transfected with a plasmid to achieve endothelial-specific overexpression of human Fis1) results in eNOS-dependent vasodilation impairment. (This can be inferred from the fact that endothelial cell-dependent vasodilation can be induced even when acetylcholine is lost when using the eNOS inhibitor L-NAME.) N=5, overall P<0.001. *P<0.05 at the appropriate dose of acetylcholine. Ach…Acetylcholine. [Figure 19]This figure shows that endothelial cell-dependent vasodilation impairment in resistance arterioles can be improved by pep213. Exposure for 1 hour to pep213 attached to a tat sequence (1 μM pep213-tat) to improve cell permeability can improve endothelial cell-dependent vasodilation impairment in healthy human resistance arterioles that exhibit Fis1 overexpression in endothelial cells (achieved by transfecting blood vessels with a plasmid designed for endothelial cell-specific human Fis1 overexpression using a lentiviral vector). A 1 μM peptide scrambled using the same amino acids as pep213 in a different order showed no effect on acetylcholine-mediated endothelial cell-dependent vasodilation. pep213-tat brings about eNOS-dependent improvement in endothelial cell-dependent vasodilation. (This can be inferred from the fact that endothelial cell-dependent vasodilation can be induced even when acetylcholine is lost when using the eNOS inhibitor L-NAME.) N=5, overall P<0.001. *P<0.05 at the appropriate dose of acetylcholine. Ach…Acetylcholine. [Figure 20] Figure showing the crystal structures of pep213 and Fis1, and the peptide mapping of pep213. (A) The co-complex structure clearly shows that pep213 is bound to Fis1 through various binding interactions, including salt bridge formation, hydrogen bonding, and van der Waals interactions. (B) Microscale thermophoresis was used to identify the pep213 residues essential for the Fis1-pep213 interaction. Each residue of pep213 was sequentially substituted with alanine to obtain a total of 14 peptides. The ΔG° of the reaction (ΔG°=-RTlnK) was determined using the binding affinity value, and the value of ΔG° was calculated using this (ΔG°pep213-ΔG°variant). As can be seen from the low ΔΔG° value, subsets of residues at each terminal of the peptide do not show a significant contribution to binding. [Modes for carrying out the invention]

[0018] The present invention provides a peptide, a nucleic acid sequence and vector encoding the peptide, a composition containing the peptide or vector, and a method for treating diseases related to endothelial dysfunction or vascular dysfunction, such as type 2 diabetes mellitus (T2DM) and vascular diseases, using these.

[0019] Excessive mitochondrial fission has been thought to be involved in various diseases, including endothelial dysfunction associated with diabetes. Mitochondrial fission is the process by which mitochondria divide into individual smaller mitochondria, and in vitro, vasodilation can be improved by inhibiting fission genetically or pharmacologically. Such inhibition includes RNAi-mediated genetic silencing of the gene encoding mitochondrial fission protein 1 (Fis1). These data suggest that inhibition of Fis1 may improve pathological conditions accompanied by impaired vasodilation. To identify Fis1 inhibitors, the inventors developed pep213 (SEQ ID NO: 1), a high-affinity 14-residue peptide that binds to recombinant Fis1 with micromolar to submicromolar affinity. When cell-permeable pep213 is applied to human endothelial blood vessels, vasodilation is restored, suggesting that this peptide can exert a therapeutic effect by inhibiting the in vivo activity of Fis1. pep213 was derived from peptides using phage display screening and is a novel peptide that binds to severely shortened Fis1, which lacks the first 32 residues. Furthermore, the inventors focused on the key amino acids in pep213 that are necessary for binding to Fis1 and conducted analyses of cocrystallization and mutation generation (see Figure 20).

[0020] Based on previous studies that demonstrated (1) increased expression of mitochondrial fission protein 1 (Fis1) in human endothelial cells collected from patients with type 2 diabetes, (2) inhibition of increased high-glucose-induced mitochondrial superoxide production and impaired phosphorylation at the Ser1177 activation site of nitric oxide synthase (eNOS) derived from endothelial cells by molecular knockdown of Fis1 or dynamin-related protein 1 (Drp1, which can bind to Fis1 and induce mitochondrial fission), and (3) improvement of low-glucose-induced endothelial dysfunction by pharmacologically and molecularly knocking down Drp1 in human resistance arterioles, targeting proteins and peptides involved in mitochondrial fission is expected to be a promising alternative. 1,8 Fis1 is attracting particular attention as a pharmacological target due to its role in mitochondrial dynamics, and is thought to be most effective as a pathological stimulus for conditions such as hypoxemia and hyperglycemia. 9-12 .

[0021] In this disclosure, as described in the examples, we conducted tests to investigate whether knockdown of Fis1 expression can improve endothelial cell-dependent vasodilation and nitric oxide (NO) production in resistant arteries collected from patients with type 2 diabetes and in blood vessels collected from healthy individuals acutely exposed to high and low glucose concentrations. Furthermore, we investigated the effects of Fis1 knockdown on endothelial cell barrier function, oxygen consumption, and glycolysis under high and low glucose conditions. Next, we designed a novel peptide that binds to Fis1 and inhibits Fis-1-mediated nuclear fission, and conducted tests to investigate whether it favorably acts on endothelial cell-dependent vasodilation in human resistant arterioles derived from patients with T2DM and in healthy human blood vessels exposed to high glucose concentrations. By targeting Fis1 pharmacologically, useful therapeutic means can be obtained for vascular disease challenges in T2DM.

[0022] [Peptides and Compositions] The present invention provides a novel peptide that binds to recombinant Fis1, preferably having an affinity of micromoles to submicromoles.

[0023] In a first embodiment of this disclosure, a mitochondrial fission protein 1 (Fis1) activity inhibitory peptide is provided, characterized in that (a) the amino acid sequence of SEQ ID NO: 38 (XLPYPZ), or a sequence having at least 80% sequence identity with SEQ ID NO: 38, wherein X and Z are peptides consisting of 0 to 30 amino acids, or optionally 1 to 20 amino acids, or optionally 1 to 10 amino acids. In another embodiment, the peptide includes the amino acid sequence of SEQ ID NO: 38, or a sequence having at least 90% sequence identity. In yet another embodiment, the inhibitory peptide of claim 1 is characterized in that (a) the peptide includes an amino acid sequence selected from SEQ ID NOs: 33 to 37, or a sequence having at least 80% or at least 90% sequence identity with SEQ ID NOs: 33 to 37, and is a peptide with an amino acid length of about 5 to 50 amino acids, or optionally 5 to 30 amino acids. Preferably, the amino acid length is about 10 to 20 amino acids, or about 12 to 16 amino acids. Other appropriate lengths may be intended. Furthermore, as described below, it is desirable that (a) is linked to the carrier peptide or tag of (b).

[0024] In further embodiments, when a 14-mer peptide pep213 (SEQ ID NOs: 1, 16-21, 26, or 29, preferably SEQ ID NO: 1 in one embodiment) or a sequence having at least 80% sequence identity, optionally at least 90%, is obtained as a cell-permeable fusion peptide (e.g., pep213-TAT, SEQ ID NO: 3, or SEQ ID NOs: 31 or 32, or a sequence having at least 80% or at least 90% sequence identity with SEQ ID NOs: 3, 31, or 32), it has the function of inhibiting Fis1 activity in vivo. This inhibitory peptide also has the function of restoring the diastolic capacity of endothelial cells and blood vessels whose diastolic capacity is reduced. Thus, because the peptide has the function of improving endothelial cell-dependent vasodilation disorders, it can be used to treat vascular diseases such as vascular dysfunction associated with type 2 diabetes. Furthermore, the results of cocrystallization and peptide mutation analysis shown in Figures 19 and 20 clearly indicate the amino acids in the 14-mer that are important for Fis1 binding. Therefore, in some embodiments, modified pep213 peptides are intended (e.g., SEQ ID NOs: 16-29, preferably SEQ ID NOs: 16-21, 26, or 29, or SEQ ID NO: 30, in which one or more amino acids X are substituted with any amino acid, preferably alanine or glycine).

[0025] In one embodiment, the mitochondrial fission protein 1 (Fis1) activity inhibitory peptide comprises or consists of an inhibitory peptide of the present disclosure linked to a carrier peptide, a tag peptide, or a cell-binding peptide. In one embodiment, the mitochondrial fission protein 1 (Fis1) activity inhibitory peptide is a peptide comprising (a) the amino acid sequence of SEQ ID NO: 38 (XPLPYPZ), or a sequence having at least 80% sequence identity to SEQ ID NO: 38, wherein X and Z consist of 0 to 30 amino acids, or optionally 1 to 20 amino acids, or optionally 1 to 10 amino acids (the amino acid peptide may contain any suitable amino acids). In another embodiment, it comprises the amino acid sequence of SEQ ID NO: 38, or a sequence having at least 90% sequence identity. In another embodiment, the inhibitory peptide of claim 1 is characterized by comprising (a) an amino acid sequence selected from SEQ ID NOs: 33 to 37, or a sequence having at least 80% or at least 90% sequence identity to SEQ ID NOs: 33 to 37, and having a length of about 5 to 50 amino acids, or optionally 5 to 30 amino acids. In another embodiment, the inhibitor comprises or consists of (a) the amino acid sequence of SEQ ID NO: 1 (SHKHDPLPYPHFLL), or a sequence having at least 90% sequence identity to SEQ ID NO: 1. In yet another embodiment, the Fis1 inhibitory peptide comprises or consists of (b) an amino acid sequence encoding a carrier peptide, tag peptide, or cell-binding peptide, or (a) the amino acid sequence of SEQ ID NO: 1 linked to a carrier, or a sequence having at least 90% sequence identity to SEQ ID NO: 1. In this application, the terms “Fis1 inhibitory peptide” and “Fis1 inhibitory peptide” are used synonymously to refer to peptides that can inhibit Fis1 activity in cells.

[0026] In one embodiment, the mitochondrial fission protein 1 (Fis1) activity inhibitory peptide is (a) the amino acid sequence of SEQ ID NOs. 1, 16-21 or a sequence having at least 80% or at least 90% sequence identity to SEQ ID NOs. 1, 16-21 or 29, or a sequence having at least 80% or at least 90% sequence identity to SEQ ID NOs. 1, 16-21 or 26 or 29, or a sequence encoding a carrier peptide, tag peptide, or cell-binding peptide, or a sequence linked to a carrier, or a sequence having at least 80% or at least 90% sequence identity to SEQ ID NOs. 1, 16-21, 26 or 29, or a sequence having at least 90% sequence identity to 29.

[0027] In another embodiment, based on the mutant peptide analysis shown in Figure 20, the mitochondrial fission protein 1 (Fis1) activity inhibitory peptide is characterized in that (a) the amino acid sequence of SEQ ID NO: 30, or contains or is composed thereof, and one or more X in the amino acid sequence is any amino acid (e.g., alanine or glycine) or the corresponding amino acid from SEQ ID NO: 1. In another embodiment, the inhibitory peptide contains or is composed of SEQ ID NO: 30, and two or more X in SEQ ID NO: 30 are any amino acids, or three or more X are any amino acids, or four or more X are any amino acids, or five or more X are any amino acids, or six or more X are any amino acids, or seven or eight X are any amino acids.

[0028] In another embodiment, the inhibitory peptide comprises or consists of (b) an amino acid sequence encoding a carrier peptide, tag peptide, or cell-binding peptide (e.g., SEQ ID NO: 32) or (a) an amino acid sequence of SEQ ID NO: 30 linked to a carrier.

[0029] In this application, the terms "Fis1 inhibitory peptide" and "Fis1 inhibitory peptide" are used as synonyms to refer to peptides that can inhibit Fis1 activity within cells.

[0030] The Fis1 inhibitory peptide further comprises a carrier or carrier peptide, tag peptide, or cell-binding peptide linked to the inhibitory peptide. Suitable carrier peptides, tag peptides, or cell-binding peptides can be those well known and accepted in the art. In one embodiment, the carrier peptide is a cell-penetrating peptide. Cell-penetrating peptides (CPPs) are peptides that can pass through the cell membrane and reach the inside of the cell. Examples of suitable carrier peptides include, for example, TAT, which has the amino acid sequence of SEQ ID NO: 2 or a sequence that has at least 90% sequence identity with SEQ ID NO: 2 and can penetrate into the cell. Other suitable CPPs can be those well known in the art, such as penetratin, R8, transportan, and Xentry. (For example, Patel, SG, Sayers, EJ, He, L. et al. ``Cell-penetrating peptide sequence and modification dependent uptake and subcellular distribution of green florescent protein in different cell lines''.SCI Rep 9, 6298(2019) / / doi.org / 10.1038 / s41598-019-42456-8 See also, and incorporated herein by reference). Other suitable carriers known in the art may be used. Examples of other carriers include, but are not limited to, polymer composites, polymer nanoparticles, lipid-based carriers, dendrimers, carbon nanotubes, and nanocarriers such as gold nanoparticles. Lipid-based carriers include both liposomes and micelles. The carriers may be linked covalently or noncovalently. In some embodiments, peptides may be conjugated to the carrier.

[0031] In some embodiments, the peptide according to the present disclosure further comprises an exogenous tag or agent. As used in the present disclosure, the terms "tag" or "agent" include any useful moiety that enables purification, identification, detection or therapeutic use of the peptide of the present invention. In the present invention, any tag or agent can be used, as long as it does not interfere with the functionality of the inhibitory peptide. Suitable tags may be those well known in the art, include but are not limited to, affinity tags and epitope tags (e.g., cMyc, HIS, FLAG, V5-tag, HA-tag, NE-tag, S-tag, Ty-tag, etc.) and fluorescent tags (RFP, GFP, etc.) can be used. Epitope tags are generally used as "purification tags", that is, tags that facilitate the isolation of a polypeptide from other non-specific proteins and peptides.

[0032] In some embodiments, the carrier peptide or tag is a polypeptide, and the inhibitory peptide and the tag are encoded by a single nucleic acid sequence and translated simultaneously. In some embodiments, the tag is cleavable and can be removed after production and purification of the peptide.

[0033] In some embodiments, the inhibitory peptide and the carrier peptide or tag are linked via a linker sequence. A suitable peptide linker may consist of a polypeptide of 3 to 10 amino acids or 3 to 25 amino acids. In some embodiments, the peptide linker may consist of a polypeptide having an amino acid sequence selected from serine and glycine (e.g., GSGSGS (SEQ ID NO: 4), etc.). As other suitable linkers, those acceptable to those skilled in the art can be used, for example, SGSG (SEQ ID NO: 11), G n (n is an integer from 1 to 10), (SGSG) nExamples include (n is an integer from 1 to 10, SEQ ID NO: 11), GSGS (SEQ ID NO: 12), SSSS (SEQ ID NO: 13), GGGS (SEQ ID NO: 14), GGC, GGS, (GGC)8), (G4S)3, and GGAAY (SEQ ID NO: 15). The peptide linker may be cleavable by a protease. In some embodiments, the peptide linker consists of a polypeptide having the amino acid sequence of SEQ ID NO: 4. In this application, it is intended to use other suitable linkers known in the art.

[0034] In one embodiment, the Fis1 inhibitory peptide may consist of SEQ ID NO: 3 (YGRKKRRQRRRGSGSGSSHKHDPLPYPHFLL) or a peptide having at least 90% sequence identity with SEQ ID NO: 3. As shown in the examples, this inhibitory peptide can inhibit Fis1 activity in vivo.

[0035] In another embodiment, the Fis1 inhibitory peptide comprises SEQ ID NO: 31 (YGRKKRRQRRRGSXSHKHDPLPYPHFLL) or a peptide having at least 80% sequence similarity or at least 90% sequence identity with SEQ ID NO: 31, where X is the linker according to the disclosure. In another embodiment, the FIs1 inhibitory peptide comprises SEQ ID NO: 32 or a sequence having at least 80% sequence similarity or at least 90% sequence identity with SEQ ID NO: 32, where X is at least one of any amino acid (e.g., alanine), and Y is the linker according to the disclosure (see Table 2). In some embodiments, SEQ ID NO: 32 is intended to have X which includes two or more amino acids selected from any amino acids (such as alanine) in the sequence, or three or more, four or more, or five or more amino acids selected from any amino acids (such as alanine) in the sequence, or six or more X which are amino acids selected from any amino acids (such as alanine) in SEQ ID NO: 32, or seven or eight X which are amino acids selected from any amino acids (such as alanine) in SEQ ID NO: 32. Y is a linker according to the disclosure, for example, SEQ ID NOs: 4, 11-15, etc. That is, SEQ ID NO: 32 is SEQ ID NO: 2-linker-SEQ ID NO: 30, and SEQ ID NO: 31 is SEQ ID NO: 2-linker-SEQ ID NO: 1. The linker may be any linker according to the disclosure.

[0036] As used in this disclosure, the terms “protein,” “peptide,” and “polypeptide” are synonymous with a series of amino acid residues interconnected by peptide bonds between an α-amino group and the carboxyl group of an adjacent residue. While “protein” and “polypeptide” often refer to relatively large polypeptides, and “peptide” often refer to small polypeptides, the meanings of these terms overlap in the art. “Protein” may include modified amino acids (such as phosphorylated, glycated, or glycosylated amino acids) and amino acid analogs.

[0037] Inhibitory peptides may be directly, indirectly, or conjugately linked to the tag or carrier peptide. As used in this disclosure, the term “conjugated bond” refers to the covalent bonding of two entities. This can be achieved by direct covalent bonding or by linking the groups using standard synthetic coupling methods. For example, two polypeptides can be linked by simultaneous expression to form a fusion protein or chimeric protein. One or more amino acids can also be inserted into the polypeptide (i.e., by incorporating the corresponding nucleic acid sequence into a vector) to function as linking groups. For example, in some embodiments, polyserine and polyglycine linkers are included between the inhibitory peptide sequence and the tag or carrier peptide. Other possible linking groups include polyethylene glycol or hydrocarbons whose terminals are substituted with amino or carboxylic acid groups to enable amide coupling with polypeptides having amino acid side chains, each containing a carboxylic acid or amino group. Alternatively, the substitution of amino or carboxylic acid groups can be performed using other binding partners such as azide or alkyne groups to form triazoles under copper catalysis.

[0038] The Disclosure also provides polynucleotides encoding the inhibitory Fis1 peptide relating to the Disclosure. In this application, the terms “polynucleotide,” “polynucleotide sequence,” “oligonucleotide,” “nucleic acid,” and “nucleic acid sequence” are used as synonyms for a nucleotide sequence or a fragment thereof. These terms refer to DNA or RNA of genomic, natural, or synthetic origin, and include single-stranded or double-stranded molecules, as well as sense or antisense strands of such molecules. In one embodiment, the polynucleotide comprises a heterologous promoter sequence and a polynucleotide sequence encoding a peptide of sequence SEQ ID NO: 1 (SHKHDPLPYPHFLL) or SEQ ID NOs. 16-21, 26, or 29, or sequence In another embodiment, the polynucleotide comprises a polynucleotide sequence encoding the peptide of SEQ ID NO: 1, which is linked to a heterogeneous promoter sequence and a carrier or tag peptide (for example, a sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 3, 31, or 32 or a sequence having at least 80% or at least 90% sequence identity with SEQ ID NO: 3, 31, or 32).

[0039] In one embodiment, the polynucleotide comprises a heterologous promoter sequence and a polynucleotide sequence encoding a peptide of sequence number 1 (SHKHDPLPYPHFLL) or a sequence having at least 80% or at least 90% sequence identity with sequence number 1. In another embodiment, the polynucleotide comprises a heterologous promoter sequence and a polynucleotide sequence encoding a peptide of sequence number 1 linked to a carrier or tag peptide (e.g., sequence number 3 or a sequence having at least 80% or at least 90% sequence identity with sequence number 3).

[0040] The term "sequence identity" as used above and throughout this application means that when the expression "at least 80% sequence identity" is used, it includes approximately 80% of any sequence identity (for example, at least 81%, 82%, 83%, 84%, 85%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity). Similarly, when the expression "at least 90% sequence identity" is used, it includes approximately 90% or more sequence identity for a particular sequence number (for example, 91%, 92%, 93%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% sequence identity).

[0041] In some embodiments, the polynucleotide is a vector. In some embodiments, the vector can express the inhibitory peptide according to the Disclosure, and the vector includes a heterologous promoter operably attached to a polynucleotide sequence encoding the inhibitory Fis1 peptide according to the Disclosure. The vector may further include a heterologous backbone sequence. A vector suitable for use in the present invention includes a promoter operably attached to a polynucleotide sequence encoding the inhibitory peptide according to the present invention. The vector may also include a suitable regulatory sequence that enables translational control in a host cell. In some embodiments, the vector further includes one or more carrier peptides or tag nucleic acid sequences. In some embodiments, the vector further includes another regulatory sequence, such as a signal sequence.

[0042] As used in this disclosure, the term “vector” refers to a nucleic acid molecule that has the function of propagating another nucleic acid to which it is linked. This term includes vectors as self-replicating nucleic acid structures and vectors that are incorporated into the genome of a host cell to which they are introduced. Certain vectors have the function of inducing the expression of the nucleic acid to which they are manipulably linked. In this application, such vectors are referred to as “expression vectors” (or simply “vectors”). The term vector includes “plasmids,” which are the most commonly used form of vector. Plasmids are circular double-stranded DNA loops to which other DNA fragments (e.g., those encoding inhibitory Fis1 peptides) may be ligated. However, other forms of expression vectors, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adena-associated viruses), may also be used in the present invention. Certain vectors (e.g., bacterial vectors with replication of bacterial origin and mammalian episomal vectors) have the function of self-replicating in the host cell to which they are introduced. Other vectors are incorporated into the genome of the host cell upon introduction and are thereby replicated together with the host genome. In one embodiment, the vector includes a viral vector that carries a peptide expressed in a host cell using a viral mechanism.

[0043] In some embodiments, the vector of the present invention further comprises a heterologous backbone sequence. As used in this disclosure, the term “heterologous nucleic acid sequence” refers to non-human nucleic acid sequences, such as bacterial, viral, or other non-human nucleic acid sequences not naturally found in humans. A heterologous backbone sequence may be required for vector propagation and / or expression of the encoded peptide. Many commonly used expression vectors and plasmids contain non-human nucleic acid sequences, such as the CMV promoter.

[0044] Suitable promoters for implementing the present invention include, but are not limited to, structural promoters, induced promoters, temporally controlled promoters, developmentally controlled promoters, chemically controlled promoters, physically controlled (e.g., controlled by light or temperature) promoters, tissue-preferential promoters, and tissue-specific promoters. The term "heterogeneous promoter" is used to describe a suitable promoter, referring to a promoter that is not naturally associated with manipulable polynucleotides. Typical promoters in mammalian cells include those for Rous sarcoma virus (RSV), human immunodeficiency virus (HIV-1), cytomegalovirus (CMV), SV40 virus, the translation elongation factor EF-1α promoter, and the ubiquitin promoter. Those skilled in the art will be well aware that a wide range of other promoters can be used depending on the type of cell.

[0045] The identification of protein and nucleic acid sequences is evaluated using the Basic Local Alignment Search Tool (BLAST), a well-known local sequence search tool in the art (Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2267-2268; Altschul et al., 1997, Nucl. Acids Res. 25: 3389-3402). The BLAST program identifies homologous sequences by identifying fragments with high similarity scores (referred to in this application as "high-scoring segment pairs") between the query amino acid or nucleic acid sequence and the test sequence (preferably obtained from a protein or nucleic acid sequence database). The statistical significance of high-scoring segment pairs is preferably evaluated using the statistical significance formula (Karlin and Altschul, 1990). The disclosure of the aforementioned statistical significance formula is incorporated herein by reference in its entirety. When using the BLAST program, default parameters or user-provided modified parameters can be used.

[0046] The "percentage of sequence identity (%)" or "similarity rate (%)" is determined by comparing two sequences that are optimally aligned on a comparison screen. In this process, in order to optimally align the two sequences on the comparison screen, there may be added or deleted parts in the displayed portion of the polynucleotide or peptide sequence when compared to a reference sequence (without any changes such as additions or deletions) (i.e., differences from the reference sequence may be observed). The method used to calculate the above percentage (%) is to determine the number of sites where the same nucleic acid base or amino acid residue occurs in both sequences, find the number of matching sites, divide the number of matching sites by the total number of sites on the comparison screen, and multiply the result by 100 to calculate the percentage of sequence identity (%).

[0047] The terms "substantial identity" or "substantial similarity" mean that a polynucleotide or peptide contains a sequence with at least 75% sequence identity. In other words, the identity rate can be any integer value between 75% and 100%. In a more preferred embodiment, the identity rate when compared with a reference sequence using the program described in this application (preferably using standard parameters in BLAST as described above) includes at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. By appropriately adjusting these values, it becomes possible to determine the identity of corresponding proteins encoded by two nucleotide sequences, taking into account codon degeneracy, amino acid similarity, and the position of the reading frame.

[0048] In the present invention, "substantial identity" of amino acid sequences usually means that the polypeptide sequences are at least 75% identical. The polypeptide identity is preferably any integer between 75% and 100%. In more preferred embodiments, the identity includes at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.7%, and 99%.

[0049] The Disclosure also provides compositions comprising an inhibitory Fis1 peptide and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier should be selected based on the chosen route of administration and standard pharmaceutical practices. The composition may be formulated into dosage forms according to standard practices in the pharmaceutical field (see Alphonso Gennaro, ed., Remington's Pharmaceutical Sciences, 18th Ed., (1990) Mack Publishing Co., Easton, Pa). Suitable dosage forms may include, for example, liquids, parenteral liquids, or suspensions. In some embodiments, the composition comprises the isolated and purified Fis1 inhibitory peptide relating to the Disclosure. In other embodiments, the composition comprises an isolated and purified polypeptide or vector comprising a nucleic acid sequence encoding the Fis1 inhibitory peptide relating to the Disclosure.

[0050] In another aspect, the present disclosure provides a host cell containing the vector relating to the present disclosure. The present invention may use any host cell that enables the expression of the vector-encoded peptide. Examples of common host cells include bacteria (e.g., Escherichia coli, Bacillus subtilis, etc.), yeasts (e.g., budding yeast, etc.), and eukaryotic cell lines. An advantage is that mRNA splicing similar to that in humans can be performed using insect or mammalian cell lines. Those skilled in the art will be aware that the peptide of the present invention can be expressed using many expression systems and cell lines (including many of which are commercially available).

[0051] [method] This disclosure provides a method for treating vascular dysfunction using a Fis1 inhibitory peptide according to this disclosure. The method comprises administering an effective dose of the Fis1 inhibitory peptide according to this disclosure to treat vascular dysfunction. In some embodiments, the vascular dysfunction is associated with type 2 diabetes. This disclosure provides, in some embodiments, a method for treating vascular complications associated with type 2 diabetes. The method comprises administering an effective dose of the Fis1 inhibitory peptide according to this disclosure to alleviate one or more vascular complications associated with type 2 diabetes.

[0052] Furthermore, this disclosure provides a method for improving vasodilatory dysfunction in subjects requiring treatment for impaired vasodilatory function, and includes administering an effective amount of the Fis1 inhibitory peptide according to this disclosure for the purpose of restoring vasodilation in the subject. In some embodiments, the subject is a patient with type 2 diabetes. Furthermore, the subject may also be a human resistance artery with endothelial cell-dependent vasodilatory dysfunction induced by hyperglucose associated with type 2 diabetes. This dysfunction may be nitric oxide synthase-dependent.

[0053] This disclosure provides a method for enhancing the bioavailability of nitric oxide (NO) in human microvascular endothelial cells in another embodiment, which includes administering an effective amount of the Fis1 inhibitory peptide according to this disclosure for the purpose of improving the bioavailability of NO in human endothelial cells. In some embodiments, the endothelial cells are in vivo endothelial cells from a subject with vascular dysfunction.

[0054] This disclosure provides, in another embodiment, a method for preventing diabetes-induced endothelial tissue cytotoxicity, comprising administering an effective amount of the Fis1 inhibitory peptide according to this disclosure. Excessive mitochondrial fission is thought to be involved in endothelial tissue dysfunction in diabetic patients, and reducing Fis1 can prevent diabetes-induced cytotoxicity.

[0055] In yet another embodiment, this disclosure provides a method for treating RAS-mediated cancers that have been found to require mitochondrial fission. The method comprises administering an effective dose of a Fis1 inhibitory peptide according to this disclosure. While we do not wish to be bound by any particular theory, it is thought that Fis1 inhibitors may exert an effective effect in suppressing cancer progression because cancer progression can be halted by imparting the ability to block mitochondrial fission.

[0056] In another embodiment, Fis1 inhibitory peptides may be used to treat neurodegenerative diseases. The method includes administering an effective dose of the Fis1 inhibitory peptide according to the Disclosure to treat a neurodegenerative disease.

[0057] The Fis1 inhibitory peptide of this embodiment is considered a preferred target over Drp for several reasons. First, if the target is Drp1, it is embryonically lethal and is the only mechanical enzyme responsible for membrane deformation and cleavage during nuclear fission, making it an unsuitable target in cases other than cancer. While Fis1 knockout is also embryonically lethal, it is only induced under stress conditions, making it a preferred target over Drp1.

[0058] Excessive mitochondrial fission is associated with endothelial tissue dysfunction in diabetic patients [Shenouda, SM, Widlansky, ME, Chen, K., Xu, G., Holbrook, M., Tabit, CE, ... & Vita, JA (2011) and Kizhakekuttu ...Widlansky et al (2012)] and in patients with acute pulmonary failure. Targeting Fis1 allows for a novel pharmacological approach focused on the Fis1-Drp1 axis to prevent vascular damage in diabetic patients. As mentioned above, targeting the Fis1 / Drp1 axis via Drp1 peptide inhibitors has shown efficacy in many cases of neurodegenerative diseases as well as cardiomyopathy.

[0059] Furthermore, the present disclosure provides a method for treating endothelial dysfunction, comprising administering an effective dose of a Fis1 inhibitory peptide according to the present disclosure to treat endothelial dysfunction. In one embodiment, endothelial dysfunction includes atherosclerosis. In another embodiment, endothelial dysfunction is associated with any of the following diseases: atherosclerosis, cerebrovascular disease, coronary artery disease, renovascular disease, and peripheral artery disease.

[0060] The terms “effective dose” or “therapeutic effective dose” refer to a sufficient amount that can produce beneficial or desirable biological and / or clinical outcomes. For example, a therapeutically effective dose of the peptide according to the present invention may be combined with a pharmaceutically acceptable carrier to form a composition. The composition may be administered in any manner recognized in the art. The dose, method of administration, and suitable pharmaceutically acceptable carriers, diluents, and excipients used in such a method will vary depending on the given circumstances, although this will be readily apparent to those familiar with the art.

[0061] For example, the appropriate dosage can be determined by estimating factors such as the patient's weight, absorption rate, half-life, and disease severity, based on the results of animal experiments or clinical trials. The number of administrations and the course of treatment may vary from person to person. In some embodiments aimed at preventing the onset or progression of autoimmune diseases, a booster effect through additional administration may be required. The appropriate timing for booster administration can be easily determined by someone familiar with the art. For example, the peptide or vector may be administered monthly, every other month, every four months, every six months, once a year, once every two years, or at any time interval in between.

[0062] The dosage form of the composition is preferably a unit dose dosage form. In the case of such a dosage form, the preparation is divided into unit doses to contain an appropriate amount of the active ingredient. A unit dose dosage form can be a package containing the preparation, a pack containing tablets or capsules divided into smaller portions, or a vial or ampoule containing powder, each containing an appropriate quantity of the preparation. Furthermore, capsules, tablets, cachets (such as oblate capsules), or lozenges themselves can be considered unit dose dosage forms, or a package containing an appropriate number of any of these can be considered a unit dose dosage form.

[0063] As used in this disclosure, the terms “subject” or “patient” refer to both mammals and non-mammals. “Mammals” include any animal belonging to the class Mammalia, such as humans, non-human primates (e.g., apes such as chimpanzees and monkeys), farm animals (e.g., cattle, horses, sheep, goats, pigs), companion animals (e.g., rabbits, dogs, cats), and laboratory animals (e.g., rats, mice, guinea pigs). Examples of non-mammals include, but are not limited to, birds. The term “subject” does not indicate a specific age or sex. In one embodiment, the subject is a human. In certain embodiments, a human is a human with vascular disease or vascular dysfunction, or a human with a disease associated with vascular dysfunction (e.g., type 2 diabetes).

[0064] As used in this disclosure, the terms “administer” and “give” refer to any method of providing a pharmaceutical preparation or composition containing the Fis1 inhibitory peptide relating to this disclosure to a subject. Such methods are well known to those skilled in the art and include, but are not limited to, oral, transdermal, inhalation, nasal, topical, vaginal, ocular, ocular, ocular, intracerebral, rectal, sublingual, oral, and parenteral administration including injections such as intravenous, intra-arterial, intramuscular, intradermal, intrathecal, and subcutaneous. Administration can be continuous or intermittent. In various embodiments, the preparation can be administered therapeutically, that is, to treat a pre-existing disease or condition.

[0065] To facilitate the administration of the peptide or vector, it may be mixed with a suitable pharmaceutically acceptable carrier known in the art. The term "pharmaceutically acceptable" refers to a composition approved for administration to a subject by a regulatory body (e.g., a federal or state government agency). The term "carrier" may refer to a diluent, excipient, or medium used to make the pharmaceutical composition administerable. Suitable pharmaceutically acceptable carriers may include, but are not limited to, those known in the art, such as diluents, preservatives, solubilizers, emulsifiers, liposomes, and nanoparticles. Examples of suitable pharmaceutical carriers are described in E.W. Martin's *Remington's Pharmaceutical Sciences*. Furthermore, such pharmaceutically acceptable carriers may be solutions, suspensions, and emulsions in aqueous or non-aqueous solvents. Examples of non-aqueous solvents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Suitable aqueous solvent carriers include isotonic solutions, alcohol / aqueous solutions, emulsions, or suspensions (including physiological saline and buffer solutions). Examples of such carriers include, but are not limited to, water, oil (such as vegetable oil), ethanol, physiological saline (such as phosphate-buffered saline and sodium chloride aqueous solution), glucose aqueous solutions (hydrated glucose) and related sugar solutions, glycerin, or glycols such as propylene glycol and polyethylene glycol. Anti-degradation agents, antioxidants, and preservatives may be added. Suitable antioxidants include sulfite, ascorbic acid, citric acid and its salts, and sodium ethylenediaminetetraacetate. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorbutanol. Compositions for parenteral administration may take the form of aqueous or non-aqueous solutions, dispersions, suspensions, or emulsions. The composition may further contain pharmaceutically acceptable substances such as pH adjusters / buffers and toxicity modifiers (such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate) as necessary to approximate physiological conditions.

[0066] The composition can also be sterilized by conventional sterilization techniques that maintain the activity of the peptide. The formulation must be selected according to the method of administration. Examples of buffers include, but are not limited to, phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, and immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine, and lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as TWEEN® brand surfactants, polyethylene glycol (PEG), and PLURONICS® surfactants. Examples of pharmaceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M, preferably 0.05 M, phosphate buffer or 0.9% physiological saline. Other suitable and pharmaceutically acceptable carriers are also being considered.

[0067] In the present invention, "to treat" or "treatment" refers to the management and care of a subject for the purpose of combating a disease, condition, or disorder. Treatment includes administering the peptides of the present invention to reduce, suppress, or prevent the onset of symptoms or complications, to alleviate or mitigate symptoms or complications, or to eliminate a disease, condition, or disorder. In one preferred embodiment, the disease is vascular dysfunction. In one specific embodiment, the disease is type 2 diabetes with vascular dysfunction.

[0068] [kit] The manner of disclosure described with respect to the former method is applicable to the latter method and kit unless otherwise specified in the context, and vice versa.

[0069] The kit may also include a suitable kit for carrying out the methods relating to this disclosure. This may include a kit containing a Fis1 inhibitory peptide relating to this disclosure, or a composition containing the Fis1 mutant peptide thereof, and instructions for use. In another embodiment, the kit may include a nucleic acid sequence encoding the Fis1 inhibitory peptide relating to this disclosure, or cells capable of producing the Fis1 inhibitory peptide for manufacture and purification. Instructions for manufacture, purification, or use may also be included in the kit. The kit may further include a pharmaceutical composition containing the Fis1 inhibitory peptide relating to this disclosure.

[0070] It should be apparent to the parties that, in addition to those already described, various modifications can be made without departing from the inventive concept of the present invention. When interpreting this disclosure, all terms should be interpreted in the broadest possible sense that is consistent with the context. The term “comprising,” and variations thereof, should be interpreted as referring non-exclusively to elements, components, or processes, and therefore, the elements, components, or processes it refers to may be combined with other elements, components, or processes not explicitly referred to. Embodiments described as “comprising” a particular element are intended to “consisting essentially of” or “consisting of” that element. The expressions “consisting essentially of” and “consisting of” should be interpreted in accordance with the interpretations of the U.S. Patent Examination Manual and the relevant Federal Circuit Court of Appeals. The transitional phrase “consisting essentially of” limits the claims to specific materials or processes of the claimed invention “and those that do not substantially affect the basic and novel characteristics.” "Consists of" is a closed term that excludes elements, processes, or components not specified in the claims. For example, a sequence consisting of refers to the sequence listed by the sequence number, and any larger sequence that may include that sequence number as part of it.

[0071] All publications, patent applications, patents, and other references referenced herein are incorporated herein by reference in their entirety. In the event of any conflict, this specification shall prevail, including its definitions.

[0072] Other features and advantages of the present invention are evident from the description of preferred embodiments and the claims. Unless otherwise specified, all technical and scientific terms used in this disclosure have the same meaning as those generally understood by a person of ordinary skill in the art to which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the present invention, but more suitable methods and materials are as follows. Furthermore, these materials, methods and examples are illustrative and not intended to limit the scope. [Examples]

[0073] [Example 1] This study aims to improve diabetes-induced and hyperglucose-induced endothelial dysfunction in human resistance arteries by inhibiting or molecularly suppressing Fis1 using the novel peptide Pep213.

[0074] In patients with type 2 diabetes mellitus (T2DM), vascular endothelial dysfunction is observed before the onset of macrovascular or microvascular diseases. Newly obtained data suggest that mitochondrial morphological and functional abnormalities are involved in the development of human vascular endothelial dysfunction originating from T2DM patients. 1,2 In endothelial cells derived from patients with T2DM, mitochondria produce excessive superoxides. This superoxide overproduction is partly caused by polarization of the mitochondrial inner membrane. 2 Overproduction of mitochondrial reactive oxygen species (mtROS) in endothelial cells causes severe epigenetic changes and activates cellular signaling pathways, which can lead to endothelial inflammation and vascular dysfunction. 3As described above, in human resistance arterioles derived from patients with T2DM, administering antioxidants or pharmacological agents that target mitochondria partially depolarizes the mitochondrial inner membrane, thereby improving endothelial cell-dependent vasodilation impairment. 2,4 .

[0075] Unfortunately, Phase III clinical trials of antioxidant therapy for the prevention and treatment of vascular disease have failed to verify the positive effects observed in separate, smaller physiological and / or non-randomized trials. Furthermore, existing agents that target the inner mitochondrial membrane to exert their pharmacological effects have toxicity profiles that prevent their clinical use. 7 .

[0076] Based on previous studies that demonstrated (1) increased expression of mitochondrial fission protein 1 (Fis1) in human endothelial cells collected from patients with type 2 diabetes, (2) inhibition of increased high-glucose-induced mitochondrial superoxide production and reduced phosphorylation capacity at the Ser1177 activation site of nitric oxide synthase (eNOS) derived from endothelial cells by molecular knockdown of Fis1 or dynamin-related protein 1 (Drp1, which can bind to Fis1 and induce mitochondrial fission), and (3) improvement of low-glucose-induced endothelial dysfunction by pharmacologically and molecularly knocking down Drp1 in human resistance arterioles, targeting proteins involved in mitochondrial fission is expected to be a promising alternative. 1,8 Fis1 is attracting particular attention as a pharmacological target due to its role in mitochondrial dynamics, and is thought to be most effective as a pathological stimulus for conditions such as hypoxemia and hyperglycemia. 9-12 .

[0077] In this embodiment, the inventors conducted tests to investigate whether knockdown of Fis1 expression could improve endothelial cell-dependent vasodilation and nitric oxide (NO) production in resistant arteries collected from patients with type 2 diabetes and in blood vessels collected from healthy individuals acutely exposed to high and low glucose concentrations. Furthermore, the inventors also investigated the effects of Fis1 knockdown on endothelial cell barrier function, oxygen consumption, and glycolysis under high and low glucose conditions. Finally, the inventors designed and tested a novel peptide (pep213 (SEQ ID NO: 1)) that binds to Fis1 and inhibits Fis-1-mediated nuclear fission, demonstrating that it favorably acts on endothelial cell-dependent vasodilation in human resistant arterioles derived from patients with T2DM and in healthy human blood vessels exposed to high glucose concentrations. As a result, findings were obtained that support the possibility of providing a useful therapeutic means for addressing vascular disease challenges in T2DM by targeting Fis1 pharmacologically.

[0078] [Summary of results] Knockdown of Fis1 improved endothelial cell-dependent vasodilation in T2DM arteries (P=0.002) and inhibited HG-induced endothelial cell-dependent vasodilation (P=0.0008) and LG-induced vasodilation (P=0.0002) in healthy vessels. Knockdown of Fis1 maintained the bioavailability of NO and improved the integrity of the endothelial layer of cells exposed to HG or LG (P<0.001). Knockdown of Fis1 had no significant effect on other mitochondrial dynamics or the expression of autophagy-related proteins, and had no effect on endothelial cell metabolism. Pep213 showed low micromolar affinity (3.3-7 μM) for Fis1. Tat sequence-linked pep213 improved endothelial cell-dependent vasodilation in T2DM and non-T2DM vessels exposed to HG.

[0079] [method] <Recruitment and selection of test subjects> We recruited 67 healthy individuals (aged 21-75) without the aforementioned cardiovascular risk factors, as well as patients with T2DM. 2,13A person diagnosed with T2DM is someone who has been taking medication for the treatment of T2DM, or who meets the diagnostic criteria for T2DM set by the American Diabetes Association. 14 After the Medical Institutional Review Board of Wisconsin reviewed the trial methodology, the trial proceeded only after all subjects signed a written informed consent form. Screening was performed to confirm that all subjects met the inclusion criteria. The height and weight of all subjects were measured, and heart rate and blood pressure were measured three times each. Subjects with a known history of any of the following, regardless of T2DM, including atherosclerosis (coronary artery disease, peripheral vascular disease, stroke, or myocardial infarction), chronic liver disease, elevated plasma creatinine (>1.5 mg / dL in men, >1.4 mg / dL in women), those diagnosed with cancer remission less than one year prior, those regularly taking anticoagulants or antiplatelet agents other than aspirin, and those who smoked within one year of enrollment were excluded from the study. Even among those without T2DM, individuals with LDL cholesterol levels ≥ 160 mg / dl, or those with hypertension (blood pressure ≥ 140 / 90 mmHg), or those taking medication for the treatment of any of these conditions were excluded. The blood vessels used in the study to compare the effects of pep213-tat and scrambled control peptides on endothelial cell-dependent vasodilation (N=5 non-T2DM patients, N=4 T2DM patients) were obtained from subcutaneous adipose tissue treated as medical waste during surgery, in accordance with a clinical trial protocol separately reviewed and approved by the Medical Institute of Wisconsin.

[0080] <Collection of human resistance arteries> As previously explained, for human resistance arteries, resistance arteries were obtained from the upper lateral quadrant of the gluteal fat pad in adipose tissue samples. 2,8,13,15,16 In short, after sterilization and anesthesia with 1% lidocaine, a 1-1.5 cm incision was made in the upper lateral quadrant of the gluteal fat pad. The volume was approximately 8 cm³. 3 The adipose tissue was removed by sharp dissection. After hemostasis, the wound was closed with 1-2 stitches of absorbable deep dermal suture, and the epidermal layer was closed using Dermabond or Steri-Strip.

[0081] <Cell culture> Immortalized human microvascular endothelial cells (HMEC-1), purchased from ATCC (Manassas, Virginia), were cultured in a medium prepared by supplementing antibiotic-free MCDB131 (Life Technologies, Carlsbad, California, USA) with 10 mM glutamine (Thermo Fisher Scientific, Waltham, Massachusetts, USA), 10% FBS (Sigma-Aldrich, St. Louis, Missouri, USA), 10 ng / mL human EGF (Thermo Fisher Scientific, USA), and 1 μg / mL hydrocortisone (Sigma-Aldrich, USA). To test for high glucose (33 mm), normal glucose (5 mm), or low glucose (2.5 mm) states, the above supplements were added to antibiotic-free MCDB131, and then glucose and sterile PBS were added to adjust the glucose concentration. Human cutaneous microvascular endothelial cells (HMECs) were purchased from Lonza (Basel, Switzerland), and supplemented with a microvascular endothelial cell growth medium 2-bullet kit (Lonza).

[0082] [Transfection procedure between cultured endothelial cells and human blood vessels] <Transfection of cultured cells with Fis1 and Drp1 siRNA and scrambled control> Structural models (sequences described below) of Fis1 and Drp1 siRNA were obtained from Origine, Inc. (Rockville, Maryland, USA). Lipofectamine RNAiMax (Thermo Fisher Scientific) was added to a 20 nM RNAi structural model in Opti-MEM (Life Technologies, Inc.). The resulting mixture was diluted in culture medium and cultured with HMEC-1 cells for 4 hours, after which it was replaced with standard culture medium. The cells were then cultured for 24 hours before treatment and analysis. After culture, treated cells were exposed to high glucose conditions for 6 hours and low glucose conditions for 2 hours, after which NO production, bioenergy synthesis, protein expression, and endothelial integrity were measured.

[0083] <Transfection of Fis1 and Drp1 siRNA and scrambled controls in human resistance arteries> Human resistance arteries were transfected with an siRNA structural model as described above. 8,13 In short, resistance arteries detached from adipose tissue were suspended in a culture myograph chamber (204 CM, DMT, Ann Arbor, Michigan, USA). One end of the vessel was sutured onto a glass pipette in a microorganism chamber. Before suturing the second end of the artery to the glass pipette, Fis1 siRNA, Drp1 siRNA (20 nM in OptiMEM using Invitrogen's Lipofectamine RNAiMax), or a control scrambled siRNA (20 nM) was slowly injected into the lumen of the vessel. The unsutured end of the vessel was then tied to another glass pipette, and the vessel was suspended in the chamber and immersed in physiological buffer at 37 °C, pressurized to 60 mmHg. After 4-6 hours of incubation, the siRNA was cultured at a low shear rate (□<5 dyn / cm 2 ) was then slowly flushed out of the lumen over a period of 24 hours.

[0084] <Measurement of endothelial cell-dependent vascular activity> Resistance arteries from healthy individuals were exposed to one of the following conditions: normal glucose state (NG, glucose level 5 mm), low glucose (LG, glucose level 2.5 mM; 2 hours), or high glucose (HG, glucose level 33 mM; 6 hours). All tests in blood vessels from T2DM patients were performed under a 5 mm glucose state. The vessels were pre-constricted to approximately 50% of their resting diameter using endothelin-1 (Sigma-Aldrich, USA). Subsequently, acetylcholine (Ach) doses were increased by 10. -10 from 10 -5 The constricted arteries were exposed to acetylcholine (Ach) while gradually increasing the dose up to M, and changes in vascular diameter were measured using a digital caliper gauge and a video microscope. -5After reaching M, the blood vessels were exposed to 200 μM papaverine and the reactivity of the smooth muscle was examined. After a 30-minute washing period, the same arteries were constricted again and cultured for 30 minutes with L-NG-nitroarginine methyl ester (L-NAME, 100 μM), a direct inhibitor of nitric oxide synthase, and then 10 -10 ~10 -5 After exposure to Ach in M, vasodilation was remeasured.

[0085] <Measurement of bioavailability efficiency of nitric oxide (NO)> (Cultured cells): The bioavailability of nitric oxide (NO) in HMEC-1 cells and arteries was measured using a fluorescent NO marker and 4,5-diaminofluorescein diacetate (DAF2-DA). Cells were cultured in a dark room for 2 hours with or without L-NAME (100 μM), followed by 15 minutes of culture at 37°C with DAF2-DA (5 μM). Fluorescence intensity was measured using a SpectraFluer Plus plate reader (Tecan, Morrisville, North Carolina, USA) with excitation and emission wavelengths set to 485 nm and 535 nm, respectively.

[0086] (Human resistance arteries): Two blood vessels were taken from a subset of healthy subjects, and each vessel was cut in half to allow for experimentation under four different conditions. These conditions included transfection with Fis1 siRNA or Drp1 siRNA with and without 30 minutes of exposure to L-NAME, and transfection with scrambled siRNA control with and without exposure to L-NAME at room temperature. Subsequently, 10 -5 M-Ach and DAF2-DA (final concentration 5 μM) were added to each blood vessel, and the vessels were incubated at 37°C for 30 minutes. Next, the arteries were washed with PBS buffer, placed on slides, and imaged with a fluorescence microscope. Untreated and unstained arteries were used as controls for fluorescence background interference. Both treated and control vessels were measured with the same gain setting, and the results were analyzed using Metamorph 7.8 software (Universal Imaging, Westchester, Pennsylvania, USA).

[0087] <Measurement of endothelial integrity> 40,000 human microvascular endothelial (HMEC-1) cells were seeded in each well and grown on a gold electrode array plate (Applied Biophysics 8W10E+) until 50% confluence. These cells were pre-transfected with Fis-1 siRNA (20 nm) or scrambled siRNA (20 nm), and on the day of the experiment, the transfected cells were exposed to different glucose conditions (6 hours in high glucose (33 mM), 2 hours in normal glucose (5 mM), and 2 hours in low glucose (2.5 mM)). Monolayer integrity was confirmed at 64,000 Hz and capacitance less than 10 nF. Next, the cells were functionally analyzed using electrocellular matrix impedance sensing (ECIS), and transepithelial / transcutaneous electrical resistance (TEER) across the monocellular layer was measured in real time using an ECIS ZTheta instrument (Applied Biophysics, Troy, New York). Resistance was measured at frequencies of 125 Hz, 250 Hz, 500 Hz, 1,000 Hz, 2,000 Hz, 4,000 Hz, 8,000 Hz, 16,000 Hz, 32,000 Hz, and 64,000 Hz, with 4,000 Hz as the reference.

[0088] <Measurement of mitochondrial bioenergy>: Using an XFe96 analyzer (Seahorse Biosciences, Northville, Massachusetts, USA), the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured to assess the bioenergy synthesis state of mitochondria. HMEC-1 cells (20,000 cells / well) were seeded into a Seahorse microplate with 96 wells over 4-6 hours, and then the cells were adhered to the plate. siRNA targeting Fis1 and siRNA scrambled controls were pre-transfected into the cells. The culture medium was aspirated and discarded, and the cells were cultured in high-glucose medium for 6 hours, or in normal-glucose medium for 2 hours. High-glucose, normal-glucose, or low-glucose media were removed and replaced with Dulbecco's Modified Eagle Minimum Essential Medium (DMEM, pH 7.40), an XF-type medium containing 600 μL of 45% glucose, 1.5 mL of L-glutamine (200 mM), and 1.5 mL of sodium pyruvate (100 mM), so that the final glucose concentration was the same as the initial medium concentration. Subsequently, cells were cultured at 37 °C for 1 hour in a carbon dioxide-free incubator to calibrate the temperature and pH. During the mitochondrial stress test, drugs such as oligomycin A (final well concentration 2.5 μM), FCCP (carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, final well concentration 1 μM), and rotenone & antimycin A (final well concentration 1 μM) were sequentially injected in 25 μL units. During the glycolysis stress test, drugs such as glucose (final well concentration 10 mm), oligomycin A (final well concentration 2.5 μM), and 2-deoxyglucose (final well concentration 50 mm) were sequentially injected in 25 μL units.

[0089] After these measurements, the cells were fixed with 4% paraformaldehyde for 15 minutes and stained with 1.5 μg / mL of DAPI (4',6-diamidino-2-phenylindole) diluted 1:500 for 24 hours. Cell counts were obtained by measuring OCR and ECAR using the automated cell counting function of a Citation 5 multimode cell imaging reader (Biotech, Vermont, USA), and normalizing the measured values.

[0090] <Measurement of mitochondrial proteins> 0.3 x 10 per well 6HMEC-1 cells were cultured in a 6-well plate and transfected with siFis1 and siRNA. After transfection over 4-6 hours, the medium was replaced with normal cell culture medium and cultured overnight. The cells were treated with HG (33 mM) for 6 hours and NG (5 mM) or LG (2.5 mM) for 2 hours. The plate was then washed twice with cell washing buffer, and 50 μL of RIPA lysis buffer (Protein Simple, San Jose, California, USA) was added to each well. The cells were gently scraped off on ice and transferred to labeled tubes. The lysates were centrifuged at 10,000 rpm for 10 minutes, flash-frozen under liquid nitrogen (snap-freeze), and left overnight at -80°C. After thawing the cell pellet and supernatant, they were briefly mixed using a vortex mixer and centrifuged at 10,000 rpm for 10 minutes. The supernatant was transferred to a labeled tube, and the protein concentration was quantified using the Bradford assay. Protein expression was also evaluated by immunoassay using an automated capillary electrophoresis system with WES (ProteinSimple). A 12-230 kDa WES separation module and 25 capillary cartridges were used for protein analysis and detection. The lysates were diluted in 0.1x sample buffer to a 4:1 ratio of sample to master oral mixture and denatured at 95°C for 5 minutes. For immunoassay, blocking buffer, primary antibody, secondary antibody, chemiluminescent substrate, sample, biotinylated size marker, and wash buffer were placed in the designated wells on the provided microplate. The plate was centrifuged at 1000Xg for 5 minutes and then loaded into the WES. Default separation parameters were used for protein detection. Data were analyzed using ProteinSimple's "Compass for SW" software (version 3.1.7), and specific antibody peaks were integrated. Total protein amount was obtained by normalizing protein expression across all samples. A WES assay was performed using biotin-based protein labeling to analyze total protein content. Total protein was also analyzed as an integral of antibody peaks using Compass for SW. Carryover of a control sample between plates was used to correct for signal intensity variability between plates. The mean of the quotient of the antibody peak integrals for each sample against total protein was calculated.

[0091] <NMR titration experiment> To determine the peptide binding affinity to Fis1, NMR titration experiments were performed in the same manner as drug fragment titration. {Egner, 2018 #10126} First, the peptide was resuspended in Fis1 dialysis buffer (100 mM HEPES pH 7.4, 200 mM NaCl, 1 mM DTT, 0.02% v / v sodium azide) to a final concentration of 6 mM. Next, 50 μM 15 220 μL of N-hFis1 and gradually increasing amounts of peptide (0 μM, 25 μM, 50 μM, 150 μM, 400 μM, 800 μM, 1600 μM, 2000 μM) were prepared and loaded into 3 mm NMR tubes. For each sample, 1 H, 15 N HSQC spectra were collected at a temperature of 25°C using a Bruker Avance II 600 MHz spectrometer equipped with a Z-axis tilted triple-resonance cryoprobe and a sample jet autosampler, enabling automatic tuning, shimming, and data acquisition for each sample. 1 H, 15 In the N HSQC experiment, 1 Eight scans were performed at complex points in H and 15N dimensions (1024 and 300 points respectively). Spectra processing was performed using an automated Python script with NMRPipe, and chemical shifts were measured using TiterView and CARA software. {Delario, 1995 #10127}{Masse, 2005 #10128} Trend analysis determined the peptide bond affinity to each spectrum in the titration series. {Xu, 2016 #10129} {Xu, 2017 #10131} Trend analysis is an analysis that reveals changes in data by performing principal component analysis, treating each concentration point as a unique data point, and in this disclosure, each of the gradually increasing amounts of peptide 1 H, 15The N spectrum is the input data. After performing principal component analysis, the value of principal component 1 (PC1) was normalized to obtain the maximum PC1 value (in the range of 0 to 1). Next, the normalized PC1 value was plotted against the peptide concentration and fitted to the ligand depletion function while keeping the protein concentration constant (Equation 1).

[0092]

number

[0093] (In the formula, △ = change in chemical shift after adjustment, d) max = Maximum chemical shift perturbation, K d = binding / dissociation constant, p = [protein], and l = [ligand].

[0094] The spectrum was overlaid and displayed using XEASY software and Adobe Illustrator.

[0095] <Synthesis of pep213>: Peptides pep213 (SHKHDPLPYPHFLL, SEQ ID NO: 1) and TAT-p213 (YGRKRQRRGSGSSHKHDPLPYPHFLL, SEQ ID NO: 3) were all purchased from GenScript, Inc. (Piscataway, New Jersey) after undergoing N-terminal acetylation and C-terminal amidation and maintaining a purity of over 95% by HPLC. The TAT-p213 fusion peptide contained a GSGSGS (SEQ ID NO: 4) linker between the TAT cell-permeable sequence (YGRKKRRQRRR, SEQ ID NO: 2) and pep213.

[0096] <Endogenous tryptophan fluorescence> Using a PTI Model #814 fluorometer, λ at 295 nm was measured within the rectangular cell of a Starnacelles 3-Q-10 quartz fluorometer. ex and λ of 300-400 nm emTryptophan fluorescence data was collected using a path length of 10 mm and excitation / emission slit widths of 4 / 6 nm. The high-concentration peptide stock solution (pep213) was resuspended in the final hFis1 dialysis suspension to prepare a concentration series with peptide points of 0 μM, 1 μM, 3 μM, 7 μM, 10 μM, 30 μM, 70 μM, 100 μM, 300 μM, 700 μM, and 1000 μM. At each titration point, the tryptophan emission spectrum was measured for the sample excluding Fis1, and then 5 μL of 400 μM hFis1 was added to the sample to recollect the final concentration of 10 μM hFis1 and the tryptophan emission spectrum. To account for buffer fluorescence from the peptide and tyrosine fluorescence background, the background fluorescence intensity was subtracted from the spectrum lacking Fis1 to generate a differential emission spectrum. The average emission wavelength at each peptide concentration was calculated according to Equation 3, plotted as a function of the natural logarithm of the peptide concentration, and then fitted to a Boltzmann S-shaped model (Equation 4). {Royer, 1993 #10132}

[0097]

number

[0098] (where 〈 λ 〉=average emission wavelength, I n = wavelength λ n The fluorescence intensity emitted and the λ of 310-370 nm n This is the sum calculated using [the formula / method].

[0099]

number

[0100] (In the formula, λ = average emission wavelength, A = pre-transition phase, B = post-transition phase, K) D = Equilibrium dissociation constant, p = natural logarithm of peptide concentration, and c = slope of the transition phase.

[0101] <Effects of pep213 on the bioavailability efficiency of endothelial cells and endothelium-dependent vasodilation> For these studies, blood vessels were randomly selected from a subset of subjects, regardless of the presence or absence of T2DM. Blood vessels from healthy subjects were pretreated with HG (33 mM) for 6 hours and then exposed to 1 μM or 10 μM pep213 attached to TAT sequences to facilitate cell uptake. Blood vessels from T2DM subjects were cultured under NG conditions (5 mM) and exposed to 1 μM or 10 μM pep213-TAT. Endothelial cell-dependent vasodilation with increasing acetylcholine dose, smooth muscle responsiveness to papaverine, and eNOS dependence of vasodilation response to acetylcholine using L-NAME were evaluated in the same manner as described above. 2,4,8,13,15 .

[0102] <Statistical analysis> Statistical analysis was performed using either GraphPad Prism V7.03 (GraphPad Prism version 8.0.0 for Windows, GraphPad Software, Inc., San Diego, California, USA) or SigmaPlot version 12.5 (Systat Software, Inc., California, USA). P<0.05 was considered statistically significant. Unless otherwise specified, data are shown as mean ± SE. All functional vascular data were analyzed using two-way ANOVA with post-hoc studies to determine differences between groups (by Dunnett's multiple comparison test) and dose-response (by Tukey's multiple comparison test). In addition, TEER assay data and bioenergetic data were analyzed using two-way ANOVA followed by post-hoc studies (Tukey's multiple comparison test) to determine differences between HG, LG, and NG treatments. DAF2-DA fluorescence intensity in human blood vessels, Fis1 knockdown efficiency in HMEC-1 cells, and mitochondrial protein levels measured by Western blotting were analyzed using one-way ANOVA, followed by Tukey's multiple comparison test to evaluate differences between groups. DAF2-DA fluorescence intensity in HMEC-1 cells transfected with siFis1 and scrambled siRNA was analyzed using two-way ANOVA and post-hoc analysis (Tukey's multiple comparison test). Production and relative ratios of P-eNOS and β-actin in HMEC-1 cells stimulated with A 23187 were analyzed using Student's paired t-test.

[0103] <Detailed results> (Characteristics of the subject) A total of 67 subjects were recruited (14 patients with T2DM and 53 healthy controls). Table 1 shows detailed characteristics of the subjects who participated in each trial of this project. Healthy controls were significantly younger than the T2DM group (39±15 years vs. 55±12 years, P=0.0002). In the healthy control group of this trial, body weight index, waist circumference, systolic blood pressure, fasting blood glucose, and glycosylated hemoglobin were significantly lower. Healthy controls also had higher HDL cholesterol levels. None of the healthy subjects had a history of chronic use of cardiometabolic drugs.

[0104] Furthermore, the subject characteristics for each study concerning human blood vessels are shown in Appendix 1 (characteristics of subjects who underwent endothelium-dependent vasodilation testing by transfecting blood vessels with Fis1 siRNA or scrambled control), Appendix 2 (characteristics of subjects who underwent NO bioavailability testing by transfecting blood vessels with Fis1 siRNA or scrambled control), Appendix 3 (characteristics of subjects who underwent endothelium-dependent vasodilation testing by transfecting blood vessels with Drp1 siRNA or scrambled control), Appendix 4 (characteristics of subjects who underwent NO bioavailability testing by transfecting blood vessels with Fis1 siRNA or scrambled control), and Appendix 5 (characteristics of subjects who underwent vasoactivity testing by exposing blood vessels to pep213).

[0105] <Effects of Fis1 or Drp1 inhibition on endothelial cell-dependent vasodilation and NO bioavailability in human blood vessels> The inventors of this application measured vasodilation in healthy human resistance vessels cultured with glucose at the same concentrations observed in T2DM patients, in accordance with the clinical trial protocol (Figure 1A-B). Consistent with previous findings, both high glucose (33 mM) and low glucose (2.5 mM) levels blunted the endothelial cell-dependent vasodilation response to acetylcholine, which appears to be primarily related to eNOS activity based on L-NAME inhibition. To investigate whether mitochondrial fission protein Fis1 may be involved in this functional impairment, healthy blood vessels were transfected with Fis1 siRNA to reduce Fis1 mRNA by approximately 30% (Figure 9). In these healthy human resistance vessels, transfection with Fis1 siRNA prevented both HG-induced and LG-induced impairment of endothelial cell-dependent vasodilation (Figure 1A-B). Since L-NAME administration completely blocked the preventive effect on both, it is suggested that the Fis1 siRNA-related improvement is eNOS-dependent.

[0106] To support this interpretation, transfection with Fis1 siRNA resulted in a significant increase in the fluorescence emission of the NO-sensitive dye DAF2-DA in healthy human blood vessels exposed to HG [Figure 2A; N=9, P=0.04 between Fis1 siRNA and all other exposures (scrambled control, scrambled control + L-NAME, and Fis1 siRNA + L-NAME)] and LG (Figure 2B, n=8, P=0.01 between Fis1 siRNA and all other exposures). In both cases, administration of L-NAME completely eliminated the DAF2-DA fluorescence-enhancing effect. These data support the idea that Fis1 silencing under the stress conditions observed in T2DM patients can improve vasodilation in a NO-dependent manner. Next, we investigated whether Fis1 siRNA treatment could improve vasodilatory activity in resistant blood vessels of T2DM patients. Treatment with Fis1 siRNA improved endothelial cell-dependent vasodilation impairment (Figure 3, n=6, P=0.002 overall). Similar to the case with vessels from healthy subjects, the favorable effect of Fis1 siRNA transfection on vessels from diabetic subjects was entirely attenuated by L-NAME. Fis1 siRNA transfection did not affect papaverine vasodilation in any of the studies (specific data omitted). Similar findings were obtained when Drp1 genetic silencing was performed on vessels from healthy subjects and diabetic patients (Figures 10-12), which is consistent with the view that excessive mitochondrial fission plays a major role in NO-dependent vasodilation impairment in diabetic endothelial cells.

[0107] <Effects of Fis1 transfection on Fis1 expression, NO production, eNOS activation, and the expression of mitochondrial proteins related to nuclear fission, fusion, and autophagy> In HMEC-1 cells transfected with Fis1 siRNA, Fis1 expression was reduced by 70% (Figure 13). When examining the responsiveness of HMEC-1 to stimuli that increase eNOS activation and NO bioavailability, it was found that calcium ionophore A23187 significantly increased both eNOS phosphorylation at the Ser1177 activation site and DAF2-DA fluorescence intensity in untransfected HMEC-1 cells (Figure 14A, N=6, P=0.03; Figure 14B, N=4, P=0.02). Furthermore, Fis1 siRNA transfection significantly increased DAF2-DA signaling from HMEC-1 cells stimulated with A23187 compared to transfection with scrambled control siRNA (Figure 15, N=7, P<0.0001). Knockdown of Fis1 expression did not significantly alter the expression of mitochondrial proteins involved in mitochondrial fission, fusion, or autophagy (such as cytochrome c, MFN1, MFN2, Drp1, GABARAP, MFF, POLG, NDUF88, P62, OPA1, or AMP kinase) (Figure 16). Under HG, LG, and NG culture conditions, there was clearly no effect on the expression of these proteins. High and low glucose conditions affected the expression of some of these proteins, but Fis1 knockdown had no effect at all in the given glucose exposure cases.

[0108] <Effects of Fis1 knockdown and endothelial cell layer integrity> Figure 4A shows the effect of Fis1 suppression on endothelial cell layer integrity under HG and NG conditions. Under NG conditions, although Fis1 expression knockdown was modest, a statistically significant decrease in endothelial cell layer resistance was observed. Under HG conditions, endothelial cell layer resistance was significantly lower than under NG conditions, regardless of whether Fis1 knockdown was present (P<0.001). Fis1 knockdown increased resistance under HG conditions, but it remained lower than the resistance measured under NG conditions. Similar findings were obtained for LG (Figure 4B).

[0109] <Effects of Fis1 knockdown on oxygen consumption and glycolysis> Since no effect was observed on extracellular acidification rate or oxygen consumption by knockdown of Fis1, regardless of glucose exposure (NG, HG, LG; n=5 for all measurements, Figure 5), it is suggested that inhibition of Fis1 does not have a significant effect on mitochondrial bioenergy synthesis.

[0110] <Measurement of the affinity of pep213 for Fis1> Previous studies have identified several peptides expected to bind to Fis1 by screening them against truncated proteins using phage display. 17 The inventors of this invention investigated the binding of these peptide subsets to Fis1 and found that the affinity was weak (high μM). Based on this data, the inventors of this invention designed a novel peptide, pep213 (SEQ ID NO: 1), which is presumed to have high affinity for Fis1 and inhibit its activity. Stable isotope 15 The NMR sample of Fis1, homogenized with N, was titrated by gradually increasing the dose of the peptide. 1 H - 15 When the N HSQC spectra were superimposed, it was found that many signals changed with the addition of pep213 (Figure 6A). The resulting data could be globally fitted to a single-site binding model, and the apparent K D =7±2 μM (Figure 6B). In the tryptophan fluorescence experiment, when the change in the tryptophan emission spectrum upon addition of p213 was fitted to the coupled isotherm, a similar apparent K was obtained. D The result was 3.3±0.1 μM, confirming the affinity mentioned above (Figure 6C). Another peptide (scrambled pep213) with the same amino acid composition as pep213 (but in no particular order) was found at 2 mM. 15 Since the addition of N-Fis1 did not cause any perturbation of the chemical shift, the Fis1-pep213 interaction is presumed to be specific (Figure 6D).

[0111] <Effects of pep213 on endothelial cell-dependent vasodilation in human resistance vessels> Facilitating cellular uptake through exposure to pep213 attached to the TAT peptide significantly improved endothelial cell-dependent vasodilation in blood vessels from healthy subjects exposed to high glucose (Figure 7A, N=6, overall P<0.0001) and from T2DM subject Yura (Figure 7B, N=4, overall P<0.05). In both cases, the improvement observed with pep213-TAT was reversed by treatment with L-NAME. Pep213-TAT improved both the reduction of endothelial cell-dependent vasodilation induced by high glucose and the impairment of endothelial cell-dependent vasodilation in blood vessels from T2DM subjects, but the scrambled peptide using the same amino acids in a random order showed no effect in either blood vessel (Figure 8, N=5, P<0.001). Since no difference in the papaverine response was observed in any of these tests (specific data omitted), it is suggested that pep213-TAT does not affect the smooth muscle response. Furthermore, human microvascular endothelial cells exposed to pep213-TAT for 1 hour showed a significant increase in DAF2-DA fluorescence emission (Figure 17, N=3, P=0.04).

[0112] [Consideration] Through these investigations, we were able to obtain several new findings. Firstly, human microvascular endothelial cells have the ability to maintain endothelial cell barrier function by producing NO and dilating in an endothelium-dependent manner, and the negative effects of high and low glucose exposure on this ability can be blocked by molecular inhibition of Fis1. Furthermore, molecular inhibition of Fis1 expression can improve impaired endothelial cell-dependent vasodilation in T2DM patients. Moreover, these desirable effects can be achieved without altering the metabolism of endothelial cells or the expression of other proteins involved in mitochondrial fusion, fission, and autolysis. In addition, based on our knowledge of the structure of a crucial binding site on Fis1, the inventors designed a peptide pep213 with low micromolar binding affinity to Fis1 and confirmed its binding using two independent methods. Finally, this disclosure demonstrates that pep213 possesses physiological activity, improving NO bioavailability in human microvascular endothelial cells and reversing high glucose-induced endothelial cell-dependent vasodilatory attenuation and type 2 diabetes-related endothelial cell-dependent vasodilatory impairment in human resistance arteries in a nitric oxide synthase-dependent manner. These findings suggest that excessive Fis1 expression and activity play a significant dynamic role in endothelial dysfunction during acute and chronic glycemic abnormalities. Furthermore, these data support the idea that Fis1-targeted pharmacological therapy is a promising means of improving vascular health in patients with type 2 diabetes.

[0113] There is rationale supporting the importance of investigating whether targeting mitochondrial proteins involved in regulating mitochondrial dynamics could have a favorable effect on human vascular endothelial function in patients with type 2 diabetes or those exposed to high glucose concentrations. Maintaining a proper balance in the dynamics of the mitochondrial reticular structure is essential for maintaining normal mitochondrial function. When mitochondrial dynamics are normal, mitochondria can move to areas of increased metabolic demand, repair damaged mitochondria, maintain normal mitochondrial energy synthesis, limit the production of reactive oxygen species, and enable autophagy by isolating irreparably damaged mitochondrial components. 18-22 Acute and chronic exposure to excessive nutrients, such as elevated glucose and free fatty acids, stimulates the mitochondrial reticular structure, leading to nuclear fission in multiple cell types, including human endothelial cells, and the production of excessive ROS within the mitochondria. 1,22-24 In semen studies using cultured endothelial cells, it has been demonstrated that high glucose levels lead to the production of excessive ROS in mitochondria, resulting in acute and chronic endothelial cell dysfunction through cellular signaling pathways and epigenomic pathways. 3,25 Furthermore, the inventors of this application have previously demonstrated that reducing mitochondrial superoxide concentration in resistance arteries derived from patients with type 2 diabetes can improve endothelial cell-dependent vasodilation impairment in human resistance arterioles. 2 .

[0114] These data suggest that targeting mitochondrial dynamics proteins to suppress excessive mitochondrial fission may be beneficial in maintaining the health of blood vessels exposed to acute or chronic abnormal glucose levels. While multiple proteins are involved in mitochondrial fusion and fission, Fis1, a Drp1 docking protein located on the outer mitochondrial membrane, has repeatedly been shown to be overexpressed during diabetes, acute exposure to high glucose, and, in some cell types, during acute exposure to excessive free fatty acids. 1,26-29Fis1 is not necessarily required for all nuclear fission processes. 30 Under cellular stress conditions such as hypoxia or excessive glucose exposure, Fis1-Drp1-mediated fission is considered preferable. 9-12 Previous studies using human vascular structures have revealed that Fis1 is overexpressed in endothelial cells derived from patients with type 2 diabetes. 1 Furthermore, in cultured human aortic endothelial cells, exposure to high glucose levels leads to overexpression of Fis1, and in human aortic endothelial cells exposed to high glucose levels, molecular silencing of Fis1 or Drp1 expression increases the phosphorylation of eNOS at its Ser1177 activation site. 1 Furthermore, the inventors of this invention have previously shown that low glucose exposure (when administered to a clinically relevant degree) leads to mitochondrial fission and overproduction of mtROS in human endothelial cells, but such fission and overproduction can be reversed by suppressing the activity of mitochondrial fission proteins. 8 New data obtained by the inventors of this application shows that molecular inhibition of Fis1 improves endothelial cell-dependent vasodilation impairment in intact human resistance arteries, enhances the bioavailability of nitric oxide in these vessels, and protects the integrity of the endothelial cell layer during the course of acute blood glucose abnormalities, significantly expanding the translational effects shown in previous studies. Furthermore, it was found that even targeting Fis1 does not affect the expression of other mitochondrial proteins involved in endothelial cell mitochondrial metabolism or its dynamics or auto-actions, thus confirming the validity of the results of this study, which was conducted to investigate the effects beyond the target expected when targeting Fis1 as part of treatment.

[0115] The inventors of this invention have found that pep213, designed to bind to Fis1 at its primary interaction surface, can improve NO bioavailability in cultured endothelial cells and mitigate both type 2 diabetes-induced endothelial cell-dependent vasodilation impairment and high glucose-induced endothelial cell-dependent vasodilation attenuation. The data obtained by the inventors using the novel pep213, together with their molecular data, support the idea that direct pharmacological inhibition of Fis1 has a positive effect on diabetic vascular structure. Interestingly, when several drugs were administered for the treatment of type 2 diabetes, two of them not only reduced the cardiovascular risk of T2DM but also inhibited mitochondrial fission as an "off-target" effect by reducing Fis1 expression and / or Drp1 expression. These drugs include empagliflozin and SGLT2 inhibitors that are effective against cardiovascular risk and mortality and microvascular renal disease in T2DM patients. 31,32 When used in a rat model of T2DM(OLEFT), it not only reduced Fis1 overexpression but also decreased mitochondrial fission and further increased the mitochondrial reactive oxygen species enzyme SOD2 in cardiomyocytes. 33 Metformin, a conventional first-line drug used for blood glucose management in T2DM and also effective for cardiovascular function, suppresses atherosclerosis formation in ApoE knockout mice by inhibiting Drp1-mediated nuclear fission. 34 Vildagliptin, a dipeptidyl peptidase 4 inhibitor, is one of a group of drugs that have been shown to increase NO production, decrease Fis1 and Drp1 expression, reduce Drp1 translocation from the cytoplasm, and decrease mitochondrial fission and ROS production in the aortic endothelium of diabetic mice. 35 Interpreting the data obtained by the present inventors within the framework, it is thought that these common diabetes medications may bring about vascular improvement effects in T2DM, partly due to off-target effects on the expression and / or interaction of Fis1 and Drp1. Further investigation is needed to determine whether these improvements are due to improved blood glucose control or direct inhibition by the interaction of Fis1 or Drp1.

[0116] The experiments conducted by the inventors of this invention have several limitations. First, previous data support the conclusion that the Fis1-Drp1 axis is the axis most strongly associated with the development of hyperglycemia and other pathological stimuli. Second, previous research by the inventors of this invention supports the pathophysiological role of Fis1 in diabetic endothelial cells. Therefore, the inventors of this invention focused primarily on the Fis1-Drp1 axis to modulate vascular effects in T2DM and abnormal glucose exposure cases. 1,9-12 It is unclear whether inhibiting Fis1 inhibits its interaction with Drp1 and other mitochondrial docking proteins (MFF, MiD49 / 51), and this warrants further investigation. Furthermore, mitochondrial fusion proteins (e.g., OPA1, Mfn1, Mfn2, etc.) were intentionally not focused on. New data suggest that Mfn2 expression is downregulated in the tissues of T2DM patients, and that Fis1 may promote fission and further reduce bioavailability by inhibiting the GTPase activity of fusion proteins. 1,36-38 Further investigation is warranted regarding the role of the fusion pathway in regulating human vascular endothelial function and its potential interaction with Fis1. While the inventors of this invention did not control osmotic pressure in this experiment, previous studies have conducted similar experiments using intact blood vessels and endothelial cells to investigate mitochondrial regulation of vascular function, and no osmotic pressure differences that could influence the inventors' results were observed. 4,8 The novelty of the findings obtained by the inventors of this application regarding human-derived tissues highly associated with disease, as well as the development of a novel therapeutic intervention method to slow down or improve the adverse effects of diabetes on human vascular structure by targeting the mitochondrial fission mechanism, are sufficient to offset the aforementioned limitations.

[0117] <Supplementary results> In previous research, the inventors of this invention have reported similar findings supporting the role of excessive mitochondrial fission in the reduction of NO-dependent vasodilation in diabetic endothelial cells, by silencing the Drp1 gene, the only mechanical enzyme responsible for membrane deformation and cleavage in mitochondrial fission. 8 The study revealed that introducing siRNA into Drp1 significantly reduced Drp1 expression in human arterioles, preventing LG-induced impairment of endothelial cell-dependent vasodilation in healthy human resistance arteries. 8 This study revealed that genetic silencing of Drp1 can also prevent HG-induced impairment of endothelial cell-dependent vasodilation (Figure 10, N=6, overall P<0.0001 compared to all exposures other than HG). This effect was completely negated by L-NAME but not by papaverine (specific data omitted). In addition, DAF2-DA fluorescence emission was significantly increased in healthy human blood vessels exposed to HG (Figure 11A, n=9, P=0.02 overall, P<0.05 between Drp1 siRNA and scrambled control, scrambled control + L-NAME, and Drp1 siRNA + L-NAME) and healthy human blood vessels exposed to LG (Figure 11B, n=9, P=0.003 overall, P<0.04 between Fis1 siRNA and other exposures). In all cases, the DAF2-DA fluorescence-enhancing effect was completely negated by administration of L-NAME. Furthermore, transfection with Drp1 siRNA tended to improve endothelial cell-dependent vasodilation in human blood vessels derived from patients with type 2 diabetes (Figure 12).

[0118] [Conclusion] The studies described herein demonstrated the critical role of Fis1 in regulating vascular endothelial function in intact resistance vessels derived from T2DM patients and in the course of acute glucose abnormalities. To demonstrate the importance of this interaction to vascular function in vessels derived from T2DM patients, the inventors used a novel peptide, pep213, specifically designed to occlude the major binding surface of Fis1. Furthermore, the inventors showed that a decrease in Fis1 concentration leads to improved endothelial function but does not affect other important mitochondrial proteins or mitochondrial oxygen consumption. Considering these data in conjunction with previous research findings, there is a need for further research on pharmacological targeting of Fis1 with the aim of mitigating T2DM-related vascular complications.

[0119] [Example 2] The inventors of this invention further demonstrated that overexpression of Fis1 in resistance arteries derived from healthy humans (resistance arteries transfected with a plasmid for 48 hours of culture time for the purpose of endothelial-specific overexpression of human Fis1) leads to impaired eNOS-dependent vasodilation (this can be determined from the fact that endothelial cell-dependent vasodilation can be induced even when acetylcholine is lost when using the eNOS inhibitor L-NAME). N=5, overall P<0.001, and as shown in Figure 18, *P<0.05 at the appropriate dose of acetylcholine. Furthermore, the peptide of the present invention, such as pep213, can improve impaired endothelial cell-dependent vasodilation in resistance arteries. Exposure for 1 hour to pep213 attached to a tat sequence (1 μM pep213-tat) for the purpose of improving cell permeability can improve impaired endothelial cell-dependent vasodilation in healthy human resistance arterioles that exhibit overexpression of Fis1 in endothelial cells (achieved by transfecting blood vessels with a plasmid aimed at endothelial cell-specific overexpression of human Fis1 using a lentiviral vector). A 1 μM scrambled peptide using the same amino acids as pep213 in no particular order showed no effect on acetylcholine-mediated endothelial cell-dependent vasodilation. pep213-tat brings about improvement of endothelial cell-dependent vasodilation in an eNOS-dependent manner. (This can be judged from the fact that endothelial cell-dependent vasodilation can be induced even when acetylcholine is lost when using the eNOS inhibitor L-NAME.) N=5, overall P<0.001, *P<0.05 at the appropriate dose of acetylcholine.

[0120] Furthermore, cocrystallization of recombinant human Fis1 and Pep213 was performed. The peptides were then resuspended in Fis1 buffer. Crystals were grown by the suspension vapor diffusion method, rapidly transferred to an antifreeze solution, and then rapidly frozen in liquid nitrogen. Multiple datasets were remotely collected at the LS-CAT beamline 21-ID-F of the US Advanced Synchrotron Radiation Facility (Argonne, Illinois) using an MD2-S microdiffractometer and a Rayonics MX300 detector. Data was collected at a detector distance of 260 mm, with a total of 180° of data collected in 0.5° increments. All data were processed using XDS. Molecular substitution was performed using Phoenix Phaser MR, followed by automated model construction using Phoenix Autobuild. Improvements and fine-tuning were performed using Phoenix Refine and Wincoot. The final structural resolution was 1.85 Å. The co-complex structure (A) shows that pep213 is bound to Fis1 through various bonding interactions, including salt bridge formation, hydrogen bonding, and van der Waals interactions.

[0121] Pep213, which is important for Fis1 binding, was identified by sequentially substituting each of the 14 amino acids with alanine, as shown in Table 2. Microscale thermophoresis was used to identify the pep213 residues essential for the Fis1-pep213 interaction. A total of 14 peptides were obtained by sequentially substituting each residue of pep213 with alanine. The peptides were then resuspended in Fis1 buffer. Microscale thermophoresis experiments were performed at 25°C using a nanotemper monolith NT.115 instrument, and a 16-point dilution series (1:1 dilution) of each peptide was quantified against a constant concentration of fluorescently labeled Fis1. Data analysis was performed using affinity analysis software attached to the nanotemper monolith. D The value was determined. The reaction ΔG° (ΔG°=-RTlnK) was calculated using the bond affinity value, and this was used to determine the ΔΔG° value (ΔG° pep213 -ΔG° 異形 The following calculation was performed. As can be seen from the low ΔΔG° value, the subset of residues at each terminal of the peptide does not show a significant contribution to binding.

[0122] [Table 1]

[0123] Table 2

[0124] Table 3

[0125] Table 4

[0126] Table 5

[0127] Table 6

[0128] Table 7

[0129] Table 8

[0130] Table 9

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Claims

1. (a) A mitochondrial fission protein 1 (Fis1) activity inhibitory peptide consisting of one amino acid sequence of SEQ ID NO: 1 (SHKHDPLPYPHFLL) or SEQ ID NOs: 16-21, 26, and 29.

2. The inhibitory peptide according to claim 1, wherein the inhibitory peptide comprises (b) an amino acid sequence including a carrier peptide, a tag peptide, or a cell-binding peptide, or (a) linked to a carrier peptide.

3. The inhibitory peptide according to claim 1 or 2, characterized in that the inhibitory peptide is linked to a carrier peptide.

4. The inhibitory peptide according to claim 2 or 3, wherein the carrier peptide is a cell-permeable peptide sequence.

5. The inhibitory peptide according to claim 4, wherein the cell-permeable peptide sequence is a sequence having at least 90% sequence identity with TAT (SEQ ID NO: 2) or SEQ ID NO:

2.

6. The inhibitory peptide according to any one of claims 2 to 4, wherein both (a) and (b) are peptides linked by a linker sequence.

7. The inhibitory peptide according to claim 6, wherein the linker sequence is sequence number 4, 11, 12, 13, 14, or 15.

8. The inhibitory peptide according to any one of claims 1 to 7, wherein the inhibitory peptide comprises the amino acid sequence of SEQ ID NO: 3 (YGRKKRRQRRRGGSGSGSSHKHDPLPYPHFLL), SEQ ID NO: 31, SEQ ID NO: 32, or SEQ ID NO:

39.

9. A polynucleotide encoding a peptide that inhibits Fis1 activity, comprising a heterologous promoter sequence and a polynucleotide sequence encoding the inhibitory peptide according to any one of claims 1 to 8.

10. The polynucleotide according to claim 9, wherein the polynucleotide is a vector.

11. A vector capable of expressing an inhibitory peptide, comprising a promoter operably connected to a polynucleotide encoding the inhibitory peptide according to any one of claims 1 to 8 or the polynucleotide according to claim 9.

12. A host cell comprising the vector described in claim 11, capable of expressing an inhibitory peptide.

13. An inhibitory peptide according to any one of claims 1 to 8, for use in the treatment of vascular complications associated with type 2 diabetes.

14. An inhibitory peptide according to any one of claims 1 to 8, for use in improving vasodilation disorders.

15. An inhibitory peptide according to any one of claims 1 to 8, for use in enhancing the bioavailability of nitric oxide (NO) in human microvascular endothelial cells.

16. The inhibitory peptide for use according to claim 15, wherein the endothelial cells are from the body of a subject having vascular dysfunction.

17. An inhibitory peptide according to any one of claims 1 to 8 for use in treating endothelial dysfunction.

18. The inhibitory peptide for use according to claim 17, wherein the endothelial dysfunction includes atherosclerosis.

19. The inhibitory peptide for use according to claim 17, wherein endothelial dysfunction is associated with any of the following diseases: atherosclerosis, cerebrovascular disease, coronary artery disease, renovascular disease, and peripheral artery disease.

20. A kit for use in inhibiting Fis1 activity, comprising an inhibitory peptide according to any one of claims 1 to 8, a polynucleotide according to claim 9 or claim 10 capable of expressing the inhibitory peptide, a vector according to claim 11, or a cell according to claim 12, and instructions for use.

21. A composition comprising an inhibitory peptide according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.

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