Inhibitors of DRP1 / FIS1 and methods of use thereof

Abstract

The present invention provides novel therapeutic agents, pharmaceutical compositions, and methods for the treatment, prevention, or amelioration of conditions associated with mitochondrial dysfunction.

Description

[0001] INHIBITORS OF DRP1 / FIS1 AND METHODS OF USE THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to novel therapeutic agents, pharmaceutical compositions, and methods for the treatment, prevention, or amelioration of conditions associated with mitochondrial dysfunction. More particularly, the invention relates peptides and peptidomimetics having drug-like properties that selectively modulate mitochondrial protein-protein interactions, as well as compositions comprising such agents and methods of using them in the management of diseases and disorders involving impaired mitochondrial function.

[0004] BACKGROUND OF THE INVENTION

[0005] Mitochondria play critical roles in many cellular activities, such as energy production, metabolism, cell signaling, aging, and cell death [1, 2], Mitochondrial dysfunction is associated with various diseases, including inflammatory, neurodegenerative [3], cancer, and cardiovascular diseases (CVDs) [4, 5], Under physiologic conditions, the cell relies on coordinated processes of fusion and fission to maintain mitochondrial quality control and function [6], Mitochondrial fission is mediated by an evolutionarily conserved dynamin-r elated GTPase, dynamin-related protein 1 (Drpl) [7], Drpl is a cytosolic protein that assembles into spiral filaments around mitochondrial tubules and induces membrane fission. Drpl is recruited to the mitochondrial outer membrane by several adaptor proteins, including Fission protein 1 (Fisl) [8], Mitochondrial fission factor (Mff) [9], and the two homologous mitochondrial dynamics proteins of 49 and 51 kDa (Mid49 and Mid51)

[0010] , The interaction between Drpl and Fisl under stress conditions has been shown to induce excessive fission, resulting in dysfunctional mitochondria, cell death, and organ damage

[0011] , As a result, we were seeking to develop an inhibitor of Drpl / Fisl protein-protein interaction (PPI)

[0012] , Small molecules, while cost-effective, cell permeability, and oral availability, often struggle to target PPIs effectively due to their flat surfaces lacking binding pockets [13, 14], Antibodies are potent but expensive and cell-impermeable [15, 16], Peptides offer a balance with better selectivity and cell permeability than small molecules and are cheaper than antibodies.

[0006] Previously, we developed a linear peptide, Pl 10, that targets the Drpl / Fisl PPI specifically and inhibits excessive mitochondrial fission. Pl 10 demonstrates versatile therapeutic potential across several disease models. In Parkinson's disease models, it effectively inhibits mitochondrial fragmentation and reduces reactive oxygen species (ROS) production, thereby improving mitochondrial membrane potential and integrity. This treatment also enhances neuronal cell viability and attenuates the loss of primary dopaminergic neurons

[0017] , In Huntington's disease models, Pl 10 mitigates excessive mitochondrial fragmentation induced by mutant huntingtin (mtHtt), leading to improved mitochondrial function and increased cell viability

[0018] , Moreover, in ischemia-reperfusion (I / R) injury models, including cardiomyocytes and in vivo myocardial infarction models in rats, Pl 10 prevents long-term cardiac dysfunction

[0019] ,

[0007] Linear peptides are known to face some challenges as drug candidates, including poor serum stability due to susceptibility to enzymatic degradation, and can have reduced specificity for their targets, leading to reduced efficacy and off-target effects. These limitations stem from their structural flexibility, which can lead to nonselective receptor binding

[0020] , To enhance peptide stability, various chemical modifications can be employed, such as N-terminal or C-terminal substitutions

[0021] , modifications of the amide bond

[0022] , incorporation of unnatural amino acids

[0023] , or cyclization [24-27], Cyclization, in particular, can be beneficial for increasing the structural rigidity and metabolic stability of peptides. Certain cyclic peptides, like Cyclosporin A derived from fungi, exemplify this approach by demonstrating long-term stability and enabling membrane permeability. Cyclosporin A's clinical success as an immunosuppressant underscores the potential of peptidomimetics (modified peptides) as promising drug candidates

[0028] ,

[0008] Recent progress in the field of mitochondrial research has yielded important insights and therapeutic approaches, yet significant challenges remain. Accordingly, there is an ongoing need for the development of novel therapeutic agents, compositions, and methods capable of addressing a wide range of conditions linked to mitochondrial dysfunction, with improved pharmacological profiles and clinical applicability.

[0009] SUMMARY OF THE INVENTION

[0010] It is the principal object of the present invention to provide novel agents, compositions, and methods for the treatment of conditions associated with mitochondrial dysfunction, including peptides and peptidomimetics with drug-like properties, offering improved therapeutic efficacy and applicability across diverse clinical indications. According to some embodiments, the invention provides a peptide or a peptidomimetic comprising amino acid sequence:

[0011] Aai-Aa2-Arg-Pro wherein

[0012] Aai is Ser, Thr, or an analogue thereof,

[0013] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth in SEQ ID NO: 1 or a portion thereof.

[0014] According to some embodiments, the invention provides a pharmaceutical composition comprising the peptide or the peptidomimetic comprising amino acid sequence:

[0015] Aai-Aa2-Arg-Pro wherein

[0016] Aai is Ser, Thr, or an analogue thereof,

[0017] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth in SEQ ID NO: 1 or a portion thereof, and at least one pharmaceutically acceptable carrier.

[0018] According to some embodiments, the invention provides a method of treating a condition associated with mitochondrial dysfunction, comprising administering to a subject in need of such treatment an effective amount of a peptide or a peptidomimetic comprising amino acid sequence: Aai-Aa2-Arg-Pro wherein

[0019] Aai is Ser, Thr, or an analogue thereof,

[0020] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth in SEQ ID NO: 1 or a portion thereof, and at least one pharmaceutically acceptable carrier.

[0021] According to some embodiments, the invention provides a method of treating a condition associated with mitochondrial dysfunction, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a peptide or a peptidomimetic comprising amino acid sequence:

[0022] Aai-Aa2-Arg-Pro wherein

[0023] Aai is Ser, Thr, or an analogue thereof,

[0024] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof, and at least one pharmaceutically acceptable carrier.

[0025] According to some embodiments, the invention provides a method of preventing / reducing mitochondrial fission, comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a peptide or a peptidomimetic comprising amino acid sequence: Aai-Aa2-Arg-Pro wherein

[0026] Aai is Ser, Thr, or an analogue thereof,

[0027] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof, and wherein the pharmaceutical composition comprises at least one pharmaceutically acceptable carrier.

[0028] According to some embodiments, the invention provides a method of preventing / reducing mitochondrial fission, comprising administering to a subject in need of such treatment an effective amount of a peptide or a peptidomimetic comprising amino acid sequence:

[0029] Aai-Aa2-Arg-Pro wherein

[0030] Aai is Ser, Thr, or an analogue thereof,

[0031] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof.

[0032] According to some embodiments, the invention provides a method of inhibiting Drpl / Fisl protein-protein interaction (PPI) in a cell, comprising applying to the cell an effective amount of a peptide or a peptidomimetic comprising amino acid sequence: Aai-Aa2-Arg-Pro wherein

[0033] Aai is Ser, Thr, or an analogue thereof,

[0034] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof.

[0035] According to some embodiments, the invention provides use of a peptide or a peptidomimetic comprising amino acid sequence:

[0036] Aai-Aa2-Arg-Pro wherein

[0037] Aai is Ser, Thr, or an analogue thereof,

[0038] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof.

[0039] According to some embodiments, the invention provides use of a peptide or a peptidomimetic comprising amino acid sequence:

[0040] Aai-Aa2-Arg-Pro wherein

[0041] Aai is Ser, Thr, or an analogue thereof, Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof, in the treatment of a disease or condition associated with mitochondrial dysfunction.

[0042] According to some embodiments, the invention provides a peptide or a peptidomimetic comprising amino acid sequence:

[0043] Aai-Aa2-Arg-Pro wherein

[0044] Aai is Ser, Thr, or an analogue thereof,

[0045] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof for use as a medicament.

[0046] According to some embodiments, the invention provides a pharmaceutical composition comprising peptide or a peptidomimetic comprising amino acid sequence:

[0047] Aai-Aa2-Arg-Pro wherein

[0048] Aai is Ser, Thr, or an analogue thereof,

[0049] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 1009 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof, and at least one pharmaceutically acceptable carrier, for use as a medicament.

[0050] According to some embodiments, the invention provides use of a pharmaceutical composition comprising peptide or a peptidomimetic comprising amino acid sequence:

[0051] Aai-Aa2-Arg-Pro wherein

[0052] Aai is Ser, Thr, or an analogue thereof,

[0053] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain; and, wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID NO: 1 or a portion thereof, and at least one pharmaceutically acceptable carrier, in the treatment of a disease or condition associated with mitochondrial dysfunction.

[0054] BRIEF DESCRIPTION OF THE DRAWINGS

[0055] Figure 1 : (A) A graphical representation of the structure-activity relationship

[0056] (SAR) studies and binding activity relationship analysis; (B) The bioactivity of linear analogues of Pl 10 was assessed through a cell viability assay following stress treatment (C0CI2, 500 pM);

[0057] Figure 2: (A) presents results of optimization of the identified bioactive amino acids that were conducted based on binding to the target protein, Fisl, using field-effect biosensing (FEB) technology. Fisl was immobilized to the chip, and the analyte was applied in a series of concentration solutions. The Y-axis corresponds to the response in biosensor units (BU), and the X-axis corresponds to the different concentrations of the analyte; (B) demonstrates Comparison between the linear long peptide Pl 10, the short linear counterpart CVP-354, and the macrocyclic compound CVP-350, binding to the target protein using FEB technology; (C) A summary of KD values of protein- peptide interactions. KD values were calculated using Prism software. Data are presented as mean ± SD (n=3); (D)Demonstrates Fluorescence polarization binding of CVP-378 (CVP-350 with FAM dye conjugated) to the target protein, Fisl.

[0058] Figure 3: (A) A prediction view of CVP-355 binding to Fisl. The prediction of peptide binding to the crystal structure of the cytosolic domain of Fisl (AlphaFold predicted model: AF-Q9Y3D6-F1) using a rigid receptor protocol (MOE). The resulting model predicts peptide binding to a region on the receptor that shares sequence homology with Drpl (Cyan); (B) A summary of KD values of protein-peptide interactions. KD values were calculated using Prism software. Data are presented as mean ± SD (n=3);

[0059] Figure 4: Presentation of circular dichroism profiles were acquired for compounds in aqueous SDS (100 mM) (A) and water (B);

[0060] Figure 5: The analysis of Pl 10, CVP-354, and CVP-350 according to Lipinski’s

[0061] Rule of Five compared with cyclosporine A, oxytocin and MK-0616;

[0062] Figure 6: (A) Competitive inhibition studies of Drpl / Fisl PPI without and with the compounds studied. Drpl was immobilized on the chip, and Fisl (Black) or Fisl with Pl 10 (blue), or Fisl with CVP-354 (purple), or Fisl with CVP-350 (green) were used as analytes; (B) Summary of KD values of the interaction PPI in the presence and absence of compounds. Data are presented as mean ± SD (n=3);

[0063] Figure 7:(A) Drpl / Mid51 binding curves with and without Pl 10 and CVP-350. Drpl was immobilized on the chip, and Mid51 (Black), or Mid51 with Pl 10 (blue), or Mid51 with CVP-350 (green) were used as analytes; (B) The summary of KD values of the interaction PPI in the presence and absence of compounds; (C) demonstrates competitive binding studies Fisl / Mid51 with Pl 10, CVP-350 or CVP-354. Fisl (500 nM) was immobilized on the chip and Mid51 (10, 100, 250, 500, 1,000, 1,500, 2,000, 2,500, 3,000, and 3,500 nM) (A), or Mid51 with Pl 10 (250 pM) (B), or Mid51 with CVP-350 (250 pM) (C) were sampled at different concentrations. FEB technology monitors experimental data in real-time. The Y-axis corresponds to the Response in biosensor units (BU), and the X-axis corresponds to the different concentrations of the analyte in the experiment; (D) Fisl / Mid51 binding curves with and without Pl 10 and CVP-350. Fisl was immobilized on the chip, and Mid51 (Black), or Mid51 with Pl 10 (blue), or Mid51 with CVP-350 (green) were used as analytes;

[0064] Figure 8:(A) demonstrates Peptides stability in Trypsin at 37 °C. Data are presented as mean ± SD (n=3); (B) demonstrates full RP-HPLC characterization profile of trypsin stability studies for Pl 10; (C) demonstrates Full RP-HPLC characterization profile of trypsin stability studies for CVP-350;

[0065] Figure 9: Impact of compounds on mitochondrial function and oxidative stress in H9c2 cells. (A-B) Pl 10, CVP-354, or CVP-350 (1 pM) significantly increased cell viability compared to controls (untreated cells or cells treated with CTRL compound (TAT peptide)) when exposed to stress (CoCh, 500 pM) (A) (n=6); and did not demonstrate any toxic effects (B) (n=6); (C) Analysis of lactate dehydrogenase (LDH) result with and without peptide treatment Lactate dehydrogenase (LDH) analysis of Pl 10, CVP- 350 and CVP-354 in H9c2 measured by LDH kit; (D-F) Pl 10, CVP-354, or CVP-350 (1 pM) significantly reduced total ROS under stress conditions (D) (n=8); and mitochondrial specific ROS levels (E) (n=3); evaluated mitochondrial markers commonly used to assess the health, function, and integrity of mitochondria. Pl 10, CVP-354, and CVP-350 (1 pM) increased MMP levels (F) (n=6). All values in the graphs are expressed as the mean ± SD;

[0066] Figure 10: Analysis of mitochondrial bioenergetics in H9c2 cells. (A) Representative data of the seahorse assay which was carried out on untreated healthy cells (Control), or cells treated with a stressor (H2O2 at 250 pM treated for 6 h) with or without CVP- 350 or CVP-354 (10 pM each). The Y-axis represents fold changes in oxygen consumption rates (OCR) normalized to total protein content from the cells measured in terms of pmol min-1 pg-1, while the X-axis depicts the time during which OCR was measured; (B - E) Bars represent changes in OCR between the different conditions as indicated. Results show that cells treated with the stressor had reduced levels of basal respiration (B), ATP-linked respiration (C), maximal respiration (D) and spare respiratory capacity (E), compared to the control, which were rescued by the treatment with CVP-350 or CVP-354. All values in the graphs are expressed as the mean ± SD (n=3);

[0067] Figure 11 : Assessment of mitochondrial morphology in H9c2 cells. (A) cells only, (B- F) stressed cells treated with PBS (B), with Pl 10 (C), with CVP-350 (D), with CVP- 354 (E), or with CTRL (TAT) (F) Results were qualitatively described as large and elongated mitochondria (marked by red circle, normal) with impaired mitochondrial membrane integrity (marked by white words), reduced number of cristae, and deformed cristae (wedged). Cells treated with Pl 10 (G) or CVP-350 (H) or CVP-354 (I) showed reduced swelling and fragmented mitochondrial morphology, circled, and marked by arrows, indicating a visible reduction in mitochondrial morphology and fragmentation compared with controls;

[0068] Figure 12 demonstrates TEM images of mitochondrial morphology in H9c2 cells with and without compounds; (A) Mitochondrial morphology comparison with cells only, stress / CoCh + PBS, and stress / CoCl2+ CVP-350. (B) Mitochondrial morphology comparison with cells only and stress / CoCh + PBS. (C) Mitochondrial morphology comparison with cells only, stress / CoCl2+ PBS, and stress / CoCh + CVP-354. (D) Mitochondrial morphology comparison with cells only, and stress / CoCl2+ CVP-350.

[0069] (E) Mitochondrial morphology comparison with stress + PBS, and stress / CoCL + TAT.

[0070] (F) Mitochondrial morphology comparison with stress / CoCl2+ PBS, and stress + CVP- 354. (G) Mitochondrial morphology comparison with stress / CoCh + PBS, and stress / CoCh + CVP-350. (H) Mitochondrial morphology comparison with cells only and stress / H2O2 + PBS. (I) Mitochondrial morphology comparison with cells only and stress / H2O2+ CVP-350;

[0071] Figure 13: CVP-350 reduced cells death and improved cells viability. (A) or during reperfusion only (B). The cells were stained with Dapi (in blue), and dead cells were stained with Ethidium Homodimer I dye (EthD-I) (in red).

[0072] Figure 14: (A) Predicted pharmacokinetics / ADMET parameters calculated using ADMETlab 2.0

[0053] , (B) Histopathological evaluation of the heart, liver, and kidney sections of rats in a 28-day toxicity study. Representative histologically stained sections of rat organs after Pl 10 and CVP-350 administration; (C) Representative histology of heart tissue after Pl 10 toxicity testing H&E-stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and Pl 10 (2 mg / kg) treated rats (right panel), presented in three magnification x20, xlOO and x200; (D) Representative histology of heart tissue after CVP-350 toxicity testing. H&E-stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and CVP- 350 (2 mg / kg) treated rats (right panel), presented in three magnification x20, xlOO, and x200; (E) Representative histology of liver tissue after Pl 10 toxicity testing. H&E- stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and Pl 10 (2 mg / kg) treated rats (right panel), presented in three magnification x20, xlOO, and x200; (F) Representative histology of liver tissue after CVP-350 toxicity testing. H&E-stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and CVP-350 (2 mg / kg) treated rats (right panel), presented in three magnification x20, xlOO; and x200; (G) Representative histology of kidney tissue after Pl 10 toxicity testing. H&E-stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and Pl 10 (2 mg / kg) treated rats (right panel), presented in three magnification x20, xlOO, and x200; (H) Representative histology of kidney tissue after CVP-350 toxicity testing. H&E-stained histological section of cardiac tissue, dissected from untreated rats (control, left panel) and CVP-350 (2 mg / kg) treated rats (right panel), presented in three magnification x20; xlOO; and x200; and,

[0073] Figure 15: Effect of CVP-350 in vivo; (A) Triphenyltetrazolium chloride (TTC) staining, with red indicating live tissue and white indicating dead tissue (B) The graph depicts infarct size compared with non-treatment and CVP-350 treatment (n=5). All values in the graphs are expressed as the mean ± SD; (C) Infract size in I / R rat after treatment with and without CVP-350. Representative images of Triphenyltetrazolium chloride (TTC) solution stained (red indicates live tissue and white indicated dead tissue heart section obtained from Normoxia, I / R + vehicle and I / R + CVP-350; (D) Lactate dehydrogenase (LDH) biochemical values after I / R with or without CVP-350. (E) Echocardiographic parameter of ejection fraction (EF) analysis after I / R with or without CVP-350. (F) Echocardiographic parameter of fractional shortening (FS) analysis after I / R with or without CVP-350. Echocardiography representation of sham (G), untreated (H) and CVP-350 treated rats after I / R (I)

[0074] DETAILED DESCRIPTION OF THE INVENTION

[0075] The present invention is now described more fully hereinafter with reference to the accompanying examples and drawings, in which embodiments of the invention are shown. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the scope of the invention to those skilled in the art.

[0076] According to some embodiments, the invention provides a peptide or a peptidomimetic. According to some embodiments, the above peptide or a peptidomimetic comprises amino acid sequence:

[0077] Aa 1 - Aa2- Arg-Pro

[0078] According to some embodiments of the above peptide or a peptidomimetic,

[0079] Aai is Ser, Thr, or an analogue thereof,

[0080] Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain. wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth in SEQ ID NO: 1 or a portion thereof.

[0081] As used herein, the term “portion” with respect to Fisl refers, without limitation, to any fragment, segment, domain, or region of the protein, whether contiguous or noncontiguous, that comprises less than the full-length sequence yet retains at least one structural and / or functional characteristic of the reference protein. A “portion” may include, without limitation, N-terminal or C-terminal truncations, internal fragments, functional motifs, epitopes, or domains, and may be naturally occurring, recombinant, or synthetically generated. Unless otherwise indicated, the term encompasses variants, derivatives, and modifications of such fragments, including, without limitation, conservative substitutions, deletions, insertions, and fusions with heterologous sequences

[0082] According to some embodiments, the above peptides or the peptidomimetics has binding affinity of between 5000 pM-4500 pM, 4750 pM-4250 pM, 4500 pM-

[0083] 4000 pM, 4250 pM-3750 pM, 4000 pM-3500 pM, 3750 pM-3250 pM, 3500 pM-

[0084] 3000 pM, 3250 pM- 2750 pM, 3000 pM-2500 pM, 2750 pM-2250 pM, 2500 pM-

[0085] 2000 pM, 2250 pM-1750 pM, 2000 pM- 1500 pM, 1750 pM-1250 pM, 1500 pM-

[0086] 1000 pM, 1250 pM-750 pM, 1000 pM-500 pM, 750 pM-250 pM, 500 pM-100 pM, 250 pM-50 pM, 100 pM-0.2 pM, 100 pM-0.1 pM, to Fisl.

[0087] According to some embodiments, the above peptides or the peptidomimetics has binding affinity of 5000 pM, 4950 pM, 4900 pM, 4850 pM, 4800 pM, 4750 pM, 4700 pM, 4650 pM, 4600 pM, 4550 pM, 4500 pM, 4450 pM, 4400 pM, 4350 pM, 4300 pM, 4250 pM, 4200 pM, 4150 pM, 4100 pM, 4050 pM, 4000 pM, 3950 pM,

[0088] 3900 pM, 3850 pM, 3800 pM, 3750 pM, 3700 pM, 3650 pM, 3600 pM, 3550 pM,

[0089] 3500 pM, 3450 pM, 3400 pM, 3350 pM, 3300 pM, 3250 pM, 3200 pM, 3150 pM,

[0090] 3100 pM, 3050 pM, 3000 pM, 2950 pM, 2900 pM, 2850 pM, 2800 pM, 2750 pM,

[0091] 2700 pM, 2650 pM, 2600 pM, 2550 pM, 2500 pM, 2450 pM, 2400 pM, 2350 pM,

[0092] 2300 pM, 2250 pM, 2200 pM, 2150 pM, 2100 pM, 2050 pM, 2000 pM, 1950 pM,

[0093] 1900 pM, 1850 pM, 1800 pM, 1750 pM, 1700 pM, 1650 pM, 1600 pM, 1550 pM,

[0094] 1500 pM, 1450 pM, 1400 pM, 1350 pM, 1300 pM, 1250 pM, 1200 pM, 1150 pM,

[0095] 1100 pM, 1050 pM, 1000 pM, 950 pM, 900 pM, 850 pM, 800 pM, 750 pM, 700 pM, 650 pM, 600 pM, 550 pM, 500 pM, 450 pM, 400 pM, 350 pM, 300 pM, 250 pM,

[0096] 200 pM, 150 pM, 100 pM, 99.5 pM, 99 pM, 98.5 pM, 98 pM, 97.5 pM, 97 pM,

[0097] 96.5 pM, 96 pM, 95.5 pM, 95 pM, 94.5 pM, 94 pM, 93.5 pM, 93 pM, 92.5 pM, 92 pM,

[0098] 91.5 pM, 91 pM, 90.5 pM, ... 5 pM, 4.5 pM, 4 pM, 3.5 pM, 3 pM, 2.5 pM, 2 pM,

[0099] 1.5 pM, 1 pM, 0.5 pM, 0.3 pM, 0.25 pM, 0.2 pM, 0.15 pM, 0.1 pM to Fisl.

[0100] As used herein the term “peptide” refers, without limitation, to a polymer of two or more amino acids joined by peptide bonds. Peptides of the invention may encompass linear or cyclic structures, naturally occurring or synthetic sequences, and any combination of such. The term includes peptides comprising L- and / or D-amino acids, non-natural amino acids, amino acid analogs, and chemically modified residues. Unless otherwise indicated, peptides of the invention can also encompass functional fragments, variants, derivatives, conjugates, and / or pharmaceutically acceptable forms thereof.

[0101] As used herein, the term “peptidomimetic” refers, without limitation, to a molecule that structurally and / or functionally mimics a peptide, but in which the peptide backbone and / or side chains are partially or wholly replaced, modified, or substituted to confer a distinctive feature / s. For example, peptidomimetic can have enhanced stability, bioavailability, binding affinity, or other desirable properties. Peptidomimetics include, without limitation, modified peptides, peptide analogues, and non-peptidic compounds that retain the biological activity of a reference peptide.

[0102] As used herein the term “binding affinity” refers, without limitation, to the strength of the interaction between two molecules, such as one protein with another. Binding affinity may be quantitatively expressed by an equilibrium dissociation constant (KD), association constant (Ka), inhibition constant (Ki), or other suitable parameter, as determined using standard techniques known in the art. Unless otherwise specified, a lower KD value denotes a higher binding affinity.

[0103] According to some embodiments of the above peptides or the peptidomimetics, Aa2 may be, without limitation, Gly, Gin, Asn, or any analogue thereof.

[0104] According to some embodiments, the above peptide or the peptidomimetic further comprises Aa3at the C-terminus, wherein Aa3is a natural, synthetic amino acid, and / or amino acid analogue. According to some embodiments, a non-limiting list of Aa3includes lysine, arginine, histidine, ornithine, cadaverine, or an analogue thereof.

[0105] According to some embodiments, the above peptide or the peptidomimetic has amino acid sequence set forth in SEQ ID NO: 4-17, 57-68, and 100-103.

[0106] According to some embodiments, the above peptide or the peptidomimetic has amino acid sequence SQRP as set forth in SEQ ID NO: 7.

[0107] According to some embodiments, the above peptide or the peptidomimetic has amino acid sequence SQRPK set forth in SEQ ID NO: 10.

[0108] Exemplary embodiments of the peptides or peptidomimetics of the present invention are set forth in Table I below, wherein each peptide or peptidomimetic listed in Table I constitutes a separate embodiment of the invention. Each such embodiment may be employed independently to achieve the desired result; in combination with additional active or inactive constituents; as part of the pharmaceutical compositions, and methods described in the foregoing and following embodiments. In the context of the present invention, the desired result comprises a reduction in binding affinity of Drpl / Fisl.

[0109] Table I: peptides or peptidomimetics of the invention

[0110]

[0111] According to some embodiments, the above peptides or the peptidomimetics are linear peptides or the peptidomimetics.

[0112] According to some embodiments, the above peptides or the peptidomimetics are cyclic peptide or the peptidomimetic.

[0113] According to some embodiments, the above peptides or the peptidomimetics are macrocyclic molecules. According to some embodiments, the above peptide or the peptidomimetic has the structure

[0114] According to some embodiments, the above peptide or the peptidomimetic is ( 14S, 17S,20S,25aS)- 17-(3 -amino-3 -oxopropyl)-20-(3 -guanidinopropyl)- 14- (hydroxymethyl)- 1,9,12,15,18,21 -hexaoxotetracosahydro- lH-pyrrolo[2, 1 - c] [ 1 ,4,7, 10, 13 , 18]hexaazacyclotricosine-3 -carboxylic acid

[0115] According to some embodiments, the above peptide or the peptidomimetic has the structure

[0116] According to some embodiments, the above peptide or the peptidomimetic is 17-(3- amino-3-oxopropyl)-20-(3-guanidinopropyl)-14-(hydroxymethyl)-l,9,12,15,18,21- hexaoxotetracosahydro-lH-pyrrolo[2,l-c] [l,4,7,10,13,18]hexaazacyclotricosine-3- carboxylic acid.

[0117] According to some embodiments, the above peptide or the peptidomimetic has the structure

[0118] According to some embodiments, the above peptide or the peptidomimetic : 6-amino- 2-(l-(2-(5-amino-2-(3-hydroxy-2-(4-oxobutanamido)propanamido)-5- oxopentanamido)-5-guanidinopentanoyl)pyrrolidine-2-carboxamido)hexanoic acid.

[0119] According to some embodiments, the above peptide or the peptidomimetic has the structure

[0120] According to some embodiments, the above peptide or the peptidomimetic 6-amino-2- ((S)-l-((S)-2-((S)-5-amino-2-((S)-3-hydroxy-2-(4-oxobutanamido)propanamido)-5- oxopentanamido)-5-guanidinopentanoyl)pyrrolidine-2-carboxamido) hexanoic acid.

[0121] For cyclization of the above peptides or the peptidomimetics, numerous cyclization methods can be used. Non-limiting examples of such techniques include using succinic and glutaric anhydrides to generate two- and three-methylene bridges; using longer linkers, such as, without limitation, Adipic acid and Pimelic acid; using natural or unnatural amino acids, such as, for example, lysine or its analogues; performing head- to-tail cyclization; through disulfide bonds using cysteine residues; and any other suitable technique which can achieve the desirable result.

[0122] According to some embodiments of the above peptides or the peptidomimetics, basic modification scans, such, for example, N-methyl and D-amino acid scans, can be performed to preserve bioactivity while improving compound stability.

[0123] According to some embodiments of the above peptides or the peptidomimetics, incorporating unnatural amino acids that mimic natural residues and might enhance stability and membrane permeability can be introduced.

[0124] As used herein, the term “amino acid analogue” refers to a compound that differs structurally from a naturally occurring amino acid yet retains at least one chemical or functional characteristic that allows it to substitute for, mimic, or modify the properties of that amino acid within a peptide, polypeptide, peptidomimetic, or protein. Amino acid analogues include, without limitation, non-naturally occurring amino acids, chemically modified amino acids, isosteres, and derivatives in which the side chain, backbone, stereochemistry, or functional groups are altered (for example, substitution with halogen, alkyl, aryl, hydroxyl, sulfhydryl, or other moieties). The term encompasses D-amino acids, P-amino acids, N-methylated residues, and other synthetic or semi-synthetic variants capable of incorporation into a peptide or peptidomimetic structure.

[0125] According to some embodiments, the above peptides or the peptidomimetics, are in the form of a pharmaceutically acceptable salt. A non-limiting list of pharmaceutically acceptable salts includes hydrochloride, hydrobromide, sulfate, bisulfate, nitrate, phosphate, hydrogen phosphate, acetate, citrate, tartrate, maleate, fumarate, succinate, gluconate, glucuronate, lactate, malate, methanesulfonate (mesylate), ethanesulfonate (esylate), benzenesulfonate (besylate), p-toluenesulfonate (tosylate), camphorsulfonate, sodium, potassium, calcium, magnesium, zinc, ammonium, and, tromethamine.

[0126] According to some embodiments, the invention provides a pharmaceutical composition comprising any of the above peptides or the peptidomimetics, and at least one pharmaceutically acceptable carrier. As used herein, the term “pharmaceutically acceptable carrier” which can be interchangeably referred to as an “excipient”, stands for, without limitation, a non-toxic material that is physiologically acceptable and suitable for formulating an active ingredient into a dosage form for administration to a subject. Such carriers include, without limitation, diluents, fillers, binders, disintegrants, lubricants, glidants, stabilizers, preservatives, antioxidants, solubilizers, surfactants, buffers, tonicity agents, flavoring agents, sweeteners, colorants, pH adjusters, viscosity enhancers, thickeners, cryoprotectants, lypoprotectants, osmotic agents, and coating agents, as well as any combination thereof, provided that the carrier does not adversely affect the biological activity or safety of the active ingredient.

[0127] According to some embodiments, the above pharmaceutical compositions may comprise more than one excipient, which may have similar or distinct properties and functions. The particular pharmaceutically acceptable carrier employed will depend on the type of formulation, including but not limited to oral, parenteral, topical, inhalable, or transdermal dosage forms, for which the composition is intended.

[0128] According to some embodiments, the above pharmaceutical compositions can be in the form of a solid composition, a semi-solid composition, or a liquid composition.

[0129] As used herein, the term “solid composition” refers to a pharmaceutical formulation in which the active ingredient is combined with one or more pharmaceutically acceptable carriers or excipients in a solid state. Solid compositions include, without limitation, powders, granules, pellets, tablets, capsules, and multi -particulate systems. As used herein, the term “semi-solid composition” refers, without limitation, to a pharmaceutical formulation in which the active ingredient is combined with one or more pharmaceutically acceptable carriers or excipients to form a preparation having a consistency between a solid and a liquid. Semi-solid compositions include, without limitation, ointments, creams, gels, pastes, and suppositories.

[0130] As used herein, the term “liquid composition” refers, without limitation, to a pharmaceutical formulation in which the active ingredient is combined with one or more pharmaceutically acceptable carriers or excipients in a fluid state. Liquid compositions include, without limitation, solutions, suspensions, emulsions, syrups, and injectable formulations.

[0131] According to some embodiments, the above pharmaceutical compositions are designed for immediate, delayed, or controlled release.

[0132] According to some embodiments, the above pharmaceutical compositions are designed for oral administration, intravenous administration, subcutaneous administration, transdermal administration, intradermal administration, and topical administration.

[0133] According to some embodiments, the invention provides a method of treating a condition associated with mitochondrial dysfunction.

[0134] According to some embodiments, the above method comprises administering to a subject in need of such treatment an effective amount of the peptides or the peptidomimetics according to one or more of the above embodiments.

[0135] According to some embodiments, the above method comprises administering to a subject in need of such treatment an effective amount of pharmaceutical composition according to one or more of the above embodiments.

[0136] According to some embodiments of the above methods, the administration route can be, without limitation, oral administration, intravenous administration, subcutaneous administration, transdermal administration, intradermal administration, and topical administration.

[0137] As used herein, the term “subject in need” refers to any individual or animal that would benefit from the prevention, treatment, or amelioration of a disease, disorder, or condition targeted by the composition or method described herein. The term encompasses subjects who are at risk of developing the condition, as well as those who have been diagnosed with or are exhibiting symptoms. Unless otherwise specified, a “subject in need” may include humans of any age or gender, and, where appropriate, non-human animals.

[0138] According to some embodiments of the above methods, the subject is a human subject.

[0139] According to some embodiments of the above methods, the subject is afflicted with or suffering from a disease or a condition associated with mitochondrial dysfunction.

[0140] As used herein, the term “mitochondrial dysfunction” refers to a condition in which mitochondria exhibit impaired function, including but not limited to defects in oxidative phosphorylation, electron transport chain activity, ATP production, mitochondrial membrane potential, reactive oxygen species (ROS) regulation, mitochondrial biogenesis, dynamics (fission / fusion), or apoptosis regulation.

[0141] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of the conditions associated with mitochondrial dysfunction includes an inflammatory disease, a neurodegenerative disease, a condition associated with abnormal cell growth, an autoimmune disease, sepsis, acute kidney injury, a metabolic disorder, disease or condition associated with aging, and a cardiovascular disease.

[0142] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of cardiovascular diseases includes ischemic heart disease, sepsis-induced cardiac injury, cardiomyopathy, stroke, heart failure, and inflammatory heart disease.

[0143] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of neurodegenerative diseases includes Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), Amyotrophic lateral sclerosis (ALS)

[0144] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of autoimmune diseases includes Rheumatoid Arthritis (RA), Multiple Sclerosis (MS)

[0145] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of inflammatory diseases includes Sjogren’s Syndrome, Primary Biliary Cholangitis (PBC). According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of conditions associated with aging includes Aging, Mitochondrial DNA Depletion Syndromes, Hutchinson-Gilford Progeria Syndrome (HGPS).

[0146] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of conditions associated with abnormal cell growth includes, lung cancer (including small cell lung carcinoma and non-small cell lung carcinoma), breast cancer (including triple-negative, HER2 -positive, ER / PR-positive, ductal and lobular carcinoma), colorectal cancer, gastric cancer, pancreatic cancer, esophageal cancer, liver cancer (including hepatocellular carcinoma and cholangiocarcinoma), kidney cancer (including renal cell carcinoma and transitional cell carcinoma), bladder cancer, prostate cancer, head and neck squamous cell carcinoma, cervical cancer, ovarian cancer, endometrial cancer, basal cell carcinoma, squamous cell carcinoma, Merkel cell carcinoma, osteosarcoma, chondrosarcoma, Ewing sarcoma, rhabdomyosarcoma, leiomyosarcoma, liposarcoma, angiosarcoma, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, Hodgkin’s lymphoma, non -Hodgkin’s lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, mantle cell lymphoma, Burkitt’s lymphoma, T-cell lymphoma, multiple myeloma, glioblastoma multiforme, astrocytoma, oligodendroglioma, medulloblastoma, ependymoma, meningioma, thyroid cancer, testicular cancer, neuroblastoma, retinoblastoma, mesothelioma, and melanoma.

[0147] According to some embodiments of the above methods, peptides, peptidomimetics and compositions, a non-limiting list of metabolic disorders includes obesity, overweight, insulin resistance, metabolic syndrome, type 2 diabetes mellitus, prediabetes, nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), dyslipidemia, hypercholesterolemia, hypertriglyceridemia, hypertension associated with metabolic dysfunction, cardiovascular disease associated with metabolic dysfunction, polycystic ovary syndrome (PCOS), lipodystrophy, impaired glucose tolerance, impaired fasting glucose, and related disorders of energy homeostasis.

[0148] According to some embodiments of the above methods, the effective amount of the peptide or the peptidomimetic is equivalent to the amount in the range of 0.01 mg / kg / day to 20mg / kg / day in a rodent, according to the BSA scaling. According to some embodiments of the above methods, the effective amount of the peptide or the peptidomimetic is equivalent to the amount in the range of 0.01-0.5 mg / kg / day, 0.05-1 mg / kg / day, 0.1-2 mg / kg / day, 0.5-3 mg / kg / day, 1-5 mg / kg / day, 2- 6 mg / kg / day, 3-8 mg / kg / day, 5-10 mg / kg / day, 6-12 mg / kg / day, 8-15 mg / kg / day, 10- 17 mg / kg / day, 12-20 mg / kg / day, 15-20 mg / kg / day in a rodent, according to the BSA scaling

[0149] According to some embodiments the effective amount of the peptide or the peptidomimetic is equivalent to the amount of 0.01 mg / kg / day, 0.02 mg / kg / day, 0.03 mg / kg / day, 0.04 mg / kg / day, 0.05 mg / kg / day, 0.06 mg / kg / day, 0.07 mg / kg / day, 0.08 mg / kg / day, 0.09 mg / kg / day, 0.10 mg / kg / day, 0.11 mg / kg / day, 0.12 mg / kg / day, 0.13 mg / kg / day, 0.14 mg / kg / day, 0.15 mg / kg / day, 0.16 mg / kg / day, 0.17 mg / kg / day, 0.18 mg / kg / day, 0.19 mg / kg / day, 0.20 mg / kg / day, 0.21 mg / kg / day, 0.22 mg / kg / day, 0.23 mg / kg / day, 0.24 mg / kg / day, 0.25 mg / kg / day, 0.50 mg / kg / day, 0.75 mg / kg / day, 1.00 mg / kg / day, 1.25 mg / kg / day, 1.50 mg / kg / day, 1.75 mg / kg / day, 2.00 mg / kg / day, 2.25 mg / kg / day, 2.50 mg / kg / day, 2.75 mg / kg / day, 3.00 mg / kg / day, 3.25 mg / kg / day, 3.50 mg / kg / day, 3.75 mg / kg / day, 4.00 mg / kg / day, 4.25 mg / kg / day, 4.50 mg / kg / day, 4.75 mg / kg / day, 5.00 mg / kg / day, 5.25 mg / kg / day, 5.50 mg / kg / day, 5.75 mg / kg / day, 6.00 mg / kg / day, 6.25 mg / kg / day, 6.50 mg / kg / day, 6.75 mg / kg / day, 7.00 mg / kg / day, 7.25 mg / kg / day, 7.50 mg / kg / day, 7.75 mg / kg / day, 8.00 mg / kg / day, 8.25 mg / kg / day, 8.50 mg / kg / day, 8.75 mg / kg / day, 9.00 mg / kg / day, 9.25 mg / kg / day, 9.50 mg / kg / day, 9.75 mg / kg / day, 10.00 mg / kg / day, 10.25 mg / kg / day, 10.50 mg / kg / day, 10.75 mg / kg / day, 11.00 mg / kg / day, 11.25 mg / kg / day, 11.50 mg / kg / day, 11.75 mg / kg / day, 12.00 mg / kg / day, 12.25 mg / kg / day, 12.50 mg / kg / day, 12.75 mg / kg / day, 13.00 mg / kg / day, 13.25 mg / kg / day, 13.50 mg / kg / day, 13.75 mg / kg / day, 14.00 mg / kg / day, 14.25 mg / kg / day, 14.50 mg / kg / day, 14.75 mg / kg / day, 15.00 mg / kg / day, 15.25 mg / kg / day, 15.50 mg / kg / day, 15.75 mg / kg / day, 16.00 mg / kg / day, 16.25 mg / kg / day, 16.50 mg / kg / day, 16.75 mg / kg / day, 17.00 mg / kg / day, 17.25 mg / kg / day, 17.50 mg / kg / day, 17.75 mg / kg / day, 18.00 mg / kg / day, 18.25 mg / kg / day, 18.50 mg / kg / day, 18.75 mg / kg / day, 19.00 mg / kg / day, 19.25 mg / kg / day, 19.50 mg / kg / day, 19.75 mg / kg / day, 20.00 mg / kg / day in a rodent, according to the BSA scaling.

[0150] In the context of the invention the term “Rodent” refers, to any member of the order Rodentia, including, but not limited to, mice, rats, hamsters, guinea pigs, and gerbils, which are commonly used as experimental or preclinical animal models in pharmaceutical research and development to evaluate the efficacy, safety, pharmacokinetics, or pharmacodynamics of a compound, composition, or therapeutic intervention

[0151] As used herein, the term BSA scaling, or Body Surface Area scaling, refers to a method used to estimate a human equivalent dose (HED) from animal doses, accounting for differences in body surface area between species.

[0152] The formula for BSA Scaling is:

[0153] HED (mg / kg) = Animal dose (mg / kg) x (Km factor of animal / Km factor of human)

[0154] Where the Km factor is the ratio of body weight (kg) to body surface area(m2) https: / / pmc.ncbi. nlm.nih.gov / articles / PMC4804402 / ?utm_source=chatgpt.com).

[0155] As used herein, the term “Treating” refers to alleviating, reducing, managing, preventing, or delaying the onset, progression, severity, or symptoms of a disease, disorder, or condition in a subject, and encompasses both therapeutic and prophylactic applications.

[0156] According to some embodiments, the invention provides a method of preventing / reducing mitochondrial fission.

[0157] According to some embodiments, the above method comprises administering to a subject in need an effective amount of the peptides or the peptidomimetics according to one or more of the above embodiments.

[0158] According to some embodiments, the above method comprises administering to a subject in need an effective amount of the pharmaceutical compositions according to one or more of the above embodiments.

[0159] In the context of the invention, the phrase “Reducing or preventing mitochondrial fission” refers, without limitation, to any method, act, or intervention that decreases, inhibits, or blocks the division of mitochondria within a cell, thereby maintaining mitochondrial integrity, morphology, or network connectivity, and can include partial or complete suppression of fission processes mediated by fission-related proteins such as Drpl, Fisl, or other regulatory factors. Mitochondrial fission can be measured or assessed by any suitable or acceptable technique known in the art, including, but not limited to, imaging-based methods, biochemical assays, fluorescence microscopy, livecell imaging, or analysis of fission-related protein activity or expression. According to some embodiments of the above methods, the subject is a human subject.

[0160] According to some embodiments of the above methods, the subject is afflicted with or suffering from a disease or a condition associated with mitochondrial dysfunction according to one or more of the above embodiments.

[0161] According to some embodiments, the invention provides a method of reducing the affinity of Drpl / Fisl binding in a cell-free system, comprising applying an effective amount of the peptides or the peptidomimetics of one or more of the above embodiments.

[0162] As used herein, the term “cell-free system” refers, without limitation, to any biochemical or molecular system that does not require intact, living cells for operation. A cell-free system may include, without limitation, purified proteins, nucleic acids, lipids, organelles, subcellular fractions, lysates, reconstituted transcription and / or translation systems, or synthetic biomolecular assemblies. In the context of the invention, cell-free systems can be employed, without limitation, for the study, detection, or measurement of biochemical activities, binding interactions, or other molecular processes in the absence of a whole cell environment.

[0163] According to some embodiments of the above method, the reduction in the affinity of Drpl / Fisl binding is reflected by an increase in the KD value of the Drpl / Fisl binding while and / or after applying the peptide or the peptidomimetic, wherein the KD value is increased by between 1.5-fold to 35-fold while compared to the KD value of the Drpl / Fisl binding before applying the peptide or the peptidomimetic.

[0164] According to some embodiments of the above method, the reduction in in binding affinity can be measured, without limitation, in vitro or in vivo. The measurements can be performed, without limitation, using any system or biochemical assay suitable for the detection and quantification of molecular interactions.

[0165] According to some embodiments of the above method, the reduction in the affinity of Drpl / Fisl binding is reflected by an increase in the KD value of the Drpl / Fisl binding while and / or after applying the peptide or the peptidomimetic by between 1.5-5 fold, 2.5-30 fold, 3-10 fold, 5-15 fold, 10-25 fold, 20-35 fold, while compared to the KD value of the Drpl / Fisl binding before applying the peptide or the peptidomimetic to the cell. According to some embodiments of the above method, the reduction in the affinity of Drpl / Fisl binding is reflected by an increase in the KD value of the Drpl / Fisl binding while and / or after applying the peptide or the peptidomimetic by 1.5 fold, 1.75 fold, 2.0 fold, 2.25 fold, 2.5 fold, 2.75 fold, 3.0 fold, 3.25 fold, 3.5 fold, 3.75 fold, 4.0 fold, 4.25 fold, 4.5 fold, 4.75 fold, 5.0 fold, 5.25 fold, 5.5 fold, 5.75 fold, 6.0 fold, 6.25 fold, 6.5 fold, 6.75 fold, 7.0 fold, 7.25 fold, 7.5 fold, 7.75 fold, 8.0 fold, 8.25 fold, 8.5 fold, 8.75 fold, 9.0 fold, 9.25 fold, 9.5 fold, 9.75 fold, 10.0 fold, 10.25 fold, 10.5 fold, 10.75 fold, 11.0 fold, 11.25 fold, 11.5 fold, 11.75 fold, 12.0 fold, 12.25 fold, 12.5 fold, 12.75 fold,

[0166] 13.0 fold, 13.25 fold, 13.5 fold, 13.75 fold, 14.0 fold, 14.25 fold, 14.5 fold, 14.75 fold,

[0167] 15.0 fold, 15.25 fold, 15.5 fold, 15.75 fold, 16.0 fold, 16.25 fold, 16.5 fold, 16.75 fold,

[0168] 17.0 fold, 17.25 fold, 17.5 fold, 17.75 fold, 18.0 fold, 18.25 fold, 18.5 fold, 18.75 fold,

[0169] 19.0 fold, 19.25 fold, 19.5 fold, 19.75 fold, 20.0 fold, 20.25 fold, 20.5 fold, 20.75 fold,

[0170] 21.0 fold, 21.25 fold, 21.5 fold, 21.75 fold, 22.0 fold, 22.25 fold, 22.5 fold, 22.75 fold,

[0171] 23.0 fold, 23.25 fold, 23.5 fold, 23.75 fold, 24.0 fold, 24.25 fold, 24.5 fold, 24.75 fold,

[0172] 25.0 fold, 25.25 fold, 25.5 fold, 25.75 fold, 26.0 fold, 26.25 fold, 26.5 fold, 26.75 fold,

[0173] 27.0 fold, 27.25 fold, 27.5 fold, 27.75 fold, 28.0 fold, 28.25 fold, 28.5 fold, 28.75 fold,

[0174] 29.0 fold, 29.25 fold, 29.5 fold, 29.75 fold, 30.0 fold, 30.25 fold, 30.5 fold, 30.75 fold,

[0175] 31.0 fold, 31.25 fold, 31.5 fold, 31.75 fold, 32.0 fold, 32.25 fold, 32.5 fold, 32.75 fold,

[0176] 33.0 fold, 33.25 fold, 33.5 fold, 33.75 fold, 34.0 fold, 34.25 fold, 34.5 fold, 34.75 fold,

[0177] 35.0 fold, while compared to the KD value of the Drpl / Fisl binding before applying the peptide or the peptidomimetic.

[0178] According to some embodiments of the above methods, the effective amount of the peptides and peptidomimetics of the invention for reducing the affinity of Drpl / Fisl binding in a cell-free system is in the range of 25 pM to 500 pM.

[0179] According to some embodiments of the above methods, the effective amount of the peptides and peptidomimetics of the invention for reducing the affinity of Drpl / Fisl binding in a cell-free system is in the range of 500 pM-400 pM, 450 pM-350 pM, 400 pM-300 pM, 350 pM-250 pM, 300 pM-200 pM, 250 pM-150 pM, 200 pM- 100 pM, 150 pM-50 pM, 100 pM-25 pM.

[0180] According to some embodiments of the above methods, the effective amount of the peptides and peptidomimetics of the invention for reducing the affinity of Drpl / Fisl binding in a cell-free system is 25 pM, 35 pM, 45 pM, 55 pM, 65 pM, 75 pM, 85 pM, 95 pM, 105 pM, 115 pM, 125 pM, 135 pM, 145 pM, 155 pM, 165 pM, 175 pM, 185 pM, 195 pM, 205 pM, 215 pM, 225 pM, 235 pM, 245 pM, 255 pM, 265 pM,

[0181] 275 pM, 285 pM, 295 pM, 305 pM, 315 pM, 325 pM, 335 pM, 345 pM, 355 pM,

[0182] 365 pM, 375 pM, 385 pM, 395 pM, 405 pM, 415 pM, 425 pM, 435 pM, 445 pM,

[0183] 455 pM, 465 pM, 475 pM, 485 pM, 495 pM, 500 pM.

[0184] According to some embodiments, the invention provides a method of reducing the affinity of Drpl / Fisl binding in a cell, comprising applying to the cell an effective amount of the peptides or the peptidomimetics of one or more of the above embodiments under conditions sufficient to reduce Drpl / Fisl binding.

[0185] According to some embodiments of the above method, wherein the half-maximal inhibitory concentration (ICso) of the peptides or the peptidomimetics of one or more of the above embodiments, as measured by a cell-based assay, is in the range of 0.3 pM to 2pM.

[0186] According to some embodiments of the above method, wherein the half-maximal inhibitory concentration (ICso) of the peptides or the peptidomimetics of one or more of the above embodiments, as measured by a cell-based assay, is in the range of 0.3 pM to 2.0 pM, from 0.3 pM to 1.5 pM, from 0.5 pM to 2.0 pM, from 0.4 pM to 1.8 pM, from 0.6 pM to 1.6 pM, from 0.7 pM to 1.2 pM, from 0.3 pM to 0.8 pM, from 0.5 pM to 1.0 pM, from 0.8 pM to 1.5 pM, or from 1.0 pM to 2.0 pM.

[0187] According to some embodiments of the above method, wherein the half-maximal inhibitory concentration (ICso) of the peptides or the peptidomimetics of one or more of the above embodiments, as measured by a cell-based assay, is .3 pM, 0.4 pM, 0.5 pM, 0.6 pM, 0.7 pM, 0.8 pM, 0.9 pM, 1.0 pM, 1.1 pM, 1.2 pM, 1.3 pM, 1.4 pM, 1.5 pM, 1.6 pM, 1.7 pM, 1.8 pM, 1.9 pM, 2.0 pM.

[0188] In the context of the invention, the term “ICso” refers to the half-maximal inhibitory concentration of a test agent or inhibitor, defined as the concentration of the inhibitor at which the interaction or binding affinity between two proteins is reduced by 50% relative to a control in the same assay conditions. ICso values may be determined, without limitation, using biochemical, biophysical, or cell-based assays suitable for quantifying protein-protein interactions, including but not limited to fluorescence resonance energy transfer (FRET), bioluminescence resonance energy transfer (BRET), surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), coimmunoprecipitation, proximity ligation assay, or reporter gene assays. According to some embodiments of the above methods, the peptides and peptidomimetics of the invention reduce the affinity of Drpl / Fisl binding in a cell and / or a cell-free system in a dose dependent manner.

[0189] According to some embodiments of the above methods, the cell is a eukaryotic cell.

[0190] According to some embodiments of the above methods, the cell is a mammalian cell.

[0191] According to some embodiments, the invention provides the peptides and peptidomimetics of the invention for use in the treatment of a disease or condition associated with mitochondrial dysfunction.

[0192] According to some embodiments, the invention provides the pharmaceutical compositions of the invention for use in the treatment of a disease or condition associated with mitochondrial dysfunction.

[0193] According to some embodiments, the invention provides the peptides and peptidomimetics of the invention for use as a medicament.

[0194] According to some embodiments, the invention provides the pharmaceutical compositions of the invention for use as a medicament.

[0195] In the context of the present invention, the term “medicament” refers to any composition or formulation intended for therapeutic use in a subject, for preventing, treating, or ameliorating a disease, disorder, or condition, wherein the composition comprises an active agent and, optionally, one or more pharmaceutically acceptable carriers, excipients, or adjuvants, and is suitable for administration by any appropriate route.

[0196] Examples / Results

[0197] Example 1 : Assessment of key amino acids contributing to biological activity using Structure-Activity Relationship (SAR) studies

[0198] To assess the contribution of individual amino acids to the bioactivity of the previously identified Pl 10, each residue of the cargo portion of the peptide was sequentially mutated to alanine as presented in Table 1. The assessment was done by evaluating how these mutations impact Pl 10’ s efficacy in improving cell viability following stress with C0CI2 (500 pM). Table 1. Alanine scan linear peptide library: ID, sequences, and calculated molecular weight

[0199] The bioactivity trends indicated that mutations at the C-terminus residues (S7A, G6A, R5A, and P4A) significantly reduced biological activity. In contrast, mutations at the N-terminus residues (L3A, L2A, and D1A) did not reduce the biological activity of Pl 10 as demonstrated on Figure 1 A-B).

[0200] Additional studies with cyclic analogues of the peptides listed in Table 1 confirmed that the four amino acids at the C-terminus (S7, G6, R5, and P4) are crucial for the peptide's bioactivity (not shown).

[0201] Example 2: Assessment of biological activity of short peptides using SAR study. A SAR study with peptides synthesized based on the previously identified four crucial amino acids, listed in Table 2 below was performed.

[0202] Table 2. Peptidomimetics library: ID, sequences, and calculated molecular weight To further assess the compounds' binding to Fisl, a real-time detection of biomolecular interactions, similar to Biocore or surface plasmon resonance (SPR) technology [35, 36] was employed. The following peptides were synthesized and evaluated: a linear peptide consisting of the amino acids PRGT and added Lys at the C-terminus for potential cyclization (CVP-249, Table 2). a peptide having altered sequence from the C-terminus to the N-terminus (CVP-244, Table 2), peptides having different substitutions of G6: Gly to Gin (CVP-245, Table 2), Gly to Glu (CVP-246, Table 2), Gly to Lys (CVP-247, Table 2); a linear peptide having the aa sequence SQRP-K (CVP- 354, Table 2), and a cyclic peptide having the aa sequence SQRP-K (CVP-350, Table 2). CVP-244, demonstrated improved binding affinity to the target protein (KD = 1,009 pM) compared to CVP-249 (KD = 48,447 pM (Figure 2A). Glycine was systematically replaced with uncharged Gin, negatively charged Glu, and positively charged Lys. In the binding study, it was evident that the use of negatively and positively charged amino acids decreased peptide binding. Specifically, the negatively charged amino acid Glu had a KD value of 85, 421 pM compared to Lys with aKo value of 4, 801 pM. In contrast, the uncharged amino acid, Gin, slightly improved binding, with a KD value of 800 pM. Next, Thr was replaced by Ser in the short peptide sequence leading to a two-fold improvement in peptide binding affinity, with KD of 1,009 pM, and KD of 437 pM, respectively (Figure 2A). Based on the above SAR studies a linear peptide SQRPK was synthesized and demonstrated enhanced binding affinity to the target protein with KD value of 356 pM). Finally, to improve peptide stability, bioactivity, and binding affinity, the linear peptide was cyclized and demonstrated the highest binding affinity with KD value of 121 pM) (Figures 2A-B).

[0203] To confirm the above findings, an alternative approach, fluorescence polarization (FP) technology, in which the peptide bound to a fluorophore serves as a tracer. This assay, evaluates peptide binding to the target protein without immobilizing any molecules, providing a sensitive technique to assess the binding interactions of the compound with the target protein, in which CVP-350 with FAM dye conjugated (aka CVP-378) demonstrated strong binding affinity to Fisl with KD value of 0.282 pM, (Figure 2C- D).

[0204] Example 3: Identifying Compound Interaction Sites Using Docking and Cross-Linking Mass Spectrometry Technology. Molecular docking techniques and Cluspro to predict the interaction site of CVP-355 (cargo of Pl 10, DLLPRGT, with lysine) with Fisl. The docking analysis results were then analyzed, and 3 -dimensional (3D) graphics were generated. The docking study indicated that the peptide binds close to the homologous conservation site of Fisl (Figure 3A). To further analyze the docking results and confirm the interaction sites between the peptide and the protein, a technique called cross-linking coupled with mass spectrometry (CL-MS) was used. For this purpose, bis(sulfosuccinimidyl)suberate (BS3) cross-linker, which primarily reacts with the epsilon-amino group of lysine residues in proteins, was used. Considering the length of the crosslinker 11 A for B S3, the side chain of the target residues (e.g., lysine), and backbone dynamics, the assumption was that these residues within a Ca-Ca distance of up to 35 A would be preferentially crosslinked

[0038] , The additional lysine adjacent to the bioactive cargo domain of the peptide was identified to be particularly reactive. As this residue is located near the cargo of the peptide, this points to a distinct role of the bioactive part of the peptide. Therefore, eleven and thirteen sequences of peptide pairs reporting a cross-link between the Fisl sequence and the peptide CVP-355 were identified. The introduced lysine adjacent to the peptide cargo was the reactive lysine in all cases at the peptide sequence, while two different lysine aa were identified on Fisl, residues 64 and 108, in 20.5 and 21.8 A, respectively. The cross-linked peptide-protein pairs identified using the CL-MS approach were 100% consistent with the docking results analysis and the rational design of the peptide. Combining the crosslinking results and bioinformatics docking analysis demonstrates a high degree of complementarity between these two orthogonal approaches (Figure 3B).

[0205] Example 4: Circular Dichroism (CD) studies

[0206] CD spectra for all peptides in the Ala-scan study (Pl 10 and CVP-357 - CVP-363), as well as for the two pentapeptides CVP-354 and C VP-350 were acquired. There are two bands in the CD profile obtained in water, one negative maximum centered around 198 nm and a positive maximum at approximately 218 nm, which indicates an unordered 3D structure

[0039] , Spectra obtained in the presence of the structure-stabilizing solvent 2,2,2-trifluoroethanol (TFE) up to 40% in water did not alter the CD spectra significantly, confirming the presence of an unordered conformation even in 40% TFE (Figure 4A-B). Changes in the CD spectra were observed for compounds dissolved in a membrane-mimetic environment consisting of sodium dodecyl sulfate (SDS, 100 mM) solution in water (Figure 4A). These changes primarily affected the positive band, which shifted from positive to negative and moved towards 220 nm. This effect may indicate a small presence of helical conformations, although the main negative maximum remains below, albeit closer to 200 nm, which is not within the typical range for helical structures (usually around 205-208 nm)

[0039] , It is noteworthy that the two compounds, CVP-354 and CVP-350, although being significantly smaller, exhibit the same CD profile as the longer peptides.

[0207] Example 5: Evaluation of Lipinski’s Rule of Five

[0208] Lipinski’s Rule of Five is a set of criteria used to assess the drug-like properties of potential drug structures based on their physicochemical properties. Generally, compounds with the following characteristics are considered likely to be orally bioavailable: LogP (the logarithm of the compound partition coefficient between n- octanol and water) < 5, hydrogen bond donors (HBD) < 5, hydrogen bond acceptors (HBA) < 10, molecular weight (MW) < 500 Da, and rotatable bonds (nRB) < 10

[0040] ,

[0209] As presented in Table 3 and Figure 5, CVP-350 fulfilled nearly four out of the five criteria specified in Lipinski’s Rule of Five. However, it is important to note that many successful drugs do not strictly adhere to Lipinski’s Rule of Five

[0041] , For example, cyclosporine A, a potent immunosuppressant derived from the fungus Tolypocladium inflatum; Oxytocin, a key neuropeptide involved in social bonding and reproductive behaviors; and MK-0616, an oral macrocyclic inhibitor of proprotein convertase subtilisin / kexin type 9 (PCSK9) that effectively lowers low-density lipoprotein cholesterol (LDL-C) levels and showed promising results in hypercholesterolemia patients during a Phase 2b study

[0042] , all violate these rules based on their chemical structures

[0043] , Given its predicted physicochemical properties, CVP-350 shows potential as a drug lead.

[0210] Table 3: Lipinski’s rule-of-five, water solubility, drug-likeness and medicinal chemistry friendliness of the compounds

[0211] Compounds

[0212] Parameters CVP- CVP- Cyclosporine > MK-

[0213] PHO

[0214] 350 354 AOxytOCm0616

[0215] Lipinski’s MW <500 2425.40 569.54 527.28 1,201.84 1007.19 1,614.76 rule of 5 HBD <5 58 10 8 5 12 9 HBA <10 70 10 8 12 13 20

[0216] LogP <5 4.95 0.56 0.22 2.92 1.21 3.8 nRB <10 93 10 15 15 20 35

[0217] Water Log S (ESOL) 4.03 0.97 3.35 -8.15 -6.13 -9.10 solubility / rem i Highly Highly Highly Poorly Poorly Poorly soluble soluble soluble soluble soluble soluble

[0218] Drug- Bioavailability 0 7 0 1? 0 17 0 17 0 17likeness score

[0219] Lead likeness (number of No; 3 No; 2 No; 2 No; 2 No; 2 No; 2

[0220] Medicinal violations) chemistty Synihettc14,8 6 4Q 10 M 8 54 1 1 20accessibility

[0221] Example 6: Peptide Inhibitors Drpl / Fisl PPI

[0222] The linear peptide Pl 10 was designed based on sequence homology between Drpl and Fisl and was previously found to inhibit excessive mitochondrial fission. To further evaluate the role of the compounds described above, inhibition of Drpl / Fisl PPI binding affinity between Drpl and Fisl was examined (Figures 6A-B). Next, the inhibition of Drpl / Fisl PPI by the designed compounds was validated. All the compounds inhibited Drpl / Fisl PPI significantly. Pl 10, the long linear peptide, inhibited Drpl / Fisl PPI by about 3 -fold, the short linear compound CVP-354 by 6-fold, and the macrocyclic compound CVP-350 by over 20-fold (Figures 6A-B).

[0223] Example 7: CVP-350 as a Selective Drpl Inhibitor

[0224] It was previously demonstrated that Pl 10 selectively inhibits the Drpl / Fisl interaction without affecting Drpl's interaction with other mitochondrial fission adaptors such as Mff, Mid49, and Mid51 [17, 29, 30], To confirm the selectivity of the designed peptides, the ability of the designed peptides to inhibit PPI of Drpl with the referenced mitochondrial fission adaptors was examined. As expected, high level of binding was observed between Drpl and Mid51, while neither Pl 10 nor CVP-350 exhibited inhibitory effects of this interaction (KD = 0.39 pM, KD = 0.45 pM, and KD = 0.38 pM, respectively, (Figures 7A-C). Then the inhibitory activity of the compounds on the interaction between Fisl and Mid51, which is also essential for mitochondrial fission was evaluated. A strong binding affinity was observed between Fisl and Mid51; however, neither Pl 10 nor CVP-350 inhibit this interaction (KD = 0.56 pM, KD = 0.50 pM, and KD = 0.48 pM, respectively, Figures 7B-D). Therefore, both compounds inhibit Drpl / Fisl interactions selectively. Example 8: Stability of Linear and Macrocyclic Compounds Against Proteolytic Digestion

[0225] Compound stability using trypsin, which cleaves peptide bonds after lysine and arginine, was evaluated and the data were analyzed using high-pressure liquid chromatography (HPLC). A complete degradation of the linear peptide, Pl 10, occurred in less than 30 minutes. In contrast, the proteolytic cleavage of macrocyclic CVP-350 was much slower. HPLC analysis showed that only 32% of macrocyclic CVP-350 was cleaved by trypsin in 2 hours (Figure 8 A-C).

[0226] Example 9: Compounds Bioactivity in Cells

[0227] It was previously demonstrated that Drpl recruitment from the cytosol to the outer mitochondrial membrane promotes mitochondrial fission, and the interaction between Drpl and Fisl has been linked to abnormal mitochondrial fragmentation in various pathological conditions, such as neurodegenerative diseases and ischemic heart disease [44-46], Based on the assumption that blocking Drpl / Fisl PPI could prevent pathological fission, the bioactivity of the peptides in H9c2 cells under hypoxic conditions was evaluated. H9c2 cardiomyocytes were treated with Cobalt (II) chloride (500 pM) and peptides (1 pM each). Cell viability was measured by XTT release assay. Cell viability assay values are from 6 independent experiments.

[0228] Table 4: Summarized results of compound treatment cell viability effects in H9c2 cardiomyocytes cells

[0229] Table 5: Summarized results of compound treatment toxicity effects in H9c2 cardiomyocytes cells All compounds significantly improved cell viability (Figure 9 A, Table 4) and showed no signs of toxicity (Figure 9B, Table 5), highlighting their cardioprotective and safety properties. We also measured the activity of lactate dehydrogenase (LDH), which is an indicator of cellular damage and oxidative stress. We observed that LDH activity was significantly reduced in the treatment group with compounds compared to the untreated group (Table 6, Figure 9C).

[0230] Table 6. Summary of lactate dehydrogenase (LDH) data with and without PHO, CVP-354, and CVP- 354 treatment

[0231] Mitochondrial dysfunction and oxidative stress are pivotal factors in the pathogenesis of various heart conditions, such as myocardial infarction (MI). This process typically begins with the production of ROS within the ischemic myocardium. High levels of ROS can cause deoxyribonucleic acid (DNA) damage, disrupt mitochondrial function, lead to further ROS production, and result in cellular injury, promoting myocardial remodeling and disease progression. While baseline level of mitochondrial fission is necessary for normal cellular function and mitochondrial quality control, excessive mitochondrial fragmentation driven by the Drpl / Fisl interaction has been shown to propagate oxidative stress and ischemic injury. Therefore, we evaluated the efficacy of Pl 10 and associated analogues in preventing oxidative stress following multiple cellular stressors (CoCh and H2O2). Results demonstrated that treatments with these compounds significantly reduced total and mitochondrial specific ROS levels under stress (Figures 9E-D, Table 7).

[0232] Table 7: Summary of ROS data with and without Pl 10, CVP-354, and CVP-354 treatment

[0233] Next, the efficacy of the designed compounds in preserving mitochondrial membrane potential (MMP, or A m) under stress with C0CI2 using the fluorescent dye JC-1 (5,5',6,6'-tetrachloro-l,l',3,3'-tetraethylbenzimidazolcarbocyanine iodide) was quantified. A m is generated by proton pumps and is crucial for energy storage during oxidative phosphorylation, as well as for many vital biological processes. JC-1 was used to evaluate mitochondrial depolarization, an indicator of early-stage mitochondrial dysfunction and apoptosis

[0047] , The results showed improved mitochondrial membrane potential in stressed cells treated with the compounds (Figure 9F, Table 8). Table 8: Summary of MMP data with and without Pl 10, CVP-354, and CVP-354 treatment.

[0234] Finally, the impact of the designed peptides which block the Drpl / Fisl PPI on cellular bioenergetics using seahorse oxygraphy was evaluated. Results demonstrated that both CVP-350 and CVP-354 were able to rescue H2O2 mediated impairments in cellular respiration (Figure 10A), represented by substantial increase in basal (Figure 10B), adenosine triphosphate (ATP) linked (Figure 10C) and maximal (Figure 10D) oxygen consumption rate (OCR) as well as spare respiratory capacity (Figure 10E). In summary, the results indicated that the designed compounds enhance cell viability, improve mitochondrial membrane potential, cellular respiration, and reduce ROS production as demonstrated on Figure 10 and tables 9-13)

[0235] Table 9: Summary of oxygen consumption rates (OCR) measured at specific time points during the ‘Seahorse’ assay

[0236] Table 10: Summary of OCR data to measure basal respiration

[0237] Table 11 : Summary of OCR data to measure ATP -linked respiration

[0238] Table 12: Summary results of OCR data to measure maximal respiration

[0239] Table 13: Summary results of OCR data to measure spare respiratory capacity

[0240] Example 10: Mitochondrial Morphology

[0241] To investigate the impact of Drpl / Fisl PPI inhibition on mitochondrial fragmentation at the cellular level, transmission electron microscopy (TEM) was used. Cardiomyocytes under stress exhibited significant mitochondrial swelling, fragmentation, and deformation compared to controls (Figure 11). However, cells treated with the compounds showed a substantial reduction in mitochondrial swelling, mitochondrial deformation (Figures 11C-E and Figure 12A-I), and mitochondrial fragmentation (Figures 11G-I). Notably, CVP-350 demonstrated the most significant reduction in mitochondrial swelling, fragmentation, and deformation.

[0242] Example 11 : CVP-350 Reduces Programmed Cell Death and Improves Cell Viability by Protecting Mitochondrial Integrity

[0243] Impairment of mitochondrial fission is closely associated with increased apoptosis and autophagic cell death triggered by various stimuli, which elevate mitochondrial depolarization and ROS production

[0048] , Moreover, hyperactivation of Drpl in mitochondria has been implicated in TNF alpha-induced necrotic cell death

[0049] , indicating that Drpl -dependent mitochondrial dysfunction may serve as a focal point for multiple programmed cell death (PCD) pathways. Therefore, reduction in cell death upon treatment with the CVP-350, during ischemia and reperfusion was investigated. Results demonstrated that CVP-350 significantly reduced the number of apoptotic H9c2 cells when co-treated at the time of ischemia and reperfusion (Figure 13 A) as well as during reperfusion alone (Figure 13B). These results are consistent with previous studies demonstrating Drpl hyperactivation's involvement in various cell death pathways. Altogether, these results demonstrate that CVP-350 effectively rescues cells from pathways associated with cell death. Example 12: In Silico Pharmacokinetic, Pharmacological, and Safety Profiling of

[0244] Identified Leads

[0245] The pharmacokinetic properties of drug candidates encompass absorption, distribution, metabolism, excretion, and toxicity (ADMET), providing crucial insights into their pharmacokinetics and potential risks to the human body [50, 51], Prediction results suggested that the designed compounds would have no potential inhibitor effect of five key cytochrome P450 enzymes (CYP1A2, CYP2C19, CYP2C9, CYP2C6, and CYP3A4), which collectively metabolize around 80% of clinically used drugs

[0052] , Additionally, the compounds exhibited a negative BBB crossing potential (BBB-) and showed no signs of toxicity (Figure 14A). Also, we predicted the physicochemical properties, and it supports the experimental values summarized in Tables 14-16).

[0246] Table 14: Physicochemical properties of PHO, CVP-350, and CVP-354

[0247] T_T_, H N Molar

[0248] Compounds Sequences L pl NC ,, _ . .

[0249] (Kcal / mol) atoms refractivity

[0250] CH3-C(O)-DLLPRGS-

[0251] P110 GG-YGRKKRRQRRR- 20 12.62 +8 +32.36 119 432

[0252] NH2

[0253] CVP-350 CH3-C(O)-SQRPK-NH25 14.00 +2 +13.55 49 189.91

[0254] CVP-354 CH3-C(O)-SQRPK-NH25 14.00 +2 +13.88 43 156.55

[0255] Table 15: Fulfilment of the rules revealing bioavailabilily (Lipinski) and safely (Pfizer and GSK) of the PHO, CVP-350, CVP-354, Cyclosporine A and MK-0616

[0256] *Lipinski Rule: MW<500; logP<5; Hacc<10; Hdon<5; *Pfizer Rule: logP > 3; TPSA < 75; *GSK Rule: MW < 400; logP < 4; *Golden Triangle: 200 <MW <50; -2 < logD <5

[0257] Table 16: Physicochemical properties of PHO, CVP-350, CVP-354, Cyclosporine A, and MK-0616

[0258] Volume: Van der Waals volume. Density: Density = MW / Volume. nRot: Number of rotatable bonds. In some situation Amide C-N bonds are not considered because of their high rotational energy barrier. Optimal:0~ll, based on Drug-Like Soft rule. TPSA: Topological polar surface area. Sum of tabulated surface contributions of polar fragments. Optimal:0~140, based on Veber rule. nRing: Number of rings. Smallest set of smallest rings. Optimal:0~6, based on Drug-Like Soft rule. MaxRing: Number of atoms in the biggest ring. Number of atoms involved in the biggest system ring. Optimal:0~18, based on Drug- Like Soft rule. nHet: Number of heteroatoms. Number of non-carbon atoms (hydrogens included). Optimal: 1—15, based on Drug Like Soft rule. fChar: Formal charge. Optimal: -4 ~4, based on Drug-Like Soft rule. nRig: Number of rigid bonds. Number of non-flexible bonds, in opposite to rotatable bonds. Optimal:0~30, based on Drug-Like Soft rule.

[0259] Example 13: Histological Evaluation of Safety Profiles for Pl 10 and CVP-350 in Vivo

[0260] To assess the safety of Pl 10 and CVP-350 in animals, we conducted evaluations for toxicity. It was shown above that both Pl 10 and CVP-350 showed no toxicity in cellular studies, indicating their safety profile in cells. Further examination revealed no structural changes in the cardiac tissues of rats treated with Pl 10 or CVP-350 compared to untreated rats. Histologically, there were no indications of muscle width alterations or cardiac fiber integrity issues, and no signs of inflammatory cell infiltration were observed (Figure 14B-D). Similarly, in terms of liver histology, the tested substances showed no toxicity, as evidenced by intact gross anatomy and hepatic acini (Figure 14B-F). Kidney histology also indicated no toxicity, with normal nephron structure, glomerular density, and tubular substrate observed. High-magnification analysis confirmed the preservation of glomerular mesangium and convoluted tubules (Figure 14B,G,Hw). Based on hematoxylin and eosin (H&E) staining, the hearts, livers, and kidneys of rats administered with Pl 10 or CVP-350 did not exhibit significant differences compared to normal rats. Histological examination of a heart tissue sample revealed no evidence of inflammation or disruption to the heart's normal architecture. The liver sections show normal liver morphology. In addition, there is no abnormal lesion or tubular dilation in the kidney's histology. The kidneys appear healthy, and the kidney histology section shows no abnormalities demonstrates schematic structure, HPLC profile and MALDI spectrum of CVP-366. These findings suggest that both Pl 10 and CVP-350 are safe for further animal studies.

[0261] Example 14: Evaluation of CVP-350 in the Myocardial Ischemia (MI) Model.

[0262] Cardiac ischemia-reperfusion (I / R) experiments using small rodents are crucial in vivo models for studying human diseases, particularly MI. These models accurately mimic clinical scenarios where periods of reduced blood supply (ischemia) are followed by restoration of blood flow to the myocardium (reperfusion), resembling medical interventions like angioplasty or thrombolysis used in acute MI treatment. By replicating these conditions, these models provide valuable insights into the pathophysiological processes involved in myocardial I / R injury, including mechanisms of ischemic damage and I / R injury. Consequently, they play a pivotal role in advancing and evaluating innovative therapeutic strategies aimed at minimizing ischemic damage and mitigating I / R injury. Herein, this model was used to validate the efficacy and bioactivity of the compounds. Rats used in the study showed no significant differences in body weight, heart weight, or heart-to-body weight ratios among the sham, untreated, and CVP-350 treated groups.

[0263] Triphenyl tetrazolium chloride (TTC) staining:

[0264] The size of the infarct area in the heart serves as a key indicator of myocardial disease severity. Under normal conditions, healthy rats exhibit no infarct area, indicating the absence of myocardial injury. However, under conditions of myocardial injury induced by I / R, a significant enlargement of the infarct area occurs, serving as a reliable measure of disease severity. Treatment with CVP-350 in rats subjected to I / R conditions markedly reduced the size of the infarct area (Figures 15A-C). This substantial reduction underscores the potential therapeutic benefit of CVP-350 in alleviating myocardial damage caused by I / R.

[0265] Blood Results

[0266] Lactate dehydrogenase (LDH) is a common indicator of tissue damage and oxidative stress following I / R injury. Rats in the untreated group showed a significantly higher concentration of LDH compared to animals treated with CVP-350 (P<0.05, Figure 15D).

[0267] Echocardiography Results

[0268] The echocardiography data post I / R for the sham, untreated, and CVP-350 treated groups showed significant differences. Severe and unresolved cardiac damage led to increased left ventricle (LV) systolic and diastolic diameters and cross-sectional areas, impacting functional parameters, such as ejection fraction (EF), and fractional shortening (FS). The echocardiography results demonstrated that following CVP-350 treatment, both left ventricular ejection fraction (LVEF) % (Figure 15E) and left ventricular fractional shortening (LVFS) % (Figure 15F) significantly increased compared to the untreated group. The CVP-350 treated group values aligned more closely with those of the sham group compared to the untreated group. The echocardiography image representation clearly showed the effect of sham (Figure 15G), untreated (Figure 15H) and CVP-350 treated rats (Figure 151).

[0269] Discussion

[0270] Mitochondria play a pivotal role in various diseases, including CVDs, neurological conditions, and cancer, largely through their influence on mitochondrial dynamics. In CVDs, mitochondrial dysfunction contributes to oxidative stress and energy depletion in cardiac cells, leading to conditions like heart failure and ischemic heart disease. Understanding and targeting mitochondrial dynamics holds promise for developing novel therapies across these diseases, aiming to restore mitochondrial health and improve patient outcomes. Pl 10, a linear peptide that was previously developed to target Drpl / Fi si PPI and inhibit excessive mitochondrial fission, has shown significant therapeutic potential across various disease models. However, linear peptides face several disadvantages as drug candidates, primarily concerning stability and selectivity. To overcome these challenges, extensive SAR studies were conducted, and series of short linear and cyclic peptides were identified. Among those was a macrocyclic peptide (CVP-350) with drug-like properties. CVP-350 demonstrated high binding affinity for the target protein (KD 282 nM) and inhibited the Drp 1 / Fis 1 PPI significantly. Importantly, it improved mitochondrial health parameters in cardiomyocytes and reduces cardiac damage by 50-70% in a rat model of heart attack, all without causing observable toxicity.

[0271] Based on the mechanism of action of CVP-350 and related peptides, and its impact on mitochondrial dynamics, which are significant in numerous human diseases, we propose it as a platform for targeting various conditions characterized by mitochondrial dysfunction.

[0272] Methods

[0273] General Chemistry

[0274] All commercially available solvents and reagents were used without further purification. Piperidine, Diethyl ether, N,N-Diisopropylethylamine (DIEA), Trifluoroacetic acid (TFA), and Water (HPLC grade) were obtained from Bio-Lab (Jerusalem, Israel); Acetonitrile (ACN) (HPLC grade) was acquired from J.T. Baker (Poland); Acetic anhydride was obtained from Daejung (Gimhae-si, Korea); Triisopropylsilane (TIS), and Succinic anhydride were purchased from Acros organics (Branchburg, NJ, USA); Dithiothreitol (DTT) was purchased from Fisher Bioreagents (Ottawa, ON, Canada); Dimethylformamide (DMF) was purchased from Carlo Erba (Vai De Reuil, France); Oxyma Pure was contributed by Luxembourg Bio Technologies Ltd. (Ness Ziona, Israel); N,N-Diisopropylcarbodiimide (DIC) was purchased from Angene International Limited (Nanjing, China); Glutaric anhydride was purchased from Alfa Aesar (Chaoyang, China); Benzotriazole-l-yl-oxy-tris- pyrrolidinophosphonium hexafluorophosphate (PyBOP) was purchased from GL Biochem Ltd. (Shanghai, China); Fmoc Rink amide MB HA resin was purchased from AnaSpec (substitution 0.50 mmol / g, Fremont, CA, USA); Fmoc-protected amino acids were purchased from Ontores Biotechnologies (Hangzhou, China). Side chains of the amino acids used in the synthesis were protected as follows: tert-Butyloxycarbonyl (BOC) (Lys / Trp), tert-butyl (tBu) (Ser / Thr / Tyr / Glu), t-butyl ester (OtBu) (Asp), 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) (Arg), 4-methyltrityl (Mtt) (Lys), and triphenylmethy (Trt) (Cys / Gln).

[0275] Peptide Synthesis

[0276] The peptides were chemically synthesized using the fluorenyl methoxycarbonyl (Fmoc) / tert-butyl (tBu) method, using a fully automated parallel peptide synthesizer (Syro I, Biotage, Uppsala, Sweden).

[0277] Fmoc deprotection was carried out in two steps at 75 °C by applying piperidine (40%) in DMF solution for 3 and 12 minutes. Coupling reactions were carried out by repetition of the following cycle conditions: 45 minutes at 75 °C (30 minutes at 50 °C for Cys AA only) with DIC (0.2 M) in DMF, Oxyma Pure (0.2 M) in DMF, and amino acids (0.2 M) in DMF. The coupling and Fmoc deprotection steps were monitored whenever necessary using small cleavages. Anhydride coupling was carried out, the following cycle conditions were repeated: 30 min at room temperature, combining anhydride (10 eq) / DIPEA (10 eq) / peptide (1 eq) in DMF.

[0278] N-methyltrityl (Mtt) deprotection was done manually on a shaker at room temperature. The resin was washed with DCM (3 x 2 min) and a solution of TFA / TIS / DCM (1 : 5 :94) was added. The resin was mixed for 5 min at room temperature and drained three times.

[0279] The resin was washed with DCM (5 x 2 min).

[0280] Cyclization was done after washing the resin with DCM (3 x 2 min) using a solution of benzotriazol- 1 -yl-oxytripyrrolidinophosphonium hexafluorophosphate

[0281] (PyBOP) / DIPEA (5: 10) in dichloromethane (DCM) (1 hr, 3 times, at room temperature). The resin was washed with DCM (3 x 2 min).

[0282] In the final step of synthesis, the peptide was cleaved from the resin. Its amino acid side chains were deprotected with a pre-cooled mixture of TFA / TIS / H2O solutions (90:2.5:2.5 v / v / v) for three hours at room temperature. A stream of compressed air was used to evaporate the solvents and remove the resin. After precipitating the crude products with diethyl ether, centrifugation was used to collect the residue. This residue was then dissolved in CH3CN / H2O (30:70) and lyophilized.

[0283] Purity of all the compounds was determined by analytical reverse-phase high-pressure liquid chromatography (RP-HPLC) (Agilent, Santa Clara, CA, USA) equipped with: CBM-20A system controller, SPD-20A detector, CTO-20A column oven, 2 x LC- 20AD solvent delivery unit, SIL-20AC autosampler, DGU-20A5 degasser using a Luna 5 pm C18(2) 100 A (250 x 4.6 mm) column (Phenomenex, Torrance, CA, USA) at 1 mL / min. The solvent systems used were A (H2O with 0.1% TFA) and B (CH3CN with 0.1% TFA). For separation, a linear gradient was applied, and detection was at 215 or 254 nm. Unpure compounds were further purified by preparative RP-HPLC. The purity of all compounds tested was > 95%.

[0284] Peptide Binding to Proteins, in Vitro

[0285] A study was conducted to determine peptide binding to immobilized proteins in vitro using the AGILE Dev Kit label-free binding assay with field-effect biosensing (FEB) technology (Cardea, San Diego, CA, USA). Manufacturer's standard protocol was followed. The peptide was applied as an analyte, and the protein was immobilized on the chip. An alteration in the current (I) was measured and recorded continuously throughout the experiment as a result of interaction. To determine the baseline equilibration response, phosphate-buffered saline (PBS) X 1 (pH 7.4) was used as a calibration solution. The analytes were diluted (50 pL) into PBS X 1 (pH 7.4) to perform the association step. After the experiment, data was exported from three transistors, averaged them, and subtracted any background drift recorded in PBS X 1.

[0286] Ethyl (dimethyl aminopropyl) carbodiimide and N-hydroxysuccinimide were used to link the capture molecules to the chip. The protein amine was covalently attached to the carboxyl on the chip using EDC (2 mg) and sulfo-NHS (6 mg) in MES buffer (1 M (pH 6.0)) for 15 minutes. A protein solution (500 nM) was incubated for 30 minutes on the chip. To quench the remaining unoccupied binding sites on the chip, Quench 1 was followed by Quench 2 for 15 minutes each. The baseline current levels for the chip were recorded for at least five minutes prior to a rinse in PBS. An aspiration in PBS was performed accompanied by the application of a droplet of the tested analyte to the sensor chip. This was followed by recording the change on the sensor chip readout. It was followed by the aspiration of the analyte, and finally the rinsing of the chip with PBS. In order to calibrate, dissociate, regenerate, and rinse the system, a PBS X 1 buffer was applied. Further measurements were conducted at varying analyte concentrations. Upon collecting data, the sensor responses on one assay chip were averaged and the background drift in PBS was subtracted. KD was determined using a Hill fit plot. A statistical analysis program, GraphPad Prism 9, was used to calculate KD values. Data presented as mean ± SD of all measurements. All samples were identical prior to treatment assignment.

[0287] For the experiment, all commercially available solvents and reagents were used without further purification. The N-hydroxysulfosuccinimide sodium salt (Sulfo-NHS) was acquired from Biosynth Carbosynth (Compton, UK); the l-(3-Dimethylaminopropyl)- 3 -ethyl carbodiimide hydrochloride (EDC-HC1) was acquired from Alfa Aesar (Kandel, Germany); Quench 1 (3.9 mM amino-PEG5-alcohol in PBS (pH 7.4)) and Quench 2 (1 M ethanolamine (pH 8.5)) were purchased from Cardea (San Diego, CA, USA); the 2- (N-morpholino)-ethane sulfonic acid (MES) was purchased from Sigma-Aldrich (Saint Louis, MO, USA); and the PBS X 10 (pH 7.4) was purchased from Hylabs (Rehovot, Israel).

[0288] Fluorescence Polarization Assay

[0289] Recombinant Fisl was serially diluted with PBS X 1 in 10 different concentrations and then 200 pM of CVP-378 ((FAM maleimide conjugated with CVP-350) diluted in PBS X I) were mixed to each fraction in 96 black well flat clear bottoms. The plates were gently shaken and incubated for 1 h at room temperature in the dark. The fluorescence polarization was measured with a Spark plate reader (Scientific Equipment Center). A statistical analysis program, GraphPad Prism 9, was used to calculate KD values. Data presented as mean ± SD of 3 independent measurements. All samples were identical prior to treatment assignment.

[0290] Docking and Virtual ADMET Prediction

[0291] Physicochemical and pharmacokinetic properties of peptides were calculated by SwissADME and ADMETlab 3.0. Using the "SMILES" feature of the NovoPro server, the amino acid sequences of the designed peptides were converted into SMILES. Several drug-like properties, including Lipinski’s rule of five and pharmacokinetic properties were calculated.

[0292] ClusPro 2.0 (Docking for proteins and peptides), an online docking system, and ChimeraX were used to visualize protein-peptide docked structures. The PDB / Alpha fold file and structural information on the target proteins were obtained from the Protein Data Bank. The chou-Fasman method was applied to calculate the predicted secondary structure and converted to peptide PDB file. Finally, ClusPro selected the most appropriate docking mode based on affinity energy and showed the socking site of the complex.

[0293] Cross-linking coupled with mass spectrometry (CL-MS)

[0294] A mixture solution of purified protein and peptide was prepared in PBS buffer (pH 7.4). Proteins and peptides’ final concentration was 10 pM. The peptide was added at a 1 : 10 or 1 : 100 protein-to-peptide ratio, and the mixture incubated with agitation for 90 min at room temperature. BS3 cross-linker was added to the proteins to a final BS3 concentration of 2 mM. The cross-linking reaction was incubated at room temperature for 30 min with agitation. The reaction was quenched by adding ammonium bicarbonate to a final concentration of 30 mM.

[0295] Mass Spectrometry

[0296] The proteins were precipitated in acetone at -80 °C for 1 h, followed by centrifugation at 13,000 x g. The pellet was resuspended in urea (20 pL, 8 M) with DTT (5 mM). After 30 min, iodoacetamide was added to a final concentration of 15 mM and the alkylation reaction proceeded for 30 min in the dark. The urea was diluted by adding 250 pL digestion buffer (25 mM TRIS (pH 8.0); 10% acetonitrile), trypsin (Promega, Fitchburg, WI, USA) was added at a 1 : 100 protease-to-protein ratio, and the protein was digested overnight at 37 °C with agitation. Following digestion, the peptides were desalted on Cl 8 stage-tips and eluted in 55% acetonitrile. The eluted peptides were dried in a SpeedVac, reconstituted in formic acid (0.1%), and measured in a mass spectrometer. The samples were analyzed by a 120 min 0-40% acetonitrile gradient on a liquid chromatography system (Acquity M UPLC, Waters, Milford, MA, USA) coupled to a Q-Exactive Plus mass spectrometer (Thermo Fisher, Waltham, MA, USA). The RAW data files from the mass spectrometer were converted into MGF format in a Proteome Discoverer (Thermo Fisher, Waltham, MA, USA), which was the input format for the analysis pipeline. The method parameters of the runs were as follows: data-dependent acquisition; full MS resolution, 70,000; MSI AGC target, le6; MSI maximum IT, 200 ms; scan range, 450-1800; dd-MS / MS resolution, 35,000; MS / MS AGC target, 2e5; MS2 maximum IT, 300 ms; loop count, top 12; isolation window, 1.1; fixed first mass, 130; HCD energy (NCE), 26; MS2 minimum AGC target, 800; charge exclusion: unassigned, 1, 2, 3, 8, >8; peptide match, off; exclude isotope, on; and dynamic exclusion, 45 s. Cross-links were identified using an established analysis application.

[0297] Circular Dichroism (CD)

[0298] CD spectra were acquired on two lasco (lapan) instruments: either a -815 or a -1500 Spectrometer. Either a cylindrical cell with 0.02 cm pathlength or a rectangular one with 0.1 cm pathlength was used for 0.5 mM or 0.1 mM peptide solutions, respectively. The following solvents were investigated: water, 10% TFE in H2O, 40% TFE in H2O and SDS 100 mM in H2O. 16 scans were acquired for each sample, at 100 nm / min scan rate.

[0299] H9c2 Cell Line

[0300] H9c2 cells obtained from embryonic rat heart tissue (CRL-1446) were obtained from the American Type Culture Collection (ATCC, Gaithersburg, MD, USA). Dulbecco's modified Eagle's medium with high glucose, fetal bovine serum, penicillinstreptomycin (10X), and combined antibiotic solutions, penicillin-streptomycin (Pen- Strep) were provided by Biological Industries (Beit-Haemek, Israel). Cell Viability Assay in H9c2

[0301] The viability of H9c2 cells was measured using sodium 3-[l-(phenylaminocarbonyl)- 3,4-tetrazolium]-bis (4-methoxy6-nitro) (XTT) benzene sulfonic acid hydrate assay, following the manufacturer's instructions and standard procedure. H9c2 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing fetal bovine serum (10% FBS), and penicillin-streptomycin (100 U / mL-100 ug / mL). A set of white (Costar 3596, Corning, NY, USA) 96-well plates with a clear bottom were seeded with H9c2 cells at a density of 1 x 104cells / well and incubated them in a humidified atmosphere at 37 °C with a supply of 5% CO2. The cells were incubated with or without mitochondrial stressors (C0CI2 (500 pM) or H2O2 (0.25-0.60 pM)) at 37 °C with 5% CO2 and peptides (1 pM) for 14 h. Following this, XTT reagent was applied (3.2 ml of XTT reagent and 64 pL XTT activator) and the results were analyzed after 6 h, the absorbance was measured at 490 nm. Cell viability analysis was conducted using a microplate spectrophotometer Tecan plate reader infinite Ml 000 PRO (NEOTEC, Scientific Instrumentation Ltd, Israel).

[0302] Cell Viability in SH-SY5Y

[0303] Human SH-SY5Y neuronal cells were treated withl-methyl-4-phenylpyridinium (MPP+) (1 mM for 30 h) following with one treatment with compounds non -treatment (NT), CTRL (TAT), Pl 10, cyclic compounds (CVP-376 and CVP-377, Table 17) and ala-scan peptides (CVP-365, CVP-366, CVP-367, CVP-368, CVP-369, CVP-370, CVP-371, Table 17) (0.5 pM each). Cell viability was measured using an in vitro toxicology assay, MTT-based kit from Sigma-Aldrich Chemicals (MO, USA), according to the manufacturers’ instructions.

[0304] Table 17. Peptidomimetics library: ID, sequences, SEQ ID Nos

[0305] In Vitro Cytotoxicity Assays

[0306] H9c2 cells were cultured in 96-well plates in DMEM supplemented with 10% FBS. The cells were seeded at a density of 1 x 104cells per well and incubated at 37 °C with 5% CO2 for 24 h. After that, the cells were treated with peptides (1 pM) and incubated at 37 °C with 5% CO2 for 14 h. Then, XTT reagent (3.2 ml of XTT reagent and 64 pL XTT activator) was added, and the results were analyzed after 6 h. Cell cytotoxicity analysis was conducted using a microplate spectrophotometer Tecan plate reader infinite M1000 PRO (NEOTEC, Scientific Instrumentation Ltd, Israel).

[0307] Measurement of JC-1 Fluorescence

[0308] H9c2 cells (6,000) were seeded in a 96-well black (Greiner, 655090, Kremsmunster, Austria) plate with a flat clear bottom and incubated overnight at 37 °C in 95% air and 5% CO2. To mimic chemical hypoxia, C0CI2 (500 pM, Thermo Fisher Scientific, Heysham, UK) was added and incubated with the cells in serum-free DMEM (without phenol red) and a peptide (1 pM) for 16-18 h at 37 °C in 95% air and 5% CO2. JC-1, a dye that accumulates in mitochondria and forms J-aggregates with red fluorescence (emission at 590 nm) and green fluorescence of J-monomers (emission at 530 nm), was added (2 pM, (Mitochondrial Membrane Potential Assay Kit (ab288313) Abeam, UK) for 10 min, washed twice with PBS X 1, and used a microplate spectrophotometer Tecan plate reader infinite M1000 PRO (NEOTEC, Scientific Instrumentation Ltd, Israel) to detect the J-monomers and J-aggregates using emission wavelengths of 530 nm and 590 nm.

[0309] ROS Assay

[0310] The generation of intracellular ROS in H9c2 cells was measured using the green fluorescence probe 2’,7’-dichlorofluorescein diacetate (DCFDA, Abeam, UK). To analyze ROS by fluorometric analysis, 4,000 cells were plated onto 96 well-black polystyrene microplates with clear top and clear bottom (Getter Group, Biomed, Israel) in phenol red-free and 10% FBS DMEM (Sartorius, Germany), and stained with 80 pM DCFDA at 37 °C for 45 min. Subsequently, the cells were washed with phenol red-free and 10% FBS DMEM and treated with 0.6 pM hydrogen peroxide (H2O2) at 37 °C and subjected to peptide (1 pM) treatment at 37 °C for 5 h. Fluorescence was detected at excitation: 485 nm, and emission: 535 nm using a fluorometer Tecan plate reader infinite M1000 PRO (NEOTEC, Scientific Instrumentation Ltd, Israel).

[0311] Mitochondrial ROS Production Assay

[0312] For the detection of mitochondrial ROS production, clear bottom black 96-well TC- treated plates (Cat # 3904 Corning; Corning, NY, USA) were seeded with H9c2 cells (Cat # CRL-1446, ATCC; Manassas, VA, USA) at around 15,000 / well and allowed to adhere overnight in 100 pl DMEM having 10% FBS. The cells were treated with 0.75 mM of hydrogen peroxide solution (Cat # H1009, Sigma; St. Louis, MO, USA) and test peptides (concentrations ranging from 1-10 pM) for about 8 h. At the end of the treatment period, the culture medium containing hydrogen peroxide and test peptides was removed, and the cells were incubated with a staining solution containing 1 : 10,000 dilution of Hoechst 33342 (H3570; Thermo Fisher, Waltham, MA, USA) and 5 pM MitoSOX Red Mitochondrial Superoxide Indicator (M36008; Invitrogen, Waltham, MA, USA), in FluoroBrite DMEM (Cat # Al 896701, Gibco, Waltham, MA, USA). The incubation was carried out for 15-20 min at 37 °C in dark conditions. The fluorescence was analyzed using SpectraMax M2e microplate reader (Molecular devices, CA, USA), at excitation / emission maxima of 510 / 580 nm for MitoSOX followed by 350 / 470 nm for Hoechst. For analysis, the fluorescence intensity of MitoSOX was normalized to the cell number determined from the fluorescence intensity of Hoechst.

[0313] Lactate Dehydrogenase (LDH) Assay

[0314] Cytotoxicity was determined using an LDH-cytotoxicity assay kit (ab 197004, Abeam, Boston, MA, USA). According to the manufacturer's protocol. A 96-well plate (Costar ref-3596, Coming, NY, USA) was seeded with 6,000 cells per well and grown in DMEM (with phenol red-free and 10% FBS) for 24 h. The cells were subjected to 200 pM H2O2 (Merck KGaA, Darmstadt, Germany) solution in DMEM without phenol red and FBS treatment (DMEM) for 4 h. Cells were treated with fresh peptide at a final concentration of 1 pM every 30 min, throughout H2O2 stress. According to the manufacturer’s instructions, 5 pl of samples medium were collected. Each well's absorbance was measured at 535 nm and 587 nm by Tecan plate reader infinite M1000 PRO (NEOTEC, Scientific Instrumentation Ltd, Israel). Cells that were exposed to H2O2 only were set at 60% LDH release.

[0315] Live-dead Assay

[0316] H9c2 cells (6,000) were seeded onto a 96-well black (Greiner, 655090, Kremsmunster, Austria) plate with a flat clear bottom and allowed to incubate overnight at 37 °C in 95% air and 5% CO2. Ischemia was simulated by switching the complete medium to ischemia buffer with (mM): KC1 4, NaCl 115, CaCl 1.8, HEPES 5, MgCL 1, NaH2PO4 0.33, sodium lactate 20, with no glucose, and pH 6.4. O2 was set to 1%. For reperfusion, O2 was increased back to 21% and medium was changed back to complete culture medium, pH 7.4. Live dead assay was performed using a Pico microscope (ImageXpress Pico, molecular devices, CA, USA) for a duration of 2 h ischemia and 50 min reperfusion. Subsequently, the cells were stained with 3 pM calcein AM and 3 pM EthD-III for 40 min, followed by the addition of 10 pL of DAPI for 5 min.

[0317] Bioenergetic Profile

[0318] H9c2 cells (22,000 per well) were plated on Seahorse XFe24 Cell Culture Microplate (Agilent, Cat#101085-004, Santa Clara, CA, USA) and treated with H2O2 (250 pM) with or without CVP-350 or CVP-354 (10 pM each) for 6 h. Untreated cells served as the control. At the end of the treatment period, the cell culture medium was replaced with Agilent Seahorse XF Media, supplemented with 1 mM pyruvate, 2 mM L- glutamine, and 2 mM D-glucose; a final volume of 500 pl was added in each well. Cells were then incubated in a 0% CO2 chamber at 37 °C for about 1 h before analysis on a Seahorse XFe24 Analyzer (Agilent, Santa Clara, CA, USA). MitoStress assays were performed by treating the cells sequentially with 1 pM oligomycin, 2 pM 2,4- Dinitrophenol (DNP), and 0.5 pM rotenone / antimycin. A total of three oxygen consumption rate (OCR) measurements were taken after each compound was administered. OCR values were normalized to total protein content from the cells in a specific group. Fold changes of the normalized OCR data were calculated with respect to the control. Each independent experiment consisted of 3-4 technical replicates per condition. Results from 3 independent experiments were compiled for analysis and representation. Statistical analysis was done using ordinary one-way ANOVA and Tukey’s multiple comparison test.

[0319] Mitochondrial Morphology using Transmission Electron Microscopy

[0320] Cultured 15,000 H9c2 cells were treated with peptides followed by treatment with CoCh (500 pM) for 14 h. Cells were fixed in 2.5 % glutaraldehyde in 0.1 M phosphate buffer at pH 7.4 for 1 h at room temperature and then washed thrice with phosphate buffer (10 min each wash). The samples were post-fixed in 1 % osmium tetroxide in 0.1 M phosphate buffer pH 7.4 for 1 h at room temperature and dehydrated in ethanol from 10 to 100 % (three times) for 10 min each step and then included in epoxy resin. The samples were sectioned with an Ultrotome V ultramicrotome (LKB Instruments, Victoria, TX, USA). Thin sections (80-100 nm) were counterstained with uranyl acetate and lead citrate and then observed with a Tecnai G2 transmission electron microscope (FEI Company, Hillsboro, OR, USA) operating at 100 kV and equipped with a Veleta (Olympus soft imaging system) digital camera.

[0321] In Vitro Competitive Study an AGILE Dev Kit (Cardea, San Diego, CA, USA) label-free binding assay was used to investigate competitive peptide inhibition activity. To determine how the peptide competitively inhibits proteins, the proteins were immobilized in vitro on chips. The chips were immobilized with one protein, and a peptide and another protein were tested as analytes. The same procedure described previously to bind the protein to the chip was followed. Next, the analyte proteins were diluted into a series of concentrations (10 points) and a constant concentration of peptide in each of the concentrations of protein. A one-hour incubation at 4 °C was performed on the protein and peptide together. To calibrate, dissociate, regenerate, and rinse, PBS X 1 was used. The KD values were calculated using Nanomedical Agile Plus software and graphical representations and KD values were generated through Prism software.

[0322] Peptide Stability

[0323] Peptide (2 mg) was dissolved in Tris buffer (800 pL, 50 mM (pH 8.0)) and was mixed with a trypsin solution (1 pL, (1 mg / mL in 50 mM Tris buffer), (pH 8.0)) (Promega, WI, USA). The peptide was incubated at 37 °C, and samples (30 pL) were taken every 30 min. 2% TFA (30 pL) and 5% ACN in water were added, and the samples were analyzed by HPLC and MS.

[0324] In Vivo Toxicity Studies

[0325] To test the toxicity of the peptide in vivo, 2 females and 2 males Sprague Dawley rats at the age of 7 months were used. The rats were housed in a cage under controlled conditions of temperature (25 °C), moisture (50%) and lighting (12 h cycles). Rats were given regular diets during the experiment, and their weight was examined weekly. Rats were treated with peptides Pl 10 or CVP-350, given via Alzet pump-delivery system at a constant rate of 2 mg / kg / day. Pumps were exchanged every 2 weeks. All procedures were performed using isoflurane 1% for ten min followed by Ketamine / Xylazine anesthesia, according to the recommended doses found in the literature (ketamine 80- 100 mg / kg Intraperitoneal (IP), Xylazine 10-12.5 mg / kg IP). After 28 days, rats were sacrificed using extended exposure to isoflurane for 30 min. Heart, kidney and liver were extracted from the rat and transferred to 4% Para formaldehyde (PF A) solution (Electron microscopy science, Hatfield, England, United Kingdom). Slides and paraffin blocks were prepared (Phatolab, Israel) and analyzed.

[0326] In Vivo Studies

[0327] All animal experiments were conducted according to the institutional animal ethical committee guidelines (ethical number: #49-07-2021), which conform to the Guide for the Care and Use of Laboratory Animals published by the US National Research Council. Sprague-Dawley male rats (Envigo Ltd, Jerusalem, Israel) were maintained at a constant temperature and relative humidity under a regular light / dark schedule (12: 12), fed with a normal rodent diet and tap water ad libitum.

[0328] Physical Measurements

[0329] Thirty-eight rats were part of the experiment. 26 (68%) of the rats reached the final measurements and were sacrificed 24 h post I / R. The initial body weight (BW) of the rats was 308 ± 37 gr. for the sham group, 259 ± 7 gr. for the untreated group, and 295 ± 10 gr. for the CVP-350 treated group (P>0.05). Heart weight (HW) and heart to body weight ratio were measured. The final body weights were 308 ± 37 gr. for the sham group, 287 ± 26 gr. for the untreated group, and 275 ± 10 gr. for the CVP-350 treated group (P>0.05). The average HWs were 931 ± 99 mg for the sham group, 904 ± 140 mg for the untreated group, and 939 ± 62 mg for the CVP-350 treated group. The HW / BW ratios were 3.05 ± 0.44 mg / gr. for the sham group, 3.16 ± 0.52 mg / gr. for the untreated group, and 3.4 ± 0.26 mg / gr. for the CVP-350 treated group (P>0.05 for all). Overall, these parameters indicated no significant change in body weight throughout the experiment, and the HW and HW / BW ratios were comparable among the groups.

[0330] Rats initially underwent a baseline assessment which included cardiac function by echocardiography followed by a tail vein blood sample for complete blood count (CBC) and biochemistry. Then rats underwent 30 min myocardial ischemia followed by reperfusion (I / R). Rats were treated with either saline or CVP-350 peptide (2 mg / kg) by intravenous (I V.) injection 5 min before reperfusion. Twenty-four hours later, rats underwent final echocardiography, and blood tests, and were sacrificed for infarct size measurement (by Triphenyltetrazolium chloride (TTC) staining).

[0331] Myocardial Infraction (MI) Procedure

[0332] Rats underwent the left anterior descending (LAD) artery ligation procedure as previously described. Briefly, under anesthesia with a combination of 87 mg / kg ketamine and 13 mg / kg xylazine (I.M.), the rat was intubated and mechanically ventilated at a rate of 80-90 cycles / min with a tidal volume of 1-2 ml / 100 gr. Subsequently, using a left thoracotomy in the third intercostal space, the chest was opened, the pericardial sac dissected, and the heart exposed. Next, a single stitch was placed through the myocardium at a depth slightly greater than the perceived level of the LAD artery, while taking care not to puncture the ventricular chamber. The suture was tightened around the LAD with a loop that enabled its fast release, thus the occluded coronary was released immediately after 30 min of occlusion. Finally, the chest was closed, the skin was stitched, and the rat was placed in its cage for recovery.

[0333] Blood Tests

[0334] Blood samples were taken for a complete blood count and biochemical analysis. The study focused on Lactate Dehydrogenase (LDH) as a relevant biomarker for assessing cardiac muscle function after I / R injury. Echocardiograph

[0335] For the echocardiographic measurements, rats were lightly sedated by an intramuscular (I.M.) injection of 29 mg / kg ketamine and 4.3 mg / kg xylazine. After sedation the rats were placed in a left decubitus position and scanned via a commercially available echoscanner (Vivid-i, GE, Israel), using a 10S phased array pediatric transducer with a cardiac application. The transmission frequency was 12 MHz, the depth was 2.5 cm and the frame rate was at least 315 frames / sec. The measurements included two parasternal short axis sections at the papillary muscle (PM) and apical (AP) level. For the echocardiography analysis parameters are derived from the echocardiographic cines at two distinct cardiac levels (AP and PM). For each level, the parameters are derived for both the systolic and diastolic states of the heart. The structural parameters are the left ventricle (LV) cross sectional area, LV internal diameter, septal and free wall widths. Using those parameters, the following functional parameters were calculated: fractional shortening (FS), and ejection fraction (EF), calculated using the following formula: (Diastolic parameter-systolic parameter) / diastolic parameter * 100. EF was calculated by the echocardiograph machine, according to the Simpson formula.

[0336] Determination of the Viable Area, Area at Risk

[0337] The viable area, area at risk (AAR) and infarct size were determined using triphenyltetrazolium chloride (TTC) staining. After sacrifice, the heart was removed, transferred to -80 °C for 5-6 min and cut manually into six to seven transverse slices. The slices were dipped in 1% TTC solution (in double-distilled water pH 7.4) at 37 °C for 20 min, rinsed in phosphate-buffered saline (PBS) and weighed. Subsequently, the slices were placed on a light table and photographed on both sides. The pictures were analyzed using the ImageJ software (NIH, Boston, MA, USA) and the different areas were delineated. Using the weights of the slices and the percentages of the different colored areas, the percentage of viable cardiac muscle (dark blue), AAR (red and white) and infarcted area (white) of the left ventricle (LV) were calculated. The analysis included rates with AAR >20%. Statistical Analysis

[0338] Data are provided as means ± standard deviation (SD) representative of six independent experiments performed in triplicate unless otherwise specified. Data were tested for significance by using the paired Student T-test. Differences were considered statistically significant when P values were <0.05. Sample sizes for in vitro experiments were estimated based on previous experience of similar assays, and the differences observed in preliminary experiments. No samples were excluded from analysis unless otherwise specified. All samples were identical prior to allocation of treatments, and the observer was blinded to the experimental conditions. All experiments were repeated at least three times, except for molecular docking results and in silico analysis calculations.

[0339] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms "a," "an" and "the" are intended to include plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" or "comprising," when used in this specification, specify the presence of stated features, integers, steps, operations, elements components and / or groups or combinations thereof, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups or combinations thereof. As used herein the terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to". The term “consisting of’ means “including and limited to”.

[0340] As used herein, the term "and / or" includes any and all possible combinations or one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative ("or").

[0341] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the specification and claims and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. Well-known functions or constructions may not be described in detail for brevity and / or clarity.

[0342] It will be understood that, although the terms first, second, etc., may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. Rather, these terms are only used to distinguish one element, component, region, layer and / or section, from another element, component, region, layer and / or section.

[0343] Certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0344] Throughout this application, various embodiments of this invention may be presented in a range format.

[0345] It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0346] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. Whenever the term “about” is used, it is meant to refer to a measurable value such as an amount, a temporal duration, and the like, and is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0347] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0348] By “patient” or “subject” is meant to include any mammal. A “mammal,” as used herein, refers to any animal classified as a mammal, including but not limited to, humans, experimental animals including monkeys, rats, mice, and guinea pigs, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, and the like.

[0349] “Treating” or “treatment” of a disease as used herein includes: preventing the disease, i.e. causing the clinical symptoms of the disease not to develop in a mammal that may be exposed to or predisposed to the disease but does not yet experience or display symptoms of the disease; inhibiting the disease, i.e., arresting or reducing the development of the disease or its clinical symptoms, or relieving the disease, i.e., causing regression of the disease or its clinical symptoms, and / or monitoring the disease and early diagnostics of the disease.

[0350] A term druggability used in drug discovery to describe a biological target such as a protein that is known to bind or is predicted to bind with high affinity to a drug. Furthermore, the binding of the drug to a druggable target alters the function of the target with a therapeutic benefit to the patient. The term "drug" herein includes small molecules (low molecular weight organic substances) but also has been extended to include biologic medical products such as therapeutic monoclonal antibodies. In at least one embodiment, the gene fusion or gene variant can be used to identify a druggable target.

[0351] All publications, patent applications, patents, and other references mentioned in the disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.

[0352] In case of conflict, the patent specification, including definitions, will prevail. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. Throughout this application various publications, published patent applications and published patents are referenced.

[0353] It will be appreciated by persons skilled in the art that the present invention is not limited to what has been particularly shown and described hereinabove. Rather the scope of the present invention is defined by the appended claims and includes both combinations and sub-combinations of the various features described hereinabove as well as variations and modifications thereof, which would occur to persons skilled in the art upon reading the foregoing description.

[0354] While certain features of the invention have been illustrated and described herein, many modifications, substitutions, changes, and equivalents may occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the invention.

[0355] Various embodiments have been presented. Each of these embodiments may of course include features from other embodiments presented, and embodiments not specifically described may include various features described herein.

[0356] REFERENCES

[0357] 1. Nunnari, J. and A. Suomalainen, Mitochondria: in sickness and in health. Cell, 2012. 148(6): p. 1145-59.

[0358] 2. Vakifahmetoglu-Norberg, H., A.T. Ouchida, and E. Norberg, The role of mitochondria in metabolism and cell death. Biochemical and biophysical research communications, 2017. 482(3): p. 426-431.

[0359] 3. Reddy, P.H., The role of mitochondria in neurodegenerative diseases: mitochondria as a therapeutic target in Alzheimer's disease. CNS Spectr, 2009. 14(8 Suppl 7): p. 8-13; discussion 16-8.

[0360] 4. Akhmedov, A.T., V. Rybin, and J. Marin-Garcia, Mitochondrial oxidative metabolism and uncoupling proteins in the failing heart. Heart failure reviews, 2015. 20(2): p. 227-249. 5. Suarez-Rivero, J.M., et al., Mitochondrial dynamics in mitochondrial diseases. Diseases, 2016. 5(1): p. 1.

[0361] 6. Detmer, S.A. and D.C. Chan, Functions and dysfunctions of mitochondrial dynamics. Nature reviews Molecular cell biology, 2007. 8(11): p. 870-879.

[0362] 7. Reddy, P.H., et al., Dynamin-related protein 1 and mitochondrial fragmentation in neurodegenerative diseases. Brain Res Rev, 2011. 67(1-2): p. 103-18.

[0363] 8. Yoon, Y., et al., The mitochondrial protein hFisl regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1. Molecular and Cellular Biology, 2003. 23(15): p. 5409-20.

[0364] 9. Otera, H., et al., Mff is an essential factor for mitochondrial recruitment of Drpl during mitochondrial fission in mammalian cells. Journal of Cell Biology, 2010. 191(6): p. 1141-58.

[0365] 10. Palmer, C.S., et al., MiD49 and MiD51, new components of the mitochondrial fission machinery. EMBO Rep, 2011. 12(6): p. 565-73.

[0366] 11. Jin, J.-y., et al., Drpl -dependent mitochondrial fission in cardiovascular disease. Acta Pharmacologica Sinica, 2021. 42(5): p. 655-664.

[0367] 12. Zerihun, M., S. Sukumaran, and N. Qvit, The Drpl-mediated mitochondrial fission protein interactome as an emerging core player in mitochondrial dynamics and cardiovascular disease therapy. International Journal of Molecular Sciences, 2023. 24(6): p. 5785.

[0368] 13. Kunig, V.B., et al., Scanning protein surfaces with DNA-encoded libraries. ChemMedChem, 2021. 16(7): p. 1048-1062.

[0369] 14. Lu, H., et al., Recent advances in the development of protein-protein interactions modulators: mechanisms and clinical trials. Signal Transduction and Targeted Therapy, 2020. 5(1): p. 213.

[0370] 15. Anand, U., et al., Translational aspect in peptide drug discovery and development: An emerging therapeutic candidate. BioFactors, 2023. 49(2): p. 251-269.

[0371] 16. Kornfeld, O.S., et al., Interaction of mitochondrial fission factor with dynamin related protein 1 governs physiological mitochondrial function in vivo. Scientific Reports, 2018. 8(1): p. 14034. 17. Qi, X., et al., A novel Drpl inhibitor diminishes aberrant mitochondrial fission and neurotoxicity. Journal of cell science, 2013. 126(3): p. 789-802.

[0372] 18. Guo, X., et al., Inhibition of mitochondrial fragmentation diminishes Huntington's disease-associated neurodegeneration. J Clin Invest, 2013. 123(12): p. 5371-88.

[0373] 19. Disatnik, M.H., et al., Acute Inhibition of Excessive Mitochondrial Fission After Myocardial Infarction Prevents Long-term Cardiac Dysfunction. J Am Heart Assoc, 2013. 2(5): p. e000461.

[0374] 20. Sato, A.K., et al., Therapeutic peptides: technological advances driving peptides into development. Current Opinion in Biotechnology, 2006. 17(6): p. 638-42.

[0375] 21. Brinckerhoff, L.H., et al., Terminal modifications inhibit proteolytic degradation of an immunogenic MART-l(27-35) peptide: implications for peptide vaccines. International Journal of Cancer, 1999. 83(3): p. 326-34.

[0376] 22. Ahn, J.M., et al., Peptidomimetics and peptide backbone modifications. Mini Rev Med Chem, 2002. 2(5): p. 463-73.

[0377] 23. Yin, H., Constrained peptides as miniature protein structures. ISRN Biochemistry, 2012. 2012: p. 1-15.

[0378] 24. Sewald, N. and H. Jakubke, Synthesis of Special Peptides and Peptide Conjugates. Peptides: chemistry and biology. 2002: VCH. 311-334.

[0379] 25. Qvit, N., et al., Design and synthesis of backbone cyclic phosphorylated peptides: The IkappaB model. Biopolymers, 2008. 91(2): p. 157-168.

[0380] 26. Qvit, N., et al., Synthesis of a Novel Macrocyclic Library: Discovery of an IGF- 1R Inhibitor. J. Comb. Chem., 2008. 10(2): p. 256-266.

[0381] 27. Qvit, N., Microwave-assisted Synthesis of Cyclic Phosphopeptide on Solid Support. Chemical Biology & Drug Design, 2015. 85(3): p. 300-305.

[0382] 28. Joo, S.H., Cyclic peptides as therapeutic agents and biochemical tools. Biomol Ther (Seoul), 2012. 20(1): p. 19-26.

[0383] 29. Osellame, L.D., et al., Cooperative and independent roles of the Drpl adaptors Mff, MiD49 and MiD51 in mitochondrial fission. Journal of cell science, 2016. 129(11): p. 2170-2181. 30. Joshi, A.U., et al., Inhibition of Drpl / Fisl interaction slows progression of amyotrophic lateral sclerosis. EMBO molecular medicine, 2018. 10(3): p. e8166.

[0384] 31. Qvit, N. and D. Mochly-Rosen, Highly specific modulators of protein kinase C localization: applications to heart failure. Drug Discovery Today: Disease Mechanisms, 2010. 7(2): p. e87-e93.

[0385] 32. Schwarze, S.R., et al., In vivo protein transduction: delivery of a biologically active protein into the mouse. Science, 1999. 285(5433): p. 1569-72.

[0386] 33. Arafiles, J.V.V., et al., Cell-Surface-Retained Peptide Additives for the Cytosolic Delivery of Functional Proteins. J Am Chem Soc, 2023. 145(45): p. 24535-48.

[0387] 34. Rochon, K., et al., Structural basis for regulated assembly of the mitochondrial fission GTPase Drpl. Nature Communications, 2024. 15(1): p. 1328.

[0388] 35. Lerner, Y., et al., Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology. J Vis Exp, 2022(181).

[0389] 36. Jacob, N.T., et al., Synthetic molecules for disruption of the MYC protein-protein interface. Bioorg Med Chem, 2018. 26(14): p. 4234-4239.

[0390] 37. Sparks, R.P., J.L. Jenkins, and R. Fratti, Use of surface plasmon resonance (SPR) to determine binding affinities and kinetic parameters between components important in fusion machinery. SNAREs: Methods and Protocols, 2019: p. 199- 210.

[0391] 38. Wang, X., et al., 3.9 A structure of the yeast Mecl-Ddc2 complex, a homolog of human ATR-ATRIP. Science, 2017. 358(6367): p. 1206-1209.

[0392] 39. Purdie, N., Circular Dichroism and the Conformational Analysis of Biomolecules Edited by Gerald D. Fasman (Brandeis University). Plenum Press: New York. 1996. PP 738 Journal of the American Chemical Society, 1996. 118(50): p. 12871- 12871.

[0393] 40. Lipinski, C.A., et al., Experimental and computational approaches to estimate solubility and permeability in drug discovery and development settings. Advanced drug delivery reviews, 1997. 23(1-3): p. 3-25. 41. Doak, B.C., et al., Oral druggable space beyond the rule of 5: insights from drugs and clinical candidates. Chemistry & biology, 2014. 21(9): p. 1115-1142.

[0394] 42. Ballantyne, C.M., et al., Phase 2b Randomized Trial of the Oral PCSK9 Inhibitor MK-0616. J Am Coll Cardiol, 2023. 81(16): p. 1553-1564.

[0395] 43. Lipinski, C.A., Rule of five in 2015 and beyond: Target and ligand structural limitations, ligand chemistry structure and drug discovery project decisions. Advanced drug delivery reviews, 2016. 101 : p. 34-41.

[0396] 44. Joshi, A.U. and D. Mochly-Rosen, Mortal engines: Mitochondrial bioenergetics and dysfunction in neurodegenerative diseases. Pharmacological Research, 2018. 138: p. 2-15.

[0397] 45. Guo, X., et al., Inhibition of mitochondrial fragmentation diminishes Huntington’s disease-associated neurodegeneration. The Journal of clinical investigation, 2013. 123(12): p. 5371-5388.

[0398] 46. Disatnik, M.H., et al., Acute inhibition of excessive mitochondrial fission after myocardial infarction prevents long-term cardiac dysfunction. Journal of the American Heart Association, 2013. 2(5): p. e000461.

[0399] 47. Marchetti, P., et al., Mitochondrial permeability transition is a central coordinating event of apoptosis. J Exp Med, 1996. 184(3): p. 1155-60.

[0400] 48. Wikstrom, J.D., G. Twig, and O.S. Shirihai, What can mitochondrial heterogeneity tell us about mitochondrial dynamics and autophagy? The international journal of biochemistry & cell biology, 2009. 41(10): p. 1914-1927.

[0401] 49. Wang, Z., et al., The mitochondrial phosphatase PGAM5 functions at the convergence point of multiple necrotic death pathways. Cell, 2012. 148(1): p. 228-243.

[0402] 50. Han, Y., et al., Multifunctional oral delivery systems for enhanced bioavailability of therapeutic peptides / proteins. Acta Pharmaceutica SinicaB, 2019. 9(5): p. 902- 922.

[0403] 51. Han, Y., et al., In silico ADME and toxicity prediction of ceftazidime and its impurities. Frontiers in pharmacology, 2019. 10: p. 449496. 52. Rostkowski, M., O. Spjuth, and P. Rydberg, WhichCyp: prediction of cytochromes P450 inhibition. Bioinformatics, 2013. 29(16): p. 2051-2052.

[0404] 53. Xiong, G., et al., ADMETlab 2.0: an integrated online platform for accurate and comprehensive predictions of ADMET properties. Nucleic Acids Research, 2021. 49(W1): p. W5-W14.

[0405] 54. Ben-Uliel, S.F., et al., De Novo Development of Mitochondria-Targeted Molecular Probes Targeting Pinkl. International Journal of Molecular Sciences, 2022. 23(11): p. 6076.

[0406] 55. Lerner, Y., et al., Exploring biomolecular interaction between the molecular chaperone Hsp90 and its client protein kinase Cdc37 using field-effect biosensing technology. JoVE (Journal of Visualized Experiments), 2022(181): p. e63495.

[0407] 56. Zerihun, M. and N. Qvit, Selective inhibitors targeting Fisl / Mid51 proteinprotein interactions protect against hypoxia-induced damage in cardiomyocytes. Frontiers in Pharmacology, 2023. 14: p. 1275370.

[0408] 57. Daina, A., O. Michielin, and V. Zoete, SwissADME: a free web tool to evaluate pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small molecules. Scientific reports, 2017. 7(1): p. 42717.

[0409] 58. Lipinski, C.A., Lead-and drug-like compounds: the rule-of-five revolution. Drug discovery today: Technologies, 2004. 1(4): p. 337-341.

[0410] 59. Pande, M., D. Kundu, and R. Srivastava, Drugs repurposing against SARS-CoV2 and the new variant B. 1.1. 7 (alpha strain) targeting the spike protein: molecular docking and simulation studies. Heliyon, 2021. 7(8).

[0411] 60. Pande, M., D. Kundu, and R. Srivastava, Drugs repurposing against SARS-CoV2 and the new variant B. 1.1. 7 (alpha strain) targeting the spike protein: molecular docking and simulation studies. Heliyon, 2021. 7(8): p. e07803.

[0412] 61. Kalisman, N., C.M. Adams, and M. Levitt, Subunit order of eukaryotic TRiC / CCT chaperonin by cross-linking, mass spectrometry, and combinatorial homology modeling. Proceedings of the National Academy of Sciences, 2012. 109(8): p. 2884-2889.

[0413] 62. Kuznetsov, A.V., et al., H9c2 and HL-1 cells demonstrate distinct features of energy metabolism, mitochondrial function and sensitivity to hypoxia- reoxygenation. Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 2015. 1853(2): p. 276-284.

[0414] 63. Sukumaran, S., et al., Rational design, synthesis and structural characterization of peptides and peptidomimetics to target Hsp90 / Cdc37 interaction for treating hepatocellular carcinoma. Computational and Structural Biotechnology Journal, 2023. 21 : p. 3159-3172.

[0415] 64. Pakkala, M., et al., Activity and stability of human kalikrein-2-specific linear and cyclic peptide inhibitors. Journal of Peptide Science, 2007. 13(5): p. 348-353.

[0416] 65. Edition, E., GUIDE LABORATORY ANIMALS. 2011.

[0417] 66. Bhindi, R., et al., Rat models of myocardial infarction. Thrombosis and haemostasis, 2006. 96(11): p. 602-610.

[0418] 67. Ertracht, O., et al., The cardioprotective efficacy of TVP1022 in a rat model of ischaemia / reperfusion. British journal of pharmacology, 2011. 163(4): p. 755- 769.

[0419] 68. Malka, A., et al., The cardioprotective efficacy of TVP 1022 against ischemia / reperfusion injury and cardiac remodeling in rats. Pharmacology Research & Perspectives, 2016. 4(6): p. e00272.

[0420] 69. Gunata, M. and H. Parlakpinar, A review of myocardial ischaemia / reperfusion injury: pathophysiology, experimental models, biomarkers, genetics and pharmacological treatment. Cell biochemistry and function, 2021. 39(2): p. 190- 217.

[0421] 70. Xin, Z., et al., Assessment of left ventricular systolic synchronicity by real-time three-dimensional echocardiography in patients with dilated cardiomyopathy. Chinese medical journal, 2006. 119(11): p. 919-924.

[0422] 71. Stein, A.B., et al., Effects of anesthesia on echocardiographic assessment of left ventricular structure and function in rats. Basic research in cardiology, 2007. 102: p. 28-41.

[0423] 72. Folland, E., et al., Assessment of left ventricular ejection fraction and volumes by real-time, two-dimensional echocardiography. A comparison of cineangiographic and radionuclide techniques. Circulation, 1979. 60(4): p. 760-766. Reinhardt, R.A., et al., Gingival fluid IL-1 and IL-6 levels in refractory periodontitis. Journal of clinical periodontology, 1993. 20(3): p. 225-231. Ojha, N., et al., Characterization of the structural and functional changes in the myocardium following focal ischemia-reperfusion injury. American Journal of Physiology -Heart and Circulatory Physiology, 2008. 294(6): p. H2435-H2443.

Claims

Claims1. A peptide or a peptidomimetic comprising amino acid sequence:Aai-Aaz- Arg-Pro whereinAai is Ser, Thr, or an analogue thereof,Aa2 is an amino acid or an analogue thereof, wherein the amino acid or an analogue thereof is selected from the group consisting of an uncharged amino acid or an analogue, amino acid or an analogue having amine side chain, and amino acid or an analogue having amide side chain. wherein the peptide or the peptidomimetic has binding affinity of KD 5000 pM or less to Fission Protein 1 (Fisl) set forth as SEQ ID 1 or a portion thereof.

2. The peptide or the peptidomimetic of claim 1, further comprising Aai at the C-terminus, wherein Aai is a natural, synthetic amino acid, and / or amino acid analogue.

3. The peptide or the peptidomimetic of claim 1 or 2, wherein Aai is selected from the group consisting of lysine, arginine, histidine, ornithine, cadaverine, or an analogue thereof.

4. The peptide or the peptidomimetic of any one of claims 1 to 3, wherein Aa2 is selected from the group consisting of Gly, Gin, Asn, or an analogue thereof as set forth in SEQ ID Nos. 4 - 17, 57-68, and 100-103.

5. The peptide or the peptidomimetic of any one of claims 1 to 4, wherein Aai is Ser, as set forth in SEQ ID Nos. 4, 6-11, 57-62, and 100-101.

6. The peptide the peptidomimetic of any one of claims 1 to 4, wherein Aai is Thr, as set forth in SEQ ID Nos:5, 12-17, 63-68, and 102-103.

7. The peptide or the peptidomimetic of any one of claims 2 to 6, wherein Aai is lysine, as set forth in SEQ ID No: 4-5, 9-11, and 15-17.

8. The peptide of claim 1 comprising amino acid sequence SQRP as set forth in SEQ ID No: 7.

9. The peptide of claim 2 comprising amino acid sequence SQRPK as set forth in SEQ ID No: 10.

10. The peptide or the peptidomimetic of any one of claims 1 to 9, comprising at least one chemical modification.

11. The peptide or the peptidomimetic of any one of claims 1 to 10, wherein the peptide or the peptidomimetic is a linear or a macrocyclic compound.

12. The peptide or the peptidomimetic of any one of claims 1 to 11, wherein the binding affinity of the peptide or the peptidomimetic to Fisl is in the range of KD 5000 pM -to 100 nM.

13. The peptide or the peptidomimetic of any one of claims 1 to 12, wherein the binding affinity of the peptide or the peptidomimetic to Fisl is in the range of KD 1100 pM -to 200 nM.

14. The peptide or the peptidomimetic of claim 1, having the structure15. The peptide or the peptidomimetic of any one of claims 1 to 14, in the form of a pharmaceutically acceptable salt.

16. A pharmaceutical composition comprising the peptide or the peptidomimetic of any one of claims 1 to 15 and at least one pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, wherein the composition is a solid composition, a semi-solid composition, or a liquid composition.

18. The pharmaceutical composition of claim 16 or 17, wherein the composition is suitable for oral administration, intravenous administration, subcutaneous administration, transdermal administration, and topical administration.

19. The pharmaceutical composition of any one of claims 16 to 18, further comprising at least one of: surfactant, solubilizer, filler, diluent, binder, disintegrant, lubricant, glidant, preservative, stabilizer, antioxidant, buffers, pH adjusters, viscosity enhancer, thickener, coating agent, cryoprotectants, lyoprotectant, osmotic agent, or any combination thereof.

20. A method of treating a condition associated with mitochondrial dysfunction, comprising administering to a subject in need of such treatment an effective amount of the peptide or the peptidomimetic of any one of claims 1 to 15.

21. A method of treating a condition associated with mitochondrial dysfunction, comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of any one of claims 16 to 19.

22. The method of claim 20, wherein the peptide or the peptidomimetic is administered orally, intravenously, subcutaneously, transdermally, or topically.

23. The method of claim 21, wherein the pharmaceutical composition is administered orally, intravenously, subcutaneously, transdermally, or topically.

24. The method of any one of claims 20 to 23, wherein the subject in need is a human subject.

25. The method of claim 24, wherein the effective amount of the peptide or the peptidomimetic is equivalent to the amount in the range of 0.01 mg / kg / day to 20 mg / kg / day in a rodent, according to the BSA scaling.

26. The method of any one of claims 20 to 25, wherein the condition associated with mitochondrial dysfunction is selected from the group consisting of an inflammatory disease, a neurodegenerative disease, a condition associated with abnormal cell growth, an autoimmune disease, sepsis, acute kidney injury, a metabolic disorder, disease or condition associated with aging, and a cardiovascular disease.

27. The method of claim 26, wherein the cardiovascular disease is selected from the group consisting of ischemic heart disease, sepsis-induced cardiac injury, cardiomyopathy, stroke, heart failure, inflammatory heart disease.

28. A method of preventing / reducing mitochondrial fission comprising administering to a subject in need an effective amount of the peptide or the peptidomimetic of any one of claims 1 to 15, or the pharmaceutical composition of any one of claims 16 to 19.

29. The method of claim 28, wherein the subject in need is a human subject afflicted with a disease or a condition selected from the group consisting of an inflammatory disease, a neurodegenerative disease, a condition associated with abnormal cell growth, an autoimmune disease, sepsis, acute kidney injury, a metabolic disorder, a disease or condition associated with aging, and a cardiovascular disease.

30. A method of reducing the affinity of Drpl / Fisl binding in a cell-free system, comprising applying an effective amount of the peptide or the peptidomimetic of any one of claims 1 to 15.

31. The method of claim 30, wherein the reduction in the affinity of Drpl / Fisl binding is reflected by an increase in the KD value of the Drpl / Fisl binding after applying the peptide or the peptidomimetic, wherein the KD value is increased by between 1.5-fold to 35-fold while compared to the KD value of the Drpl / Fisl binding before applying the peptide or the peptidomimetic.

32. The method of claim 31, wherein the KD value is increased by between 2.5-fold to 30- fold while compared to the KD value of the Drpl / Fisl binding before applying the peptide or the peptidomimetic to the cell.

33. The method of claim 30 or 32, wherein the effective amount is in the range of 25 pM to 500 pM.

34. A method for reducing the affinity of Drpl / Fisl binding in a cell, wherein the method comprises applying to the cell an effective amount of the peptide or the peptidomimetic of any one of claims 1 to 15, under conditions sufficient to reduce Drpl / Fisl binding.

35. The method of claim 34, wherein the half-maximal inhibitory concentration (ICso) of the peptide or the peptidomimetic as measured by a cell -based assay, is in the range of 0.3 pM to 2pM.

36. The method of claim 34 or 35, wherein the cell is mammalian cell.

37. The peptide or the peptidomimetic of any one of claims 1 to 15 for use in the treatment of a disease or condition associated with mitochondrial dysfunction.

38. The peptide or the peptidomimetic for use of claim 37, wherein the disease or a condition is selected from the group consisting of an inflammatory disease, a neurodegenerative disease, a condition associated with abnormal cell growth, an autoimmune disease, sepsis, acute kidney injury, a metabolic disorder, a disease or a condition associated with aging, and a cardiovascular disease.

39. The pharmaceutical composition of any one of claims 16 to 20 for use in the treatment of a disease or condition associated with mitochondrial dysfunction.

40. The pharmaceutical composition for use of claim 39, wherein the disease or a condition is selected from the group consisting of an inflammatory disease, a neurodegenerative disease, a condition associated with abnormal cell growth, an autoimmune disease, sepsis, acute kidney injury, a metabolic disorder, a disease or a condition associated with aging, and a cardiovascular disease.

41. The peptide or the peptidomimetic of any one of claims 1 to 15 for use as a medicament.

42. The pharmaceutical composition of any one of claims 16 to 20 for use as a medicament.

Images