BNIP3 peptides for the treatment of reperfusion injury

Abstract

The present invention provides peptides capable of inhibiting the individual activities and interpathway communication of BNIP3, BAX, and mitochondria. These peptides can be used in methods for treating diseases or conditions in subjects in which it is desirable to prevent cell damage and cell death. The present invention provides peptides that bind to BNIP3 and BAX as monomers and their homo- and hetero-oligomers, which exhibit broad sequential activity by bypassing the individual and oligomeric activities of BNIP3 and BAX. Efficacy is not limited to organs or species, as evidenced by the protection of heart and brain tissue and human ventricular cardiomyocytes derived from human induced pluripotent stem cells from reperfusion injury. Myocardial infarct size was also significantly reduced in pigs.

Description

[Technical Field]

[0001] The present invention relates to the treatment of reperfusion injury. In particular, the present invention provides BNIP3-derived peptides that prevent cell damage and death by reducing the activity of BNIP3 and BAX in mitochondria. [Background technology]

[0002] Vascular occlusion causes the cessation of blood flow to a portion of the tissue, which, among other things, results in inadequate oxygen supply, reduced availability of nutrients, and inadequate removal of metabolic waste products, resulting in significant cell destruction and subsequent cell death. Although acute occlusion is neither predictable nor avoidable, restoration of vascular patency is feasible and essential for patient outcome. 1 Although a timely reperfusion regimen is the recommended treatment, rapid restoration of blood and especially O2 supply inflicts tissue injury for which no treatment is currently available. The early phase of reperfusion is characterized by high levels of oxygen, resulting in hyperoxia, rapid generation of reactive oxygen species, and increased calcium levels without acidosis. The pathology of reperfusion injury has been recognized in the heart, brain, liver, and kidney and is associated with severe clinical findings, including myocardial hibernation, acute heart failure, cerebral dysfunction, gastrointestinal dysfunction, renal dysfunction, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome. Therefore, reperfusion injury is a serious medical condition that poses significant therapeutic challenges. Myocardial infarction (MI) is a sudden, temporally unpredictable event in which reperfusion, essential for survival, determines up to 50% of the final infarct size. 2 This fate also applies to transplanted organs. MI is the most common cause of heart failure cases, and therefore, therapeutic interventions to mitigate reperfusion injury offer an opportunity to salvage viable myocardium, limit MI size, preserve cardiac function, and impact the incidence of heart failure. 3Two forms of cell death, necrosis and apoptosis, play essential roles in the progression of reperfusion-induced infarction. Necrotic myocardial cell death can be observed primarily in the early infarcted area. Necrosis induces downstream tissue responses, such as inflammation, matrix remodeling, and subsequent fibrosis. 4 Apoptosis occurs in the infarct and peri-infarct regions and is a major component of early post-infarct remodeling. 5 .

[0003] Cardiac damage is also a significant problem for cancer patients. Advances in screening and treatment strategies have led to a steady increase in the cancer survivor population over the past 30 years. The 5-year overall survival rate has improved to 50–70% after 10 years of follow-up. As a result, the prevalence of cancer treatment side effects, particularly cardiovascular toxicity, is increasing. Conventional chemotherapy (e.g., anthracyclines) is a commonly used treatment for many cancers that has been widely recognized as a cause of asymptomatic and symptomatic reductions in left ventricular ejection fraction (LVEF), cardiomyopathy, and heart failure (HF). Cancer-mediated cardiomyopathy is characterized by a dose-dependent decrease in LV systolic function mediated by reactive oxygen species (ROS), which is typically irreversible. Currently, no preventive approaches or treatments exist to effectively reduce cardiotoxic side effects (e.g., cardiac function decline, cardiomyopathy, etc.) in patients receiving anthracycline chemotherapy or other cancer treatments. Several studies aimed at addressing this medical need have yielded incomplete results / benefits. The CECCY trial failed to demonstrate benefit of carvedilol in breast cancer patients, but did demonstrate protection through troponin reduction. 6 The PRADA trial evaluated candesartan and metoprolol and found a significant benefit of candesartan in preventing cardiomyopathy, but the trial was underpowered according to current definitions of cardiac toxicity. 7 The primary outcome of the MANTICORE trial was change in left ventricular diameter. Neither beta-blockers nor ACE inhibitors had a significant effect on this outcome measure, but they significantly prevented heart failure as a secondary outcome. 8In summary, although the existing literature indicates the potential benefits of heart failure therapies, there are currently no studies that have thoroughly evaluated the value of state-of-the-art heart failure treatments, as defined by current guidelines, in preventing cardiotoxicity in cancer patients.

[0004] Mitochondria are central to both necrotic and apoptotic signaling 9 These include disruption of electron transport, oxidative phosphorylation and ATP synthesis, DNA fragmentation, protein and lipid damage, and excessive production of ROS.

[0005] The defining event in mitochondrial necrosis is the opening of a hole in the inner mitochondrial membrane (MIM), the so-called mitochondrial permeability transition pore (mPTP). This leads to energy disruption and the rapid exchange of osmolytes and solutes into the mitochondria. The subsequent swelling of the matrix leads to rupture of the outer mitochondrial membrane, cell swelling, and cell destruction. 10 Ca 2+ Necrotic stimuli such as erythrocyte sedimentation (e.g., erythrocyte stimuli) have been suggested to induce mPTP opening, which can be enhanced by ROS. 10 Despite extensive investigation, transgenic animal studies have not shown that adenine nucleotide translocase 11 , voltage-dependent anion channels 12 , mitochondrial phosphate carrier 13 (SLC25A3) and cyclophilin D 14 The components of the mPTP remain unknown, with the exception of several putative components, including the c-subunit of ATP synthase. More recently, it has been suggested that the c-subunit of ATP synthase forms pores in the inner membrane. 15、16 Prevention of mPTP opening using pharmacological inhibitors such as cyclosporine A has been reported to reduce infarct size in preclinical models of I / R injury. 17、18 In larger clinical trials, the effect was neutral. 19Animal studies have demonstrated that the mitochondrial-targeted peptide elamipretide (previously known as Bendavia or MTP-131) and the mitochondrial-targeted drug TRO40303, when administered at the onset of reperfusion, reduce infarct size by attenuating the production of mitochondrially derived ROS. 20~22 However, in a study of STEMI patients, intravenous elamipretide administered before PPCI 20 Neither TRO40303 nor TRO40303 were able to reduce infarct size. 23 Notably, more adverse events were reported in patients receiving TRO40303 compared with those receiving placebo, thereby limiting the clinical application of this therapeutic approach. + / H + Exchange inhibitors 24 , antioxidants such as superoxide dismutase 25 and various antineutrophil antibodies 26、27 also seemed to have no effect.

[0006] Apoptotic cell death in the infarct and peri-infarct regions is initiated by mitochondrial outer membrane (MOM) permeabilization, which allows the release of proapoptotic proteins, such as cytochrome c, apoptosis-inducing factor, SMAC / DIABLO (Second Mitochondria-derived Activator of Caspases / Direct IAP Binding Protein with Low PI), and endonuclease G, from the intermembrane space into the cytosol, leading to the initiation of a cell death cascade via caspases and DNA fragmentation. 28~30 .

[0007] The pro-death BCL-2 proteins BNIP3 (BCL-2 adenovirus E1B 19-kDa interacting protein-3) and BAX (BCL-2-associated X protein) are representative mediators and downstream effectors that mediate mitochondrial apoptosis by inducing MOM permeabilization and translocating into the MOM to form heterodimers.31~35 Furthermore, BNIP3 and BAX regulate MIM perturbation, thereby functioning as key activators of necrosis. 5、36 .

[0008] The present invention addresses the need to optimally ameliorate both acute injury in the core infarct region and subsequent cell death in the immediate surrounding region by providing inhibitors of BNIP3 and BAX interactive activity, where intra- and inter-pathway communication between BNIP3, BAX, and mitochondria, individually or as a triad, is disrupted to treat reperfusion injury in the heart, brain, liver, and kidney, and other indications where mitochondrial perturbations cause, for example, cell damage and death resulting from heart failure, organ transplantation, cardiac arrest, or surgical and pharmacological interventions, as well as cardiac damage induced by stroke, cancer, and cancer therapy. Summary of the Invention [Means for solving the problem]

[0009] The present invention provides peptides that bind to BNIP3 and BAX as monomers and their homo- and hetero-oligomers, demonstrating broad sequential activity by bypassing the individual and oligomeric activities of BNIP3 and BAX. Efficacy is not limited to organs or species, as demonstrated by protecting heart and brain tissue and human ventricular cardiomyocytes derived from human induced pluripotent stem cells from reperfusion injury. Myocardial infarct size was also significantly reduced in pigs.

[0010] The peptides were derived from the N-terminal portion of BNIP3, and an eight-amino acid section consisting of amino acids 13–20 of BNIP3 proved to be the most active. It was quite surprising that such a short peptide was able to inhibit BNIP3 and BAX activity, block the formation of homo- and hetero-oligomers of these proteins, and induce conformational changes of these homo- and hetero-oligomers in cells. Certain mutations in the peptide sequence further enhanced its efficacy.

[0011] In light of these results, the present invention provides, in a first aspect, (i) a cellular uptake signal; and (ii) a BNIP3 fragment containing positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom; The present invention provides a peptide comprising:

[0012] The peptides in particular have a length of 50, in particular 40, amino acids or less.

[0013] In a second aspect, the present invention provides a pharmaceutical composition comprising a peptide according to the first aspect and its use in the treatment and prevention of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity.

[0014] In a third aspect, the present invention provides a method for preventing cell damage or cell death, comprising contacting said cells with a peptide according to the first aspect.

[0015] Furthermore, in a fourth aspect, the present invention provides a method for screening a compound suitable for preventing reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising the steps of: (i) providing one or more candidate compounds; (ii) determining the ability of the candidate compound to interfere with the binding of BNIP3 to BAX; (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX; The present invention is directed to a method, including:

[0016] Other objects, features, advantages, and aspects of the present invention will become apparent to those skilled in the art from the following description and appended claims. It should be understood, however, that the following description, appended claims, and specific examples indicating preferred embodiments of the present application are given by way of example only. Various changes and modifications within the spirit and scope of the disclosed invention will become readily apparent to those skilled in the art from reading the following. [Brief explanation of the drawings]

[0017] [Figure 1] BNIP3 deficiency in mice reduces myocardial infarct size in vivo. A. Schematic of the in vivo ischemia / reperfusion model. B. Schematic of a heart section illustrating the non-ischemic area (remote), ischemic area (area at risk, AAR), and infarct area (white, embedded in the AAR). C. Infarct size after 24 hours of reperfusion in wild-type, BNIP3-deficient (Bnip3- / -), and Bnip3- / - mice treated with the indicated TAT-BNIP3 doses (n = 3-7 mice). AAR = area at risk; Inf = infarct. Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction.

[0018] [Figure 2]BNIP3 is a mediator of BAX activity in myocardial reperfusion injury. After vascular occlusion, mice were exposed to the indicated reperfusion times in vivo. Increased mitochondrial BNIP3 levels (A) and mitochondrial BAX concentrations (B) were observed in the risk zone at baseline and after 10 minutes of reperfusion (n = 5–7 mice). C Western blot monitoring revealed that BNIP3 co-immunoprecipitated with BAX at baseline and after 10 and 30 minutes of reperfusion. D Quantification of mitochondrial BAX levels in the risk zone of untreated and treated BNIP3-deficient (Bnip3- / -) mice (n = 3 mice) at baseline and after 10 minutes of reperfusion. BAX translocation depends on the presence of BNIP3. Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction.

[0019] [Figure 3-1] Interaction sites, secondary structure, and in silico docking. A Schematic diagram of the experimental setup (JPT, Berlin, Germany). B Illustrated heat map of BNIP3 incubation with the BAX peptide library displaying helices α5, α6, and α7+α8 as interaction sites. Color coding ranges from white (0 or low intensity) through light gray (medium intensity) to dark gray (high intensity). C Three-dimensional structural model of BNIP3 obtained by homology modeling with Modeller 9.15. D Circular dichroism (CD) spectroscopy analysis of BNIP3. E, F Schematic representation of the BAX / BNIP3 interaction with the indicated binding sites obtained from in silico docking experiments using HADDOCK. G Structure of the TAT sequence. H Structure of the BNIP3-20A structure. I Structure of the BNIP3-20C structure. J Representative image of the transmural distribution of TAT-BNIP3-20A (green) after 10 min of reperfusion in vivo. Scale bar, 1 mm. K Western blot monitoring of TAT-BNIP3-20A co-immunoprecipitated with BNIP3 after 5 min of reperfusion following vascular occlusion in vivo. [Figure 3-2]Same as above. [Figure 3-3] Same as above. [Figure 3-4] Same as above. [Figure 3-5] Same as above.

[0020] [Figure 4] TAT-BNIP3-20A attenuates myocardial reperfusion injury in vivo. A. Schematic of an in vivo myocardial infarction model. After vascular occlusion in vivo, mice were exposed to the indicated reperfusion times. Peptides were administered into the left ventricle 5 minutes before reperfusion. B. Infarct size after 24 hours of reperfusion in wild-type mice treated with vehicle, control peptide TAT-BNIP3-20C, and TAT-BNIP3-20A (n = 7-10 mice). TAT-BNIP3-20A significantly reduces infarct size. BNIP3-20C and vehicle are ineffective at attenuating infarction. TAT-BNIP3-20A inhibits BNIP3 interaction with mitochondria after 10 min of reperfusion (C) and caspase-3 activity after 4 h of reperfusion (D), whereas the control peptide TAT-BNIP3-20C does not (n = 6 mice). Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction.

[0021] [Figure 5-1]A Schematic of in vitro reoxygenation (study design in human ventricular cardiomyocytes (human CMs) derived from human induced pluripotent stem cells). Human CMs were exposed to normoxia and hypoxia followed by 2 hours of reoxygenation and treated with TAT-BNIP3-20A and the control peptide BNIP3-20C. B TAT-BNIP3-20A significantly inhibits BNIP3 interaction with mitochondria. C Representative images of apoptotic, necrotic, and healthy human CMs. Scale bar, 1 mm (left), 200 μm (right). TAT-BNIP3-20A potently prevents human CM death upon reoxygenation. D Representative images of depolarized mitochondria, healthy mitochondria, and nuclei. TAT-BNIP3-20A attenuates the loss of mitochondrial inner membrane potential induced by reoxygenation. Scale bar, 400 μm (left), 100 μm (right). The control peptide TAT-BNIP3-20C was ineffective in inhibiting BNIP3 interaction with mitochondria, loss of mitochondrial inner membrane potential, and cell death. Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction. [Figure 5-2] Same as above.

[0022] [Figure 6] A. Structure of the BNIP3-8B sequence. B. Structure of the BNIP3-8C sequence. C. Circular dichroism (CD) spectroscopy of BNIP3-8B.

[0023] [Figure 7] A. Uptake of fluorescently labeled TAT-BNIP3-8B in different organs after 5 minutes of reperfusion following vascular occlusion. TAT-BNIP3-8B was administered 5 minutes before the start of reperfusion. B. Viability of isolated adult cardiomyocytes 24 hours after treatment with TAT-BNIP3-8B and the TAT-BNIP3-8C control peptide.

[0024] [Figure 8]TAT-BNIP3-8B pharmacokinetics. Fluorescently labeled BNIP3-8B was incubated in human serum (A), plasma (B), and whole blood (C) at 37°C for the indicated periods and monitored by Western blot. Proteinase K treatment served as a control.

[0025] [Figure 9-1] TAT-BNIP3-20A reduces myocardial infarct size in vivo. A. Schematic of the in vivo myocardial infarction model. Mice were exposed to 5 minutes and 24 hours of reperfusion after vascular occlusion, respectively. The peptide was administered into the left ventricle 5 minutes before the start of reperfusion. B. Western blot monitoring of TAT-BNIP3-8B co-immunoprecipitated with BNIP3 and BAX 5 minutes after reperfusion. C. Immunoblot for cytosolic BNIP3 and BAX in the risk zone after blue native-PAGE at baseline and 10 minutes of reperfusion. Mice were treated with vehicle (NaCl) and TAT-BNIP3-8B. Sham-operated mice served as controls. D, E. Infarct size after 24 hours of reperfusion in wild-type mice treated with vehicle, TAT-β-Gal, control peptide TAT-BNIP3-8C, and the indicated doses of TAT-BNIP3-8B (n = 7–10 mice). TAT-BNIP3-8B significantly reduced infarct size in a dose-dependent manner. Vehicle, TAT-β-Gal, and TAT-BNIP3-8C were not effective in attenuating infarction. [Figure 9-2] Same as above.

[0026] [Figure 10-1]BNIP3-8B reduces myocardial reperfusion injury in vivo. After in vivo vascular occlusion, mice were exposed to the indicated reperfusion times. The peptides were administered into the left ventricle 5 min before the start of reperfusion. TAT-BNIP3-8B, but not the control peptide TAT-BNIP3-8C, inhibited the interaction of mitochondria with BNIP3 (A) and BAX (B) after 10 min of reperfusion, mitochondrial swelling after 10 min of reperfusion (C), BAX activation after 30 min of reperfusion (D), cytochrome c release after 30 min of reperfusion (E), and caspase-3 activity after 4 h of reperfusion (F) (n = 5–12 mice). Sham-operated mice served as controls (n = 5–8 mice). Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction. [Figure 10-2] Same as above. [Figure 10-3] Same as above.

[0027] [Figure 11] A. Schematic of the in vitro reoxygenation study design in human ventricular cardiomyocytes (human CMs) derived from human induced pluripotent stem cells. Human CMs were exposed to normoxia and 2 hours of reoxygenation following hypoxia and treated with TAT-BNIP3-8B and the control peptide TAT-BNIP3-8C. B. TAT-BNIP3-8B potently inhibits human CM death during reoxygenation. Representative images of apoptotic, necrotic, and healthy human CMs. C. TAT-BNIP3-8B attenuates reoxygenation-induced loss of mitochondrial inner membrane potential. Representative images of depolarized mitochondria, healthy mitochondria, and nuclei. Scale bar, 200 μm. The control peptide BNIP3-8C was not effective in inhibiting cell death and loss of mitochondrial inner membrane potential. Data are means ± standard error of the mean. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni's correction.

[0028] [Figure 12]TAT-BNIP3-8B reduces cerebral infarct size compared with vehicle treatment after 24 hours of reperfusion following transient middle cerebral artery occlusion. TAT-BNIP3-8B and vehicle were administered immediately before reperfusion. Data are means ± standard error of the mean. An unpaired Student's t-test was used, and statistical significance was set at a level of P<0.05.

[0029] [Figure 13] TAT-BNIP3-8B reduces myocardial infarct size compared with vehicle treatment in pigs after 4 hours of reperfusion following left coronary artery occlusion. TAT-BNIP3-8B and vehicle were given 5 minutes before reperfusion. Data are means ± standard error of the mean. An unpaired Student's t-test was used, and statistical significance was set at a level of P<0.05.

[0030] [Figure 14] TAT-BNIP3-8B protects against doxorubicin-induced mitochondrial damage by preventing mitochondrial swelling. HL-1 cells were treated with 5 μM doxorubicin without or with TAT-BNIP3-8B, and mitochondrial swelling was determined by optical density (OD), where swollen mitochondria have a lower OD540. Untreated cells were used as a control. DETAILED DESCRIPTION OF THE INVENTION

[0031] The present invention provides methods, compounds, and compositions for treating diseases or conditions in a subject in which it is desirable to inhibit the individual activities and inter-pathway communication of BCL-2 adenovirus E1B 19 kDa interacting protein-3 (BNIP3), BCL-2 associated X protein (BAX), and mitochondria to prevent cell damage and death.

[0032] In one aspect, the present invention relates to a peptide that inhibits BNIP3, BAX, and mitochondria, which activate the cell damage and cell death cascade. The peptide of the present invention comprises a cellular uptake signal and a BNIP3 fragment comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom, and is particularly 50 amino acids long, particularly 40 amino acids or less long. BNIP3 fragment

[0033] The BNIP3 fragment in the peptide according to the present invention is particularly capable of binding to BAX and / or BNIP3. In particular, the BNIP3 fragment is capable of binding to the BNIP3-binding region of BAX, such as the region of amino acids 108 to 164 of BAX. The BNIP3 fragment is particularly capable of interfering with or inhibiting the interaction between BNIP3 and BAX.

[0034] In certain embodiments, the BNIP3 fragment has a length of 20 amino acids or less. In particular, the BNIP3 fragment has a length of 15 amino acids or less, or 10 amino acids or less. In certain embodiments, the BNIP3 fragment has a length of 8 amino acids.

[0035] In a specific embodiment, the BNIP3 fragment has the amino acid sequence of the BNIP3 protein. As used herein, the term "BNIP3" particularly refers to the murine BCL-2 adenovirus E1B 19 kDa interacting protein-3 having the amino acid sequence of SEQ ID NO: 1. Thus, a BNIP3 fragment may have an amino acid sequence identical to a contiguous portion of the amino acid sequence of SEQ ID NO: 1, including the sequence of amino acid positions 13 to 20. For example, a BNIP3 fragment may have the amino acid sequence of positions 1 to 20 of SEQ ID NO: 1. In certain embodiments, a BNIP3 fragment has an amino acid sequence selected from the group consisting of positions 4 to 20 of SEQ ID NO: 1, positions 11 to 20 of SEQ ID NO: 1, positions 12 to 20 of SEQ ID NO: 1, and positions 13 to 20 of SEQ ID NO: 1. In a specific embodiment, a BNIP3 fragment consists of the amino acid sequence of positions 13 to 20 of SEQ ID NO: 1. In these embodiments, a BNIP3 fragment particularly does not contain any additional amino acid residues. The amino acid sequence of positions 13 to 20 of mouse BNIP3 is identical to the amino acid sequence of positions 73 to 80 of human BNIP3 (SEQ ID NO: 2). Instead of the amino acid sequence of mouse BNIP3 referred to herein, the corresponding amino acid sequence of human BNIP3 can also be used.

[0036] In a further embodiment, the BNIP3 fragment has an amino acid sequence derived from BNIP3. A target amino acid sequence is "derived from" or "corresponding to" a reference amino acid sequence if the target amino acid sequence shares at least 60%, more preferably at least 70%, at least 80%, at least 90%, or at least 95% homology or identity with the corresponding portion of the reference amino acid sequence over its entire length. In certain embodiments, a target amino acid sequence "derived from" or "corresponding to" a reference amino acid sequence is 100% homologous, or particularly 100% identical, to the corresponding portion of the reference amino acid sequence over its entire length. According to the present invention, the "homology" or "identity" of an amino acid sequence or nucleotide sequence is preferably determined over the entire length of the reference sequence or over the entire length of the corresponding portion of the reference sequence that corresponds to the sequence to which homology or identity is defined. In particular, the BNIP3 fragment may be derived from one of the BNIP3 fragments described above. For example, the BNIP3 fragment may have an amino acid sequence that is at least 60% identical, particularly at least 70% identical, to an amino acid sequence selected from the group consisting of positions 1 to 20 of SEQ ID NO: 1, positions 4 to 20 of SEQ ID NO: 1, positions 11 to 20 of SEQ ID NO: 1, positions 12 to 20 of SEQ ID NO: 1, and positions 13 to 20 of SEQ ID NO: 1. In a specific embodiment, the BNIP3 fragment comprises an amino acid sequence that is at least 60% identical to positions 13 to 20 of SEQ ID NO: 1.

[0037] In certain embodiments, the BNIP3 fragment comprises positions 13-20 of BNIP3, optionally containing one, two, or three amino acid substitutions relative to positions 13-20 of BNIP3. BNIP3 has, in particular, the amino acid sequence of SEQ ID NO: 1. In these embodiments, the one, two, or three amino acid substitutions are preferably present at one or more of the positions corresponding to positions 13, 15, 17, 18, 19, and 20 of BNIP3, in particular positions 15, 17, and 19 of BNIP3. In particular, the amino acid substitutions are (i) substitution of glutamic acid at position 15 of BNIP3 with phenylalanine, isoleucine, leucine, valine, tyrosine, cysteine, histidine, arginine, or threonine; (ii) a substitution of histidine at position 17 of BNIP3 with valine, and (iii) substitution of serine at position 19 of BNIP3 with tyrosine, cysteine, phenylalanine, or histidine; is selected from the group consisting of:

[0038] In a specific embodiment, the BNIP3 fragment comprises positions 13-20 of BNIP3 comprising one or two amino acid substitutions compared to positions 13-20 of BNIP3, wherein the amino acid substitutions are (i) a substitution of glutamic acid at position 15 of BNIP3 with histidine, isoleucine, leucine, valine, or tyrosine; and (ii) substitution of serine at position 19 of BNIP3 with tyrosine, cysteine, or phenylalanine; is selected from the group consisting of:

[0039] In certain embodiments, the serine at position 19 of BNIP3 is substituted with tyrosine, cysteine ​​or phenylalanine, particularly phenylalanine.

[0040] In a specific embodiment, the BNIP3 fragment comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 17. In a particular embodiment, the BNIP3 fragment consists of the amino acid sequence of SEQ ID NO: 7 or 8, particularly SEQ ID NO: 8.

[0041] A BNIP3 fragment may optionally contain additional amino acid residues derived from BNIP3. In particular, the entire BNIP3 fragment is derived from the continuous amino acid sequence of BNIP3. In particular, the entire BNIP3 fragment is derived from amino acid positions 1 to 20 of BNIP3, or a portion thereof comprising at least positions 13 to 20 of BNIP3. A BNIP3 fragment may have the same amino acid sequence as the corresponding portion of BNIP3, or may have 1 to 8 amino acid substitutions, particularly 1 to 6 amino acid substitutions. A BNIP3 fragment may, for example, comprise positions 12 to 20 of BNIP3, optionally containing 1, 2, 3, or 4 amino acid substitutions relative to positions 12 to 20 of BNIP3, or a BNIP3 fragment may comprise positions 4 to 20 of BNIP3, optionally containing 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to positions 4 to 20 of BNIP3, or a BNIP3 fragment may comprise positions 1 to 20 of BNIP3, optionally containing 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to positions 1 to 20 of BNIP3. In particular, 1, 2, or 3 of these amino acid substitutions are at positions 13 to 20, and the remaining amino acid substitutions are at positions 1 to 12. In these embodiments, the amino acid substitutions are preferably at one or more of positions 4, 11, 12, 13, 15, 17, 18, 19 and 20 of BNIP3, particularly positions corresponding to positions 4, 11, 12, 15, 17 and 19 of BNIP3.

[0042] In a specific embodiment, the BNIP3 fragment comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 18-30 and 70-75.

[0043] A substituted amino acid residue is specifically substituted with another naturally occurring amino acid residue. As used herein, the term "substituted" also includes the use of a chemically derivatized residue in place of an underivatized residue, provided that such polypeptide exhibits the required activity. The term "derivative" as used herein refers to a peptide having one or more residues chemically derivatized by reaction of a functional side chain. Such derivatized molecules include, for example, molecules in which a free amino group is derivatized to form an amine hydrochloride, p-toluenesulfonyl group, carbobenzoxy group, t-butyloxycarbonyl group, chloroacetyl group, or formyl group. Free carboxyl groups can be derivatized to form salts, methyl and ethyl esters or other types of esters, or hydrazides. Free hydroxyl groups can be derivatized to form O-acyl or O-alkyl derivatives. The imidazole nitrogen of histidine can be derivatized to form N-im-benzylhistidine. Peptides containing one or more naturally occurring amino acid derivatives of the 20 standard amino acids are also included as derivatives. For example, proline may be substituted with 4-hydroxyproline; lysine may be substituted with 5-hydroxylysine; histidine may be substituted with 3-methylhistidine; serine may be substituted with homoserine; lysine may be substituted with ornithine.

[0044] In some embodiments, the term "substituted" includes linking two or more substituted amino acid residues. In particular, two or more amino acid residues may be substituted and / or linked with a crosslinkable moiety, each optionally containing an additional α-carbon substitution selected from substituted, optionally hetero-lower alkyl, particularly optionally substituted, optionally hetero-methyl, ethyl, propyl, and butyl. Furthermore, two substituted amino acid residues may be substituted with homocysteine ​​connected via a disulfide bridge to generate a ring and tail cyclic peptide. Alternatively, two or more substituted amino acid residues may be replaced by a linker. Suitable linkers in this regard include, for example, -(CH2) to form a lactam peptide. n ONHCO X (CH2) m - (wherein X is CH2, NH or O, and m and n are integers from 1 to 4); -CH2OCH2CHCHCH2OCH2- which forms an ether peptide; or -(CH2)nCHCH(CH2)m- which forms a stapled peptide.

[0045] In certain embodiments, the BNIP3 fragment comprises at least one of the substitutions E15Y, S19F, and S19Y relative to the sequence of BNIP3. Cellular uptake signals

[0046] The cellular uptake signals within the peptides according to the invention are particularly capable of mediating uptake of the peptide into target cells, in particular the cellular uptake signals are capable of mediating uptake into mammalian cells, in particular human cells.

[0047] In certain embodiments, the cellular uptake signal is a peptide. In particular, the cellular uptake signal is a cell-penetrating peptide or a protein transduction domain. The cellular uptake signal may have a length of 5 to 30 amino acids, particularly 8 to 20 amino acids or 10 to 16 amino acids.

[0048] The cellular uptake signal may be, for example, a hydrophilic or amphipathic peptide. Examples of cellular uptake signals include the protein transduction domain of the HIV TAT protein (particularly amino acid residues 48-59), penetratin, Antennapedia PTD, SynB1, SynB3, PTD-4, PTD-5, FHV coat-(35-49), BMV Gag-(7-25), HTLV-II Rex-(4-16), D-Tat, R9-Tat, transportan, MAP, SBP, FBP, MPG, and MPG. (ΔNLS) , Pep-1, Pep-2, polyarginine, and polylysine. In certain embodiments, the cellular uptake signal is a peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-50.

[0049] The cell uptake signal may be composed of naturally occurring amino acid residues, or may be a peptide derivative containing chemically derivatized amino acid residues as described herein. Furthermore, the cell uptake signal may be a peptidomimetic or may contain a D-retro-inverso sequence. In certain embodiments, the cell uptake signal comprises a D-retro-inverso sequence of the cell-penetrating peptide disclosed herein. peptide

[0050] The peptide according to the present invention comprises a cell uptake signal and a BNIP3 fragment. As used herein, the term "comprise" not only means literally, but also includes and specifically refers to the terms "consist essentially of" and "consist of." Thus, the term "comprise" refers not only to embodiments in which the subject matter "comprising" the specifically listed elements can and / or actually includes additional elements, but also to embodiments in which the subject matter "comprising" the specifically listed elements does not include additional elements. Similarly, the term "have" should be understood as the term "comprise," and also includes and specifically refers to the terms "consist essentially of" and "consisting of." The term "essentially consisting of," when possible, particularly refers to embodiments in which the subject matter contains 20% or less, particularly 15% or less, 10% or less, or particularly 5% or less, of additional elements in addition to the specifically listed elements from which the subject matter essentially consists.

[0051] In certain embodiments, the peptide according to the invention consists of a cell uptake signal and a BNIP3 fragment.

[0052] In a further embodiment, the peptide according to the present invention comprises a cellular uptake signal, a BNIP3 fragment, and a linker between the cellular uptake signal and the BNIP3 fragment. In particular, the peptide according to the present invention comprises a cellular uptake signal, a BNIP3 fragment, and a linker between the cellular uptake signal and the BNIP3 fragment. The linker can be a peptide linker composed of amino acids or a chemical linker. The linker is particularly small in size. For example, the linker is a peptide linker having 10 or fewer amino acids, such as 8 or fewer, 6 or fewer, or 5 or fewer amino acids. The chemical linker has, for example, a molecular weight of 1,500 Da or less, such as 1,000 Da or less, 750 Da or less, or 500 Da or less. In an embodiment in which the cellular uptake signal is a peptide moiety, the linker is preferably a peptide linker.

[0053] In certain embodiments, the peptides according to the present invention have a length of 40 amino acids or less. In a specific embodiment, the peptides according to the present invention have a length of 35 amino acids or less, particularly 30 amino acids or less. The peptides according to the present invention may have a length of, for example, 25 amino acids or less, for example, about 20 amino acids. Shorter peptides are particularly desirable because they are easier to produce, formulate, and handle. Therefore, the peptides according to the present invention preferably have a length of 35 amino acids or less, particularly 25 amino acids or less. This particularly refers to an embodiment in which the cellular uptake signal is a peptide moiety. In a specific embodiment, the peptides according to the present invention have a molecular weight of 10,000 Da or less, particularly 5,000 Da or less, 4,000 Da or less, or 3,000 Da or less.

[0054] In certain embodiments, a peptide according to the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 51-67.

[0055] Generally, peptides according to the present invention are composed of naturally occurring L-amino acids. In certain embodiments, peptides may also contain artificial amino acids. For example, peptides may contain or consist of one or more D-amino acids, E-beta-homo amino acids, and / or N-methylated amino acids. In certain embodiments, the cell uptake signal and / or BNIP3 fragment is a D-retro-inverso sequence, particularly a D-retro-inverso sequence of an amino acid sequence described herein. For example, the peptide has the D-retro-inverso sequence QPRRRQRRKKRG-NSFHLEVWSGQLNEEGSQSM (SEQ ID NO: 68) or QPRRRQRRKKRG-NSFHLEVW (SEQ ID NO: 69).

[0056] In certain embodiments, the peptides are acetylated, acylated, formylated, amidated, phosphorylated, sulfated, nitrosated, glycosylated, suomylated, hydroxylated, alkylated, and / or isomerized. For example, the peptides may contain an N-terminal acetyl, formyl, myristyl, palmitoyl, carboxyl, or 2-furosyl group, and / or a C-terminal hydroxyl, amide, ester, or thioester group. Furthermore, the peptides may be cyclized.

[0057] In a specific embodiment, the peptide according to the present invention is a peptidomimetic of any of the peptides described herein. As used herein, the term "peptidomimetic" refers to a structure that functions as a substitute for a peptide in molecular interactions. Peptidomimetics include synthetic structures that may or may not contain amino acids and / or peptide bonds, but that retain the structural and functional characteristics of BNIP3 peptides. Peptidomimetics also include molecules that incorporate peptides into larger molecules with other functional elements, peptoids, oligopeptoids, and peptide libraries containing designed lengths of peptides representing all possible sequences of amino acids corresponding to the peptides of the present invention. All of these peptides, as well as molecules that are substantially homologous, complementary, or otherwise functionally or structurally equivalent to these peptides, can be used for the present invention.

[0058] In certain embodiments, the peptides of the present invention are present in a composition further comprising nanoparticles. In particular, the peptides are present within the nanoparticles. The nanoparticles may be any nanoparticles suitable for encapsulating peptides. Exemplary nanoparticles include liposomes, nanoemulsions, solid-liquid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles, and dendrimers. In certain embodiments, the nanoparticles comprise targeting molecules, such as peptides, ligands, or antibodies, on the outer surface of the nanoparticles, which enable the peptides of the present invention to be targeted to desired cells or tissues.

[0059] In specific embodiments, the nanoparticles allow the peptides of the present invention to be taken up into target cells. In these embodiments, the nanoparticles act as a cell uptake signal, and may in particular be a cell uptake signal, or may replace a cell uptake signal. Thus, the present invention also provides nanoparticles comprising the BNIP3 fragments defined herein. Therapeutic Uses of Peptides

[0060] The present invention provides methods, compounds and compositions for treating a disease or condition in a subject in which it is desirable to inhibit the individual activities and inter-pathway communication of BNIP3, BAX and mitochondria, comprising administering to the subject an amount of the compound effective to treat the disease or condition in the subject.

[0061] The present invention also provides a pharmaceutical composition comprising a peptide according to the present invention. In particular, the pharmaceutical composition comprises a peptide according to the present invention in a unit dose, administrable form. The present invention further provides a method for inhibiting cell damage and cell death, comprising administering an effective amount of a peptide according to the present invention to a human in need thereof. The present invention also provides the use of a peptide according to the present invention or a pharmaceutical composition comprising a peptide according to the present invention in medicine, in particular in the treatment and prevention of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity, respectively.

[0062] The present invention includes all combinations of the specific embodiments recited as if each combination were painstakingly recited separately.

[0063] The present invention also provides a method of inhibiting BNIP3 in a subject, comprising contacting the BNIP3 with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit BNIP3. Preferably, the BNIP3 is in the subject, and one or more of the peptides or compositions are administered to the subject.

[0064] The present invention also provides a method of inhibiting BAX in a subject, comprising contacting BAX with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit BAX. Preferably, the BAX is in the subject and one or more of the peptides or compositions are administered to the subject.

[0065] The present invention also provides methods for inhibiting BNIP3 / BAX dimer and / or oligomer activity in a subject, comprising contacting the BNIP3 / BAX dimer and / or oligomer with one or more of any of the peptides or pharmaceutical compositions disclosed herein in an amount effective to inhibit BNIP3 / BAX dimer / oligomer activity. Preferably, the BNIP3 and BAX are in the subject, and one or more of the peptides or compositions are administered to the subject.

[0066] The present invention also provides a method for treating a reperfusion-related and / or mitochondrial-related disorder in a subject, comprising administering to the subject a therapeutically effective amount of a peptide or pharmaceutical composition according to the present invention.

[0067] The present invention also provides a method for treating or preventing tissue damage due to mitochondrial-induced apoptosis or necrosis in a subject, comprising administering to the subject a therapeutically effective amount of a peptide or pharmaceutical composition according to the present invention. The peptide may be administered to the subject before, during, and / or after the onset of cell death or damage.

[0068] The subject to whom the peptide or pharmaceutical composition is administered and treated may have a disease or condition selected from the group consisting of, for example, hypoxic and / or ischemic cells; ischemia of the heart, brain, liver, kidneys, intestines, limbs, vascular occlusion in the limbs; reperfusion injury of the heart, brain, liver and kidneys, intestines, limbs; myocardial infarction and reperfusion injury; chemotherapy, radiation therapy, targeted therapy, and immunotherapy-induced cardiotoxicity; atherosclerosis; heart failure; heart, liver, kidney transplant; aneurysm; chronic lung disease; ischemic heart disease; hypertension; pulmonary hypertension; embolism; thrombosis; cardiomyopathy; stroke; neurodegenerative disease or disorder; immunological disorder; renal hypoxia; hepatitis; liver disease; kidney disease; cerebellar degeneration; organ transplant rejection, and diseases or disorders involving cell death and / or tissue damage. In certain embodiments, the subject suffers from ischemia, reperfusion and / or mitochondrial-related disorders, particularly after vascular occlusion, especially myocardial infarction, ischemic stroke, acute kidney injury, trauma, circulatory arrest, and ischemia during organ transplantation.In further embodiments, the subject suffers from cancer treatment-induced cardiotoxicity, such as cardiotoxicity induced by chemotherapy, radiotherapy, immunotherapy, and / or targeted therapy.Alternatively, the subject may undergo any type of cancer treatment, and the treatment may be applied to prevent cardiotoxicity.Chemotherapy particularly refers to anthracycline-based chemotherapy, such as doxorubicin-based chemotherapy.In a specific embodiment, the subject suffers from cardiotoxicity induced by anthracycline-based chemotherapy, such as doxorubicin-based chemotherapy.

[0069] Reperfusion-related injury can generally refer to injury associated with reperfusion injury.Reperfusion injury is, for example, tissue damage caused when blood supply returns to tissue after a period of ischemia, and the return of circulation leads to inflammation and oxidative damage through the induction of oxidative stress, rather than the restoration of normal function.Therefore, the present invention provides a method for treating reperfusion injury in a subject, comprising administering to the subject a therapeutically effective amount of the peptide or pharmaceutical composition according to the present invention.

[0070] The present invention also provides methods for treating acute myocardial infarction, myocardial reperfusion injury, or heart failure in a subject, comprising administering to the subject one or more of the peptides or pharmaceutical compositions disclosed herein in an amount effective to treat acute myocardial infarction, myocardial reperfusion injury, or heart failure in a subject in need thereof. Preferably, the one or more peptides or pharmaceutical compositions are administered in an amount effective to inhibit BNIP3, BAX, or BNIP3 / BAX dimer / oligomer activity, respectively, in the subject.

[0071] In certain embodiments, treatment involves mitigating and / or preventing reperfusion and mitochondrial-related injury. In further embodiments, treatment involves mitigating and / or preventing cancer therapy-induced cardiotoxicity.

[0072] The subject can be, for example, a mammal, preferably a human.

[0073] As used herein, "treating" or "treat" a disease or disorder means alleviating or ameliorating or eliminating the signs or symptoms of the disease or disorder being treated. When a peptide or composition is administered to a subject before or at the onset of a disease or disorder, the peptide or composition can suppress or reduce the severity of the disease or disorder. For example, administering a peptide or composition to a subject can suppress or reduce the severity of cancer therapy-induced cardiotoxicity, such as chemotherapy-, radiation therapy-, targeted therapy-, or immunotherapy-induced cardiotoxicity. In these embodiments, the peptides according to the present invention can be administered before, during, and / or after cancer treatment. Administration of the peptide can include prophylactic and / or therapeutic administration.

[0074] The peptides and compositions of the present invention can be administered to a subject using administration routes known in the art. Administration can be systemic or localized to a specific site. Administration routes include, but are not limited to, intravenous, intramuscular, intracardiac, intrathecal or subcutaneous injection, oral or rectal administration, and injection into a specific site.

[0075] In a specific embodiment, the peptide or pharmaceutical composition according to the present invention is administered to a subject during or after the occurrence of blood supply disorder, ischemia or vascular obstruction, particularly before the reperfusion of the tissue affected by vascular obstruction.In a specific embodiment, the peptide or pharmaceutical composition is administered within 6 hours before reperfusion, particularly within 4 hours, 2 hours or 1 hour before reperfusion.In a specific embodiment, the peptide or pharmaceutical composition is administered within 45 minutes, particularly within 30 minutes before reperfusion.

[0076] All combinations of the various elements described herein are within the scope of the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0077] The method may include expressing an effective amount of a peptide according to the present invention in a cell, wherein apoptosis, necrosis, or a combination thereof is altered in the cell compared to a control cell. The expression may include, for example, introducing a polynucleotide encoding the peptide into the cell. The cell may be ex vivo or in vivo, and may be a cardiac cell. Apoptosis, necrosis, or a combination thereof may be reduced in the cell.

[0078] The present invention provides a method comprising administering an effective amount of a composition comprising a polynucleotide encoding a peptide of the present invention to a subject in need thereof, wherein apoptosis, necrosis, or a combination thereof is increased in the subject. The administration may include delivery of the polynucleotide to cardiac tissue, brain tissue, liver tissue, and kidney tissue. The subject may have or be at risk for a disease selected from acute infarction, hypoxia, ischemia, stroke, or vascular disease. The method may result in alleviation of the disease symptoms.

[0079] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxynucleotides, and includes both double-stranded and single-stranded RNA and DNA. Polynucleotides can be obtained directly from natural sources or prepared using recombinant, enzymatic, or chemical techniques. The topology of a polynucleotide can be linear or circular. A polynucleotide can be part of, or a fragment of, a vector, such as an expression vector or a cloning vector. A polynucleotide can contain nucleotide sequences with different functions, including, for example, coding regions and non-coding regions, such as regulatory regions.

[0080] As used herein, "gene" refers to a nucleotide sequence encoding an mRNA. A gene has a transcription initiation site at its 5' end and a transcription terminator at its 3' end. As used herein, "target gene" refers to a specific gene whose expression is inhibited by a polynucleotide as described herein. As used herein, "target mRNA" refers to the mRNA encoded by the target gene. Unless otherwise specified, a target gene can produce multiple mRNAs that are distinguished by the use of different combinations of exons. Such related mRNAs are called splice variants or transcript variants of the gene.

[0081] As used herein, the terms "coding region" and "coding sequence" are used interchangeably and refer to a nucleotide sequence that encodes a polypeptide and that, when placed under the control of appropriate regulatory sequences, expresses the encoded polypeptide. The boundaries of a coding region are generally determined by a translation start codon at its 5' end and a translation stop codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that controls the expression of a coding sequence to which it is operably linked. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription initiation sites, translation start sites, translation stop sites, and transcription terminators. The term "operably linked" refers to the juxtaposition of components such that the components are in a relationship permitting them to function in their intended manner. A regulatory sequence is "operably linked" to a coding region when the regulatory sequence is joined in such a way that expression of the coding region is achieved under conditions compatible with the regulatory sequence.

[0082] A polynucleotide comprising a coding region may contain heterologous nucleotides flanking one or both sides of the coding region. As used herein, "heterologous nucleotides" refers to nucleotides that are not normally present adjacent to the coding region present in wild-type cells. For example, a coding region present in a wild-type microorganism that encodes a polypeptide is flanked by homologous sequences, and any other nucleotide sequence flanking the coding region is considered heterologous. Examples of heterologous nucleotides include, but are not limited to, regulatory sequences. Typically, heterologous nucleotides are present in the polynucleotides of the present invention by using standard genetic and / or recombinant methodologies well known to those skilled in the art. The polynucleotides of the present invention may be contained in an appropriate vector. The presence of heterologous nucleotides flanking one or both sides of the polynucleotides described herein results from human manipulation.

[0083] As used herein, the terms "complement" and "complementary" refer to the ability of two single-stranded polynucleotides to base pair with each other, such that an adenine on one strand of a polynucleotide base pairs with a thymine or uracil on a second strand of a polynucleotide, and a cytosine on one strand of a polynucleotide base pairs with a guanine on a second strand of a polynucleotide. Two polynucleotides are complementary to each other if the nucleotide sequence in one polynucleotide can base pair with the nucleotide sequence in the second polynucleotide. For example, 5'-ATGC and 5'-GCAT are complementary. As used herein, the term "substantial complement" and related terms refer to a polynucleotide that can selectively hybridize to a specified polynucleotide under stringent hybridization conditions. Stringent hybridization can be performed under a variety of pH, salt, and temperature conditions. The pH can range from 6 to 9, preferably 6.8 to 8.5. The salt concentration can vary from 0.15M sodium to 0.9M sodium, and other cations can be used as long as the ionic strength is equivalent to that specified for sodium. The temperature of the hybridization reaction can vary from 30°C to 80°C, preferably 45°C to 70°C. Furthermore, other compounds can be added to the hybridization reaction to promote specific hybridization at lower temperatures, such as at or near room temperature. A compound that can be envisioned to lower the temperature requirement is formamide. Thus, if hybridization occurs between a polynucleotide and a second polynucleotide, the polynucleotide is typically substantially complementary to the second polynucleotide. As used herein, "specific hybridization" refers to hybridization between two polynucleotides under stringent hybridization conditions.

[0084] A polynucleotide comprising a coding region may contain heterologous nucleotides flanking one or both sides of the coding region. As used herein, "heterologous nucleotides" refers to nucleotides that are not normally present adjacent to the coding region present in wild-type cells. For example, a coding region present in a wild-type microorganism that encodes a polypeptide is flanked by homologous sequences, and any other nucleotide sequence flanking the coding region is considered heterologous. Examples of heterologous nucleotides include, but are not limited to, regulatory sequences. Typically, heterologous nucleotides are present in the polynucleotides of the present invention by using standard genetic and / or recombinant methodologies well known to those skilled in the art. The polynucleotides of the present invention may be contained in an appropriate vector. The presence of heterologous nucleotides flanking one or both sides of the polynucleotides described herein results from human manipulation.

[0085] The present invention further provides a method for preventing cell damage or cell death, comprising contacting said cells with a peptide according to the invention, which method may be performed in vitro or in vivo, in particular ex vivo.

[0086] Furthermore, the present invention provides a method for screening compounds suitable for preventing reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising the steps of: (i) providing one or more candidate compounds; (ii) determining the ability of the candidate compound to interfere with the binding of BNIP3 to BAX; (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX; The present invention is directed to a method, including:

[0087] Candidate compounds can be any suitable compound, including, inter alia, peptides and small molecule compounds. [Example]

[0088] Example 1: BNIP3 is a representative therapeutic target for I / R injury The combination of necrotic and apoptotic cardiomyocyte death is a hallmark of the early stages of reperfusion injury. Mitochondria play a key role in both processes. BNIP3, a BH-exclusive BCL2 family member, may be an activator of the mitochondrial-driven necrotic and apoptotic cell death cascade in cell culture and isolated rat hearts. 31、32、36~38 BNIP3 has previously been implicated in left ventricular remodeling and heart failure with preserved ejection fraction after acute myocardial infarction. 33、39、40 To investigate the involvement of BNIP3 in I / R injury and whether BNIP3 represents an attractive target for therapeutic intervention, wild-type and BNIP3-deficient mice were subjected to 24 hours of reperfusion after occlusion of the left anterior descending coronary artery in a clinically relevant in vivo model. 41~44 (Figure 1A). To delineate the affected area from the non-affected zone, Evans blue dye was injected into the aorta and coronary arteries. To demarcate the non-viable myocardium, the infarcted area within the risk zone, cardiac sections were stained with triphenyltetrazolium chloride (Figure 1B). Previous studies in isolated rat hearts using a well-established dominant-negative inhibitor of BNIP3 31 Consistent with these findings, genetic ablation of BNIP3 significantly reduced infarct size by 46% compared to wild-type mice (Figure 1C). Correspondingly, reinstatement of BNIP3 to BNIP3-deficient mice dose-dependently induced infarct size comparable to that of wild-type mice, eliminating potential side effects of genetic deletion of BNIP3 (Figure 1C). These results indicate that BNIP3 plays an important role in infarct development in vivo. Example 2: BNIP3 is a mediator of BAX-induced cell death in I / R injury

[0089] BAX, a pro-death BCL-2 family member, appears to be an effector protein at the intersection of mitochondria-dependent necrosis and apoptosis. 5Therefore, translocation of BAX from the cytosol to mitochondria is a critical step. 37、45、46 We recently demonstrated a basal interaction between BAX and BNIP3 in the MOM of cardiomyocytes in vivo. 35 To determine whether BAX translocation to mitochondria, the most critical step in cell death, occurs during I / R and is dependent on BNIP3, we investigated the initial changes in mitochondrial BAX and BNIP3 concentrations. After 10 min of reperfusion following vascular occlusion, BAX levels in the MOM significantly increased, along with increased mitochondrial BNIP3 concentrations (Figure 2A and B). Coimmunoprecipitation revealed that BNIP3 and BAX form heterodimers in the MOM (Figure 2C). Next, by utilizing BNIP3-deficient mice, we investigated whether BNIP3 is an upstream mediator of BAX for this translocation during reperfusion injury. Increases in mitochondrial BAX were not observed within the early phase of reperfusion with genetic ablation of BNIP3, demonstrating the direct influence of BNIP3 (Figure 2D). To verify that this effect was indeed achieved by BNIP3, we administered BNIP3 to the hearts of BNIP3-deficient mice 5 min before initiating the myocardial infarction procedure. Addition of BNIP3 restored BAX translocation in BNIP3-deficient mouse hearts during the early phase of reperfusion, eliminating a possible side effect of genetic deletion of BNIP3 (Fig. 2D). Example 3 Identification of key BNIP3 sequences required for BNIP3 inhibition in I / R injury

[0090] Since the interaction of BNIP3 with BAX was recognized as a substantial activity in reperfusion injury in vivo, as assessed by co-immunoprecipitation, we identified helices α5, α6, α7, and α8 in BAX as potential binding sites by evaluating a peptide microarray with a library of 13 synthesized BAX peptides (Figure 3A and B). For this study, we predicted the 3D structure of BNIP3 in silico by homology modeling using Modeller 9.15. 47(Figure 3C). The generated model was energy-minimized using NAMD2.9 and the CHARMM36 force field. The BNIP3 model showed nine α-helices of various lengths, accounting for 64% of the BNIP3 structure, followed by 19% random coils and 17% unidentified structures, as confirmed by circular dichroism spectroscopy (Figure 3D). Computational docking simulations suggested helices α5, α6, α7, and α8 of BAX and the BNIP3 sequence MSQSGEENLQGSWVELHFSN (amino acids 1–20; SEQ ID NO: 20) as the interaction sites (Figure 3E). However, the first 10 amino acids alone were unable to bind to BAX (Figure 3F). The inventors hypothesized that amino acids 1-20 of BNIP3 might be sufficient to antagonize the activity of BNIP3, and therefore, synthesized an HIV-1 TAT protein transduction domain (PTD, GRKKRRQRRRPQ (SEQ ID NO: 31), Figure 3G) covalently linked to 20 amino acids derived from amino acids 1-20 of BNIP3. 48、49 We designed a cell-penetrating peptide, TAT-BNIP3-20A peptide, consisting of BNIP3 (Figure 3H). Amino acids 42–61 of BNIP3 were used to generate a control peptide, TAT-BNIP3-20C (Figure 3I). The N-terminus of the peptide fragment was capped with an acetyl group and the C-terminus with an amide group. Treatment of wild-type mice with TAT-BNIP3-20A demonstrated that this peptide was taken up by the myocardium (Figure 3J), where it interacted with endogenous BNIP3.

[0091] To investigate whether TAT-BNIP3-20A administered 5 minutes before reperfusion, a time point appropriate for clinical practice, has the ability to antagonize BNIP3 activity and reduce reperfusion injury in vivo, we used a mouse model of myocardial infarction (Figure 4A). Treatment with TAT-BNIP3-20A resulted in a 37% reduction in infarct size, whereas treatment with vehicle or TAT-BNIP3-20C did not (Figure 4B). This was due to the inhibition of BNIP3 translocation to mitochondria by the TAT-BNIP3-20A peptide fragment (Figure 4C), resulting in significantly inhibited caspase-3 activity, a key event in mitochondrial membrane perturbation following I / R (Figure 4D). Example 4: TAT-BNIP3-20A peptide fragment inhibits apoptotic and necrotic human cardiomyocyte death

[0092] To address this translational need, we performed reoxygenation experiments in human ventricular cardiomyocytes (human CMs) derived from human induced pluripotent stem cells (Figure 5A). Following hypoxia, human CMs were exposed to 2 hours of reoxygenation. Notably, even in human CMs, TAT-BNIP3-20A demonstrated its protective properties. BNIP3-20A was able to inhibit human BNIP3 translocation to mitochondria (Figure 5B), resulting in significant mitochondrial protection as evidenced by reduced mitochondrial inner membrane depolarization (Figure 5C) and fewer apoptotic and necrotic cells (Figure 5D). Example 5: Identification and design of peptide fragment inhibitors of BNIP3 / BAX activity

[0093] N-terminal truncation of the BNIP3-20A peptide sequence followed by a single residue exchange revealed that a peptide containing amino acids 13–20 in combination with a Ser to Phe substitution at position 19 exhibited significantly higher BNIP3 binding in peptide microarrays (see Table 1 ). Table 1: Truncated BNIP3-20A fragments showing the highest binding ability to BNIP3 [Table 1]

[0094] Based on these results, we designed the BNIP3-8B peptide, which consists of a PTD covalently linked to eight amino acids derived from amino acids 13–20 of BNIP3 (WVELHFFN (SEQ ID NO: 8); Figure 6A) with a Ser-19 to Phe-19 substitution. To demonstrate the necessity of the phenylalanine residue in the peptide sequence, we substituted Phe-18 with Ala-18 and His-17 with Ala-17 to generate the BNIP3-8C peptide (Figure 6B). Circular dichroism spectra of BNIP3-8B indicated that this peptide exhibited a random coil conformation (Figure 6C).

[0095] Furthermore, we performed BNIP3 / BNIP3 interaction studies using the BNIP3-20A peptide, in which a single residue in the wild-type sequence of BNIP3 1-20 was replaced with 18 neutral amino acids. BNIP3 peptides with specific amino acid substitutions showed increased binding ability to BNIP3 (exemplary data are shown in Table 2). Table 2: Substituted BNIP3-20A peptides showing the highest binding ability to BNIP3 [Table 2] Example 6: In vitro and in vivo effects of uptake, toxicity, and stability of TAT-BNIP3-8B peptide fragment

[0096] First, we evaluated the distribution and presence of BNIP3-8B at the time of the desired in vivo effect after intracardiac injection. Next, we evaluated the pharmacokinetic profile of TAT-BNIP3-8B in human serum, plasma, and whole blood, and its toxicity in isolated adult cardiomyocytes. TAT-BNIP3-8B is taken up by the heart, spleen, and liver and present in plasma (Figure 7A). Thus, TAT-BNIP3-8B is present in the heart 10 min after reperfusion, the time point at which the BNIP3-BAX-mitochondrial triangle cell death cascade occurs. Cardiomyocytes showed no obvious signs of toxicity (Figure 7B), and the half-life of TAT-BNIP3-8B in human serum, plasma, and whole blood confirmed its in vivo inhibitory potential (Figures 8A-C). Incubation of TAT-BNIP3-8B with proteinase K served as a control. [Example 7] Mechanism of action of TAT-BNIP3-8B peptide

[0097] We hypothesized that TAT-BNIP3-8B binds to BNIP3 and BAX monomers and homodimers, as well as BNIP3 / BAX heterodimers and heterooligomers, and inhibits their activity. To evaluate the interaction behavior of TAT-BNIP3-8B, we performed BNIP3 / TAT-BNIP3-8B and BAX / TAT-BNIP3-8B overlay assays and docking simulations. Both results suggested that TAT-BNIP3-8B binds to BNIP3 and BAX (data not shown).

[0098] Notably, in a myocardial infarction model (Figure 9A), endogenous BNIP3 and BAX mono-, homo-, and heterodimers were co-immunoprecipitated with fluorescently labeled TAT-BNIP3-8B 5 min after reperfusion (Figure 9B). Furthermore, reperfusion induced BNIP3 and BAX oligomerization, as evidenced by the large-scale formation of oligomers consisting of BNIP3 and three BAX units (Figure 9C). SDS-PAGE and co-immunoprecipitation experiments revealed BNIP3 / BAX heterointeraction in higher-order oligomeric complexes, and this oligomerization was strongly inhibited by treatment with TAT-BNIP3-8B (Figure 9C). Example 8: TAT-BNIP3-8B reduces myocardial infarct size

[0099] We then investigated the efficacy and effectiveness of TAT-BNIP3-8B treatment given 5 minutes before the start of reperfusion in a suitable myocardial infarction model (Figure 9A). Notably, TAT-BNIP3-8B dose-dependently reduced infarct size by up to 40% compared with vehicle and TAT-β-Gal treatment (Figures 9D and 9E). TAT-BNIP3-8C treatment did not significantly affect infarct size compared with TAT-BNIP-8B, highlighting the importance of the phenylalanine residue (Figure 9D). Example 9: TAT-BNIP3-8B reduces mitochondrial perturbations and cell death cascade

[0100] Mitochondrial damage can occur through perturbations of the mitochondrial inner membrane (MIM) and MOM. A key mitochondrial event in necrosis is the early opening of the mitochondrial permeability transition pore (mPTP) in the MIM. 50 This causes a time-dependent dissipation of the potential difference across the MIM, followed by mitochondrial swelling and cell destruction. Key mitochondrial events in apoptosis are BAX activation, which induces exposure of the BAX transmembrane domain, and MOM permeabilization, which allows the release of apoptogens (e.g., cytochrome c), followed by caspase activation. 51In a defined in vivo I / R model, TAT-BNIP3-8B injected into the left ventricle 5 min before reperfusion suppressed BNIP3 and BAX translocation to mitochondria, resulting in inhibition of downstream cell death mechanisms, including mitochondrial swelling, BAX activation, cytochrome c release, and caspase-3 activity. Example 10: TAT-BNIP3-8B inhibits apoptotic and necrotic human cardiomyocyte death

[0101] We tested whether TAT-BNIP3-8B could inhibit cell death in human CM. Human CM were exposed to 2 hours of reoxygenation after hypoxia (Figure 11A). BNIP3-8B significantly inhibited necrotic and apoptotic cell death (Figure 11B) and loss of mitochondrial inner membrane potential (Figure 11C). [Example 11] TAT-BNIP3-8B reduces cerebral infarct size

[0102] BNIP3 has been suggested to play an important role in cerebral ischemia. 38 We also evaluated the effect of BNIP3-8B peptide on clinical outcomes in a mouse model of regional cerebral reperfusion. We subjected mice to 30 minutes of transient middle cerebral artery occlusion (tMCAO) followed by 24 hours of reperfusion. BNIP3-8B treatment immediately after tMCAO significantly reduced infarct size by 52% (Figure 12). Example 12: TAT-BNIP3-8B reduces myocardial infarct size in pigs

[0103] We then investigated the efficacy and effectiveness of TAT-BNIP3-8B treatment given 5 minutes before the start of reperfusion in a myocardial infarction model in pigs. Pigs were subjected to 60 minutes of occlusion of the left anterior descending coronary artery, followed by 4 hours of reperfusion. Notably, TAT-BNIP3-8B significantly reduced infarct size by 56% compared with vehicle (Figure 13). Example 13 TAT-BNIP3-8B protects against doxorubicin-induced mitochondrial damage.

[0104] We evaluated the effect of BNIP3-8B peptide on doxorubicin-induced mitochondrial damage in HL-1 cells. To monitor mitochondrial swelling, HL-1 cells were treated with 5 μM doxorubicin, reproducing the peak plasma concentration achieved by standard infusion in patients. Mitochondrial swelling was measured by optical density at 540 nm (increased mitochondrial volume due to swelling results in decreased optical density). TAT-BNIP3-8B given simultaneously with doxorubicin increased OD 540 As shown by the α- and β-blockers, TAT-BNIP3-8B inhibits mitochondrial swelling (Figure 14). These data indicate that TAT-BNIP3-8B can protect mitochondria from damage mediated by chemotherapy, such as anthracyclines. Example 14 Method

[0105] Chemicals were obtained from Sigma-Aldrich. Antibodies against BNIP3, tubulin, cytochrome c, and BAX were obtained from Abcam, and activated BAX was obtained from Enzo.

[0106] Animals. Male mice of similar age (12 ± 3 weeks) and an average body weight of 30 g were used. C57BL / 6 wild-type mice were obtained from Jackson Laboratory (Bar Harbor, ME, USA) and housed in a local animal facility for 1 week for acclimatization.

[0107] C57BL / 6J-TgH(Bnip3 - / - ) mice were obtained from Professor Gerald W. Dorn at the Pediatric Molecular Cardiovascular Research and Development Center (University of Cincinnati, Cincinnati, OH, USA). Mice were generated by replacing exons 2 and 3 with a neomycin resistance cassette. 33Mice were bred in the local animal house at the University Hospital of Essen. All experiments were approved by the local ethical committee in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes (Directive 2010 / 63 / EU).

[0108] In vivo myocardial infarction model in mice. Intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg) induced myocardial infarction in wild-type and Bnip3-deficient (Bnip3) mice. - / - Mice were anesthetized and intubated. Using a mouse mini-ventilator, ventilation parameters were set at a tidal volume of 2.1–2.5 ml and a respiratory rate of 140 breaths / min. Deep anesthesia was maintained by adding 2% vol isoflurane to the ventilation gas. The chest was opened by a lateral thoracotomy (a 1-cm left-sided lateral incision between the third and fourth ribs). A 6-0 prolene suture was placed around the left coronary artery (LCA), and a piece of soft silicone tubing was placed over the artery. Coronary artery occlusion was achieved by tightening and tying off the suture. After 30 minutes of occlusion, the silicone tubing was removed, leaving the suture in place. For longer reperfusion times, the chest was closed with 4-0 prolene. Five minutes before vascular occlusion, 2, 6, and 9 nmol of BNIP3 (in 50 μl of 0.9% sodium chloride) were injected into the left ventricular cavity. Peptides with 20 amino acids (2 nmol / 50 μl) and 8 amino acids (8 nmol / 50 μl) in NaCl were injected 5 min before reperfusion. Sodium chloride injection (50 μl) served as the control treatment.

[0109] In vivo myocardial infarction model in swine. After induction of anesthesia, small incisions were made over the femoral artery and femoral vein to isolate the vessels. A small opening was made in the artery to introduce a sheath. Additionally, a sheath was placed in the femoral vein or other appropriate vein to allow emergency drug administration, if necessary. A blood sample was drawn before heparin administration and used to establish a recorded baseline activated clotting time (ACT). Subsequently, heparin (250–350 IU / kg) was administered as needed, as directed by the surgeon, to achieve and maintain an ACT twice the level of the baseline ACT. After the first bolus of heparin, the ACT was again recorded and subsequently monitored at least every 60 min until the completion of the surgery.

[0110] Using visual guidance provided by fluoroscopy, the appropriate guide catheter was advanced into the ostium of the left anterior descending artery (LAD). Non-ionic contrast media was used throughout the procedure. The balloon catheter was introduced by advancing it through the guide catheter into the left anterior descending (LAD) coronary artery. The balloon was advanced into the coronary artery via the guide catheter to the appropriate location above the first diagonal branch of the LAD. The balloon was then inflated to a pressure sufficient to ensure complete occlusion of the artery. Occlusion of the artery was confirmed using fluoroscopy. After confirmation of occlusion, the balloon remained inflated within the artery for 60 minutes. Supportive medication and defibrillation were recorded in the study record. Five minutes before reperfusion, the peptide and vehicle were each administered intravenously. At the end of the vascular occlusion period, the balloon was deflated, allowing the ischemic area to reperfuse. Complete balloon deflation was confirmed by fluoroscopy. At the end of the procedure, all catheters were removed, the artery and vein were ligated, and the incision was closed in a standard fashion. Four hours after reperfusion, the animals were euthanized.

[0111] Measurement of infarct size. For infarct size analysis, mice were euthanized after 24 h of reperfusion, and the hearts were excised and perfused with PBS for 5 min. After perfusion, the LCA was religated at the same location as previously described. To delineate the ischemic AAR from the non-ischemic zone, Evans blue dye (1 ml of a 1% solution) was injected into the aorta and coronary arteries. The tissue was wrapped in clean food wrap and stored in a -20°C freezer for 1 h. The hearts were then serially sectioned perpendicular to the long axis into 1 mm slices, and each slice was weighed. To demarcate the boundary between viable and non-viable myocardium within the risk zone, the slices were incubated in 1% TTC at 37°C for 5 min. The infarct, AAR, and non-ischemic left ventricle were assessed using computer-assisted planimetry by an observer blinded to the identity of the samples. Myocardial infarct size was expressed as a percentage of the AAR.

[0112] Immunoblotting. Tissues and human CM cells were lysed in RIPS buffer (50 mM Tris-HCl, 150 mM NaCl, 0.5 mM EDTA, 1% NP-40, and protease and phosphatase inhibitors, pH 7.4). Isolated mitochondria were lysed in Mito-lyse buffer (200 mM sucrose, 10 mM HEPES, 1 mM EGTA, 1% Triton®-X 100, and protease and phosphatase inhibitors, pH 7.4). The lysate was clarified by centrifugation (20,000 x g, 15 min, 4°C). Protein concentration in the supernatant was measured using a DC protein assay (Bio-Rad). Samples were diluted in 4x LDS sample buffer and 10x reducing agent (Invitrogen) and prepared for SDS-PAGE by heating to 95°C for 5 min. Equal amounts of protein were separated using 4-12% Bis-Tris gels (Invitrogen), transferred to nitrocellulose, and immunoblotted with primary antibodies. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (Invitrogen). Immunoblots were detected by ECL (Thermo Scientific) and imaged on an Imager 600 (Amersham).

[0113] Interaction studies. For protein-peptide interaction studies, peptide libraries were synthesized and immobilized on microarray slides. Recombinant BNIP3 was used at a concentration of 1 μg / ml. Peptides were synthesized and immobilized for (i) BNIP3 / BAX interaction studies; (ii) BNIP3 / BNIP3 interaction studies with C-, N-, or C / N-terminal truncations of the wild-type sequence of BNIP3 1-20; and (iii) BNIP3 / BNIP3 interaction studies with single residues of the wild-type sequence of BNIP3 1-20 replaced with 18 neutral amino acids.

[0114] Microarray. For protein-peptide binding studies, recombinant BNIP3 (Cusabio) and BAX (MyBioSource) were used at concentrations of 10 μg / ml and 1 μg / ml, respectively. The labeling kit used was the DyLight Microscale Antibody Labeling Kit (Thermo), containing the label Dylight 650. The assay was performed using an automated TECAN HS4800 microarray processing station. Microarrays were incubated with customer-provided samples diluted in blocking buffer for 2 h at 30 °C. Before each step, the microarrays were washed with washing buffer. Microarrays were scanned using a high-resolution fluorescence scanner. The laser settings and applied resolution were identical for all measurements performed. The resulting images were analyzed and quantified using the spot recognition software GenePix (Molecular Devices). For each spot, the mean signal intensity was extracted (between 0 and 65535 arbitrary units). For further data evaluation, the so-called MMC2 value was determined. MMC2 is equal to the mean value of all three cases on the microarray unless the coefficient of variation (CV) (standard deviation divided by the mean) is greater than 0.5. In this case, the mean of the two closest values ​​(MC2) is assigned to MMC2. All steps were performed by JPT Peptide Technologies (Berlin, Germany).

[0115] In silico three-dimensional (3D) structural modeling of BNIP3. The predicted in silico 3D structure of BNIP3 was obtained by homology modeling using Modeller 9.15, and the resulting model was energy minimized using NAMD 2.9 and the CHARMM 36 force field.

[0116] Circular dichroism (CD) spectroscopy. CD spectra of BNIP3 protein and BNIP3-8B peptide were recorded on a Jasco J-715 spectropolarimeter at 37 °C, pH 7.4, and 1× PBS.

[0117] Docking simulation. Experiments were performed using Autodock Vina 52 and HADDOCK 53 The structure of BAX (pdb-ID: 4S0O) was obtained from the Protein Data Bank, while the structures of BNIP3 and peptide BNIP3-8B were modeled using Modeller 9.15. 47 The template structure corresponds to PDB codes 2k7w and 2ka1. NAMD 2.9 54 The generated model was energy minimized using the CHARMM36 force field.

[0118] Immunoprecipitation. Immunoprecipitation was performed using Protein G-conjugated Dynabeads (Invitrogen). 500 μg of dissolved protein was incubated with 2 μg of antibody overnight at 4°C with shaking in PBS buffer containing 1 mM DTT, 0.005% Brij35, and protease-phosphatase inhibitors. The next day, 20 μl of Dynabeads was added, and the solution was again incubated for 1 h. The precipitated immune complexes were washed twice and then resuspended in elution buffer containing LDS-sample buffer (1:4) and reducing agent (1:10) (Invitrogen) in PBS and heated at 95°C for 5 min. After removing the Dynabeads, the eluate was analyzed by immunoblotting.

[0119] Caspase-3 activity was measured using a caspase-3 assay kit (#ab39401) from Abcam. Rodent hearts were harvested after 30 minutes of ischemia and 4 hours of reperfusion, and the risk zone was isolated and lysed in the containing buffer. The assay was performed according to the manufacturer's instructions.

[0120] Cell culture. Human iPSC-derived ventricular cardiomyocytes (human CMs) were obtained from (axol) and cultured according to the manufacturer's specifications.

[0121] To simulate vascular occlusion, cells were incubated in a buffer solution (113 mM NaCl, 4.7 mM KCl, 12 mM HEPES, 1.2 mM MgSO, 30 mM taurine, 1.3 mM CaCl, pH 7.4) at 1% O at 37°C. Reoxygenation was performed in a buffer solution supplemented with 5.5 mM glucose at 21% O at 37°C.

[0122] HL-1 cells were cultured in Claycomb medium according to the manufacturer's protocol. Cells were treated with 5 μM doxorubicin and 2 nmol TAT-BNIP3-8B for 30 min.

[0123] JC-1 assay. To analyze the mitochondrial inner membrane potential in human CM cells, staining was performed with 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). Cells were incubated with 6 μM JC-1 in culture medium at 37°C for 30 minutes. After washing with PBS buffer at 37°C, cells were fixed with 4% PFA at room temperature for 15 minutes. DAPI staining was performed, and cells were analyzed using EVOS FL (Life Technologies).

[0124] Swelling assay. Mitochondrial swelling was measured by light scattering at 540 nm in a microplate absorbance reader FLUOstar Omega (BMG Labtech) at room temperature. The final assay volume was 200 μl and contained mitochondria at 0.5 mg / ml in a buffer containing 250 mM sucrose, 10 mM HEPES, 1 mM EGTA, pH 7.4.

[0125] Peptides. Peptides were generated by a resin synthesis procedure (JPT International, Berlin, Germany). The N-terminus of the peptide was capped with an acetyl group and the C-terminus with an amide. For delivery, the peptide was covalently attached to the TAT sequence GRKKRRQRRRPQ (SEQ ID NO: 31). For uptake and binding studies, the peptide was labeled with a fluorophore. A complete peptide list is attached as Appendix 1. In certain embodiments, for example, the following are provided: (Item 1) A peptide, (i) a cellular uptake signal; and (ii) a BNIP3 fragment containing positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom; Including, The peptide has a length of 50 amino acids or less. (Item 2) 2. The peptide according to item 1, wherein the BNIP3 fragment has a length of 12 amino acids or less, in particular 10 amino acids or less, in particular 8 amino acids. (Item 3) 3. The peptide according to item 1 or 2, wherein the BNIP3 fragment comprises positions 13 to 20 of BNIP3, optionally containing 1, 2 or 3 amino acid substitutions compared to positions 13 to 20 of BNIP3. (Item 4) 4. The peptide of item 3, wherein the amino acid substitutions are present at one or more of the positions corresponding to positions 15, 17, and 19 of BNIP3. (Item 5) The amino acid substitution is (i) glutamic acid at position 15 of BNIP3 to phenylalanine, isoleucine, leucine, valine, tyrosine, cysteine, histidine, arginine, or threonine, and (ii) histidine to valine at position 17 of BNIP3, and (iii) serine to tyrosine, cysteine, phenylalanine, or histidine at position 19 of BNIP3; 5. The peptide according to item 3 or 4, selected from: (Item 6) 6. The peptide according to any one of items 1 to 5, wherein the BNIP3 fragment comprises a phenylalanine residue at a position corresponding to position 19 of BNIP3. (Item 7) the BNIP3 fragment (i) comprising positions 12-20 of BNIP3, optionally containing 1, 2, 3 or 4 amino acid substitutions relative to positions 12-20 of BNIP3; (ii) positions 4 to 20 of BNIP3, optionally containing 1, 2, 3, 4, 5, or 6 amino acid substitutions relative to positions 4 to 20 of BNIP3; (iii) comprising positions 1 to 20 of BNIP3, optionally containing 1, 2, 3, 4, 5, or 6 amino acid substitutions compared to positions 1 to 20 of BNIP3; or (iv) consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 3 to 30; 7. The peptide according to any one of items 1 to 6. (Item 8) Item 1. The peptide according to Item 1, wherein the BNIP3 fragment comprises a D-retro-inverso sequence of positions 13 to 20 of BNIP3 or a D-retro-inverso sequence of any of the amino acid sequences according to Items 2 to 7. (Item 9) the cellular uptake signal is (i) a peptide having a length of 5 to 30 amino acids, particularly 8 to 20 amino acids or 10 to 16 amino acids, particularly 12 amino acids; (ii) the protein transduction domain of the TAT protein of HIV; and / or (iii) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 50; 9. The peptide according to any one of items 1 to 8. (Item 10) 9. The peptide according to any one of items 1 to 8, wherein the cell uptake signal comprises a D-retro-inverso sequence of a cell-penetrating peptide, particularly a cell-penetrating peptide according to item 9. (Item 11) 11. The peptide according to any one of items 1 to 10, comprising the cellular uptake signal and the BNIP3 fragment, and optionally containing a linker between the cellular uptake signal and the BNIP3 fragment. (Item 12) 12. The peptide according to any one of items 1 to 11, having a length of 40 amino acids or less, in particular 35 amino acids or less, in particular 30 amino acids or less. (Item 13) 13. A pharmaceutical composition comprising the peptide according to any one of items 1 to 12. (Item 14) 13. The peptide according to any one of items 1 to 12 for use in the treatment of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity. (Item 15) 15. The peptide for use according to item 14, wherein the reperfusion-related and / or mitochondrial-related disorder is selected from the group consisting of myocardial infarction, stroke, acute kidney injury, trauma, circulatory arrest and cessation of blood flow during organ transplantation. (Item 16) 16. Peptide for use according to item 14 or 15, wherein the peptide is administered to a patient during or after the occurrence of a blood supply disorder, in particular before reperfusion of tissues affected by ischemia. (Item 17) 17. Peptide for use according to item 16, wherein the peptide is administered to the patient within 2 hours before reperfusion, in particular within 30 minutes before reperfusion. (Item 18) 13. The peptide according to any one of items 1 to 12 for use in the treatment or prevention of tissue damage due to mitochondria-induced apoptosis or necrosis. (Item 19) 1. A method for screening for compounds suitable for preventing reperfusion injury and / or mitochondrial-induced damage and / or cancer therapy-induced cardiotoxicity, comprising: (i) providing one or more candidate compounds; (ii) determining the ability of the candidate compound to interfere with the binding of BNIP3 to BAX; (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX; A method comprising:

[0126] List of references

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Claims

1. A peptide, (a)(i) a cellular uptake signal; (ii) a BNIP3 fragment consisting of the amino acid sequence of SEQ ID NO: 8; The peptide has a length of 50 amino acids or less. or (b)(i) a cellular uptake signal; and (ii) a BNIP3 fragment consisting of the amino acid sequence of SEQ ID NO: 8; (iii) a linker between the cellular uptake signal and the BNIP3 fragment; The peptide has a length of 50 amino acids or less.

2. the cellular uptake signal is (i) a peptide having a length of 5 to 30 amino acids, particularly 8 to 20 amino acids or 10 to 16 amino acids, particularly 12 amino acids; (ii) the protein transduction domain of the TAT protein of HIV; and / or (iii) comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31-50; The peptide of claim 1.

3. 3. The peptide according to claim 1 or 2, having a length of 40 amino acids or less, in particular 35 amino acids or less, in particular 30 amino acids or less.

4. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 3.

5. A peptide according to any one of claims 1 to 3 for use in the treatment of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity.

6. 6. The peptide for use according to claim 5, wherein the reperfusion-related and / or mitochondrial-related disorder is selected from the group consisting of myocardial infarction, stroke, acute kidney injury, trauma, circulatory arrest and cessation of blood flow during organ transplantation.

7. 7. A peptide for use according to claim 5 or 6, wherein said peptide is administered to a patient during or after the occurrence of a blood supply disorder, in particular before reperfusion of tissue affected by ischemia.

8. 8. The peptide for use according to claim 7, wherein the peptide is administered to the patient within 2 hours before reperfusion, in particular within 30 minutes before reperfusion.

9. A peptide according to any one of claims 1 to 3 for use in the treatment or prevention of tissue damage due to mitochondria-induced apoptosis or necrosis.

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