BNIP3 peptides for treatment of reperfusion injury

Abstract

The present invention provides peptides capable of inhibiting the individual activity and pathway-to-pathway communication of BNIP3, BAX and mitochondria. The peptides may be used in a method of treating a disease or condition in a subject, wherein it is desirable to prevent cell damage and cell death.

Description

[0001] This divisional application is a divisional application based on the original Chinese patent application with application number 202080044457.6 and invention name “BNIP3 peptide for treating reperfusion injury”. Technical Field

[0002] The present invention relates to the treatment of reperfusion injury. In particular, the present invention provides BNIP3-derived peptides that prevent cell damage and cell death by reducing the activity of BNIP3 and BAX at mitochondria. Background Art

[0003] The blockage of a blood vessel results in cessation of blood flow to a portion of tissue, which results in, among other things, inadequate oxygen supply, reduced availability of nutrients, and inadequate removal of metabolic waste products, which inflicts severe damage to cells and subsequently leads to cell death. While acute occlusion cannot be predicted or avoided, restoration of vascular patency is feasible and essential to the patient's prognosis. 1 . Timely reperfusion protocols are the recommended treatment, but rapid restoration of blood, especially O2, supply causes tissue damage for which no treatment is currently available. The early phase of reperfusion is characterized by high levels of oxygen leading to a hyperoxic state, a burst of reactive oxygen species, and elevated calcium levels without acidosis. The pathology of reperfusion injury has been recognized in the heart, brain, liver, and kidneys and is associated with severe clinical manifestations including myocardial hibernation, acute heart failure, brain dysfunction, gastrointestinal dysfunction, renal dysfunction, systemic inflammatory response syndrome, and multiple organ dysfunction syndrome. Therefore, reperfusion injury is a serious medical condition that presents significant therapeutic challenges. Myocardial infarction (MI) is a sudden, temporally unpredictable event in which reperfusion is critical for survival but 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, so therapeutic interventions to reduce reperfusion injury offer the opportunity to salvage viable myocardium, limit MI size, preserve cardiac function, and influence the incidence of heart failure. 3 In the progression of reperfusion-induced infarction, two forms of cell death, necrosis and apoptosis, play a crucial role. A predominance of necrotic cardiomyocyte death can be observed in the initial infarct area. Necrosis induces downstream tissue responses such as inflammation, matrix remodeling, and later fibrosis. 4 Apoptosis occurs in both the infarct and peri-infarct regions and is a major component of early post-infarction remodeling 5 .

[0004] Cardiac damage is also a key issue for cancer patients. Due to advances in screening and treatment strategies, the number of cancer survivors has steadily increased over the past three decades. The 5-year survival rate has increased to 50-70% with a ten-year follow-up. As a result, side effects of cancer treatment, especially cardiovascular toxicity, are becoming increasingly common. Conventional chemotherapy (e.g., anthracyclines) is a common treatment for many cancers and is widely considered to be a contributing factor to asymptomatic and symptomatic declines 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 often irreversible. Currently, there is neither a preventive approach nor an effective therapy to reduce cardiotoxic side effects (e.g., decreased cardiac function, cardiomyopathy, etc.) in patients receiving anthracycline chemotherapy or other cancer therapies. Several studies have been designed to address this medical need, but have revealed only partial results / benefits. The CECCY trial failed to show a benefit of carvedilol in breast cancer patients, but showed a protective effect by lowering troponin 6 The PRADA trial evaluated candesartan and metoprolol and revealed a significant benefit of candesartan in preventing cardiomyopathy, although the study was underpowered based on current definitions of cardiotoxicity. 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 significantly prevented heart failure as a secondary outcome. 8 In summary, although the existing literature points to the potential benefits of heart failure therapies, no studies have yet thoroughly evaluated the value of current guideline-defined state-of-the-art heart failure therapies in preventing cardiotoxicity in cancer patients.

[0005] Mitochondria are the center of necrosis and apoptosis signaling 9 These include disruption of electron transport, oxidative phosphorylation and ATP synthesis, DNA fragmentation, protein and lipid damage, and excessive production of ROS.

[0006] The decisive event in mitochondrial necrosis is the opening of a hole in the mitochondrial inner membrane (MIM), the so-called mitochondrial permeability transition pore (mPTP). This causes an energy collapse and a rapid exchange of solutes, with permeates flooding into the mitochondria. Subsequent expansion of the matrix leads to rupture of the mitochondrial outer membrane, cell swelling, and cell rupture. 10 Necrotic stimuli, such as Ca 2+ , which is thought to trigger the opening of mPTP and can be enhanced by ROS 10Despite extensive research, the components of the mPTP remain unknown, and transgenic animal studies have ruled out several putative components, including adenine nucleotide translocase. 11 , voltage-dependent anion channels 12 、mitochondrial phosphotransferase 13 (SLC25A3) and cyclophilin D 14 Recently, the c subunit of ATP synthase has been proposed to form a pore in the inner membrane. 15,16 Preventing 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 a larger clinical trial, the effect was neutral 19 The mitochondrial-targeted peptide elamipretide (formerly known as Bendavia or MTP-131) as well as the mitochondrial-targeted drug TRO40303 have been shown in animal studies to reduce infarct size by reducing the production of mitochondrial-derived ROS when administered at the onset of reperfusion. 20-22 However, in studies of STEMI patients, intravenous elamipretide 20 and TRO40303 (both administered before PPCI) failed to reduce infarct size 23 Of note, patients receiving TRO40303 reported more adverse events when compared with placebo, limiting the clinical utility of this treatment approach. + / H + Exchange inhibitors 24 , antioxidants such as superoxide dismutase 25 and various anti-neutrophil antibodies26,27.

[0008] Apoptotic cell death occurring in the infarct and peri-infarct regions is initiated by permeabilization of the mitochondrial outer membrane (MOM), which enables the release of pro-apoptotic proteins such as cytochrome c, the 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 .

[0009] The pro-death BCL-2 proteins BNIP3 (BCL-2 adenovirus E1B 19 kDa interacting protein-3) and BAX (BCL-2 associated X protein) induce MOM permeabilization and represent mediators and downstream effectors of mitochondrial apoptosis by translocating into the MOM and forming heterodimers 31-35In addition, BNIP3 and BAX regulate the perturbation of MIM, thereby acting as key activators of necrosis 5,36 .

[0010] The present invention addresses the need for optimal amelioration of acute injury in the central infarct zone and subsequent cell death in the immediate surrounding areas by providing inhibitors of the interactive activity of BNIP3 and BAX that interrupt intra- and inter-pathway communication between BNIP3, BAX and mitochondria as individuals or triangles to treat reperfusion injury in the heart, brain, liver and kidneys and other indications in which perturbations of mitochondria lead to cell injury and cell death, such as heart failure, organ transplantation, cardiac arrest or cardiac injury due to surgical and drug interventions as well as stroke, cancer and cancer therapy.

[0011] Summary of the Invention

[0012] The present invention provides peptides that bind to BNIP3 and BAX as monomers and to their homo- and hetero-oligomers, which represent a broad spectrum of activity by circumventing the activity of the individual and the activity of the oligomers. The efficacy is not limited to one organ or one species, as demonstrated by protecting heart and brain tissue and human ventricular cardiomyocytes derived from human induced pluripotent stem cells from reperfusion injury. Myocardial infarction size in pigs was also significantly reduced.

[0013] These peptides are derived from the N-terminal part of BNIP3 and an 8-amino acid segment consisting of amino acids 13-20 of BNIP3 that have proven to be most active. Quite surprisingly, this short peptide is 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 even enhance its efficacy.

[0014] In view of these results, the present invention provides, in a first aspect, a peptide comprising

[0015] (i) a cellular uptake signal; and

[0016] (ii) a BNIP3 fragment comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom.

[0017] The peptide is especially 50, in particular 40 or fewer amino acids in length.

[0018] 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 of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity and in the prevention of such diseases.

[0019] In a third aspect, the present invention provides a method for preventing cell damage or cell death, comprising contacting a cell with a peptide according to the first aspect.

[0020] The present invention also relates in a fourth aspect to a method for screening compounds suitable for preventing reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising

[0021] (i) providing one or more candidate compounds;

[0022] (ii) determine the ability of candidate compounds to interfere with the binding of BNIP3 to BAX;

[0023] (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX.

[0024] According to the following description and the appended claims, other objects, features, advantages and aspects of the present invention will become apparent to those skilled in the art. However, it should be understood that the following description, the appended claims and the specific examples are given only as illustrations, and the description, the appended claims and the specific examples indicate the preferred embodiments of the present application. By reading the following, those skilled in the art will easily understand various changes and modifications within the spirit and scope of the disclosed invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 BNIP3 deficiency in mice reduces myocardial infarction size in vivo. A Schematic diagram of the in vivo ischemia / reperfusion model. B Schematic diagram of cardiac sections showing the non-ischemic area (distal), ischemic area (area at risk, AAR), and infarct area (white, embedded in AAR). C Schematic diagram of wild-type, BNIP3-deficient (Bnip3) mice treated with the indicated TAT-BNIP3 doses. - / - ) and Bnip3 - / - Infarct size after 24 hours of reperfusion in mice (n=3-7 mice). AAR-area at risk; INF-infarct. Data are mean±sem. Statistical analysis was two-way analysis of variance (ANOVA) with Bonferroni correction.

[0026] Figure 2.BNIP3 is a mediator of BAX activity in myocardial reperfusion injury. After vascular occlusion, mice were exposed to the specified reperfusion durations in vivo. Increased mitochondrial BNIP3 levels (A) and mitochondrial BAX concentrations (B) in the risk zone were observed at baseline and 10 minutes after reperfusion (n=5-7 mice). C Western blot monitoring showed that BNIP3 was co-immunoprecipitated with BAX at baseline and after 10 and 30 minutes of reperfusion. D BNIP3-deficient (Bnip3) mice not treated with BNIP3 and treated with BNIP3 at baseline and 10 minutes after reperfusion. - / - Quantification of mitochondrial BAX levels in the risk region of ) mice (n=3 mice). Translocation of BAX is dependent on the presence of BNIP3. Data are mean±sem. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni correction.

[0027] Figure 3 .Interaction sites, secondary structure, and in silico docking. A Schematic representation of the experimental setup (JPT, Berlin, Germany, Berlin). B Heat map depiction of BNIP3 incubated with the BAX peptide library showing helices α5, α6, and α7+α8 as interaction sites. Color coding ranges from white (0 or low intensity) to light gray (medium intensity) to dark gray (high intensity). C Three-dimensional structural model of BNIP3 obtained by homology modeling using Modeller 9.15. D Circular dichroism (CD) spectral analysis of BNIP3. E, F Cartoon representation of BAX / BNIP3 interactions with the indicated binding sites generated by 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 images of the transmural distribution of TAT-BNIP3-20A (green) after 10 min of in vivo reperfusion. Scale bar, 1 mm. K Western blot monitoring of co-immunoprecipitation of TAT-BNIP3-20A with BNIP3 after 5 min of reperfusion following in vivo vascular occlusion.

[0028] Figure 4.TAT-BNIP3-20A reduces myocardial reperfusion injury in vivo. A Schematic diagram of the in vivo myocardial infarction model. Mice were exposed to the specified reperfusion duration after in vivo vascular occlusion. The peptide was injected 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 reduced infarct size. BNIP3-20C and vehicle were ineffective in reducing infarction. TAT-BNIP3-20A inhibited the interaction of BNIP3 with mitochondria after 10 minutes of reperfusion (C) and the activity of caspase-3 after 4 hours of reperfusion (D), while the control peptide TAT-BNIP3-20C did not (n=6 mice). Data are mean ± sem. Statistical analysis was by two-way analysis of variance (ANOVA) with Bonferroni correction.

[0029] Figure 5 .A Schematic diagram of in vitro reoxygenation (study design in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (human CMs)). Human CMs were exposed to normoxia and 2 h of reoxygenation after hypoxia and treated with TAT-BNIP3-20A and control peptide BNIP3-20C. B TAT-BNIP3-20A significantly inhibits the interaction of BNIP3 with mitochondria. C Representative images of apoptotic, necrotic, and healthy human CMs. Scale bars, 1 mm (left), 200 μm (right). TAT-BNIP3-20A potently prevents human CM death during reoxygenation. D Representative images of depolarized mitochondria, healthy mitochondria, and nuclei. TAT-BNIP3-20A reduces the reoxygenation-induced decrease in mitochondrial inner membrane potential. Scale bars, 400 μm (left), 100 μm (right). The control peptide TAT-BNIP3-20C could not effectively inhibit the interaction of BNIP3 with mitochondria, the decrease of mitochondrial inner membrane potential and cell death. Data are mean ± sem. Statistical analysis was performed by two-way analysis of variance (ANOVA) with Bonferroni correction.

[0030] Figure 6 .A Structure of the BNIP3-8B sequence. B Structure of the BNIP3-8C sequence. C Circular dichroism (CD) spectrum analysis of BNIP3-8B.

[0031] Figure 7.A Uptake of fluorescently labeled TAT-BNIP3-8B in different organs 5 min after reperfusion following vascular occlusion. TAT-BNIP3-8B was administered 5 min before the start of reperfusion. B Survival of isolated adult cardiomyocytes treated with TAT-BNIP3-8B and TAT-BNIP3-8C control peptides 24 h later.

[0032] 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 durations and monitored by Western blotting. Proteinase K treatment served as a control.

[0033] Fig. 9 .TAT-BNIP3-20A reduces myocardial infarction size in vivo. A Schematic diagram of the in vivo myocardial infarction model. Mice were exposed to reperfusion for 5 minutes and 24 hours after vascular occlusion. The peptide was injected 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 after 5 minutes of reperfusion. C Immunoblotting of cytoplasmic 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. Mice subjected to sham surgery 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 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 ineffective in reducing infarct size.

[0034] Fig.10 .BNIP3-8B reduces myocardial reperfusion injury in vivo. Mice were exposed to the indicated reperfusion durations after vascular occlusion in vivo. The peptides were injected into the left ventricle 5 minutes before the start of reperfusion. TAT-BNIP3-8B inhibited the interaction of BNIP3 (A) and BAX (B) with mitochondria 10 minutes after reperfusion, mitochondrial swelling 10 minutes after reperfusion (C), BAX activation 30 minutes after reperfusion (D), cytochrome C release 30 minutes after reperfusion (E), and caspase-3 activity 4 hours after reperfusion (F), while the control peptide TAT-BNIP3-8C did not (n = 5 to 12 mice). Mice that underwent sham surgery served as controls (n = 5-8 mice). Data are mean ± sem. Statistical analysis was two-way analysis of variance (ANOVA) with Bonferroni correction.

[0035] Fig.11 .A Schematic diagram of the design of in vitro reoxygenation studies in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (human CMs). Human CMs were exposed to normoxia and 2 h of reoxygenation after hypoxia and treated with TAT-BNIP3-8B and 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 mitigates reoxygenation-induced decrease in 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 decrease in mitochondrial inner membrane potential. Data are mean ± sem. Statistical analysis was two-way analysis of variance (ANOVA) with Bonferron's correction.

[0036] Fig.12 Compared with vehicle treatment, TAT-BNIP3-8B reduced cerebral infarct size 24 hours after reperfusion after transient middle cerebral artery occlusion. TAT-BNIP3-8B and vehicle were given immediately before reperfusion. Data are mean ± sem. Unpaired Student's t-test was used, and statistical significance was set at P < 0.05.

[0037] Fig.13 In pigs, TAT-BNIP3-8B reduced myocardial infarct size 4 h after reperfusion following left coronary artery occlusion compared with vehicle treatment. TAT-BNIP3-8B and vehicle were administered 5 min before reperfusion. Data are mean ± sem. Unpaired Student's t-test was used, and statistical significance was set at P < 0.05.

[0038] Fig.14 TAT-BNIP3-8B prevents 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 measured by optical density, where swollen mitochondria have a lower OD 540 Untreated cells were used as control.

[0039] Detailed description of the invention

[0040] The present invention provides methods, compounds and compositions for treating a disease or condition in a subject where it is desired 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 cell death.

[0041] One aspect of the present invention is a peptide that inhibits BNIP3, BAX and mitochondrial triangle, which activates cell damage and cell death cascades. The peptide according to the present invention comprises a cellular uptake signal; and an amino acid sequence comprising a BNIP3 fragment at positions 13-20 or derived therefrom, and in particular a length of 50, in particular 40 amino acids or less.

[0042] BNIP3 fragment

[0043] The BNIP3 fragments in the peptides according to the invention are particularly capable of binding to BAX and / or BNIP3. In particular, the BNIP3 fragments are capable of binding to the BNIP3 binding region of BAX, such as the region of amino acids 108 to 164 of BAX. The BNIP3 fragments are particularly capable of interfering with or inhibiting the interaction of BNIP3 with BAX.

[0044] In certain embodiments, the BNIP3 fragment is 20 amino acids or less in length. In particular, it is 15 amino acids or less or even 10 amino acids or less in length. In a specific embodiment, the BNIP3 fragment is 8 amino acids in length.

[0045] In a specific embodiment, the BNIP3 fragment has the amino acid sequence of the BNIP3 protein. The term "BNIP3" as used herein refers in particular to the mouse BCL-2 adenovirus E1B 19kDa interacting protein-3, which has the amino acid sequence SEQ ID NO: 1. Thus, the BNIP3 fragment may have an amino acid sequence that is identical to a contiguous portion of the amino acid sequence of SEQ ID NO: 1 comprising the sequence at amino acid positions 13 to 20. For example, the BNIP3 fragment may have an amino acid sequence at positions 1 to 20 of SEQ ID NO: 1. In certain embodiments, the BNIP3 fragment has an amino acid sequence selected from 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 consists of the amino acid sequence at positions 13 to 20 of SEQ ID NO: 1. In these embodiments, the BNIP3 fragment specifically does not contain any other amino acid residues. The amino acid sequence at positions 13-20 of mouse BNIP3 is identical to the amino acid sequence at positions 73-80 of human BNIP3 (SEQ ID NO: 2). As an alternative to the amino acid sequence of mouse BNIP3 mentioned herein, the corresponding amino acid sequence of human BNIP3 can also be used.

[0046] In a further embodiment, the BNIP3 fragment has an amino acid sequence derived from BNIP3. A target amino acid sequence is "derived from" or "corresponds 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 part of the reference amino acid sequence over its entire length. In a particular embodiment, a target amino acid sequence "derived from" or "corresponding to" a reference amino acid sequence has 100% homology, or in particular 100% identity, with the corresponding part of the reference amino acid sequence over its entire length. The "homology" or "identity" of an amino acid sequence or a nucleotide sequence is preferably determined according to the invention over the entire length of the reference sequence or over the entire length of the corresponding part of the reference sequence, which corresponds to the sequence for which the homology or identity is defined. A BNIP3 fragment may in particular be derived from one of the above-mentioned BNIP3 fragments. 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 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.

[0047] In certain embodiments, the BNIP3 fragment comprises positions 13 to 20 of BNIP3, optionally comprising 1, 2 or 3 amino acid substitutions (compared to positions 13 to 20 of BNIP3). BNIP3 in particular has the amino acid sequence of SEQ D I NO: 1. In these embodiments, 1, 2 or 3 amino acid substitutions are preferably present at one or more positions corresponding to positions 13, 15, 17, 18, 19 and 20 of BNIP3, in particular at positions 15, 17 and 19 of BNIP3.

[0048] In particular, the amino acid substitutions are selected from

[0049] (i) substitution of glutamic acid at position 15 of BNIP3 with phenylalanine, isoleucine, leucine, valine, tyrosine, cysteine, histidine, arginine or threonine,

[0050] (ii) substitution of histidine at position 17 of BNIP3 with valine, and

[0051] (iii) Substitution of serine at position 19 of BNIP3 by tyrosine, cysteine, phenylalanine or histidine.

[0052] In certain embodiments, the BNIP3 fragment comprises positions 13 to 20 of BNIP3 comprising 1 or 2 amino acid substitutions (compared to positions 13 to 20 of BNIP3), wherein the amino acid substitutions are selected from

[0053] (i) substitution of glutamic acid at position 15 of BNIP3 with histidine, isoleucine, leucine, valine or tyrosine, and

[0054] (ii) Substitution of serine at position 19 of BNIP3 by tyrosine, cysteine ​​or phenylalanine.

[0055] In certain embodiments, the serine at position 19 of BNIP3 is substituted with tyrosine, cysteine ​​or phenylalanine, in particular with phenylalanine.

[0056] In a specific embodiment, the BNIP3 fragment comprises or consists of an amino acid sequence selected from SEQ ID NO: 3 to 17. In a specific embodiment, the BNIP3 fragment consists of the amino acid sequence of SEQ ID NO: 7 or 8, in particular 8.

[0057] The BNIP3 fragment may optionally further comprise amino acid residues derived from BNIP3. In particular, the entire BNIP3 fragment is derived from a continuous amino acid sequence of BNIP3. In particular, the entire BNIP3 fragment is derived from amino acids 1-20 of BNIP3 or a portion thereof comprising at least positions 13-20 of BNIP3. The BNIP3 fragment may have an amino acid sequence identical to the corresponding portion of BNIP3, or may have 1 to 8 amino acid substitutions, in particular 1 to 6 amino acid substitutions. The BNIP3 fragment may, for example, comprise positions 12 to 20 of BNIP3, optionally comprising 1, 2, 3 or 4 amino acid substitutions (compared to positions 12 to 20 of BNIP3), or it may comprise positions 4 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions (compared to positions 4 to 20 of BNIP3), or it may comprise positions 1 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions (compared 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 present at one or more positions corresponding to positions 4, 11, 12, 13, 15, 17, 18, 19 and 20 of BNIP3, in particular positions 4, 11, 12, 15, 17 and 19 of BNIP3.

[0058] In a specific embodiment, the BNIP3 fragment comprises an amino acid sequence selected from SEQ ID NOs: 18 to 30 and 70 to 75.

[0059] The amino acid residue that is substituted is particularly a replacement of another naturally occurring amino acid residue. As used herein, the phrase "substituted" also includes the use of chemically derived residues to replace non-derivatized residues, provided that such polypeptides exert the necessary activity. As used herein, the term "derivative" refers to a peptide having one or more residues that are chemically derivatized by reaction of functional side groups. Such derived molecules include, for example, those in which the free amino group has been derivatized to form an amine hydrochloride, p-toluenesulfonyl, benzyloxycarbonyl, tert-butoxycarbonyl, chloroacetyl or formyl. 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. Also included as derivatives are those peptides containing one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example: 4-hydroxyproline can replace proline; 5-hydroxylysine can replace lysine; 3-methylhistidine can replace histidine; homoserine can replace serine; and ornithine can replace lysine.

[0060] In some embodiments, the term "substituted" includes the linkage of two or more substituted amino acid residues. In particular, two or more amino acid residues may be substituted and / or linked by a cross-linkable moiety, and each optionally comprises an additional α-carbon substitution selected from substituted, optionally heterogeneous lower alkyl, in particular optionally substituted, optionally heterogeneous methyl, ethyl, propyl and butyl. In addition, two substituted amino acid residues may be substituted by homocysteine ​​linked by a disulfide bond to produce ring and tail cyclic peptides. Alternatively, two or more substituted amino acid residues may be replaced by a linker. Suitable linkers in this regard include, for example, -(CH2) n ONHCO X (CH2) m -, wherein X is CH2, NH or O, m and n are integers 1-4, forming a lactam peptide; -CH2OCH2CHCHCH2OCH2-, forming an ether peptide; or -(CH2)nCHCH(CH2)m-, forming a rivet peptide.

[0061] In certain embodiments, the BNIP3 fragment comprises at least one of the substitutions E15Y, S19F, and S19Y relative to the sequence of BNIP3.

[0062] Cellular uptake signal

[0063] The cellular uptake signal in the peptide according to the invention is particularly capable of mediating uptake of the peptide into target cells. In particular, the cellular uptake signal is capable of mediating uptake into mammalian cells, in particular human cells.

[0064] 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. Its length may be 5 to 30 amino acids, in particular 8 to 20 amino acids or 10 to 16 amino acids.

[0065] Cellular uptake signal can be, for example, a hydrophilic peptide or an amphipathic peptide. Examples of cellular uptake signals include protein transduction domains (particularly amino acid residues 48-59) of the TAT protein of HIV, 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, transport peptides (transportan), MAP, SBP, FBP, MPG, 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 SEQ ID NOs: 31 to 50.

[0066] The cellular uptake signal may consist of naturally occurring amino acid residues, and may optionally be a peptide derivative comprising chemically derivatized amino acid residues as described herein. In addition, it may be a peptide mimetic or comprise a D-retro-inverso sequence. In certain embodiments, the cellular uptake signal comprises a D-retro-inverso sequence of a cell penetrating peptide disclosed herein.

[0067] Peptides

[0068] The peptides according to the present invention comprise a cellular uptake signal and a BNIP3 fragment. As used herein, the expression "comprising", in addition to its literal meaning, also includes and specifically refers to the expressions "consisting essentially of" and "consisting of". Therefore, the expression "comprising" refers to an embodiment in which the subject matter "comprising" the specifically listed elements does not contain other elements, and an embodiment in which the subject matter "comprising" the specifically listed elements may contain and / or does include other elements. Similarly, the expression "having" should be understood as the expression "comprising", and also includes and specifically refers to the expressions "consisting essentially of" and "consisting of". Where possible, the term "consisting essentially of" refers in particular to an embodiment in which, in addition to the specifically listed elements of the subject matter that are essentially composed thereof, the subject matter also contains 20% or less, in particular 15% or less, 10% or less or especially 5% or less of other elements.

[0069] In certain embodiments, the peptides according to the invention consist of a cellular uptake signal and a BNIP3 fragment.

[0070] In another 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 consists of 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 consisting of amino acids, or a chemical linker. The linker is particularly small in size. For example, it is a peptide linker with 10 or less amino acids, such as 8 or less, 6 or less or 5 or less amino acids. The molecular weight of the chemical linker is, for example, 1500Da or less, such as 1000Da or less, 750Da or less or 500Da or less. In the embodiment in which the cellular uptake signal is a peptide portion, the linker is preferably a peptide linker.

[0071] In certain embodiments, the length of the peptide according to the invention is 40 amino acids or less. In a specific embodiment, the length of the peptide according to the invention is 35 amino acids or less, in particular 30 amino acids or less. The length of the peptide according to the invention can be, for example, 25 amino acids or less, such as about 20 amino acids. Shorter peptides are particularly desirable because they can be more easily produced, formulated and handled. Therefore, the length of the peptide according to the invention is preferably 35 amino acids or less, in particular 25 amino acids or less. This refers in particular to embodiments in which the cellular uptake signal is a peptide moiety. In a specific embodiment, the molecular weight of the peptide according to the invention is 10,000 Da or less, in particular 5,000 Da or less, 4,000 Da or less or 3,000 Da or less.

[0072] In certain embodiments, the peptide according to the present invention has an amino acid sequence selected from SEQ ID NOs: 51 to 67.

[0073] Typically, the peptides according to the present invention are composed of naturally occurring L-amino acids. In certain embodiments, the peptides may also include artificial amino acids. For example, the peptide may contain one or more D-amino acids, E-β-isoamino acids and / or N-methylated amino acids; or it may consist of them. In certain embodiments, the cellular uptake signal and / or the BNIP3 fragment is a D-reverse-flip sequence, in particular a D-reverse-flip sequence of the amino acid sequence described herein. For example, the peptide has a D-reverse-flip sequence of QPRRRQRRKKRG-NSFHLEVWSGQLNEEGSQSM (SEQ ID NO: 68) or QPRRRQRRKKRG-NSFHLEVW (SEQ ID NO: 69).

[0074] In certain embodiments, the peptide is acetylated, acylated, formylated, amidated, phosphorylated, sulfated, nitrosylated, glycosylated, sumo-ized, hydroxylated, alkylated, and / or isomerized. For example, the peptide may contain an N-terminal acetyl, formyl, myristoyl, palmitoyl, carboxyl, or 2-fucosyl group and / or a C-terminal hydroxyl, amide, ester, or thioester group. In addition, the peptide may be cyclized.

[0075] In a specific embodiment, the peptide according to the present invention is a peptide mimetic of any peptide described herein. As used herein, the term "peptide mimetic" refers to a structure as a peptide substitute in intermolecular interactions. Peptide mimetic includes a synthetic structure that may or may not contain amino acids and / or peptide bonds but retains the structural and functional characteristics of the BNIP3 peptide. Peptide mimetic also includes a molecule in which the peptide is incorporated into a larger molecule with other functional elements, peptoids, oligopeptoids, and a peptide library comprising a peptide with a designed length, the peptide library representing all possible amino acid sequences corresponding to the peptide of the present invention. All of these peptides and molecules that are substantially homologous, complementary, or otherwise equivalent in function or structure to these peptides can be used for purposes of the present invention.

[0076] In certain embodiments, the peptide according to the present invention is present in a composition further comprising nanoparticles. In particular, the peptide is present in nanoparticles. The nanoparticles can be any nanoparticles suitable for encapsulating the peptide. Exemplary nanoparticles include liposomes, nanoemulsions, solid-liquid nanoparticles, nanostructured lipid carriers, polymeric nanoparticles and dendrimers. In certain embodiments, the nanoparticles include targeting molecules, such as peptides, ligands or antibodies, on their outer surfaces, which enable the peptide of the present invention to be targeted to desired cells or tissues.

[0077] In a specific embodiment, the nanoparticle enables the uptake of the peptide according to the invention into a target cell. In these embodiments, the nanoparticle can perform the task of a cellular uptake signal, and in particular a cellular uptake signal or a surrogate cellular uptake signal. Therefore, the present invention also provides a nanoparticle comprising a BNIP3 fragment as defined herein.

[0078] Therapeutic uses of peptides

[0079] The present invention provides methods, compounds and compositions for treating a disease or condition in a subject where inhibition of the individual activities and inter-pathway communication of BNIP3, BAX and mitochondria is desired, comprising administering to the subject the compound in an amount effective to treat the disease or condition in the subject.

[0080] The present invention also provides a pharmaceutical composition comprising a peptide according to the present invention. Specifically, the pharmaceutical composition comprises the peptide according to the present invention in an administrable form of a unit dose. The present invention also 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 person in need thereof. The present invention also provides the use of a peptide according to the present invention or a pharmaceutical composition comprising the peptide in medicine, in particular in the treatment of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity and the prevention of such diseases, respectively.

[0081] The invention includes all combinations of the specific embodiments recited, as if each combination had been expressly recited individually.

[0082] The present invention also provides a method of inhibiting BNIP3 in a subject, the method comprising contacting BNIP3 with one or more of any peptides or pharmaceutical compositions disclosed herein (in an amount effective to inhibit BNIP3). Preferably, BNIP3 is in a subject, and one or more peptides or compositions are administered to the subject.

[0083] The present invention also provides a method of inhibiting BAX in a subject, comprising contacting BAX with one or more of any peptides or pharmaceutical compositions disclosed herein (in an amount effective to inhibit BAX). Preferably, BAX is in a subject, and one or more peptides or compositions are administered to the subject.

[0084] The present invention also provides a method for inhibiting the activity of BNIP3 / BAX dimers and / or oligomers in a subject, comprising contacting BNIP3 / BAX dimers and / or oligomers with one or more of any peptides or pharmaceutical compositions disclosed herein (in an amount effective to inhibit the activity of BNIP3 / BAX dimers / oligomers). Preferably, BNIP3 and BAX are in a subject, and one or more peptides or compositions are administered to the subject.

[0085] The present invention also provides a method of treating a reperfusion-related and / or mitochondrial-related disorder in a subject, comprising administering to the subject a peptide or a pharmaceutical composition according to the present invention in a therapeutically effective amount.

[0086] The present invention also provides a method for treating or preventing tissue damage caused by mitochondrial-induced apoptosis or necrosis in a subject, comprising administering a peptide or pharmaceutical composition according to the present invention to the subject in a therapeutically effective amount. The peptide can be administered to the subject before, during and / or after cell death or damage occurs.

[0087] The subject to be administered with the peptide or pharmaceutical composition and treated may suffer from a disease or condition, for example, selected from the group consisting of: hypoxia and / or ischemic cells; heart, brain, liver, kidney, intestine, limb ischemia, limb vascular occlusion; heart, brain, liver and kidney, intestine, limb reperfusion injury; myocardial infarction and reperfusion injury; chemotherapy, radiotherapy, targeted therapy and immunotherapy-induced cardiotoxicity; atherosclerosis; heart failure; heart, liver, kidney transplantation; aneurysm; chronic lung disease; ischemic heart disease; hypertension; pulmonary hypertension; embolism; thrombosis; cardiomyopathy; stroke; neurodegenerative diseases or disorders; immune disorders; 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, especially after vascular occlusion, ischemia-related disorders, reperfusion-related disorders and / or mitochondrial-related disorders, especially myocardial infarction, ischemic stroke, acute kidney injury, trauma, circulatory arrest and ischemia during organ transplantation. In other embodiments, the subject suffers from cardiotoxicity induced by cancer therapy, such as cardiotoxicity induced by chemotherapy, radiotherapy, immunotherapy and / or targeted therapy. Or the subject may receive any type of cancer therapy, and the therapy can be used to prevent cardiotoxicity. Chemotherapy refers in particular to chemotherapy based on anthracyclines, such as therapy using doxorubicin. In specific embodiments, the subject suffers from cardiotoxicity induced by anthracycline-based therapy (such as chemotherapy using doxorubicin).

[0088] Reperfusion-related disorders may generally refer to disorders involving reperfusion injury. Reperfusion injury is tissue damage caused when, for example, blood supply is restored to a tissue after a period of ischemia, wherein the restoration of circulation leads to inflammation and oxidative damage by inducing oxidative stress rather than restoring 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 a peptide or pharmaceutical composition according to the present invention.

[0089] The present invention also provides a method for treating acute myocardial infarction, myocardial reperfusion injury or heart failure in a subject, comprising administering to the subject one or more 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 the activity of BNIP3, BAX or BNIP3 / BAX dimers / oligomers in the subject, respectively.

[0090] In certain embodiments, treatment comprises reducing and / or preventing reperfusion and mitochondrial related damage. In further embodiments, treatment comprises reducing and / or preventing cancer therapy induced cardiotoxicity.

[0091] The subject can be, for example, a mammal, and is preferably a human.

[0092] As used herein, "treating" or "treating" a disease or condition means to reduce or improve or eliminate the signs or symptoms of the disease or condition being treated. When the peptide or composition is administered to a subject before or at the onset of a disease or condition, the peptide or composition can prevent or reduce the severity of the disease or condition. For example, administering the peptide or composition to a subject can prevent or reduce the severity of cardiotoxicity induced by cancer therapies such as chemotherapy, radiotherapy, targeted therapy, or immunotherapy. In these embodiments, the peptide according to the present invention can be administered before, during, and / or after cancer therapy. Administration of the peptide may include preventive and / or therapeutic administration.

[0093] The peptides and compositions of the present invention can be administered to a subject using routes of administration known in the art. Administration can be systemic or confined to a specific site. Routes of administration include, but are not limited to, intravenous, intramuscular, intracardiac, intrathecal or subcutaneous injection, oral or rectal administration, and injection into a specific site.

[0094] In a specific embodiment, the peptide or pharmaceutical composition according to the invention is administered to a subject during or after the occurrence of impaired blood supply, ischemia or vascular occlusion, in particular before reperfusion of the tissue affected by the vascular occlusion. In certain embodiments, the peptide or pharmaceutical composition is administered within 6 hours before reperfusion, in particular within 4 hours, 2 hours or 1 hour before reperfusion. In a specific embodiment, the peptide or pharmaceutical composition is administered within 45 minutes, in particular within 30 minutes before reperfusion.

[0095] 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.

[0096] The method may include expressing an effective amount of a peptide according to the invention in a cell, wherein apoptosis, necrosis, or a combination thereof in the cell is altered compared to a control cell. 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 cardiomyocyte. Apoptosis, necrosis, or a combination thereof in the cell may be reduced.

[0097] The present invention provides methods, comprising administering to a subject in need thereof an effective amount of a composition comprising a polynucleotide encoding a peptide according to the present invention, wherein apoptosis, necrosis or a combination thereof in the subject is increased. Administration may include delivering the polynucleotide to cardiac tissue, brain tissue, liver tissue and kidney tissue. The subject may have symptoms of a disease selected from acute infarction, hypoxia, ischemia, stroke or vascular disease or may have a risk of suffering from the disease. The method may reduce signs of the disease.

[0098] As used herein, the term "polynucleotide" refers to a polymeric form of nucleotides (whether ribonucleotides or deoxynucleotides) of any length, and includes double-stranded and single-stranded RNA and DNA. Polynucleotides can be obtained directly from natural sources, or can be prepared by recombinant, enzymatic or chemical techniques. Polynucleotides can be linear or circular in topology. Polynucleotides can be, for example, a part of a vector such as an expression or cloning vector, or a fragment. Polynucleotides can include nucleotide sequences with different functions, including, for example, coding regions and non-coding regions such as regulatory regions.

[0099] As used herein, "gene" refers to a nucleotide sequence encoding mRNA. A gene has a transcription start 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 the polynucleotides described herein. As used herein, "target mRNA" is an mRNA encoded by a target gene. Unless otherwise specified, a target gene can produce a variety of distinguishable mRNAs by using different exon combinations. Such related mRNAs are referred to as splice variants or transcript variants of a gene.

[0100] As used herein, the terms "coding region" and "coding sequence" are used interchangeably and refer to a nucleotide sequence encoding a polypeptide, and when placed under the control of an appropriate regulatory sequence, the encoded polypeptide is expressed. The boundaries of a coding region are usually determined by the translation initiation codon at its 5' end and the translation termination codon at its 3' end. A "regulatory sequence" is a nucleotide sequence that regulates the expression of a coding sequence that is operably connected thereto. Non-limiting examples of regulatory sequences include promoters, enhancers, transcription start sites, translation start sites, translation termination sites, and transcription terminators. The term "operably connected" refers to the juxtaposition of parts so that they are in a relationship that allows them to function in their intended manner. When a regulatory sequence is connected in a manner that achieves expression of the coding region under conditions compatible with the regulatory sequence, the regulatory sequence is "operably connected" to the coding region.

[0101] The polynucleotide comprising the coding region may comprise heterologous nucleotides flanking one or both sides of the coding region. As used herein, "heterologous nucleotides" refer to nucleotides that are not usually flanked by the coding region present in wild-type cells. For example, the flanks of the coding region that is present in wild-type microorganisms and encodes a polypeptide are homologous sequences, and any other nucleotide sequences flanking the coding region are 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 methods well known to those skilled in the art. The polynucleotides of the present invention may be contained in a suitable vector. The presence of heterologous nucleotides flanking one or both sides of the polynucleotides described herein originates from human manipulation.

[0102] As used herein, the terms "complementary" and "complementary" refer to the ability of two single-stranded polynucleotides to base pair with each other, wherein an adenine on one strand of a polynucleotide will base pair with a thymine or uracil on one strand of a second polynucleotide, and a cytosine on one strand of a polynucleotide will base pair with a guanine on one strand of a second polynucleotide. Two polynucleotides are complementary to each other when a nucleotide sequence in one polynucleotide can base pair with a nucleotide sequence in a second polynucleotide. For example, 5'-ATGC is complementary to 5'-GCAT. As used herein, the term "substantially complementary" and its homologues refer to polynucleotides that can selectively hybridize with a specified polynucleotide under stringent hybridization conditions. Stringent hybridization can be performed under many pH, salt and temperature conditions. The pH value can vary from 6 to 9, preferably from 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 equal to the specified ionic strength of sodium. The temperature of the hybridization reaction can be varied from 30°C to 80°C, preferably from 45°C to 70°C. In addition, other compounds can be added to the hybridization reaction to promote specific hybridization at relatively low temperatures (such as at room temperature or a temperature close to room temperature). Formamide is one of the compounds expected to be used to reduce the temperature requirement. Therefore, if hybridization occurs between a polynucleotide and a second polynucleotide, the polynucleotide is generally complementary to the second polynucleotide. As used herein, "specific hybridization" refers to hybridization between two polynucleotides under stringent hybridization conditions.

[0103] The polynucleotide comprising the coding region may comprise heterologous nucleotides flanking one or both sides of the coding region. As used herein, "heterologous nucleotides" refer to nucleotides that are not usually flanked by the coding region present in wild-type cells. For example, the flanks of the coding region that is present in wild-type microorganisms and encodes a polypeptide are homologous sequences, and any other nucleotide sequences flanking the coding region are 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 methods well known to those skilled in the art. The polynucleotides of the present invention may be contained in a suitable vector. The presence of heterologous nucleotides flanking one or both sides of the polynucleotides described herein originates from human manipulation.

[0104] The present invention also provides a method for preventing cell damage or cell death, which comprises contacting cells with a peptide according to the present invention. The method can be performed in vitro or in vivo, in particular ex vivo.

[0105] The present invention also provides a method for screening compounds suitable for preventing reperfusion injury and / or mitochondrial-related disorders and / or cancer therapy-induced cardiotoxicity, comprising

[0106] (i) providing one or more candidate compounds;

[0107] (ii) determine the ability of candidate compounds to interfere with the binding of BNIP3 to BAX;

[0108] (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX.

[0109] The candidate compound can be any suitable compound, including, among others, peptides and small molecule compounds. DETAILED DESCRIPTION

[0110] Example 1: BNIP3 represents a therapeutic target for I / R injury

[0111] A combination of necrotic and apoptotic cardiomyocyte death is a hallmark of the early stages of reperfusion injury. Mitochondria play a major role in both processes. The BH-only BCL2 family member BNIP3 is a potential activator of mitochondria-driven necrotic and apoptotic cell death cascades 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 provides an attractive target for therapeutic intervention, we used a clinically relevant in vivo model. 41-44 In this study, wild-type and BNIP3-deficient mice were subjected to 24-hour reperfusion after occlusion of the left anterior descending coronary artery. Figure 1 A). To distinguish affected from non-affected areas, Evans blue dye was injected into the aorta and coronary arteries. To delineate infarcted areas within the non-viable myocardium, risk zone, cardiac sections were stained with triphenyltetrazolium chloride ( Figure 1 B) Consistent with previous studies using a recognized major negative inhibitor of BNIP3 in isolated rat hearts 31 Genetic ablation of BNIP3 produced a significant 46% reduction in infarct size compared with wild-type mice ( Figure 1 C). Accordingly, restoration of BNIP3 to BNIP3-deficient mice resulted in infarct size comparable to that of wild-type mice in a dose-dependent manner and ruled out possible side effects of BNIP3 gene deletion ( Figure 1 C) These results suggest that BNIP3 plays an important role in the evolution of infarction in vivo.

[0112] Example 2: BNIP3 is a regulator of BAX-induced cell death in I / R injury

[0113] The pro-death BCL-2 family member BAX appears to act as an effector protein at the intersection of mitochondria-dependent necrosis and apoptosis 5 To this end, the translocation of BAX from the cytosol to the mitochondria is a key step 37,45,46 We recently demonstrated that the basal interaction of BAX with BNIP3 occurs in vivo at the MOM in cardiomyocytes 35 To determine whether the translocation of BAX to mitochondria, the most relevant step in cell death, occurs in I / R and is dependent on BNIP3, we first investigated changes in mitochondrial BAX and BNIP3 concentrations. After 10 min of reperfusion following vascular occlusion, BAX levels in MOM increased significantly, accompanied by an increase in mitochondrial concentrations of BNIP3 ( Figure 2 A and Figure 2 B). Co-immunoprecipitation showed that BNIP3 and BAX formed heterodimers in MOM ( Figure 2 C). By utilizing BNIP3-deficient mice, we next examined whether BNIP3 is an upstream regulator of BAX in this translocation during reperfusion injury. Upon genetic ablation of BNIP3, no increase in mitochondrial BAX was observed during the early phases of reperfusion, demonstrating a direct effect of BNIP3 ( Figure 2 D). To verify that BNIP3 can indeed achieve this effect, we administered BNIP3 to the hearts of BNIP3-deficient mice 5 minutes before starting the myocardial infarction procedure. The addition of BNIP3 restored BAX translocation in the hearts of BNIP3-deficient mice in the early stages of reperfusion and ruled out possible side effects of genetic deletion of BNIP3 ( Figure 2 D).

[0114] Example 3: Identification of critical BNIP3 sequences required for BNIP3 inhibition in I / R injury

[0115] Since the interaction of BNIP3 with BAX is thought to be a substantive activity in reperfusion injury in vivo (assessed by coimmunoprecipitation), we identified helices α5, α6, α7, and α8 in BAX as potential binding sites by evaluating a peptide microarray with a library of 13 synthetic BAX peptides ( Figure 3 A and Figure 3 B) For this study, we used Modeller 9.15 47 The 3D structure of BNIP3 was predicted in silico by homology modeling ( Figure 3 C). The created model was energy minimized using NAMD2.9 and CHARMM36 force fields. The BNIP3 model depicts 9 α-helices of variable length representing 64%, followed by 19% random coils and 17% unidentified structures, which was confirmed by circular dichroism spectroscopy ( Figure 3 D). Computational docking simulations indicated that BAX helices α5, α6, α7, and α8 and the BNIP3 sequence MSQSGEENLQGSWVELHFSN (amino acids 1-20; SEQ ID NO: 20) serve as interaction sites ( Figure 3 E), while the first 10 amino acids alone failed to bind to BAX ( Figure 3 F). We hypothesized that amino acids 1-20 of BNIP3 may be sufficient to antagonize the activity of BNIP3, so we designed a cell permeable peptide (TAT-BNIP3-20A peptide) consisting of a 20-amino acid HIV-1 TAT protein transduction domain (PTD, GRKKRRQRRRPQ (SEQ ID NO: 31) covalently attached to amino acids 1-20 of BNIP3, Figure 3 G) 48,49 composition( Figure 3 H). Amino acids 42-61 of BNIP3 were used to generate the control peptide TAT-BNIP3-20C ( Figure 3 I). The peptide fragment was capped with an acetyl group at the N-terminus and an amide group at the C-terminus. Treatment of wild-type mice with TAT-BNIP3-20A demonstrated that the peptide was taken up by the myocardium ( Figure 3 J), where it interacts with endogenous BNIP3.

[0116] To examine whether TAT-BNIP3-20A, administered 5 min before reperfusion (a time point relevant to clinical practice), has the ability to antagonize BNIP3 activity and reduce reperfusion injury in vivo, we used a given myocardial infarction model in mice ( Figure 4 A). Treatment with TAT-BNIP3-20A resulted in a 37% reduction in infarct size, but neither vehicle treatment nor treatment with TAT-BNIP3-20C resulted in a 37% reduction in infarct size ( Figure 4 B). This is because the TAT-BNIP3-20A peptide fragment prevents BNIP3 from translocating to mitochondria ( Figure 4 C), resulting in a significant inhibition of caspase-3 activity, a key event in I / R following mitochondrial membrane perturbation ( Figure 4 D).

[0117] Example 4: TAT-BNIP3-20A peptide fragment inhibits apoptotic and necrotic human cardiomyocyte death

[0118] To meet translational needs, reoxygenation experiments were performed in human ventricular cardiomyocytes derived from human induced pluripotent stem cells (human CMs) ( Figure 5A). Human CM were exposed to 2 h of reoxygenation after hypoxia. Remarkably, even in human CM, TAT-BNIP3-20A showed its protective properties. BNIP3-20A was able to prevent the translocation of human BNIP3 to mitochondria ( Figure 5 B), leading to depolarization of the inner mitochondrial membrane through low Figure 5 C) and fewer apoptotic and necrotic cells ( Figure 5 D) Considerable protection of mitochondria was demonstrated.

[0119] Example 5: Identification and design of peptide fragment inhibitors of BNIP3 / BAX activity

[0120] N-terminal truncation of the BNIP3-20A peptide sequence followed by exchange of single residues revealed that a peptide containing amino acids 13-20 in combination with a Ser to Phe substitution at position 19 showed significantly higher BNIP3 binding in the peptide microarray (see Table 1).

[0121] Table 1: Truncated BNIP3-20A fragments showing the highest binding capacity to BNIP3

[0122]

[0123] Based on these results, we designed the BNIP3-8B peptide, which consists of a PTD covalently attached to eight amino acids derived from amino acids 13-20 of BNIP3 with a Ser-19 to Phe-19 substitution (WVELHFFN (SEQ ID NO: 8); Figure 6 A). To demonstrate the need for a phenylalanine residue in the peptide sequence, we replaced Phe-18 with Ala-18 and His-17 with Ala-17 to generate the BNIP3-8C peptide ( Figure 6 B) Circular dichroism spectrum of BNIP3-8B shows that the peptide is in a random coil conformation ( Figure 6 C).

[0124] In addition, BNIP3 / BNIP3 interaction studies were performed using the BNIP3-20A peptide, in which a single residue of the wild-type sequence of BNIP3 1-20 was exchanged for 18 neutral amino acids. The BNIP3 peptides with specific amino acid substitutions showed increased binding ability to BNIP3 (exemplary data are shown in Table 2).

[0125] Table 2: Substituted BNIP3-20A peptides showed the highest binding ability to BNIP3

[0126]

[0127] Example 6: In vivo and in vitro effects of uptake, toxicity and stability of TAT-BNIP3-8B peptide fragments

[0128] First, we evaluated the distribution and presence of BNIP3-8B following intracardiac injection in vivo to achieve the desired effect. Next, we evaluated the pharmacokinetic profile of TAT-BNIP3-8B in human serum, plasma, and whole blood and assessed toxicity in isolated adult cardiomyocytes. TAT-BNIP3-8B is taken up by the heart, spleen, and liver and is present in plasma ( Figure 7 A). Thus, 10 minutes after reperfusion, the time point when the BNIP3-BAX-mitochondrial triangular cell death cascade is set in motion, TAT-BNIP3-8B is present in the heart; cardiomyocytes show no overt signs of toxicity ( Figure 7 B), the half-life of TAT-BNIP3-8B in human serum, plasma and whole blood enables its in vivo inhibitory ability ( Figure 8 AC). TAT-BNIP3-8B was incubated with proteinase K as a control.

[0129] Example 7: Mechanism of action of TAT-BNIP3-8B peptide

[0130] It is assumed that TAT-BNIP3-8B binds to BNIP3 and BAX monomers and homodimers, and binds to BNIP3 / BAX heterodimers and heterooligomers to interrupt their activity. To evaluate the interaction behavior of TAT-BNIP3-8B, BNIP3 / TAT-BNIP3-8B, and BAX / TAT-BNIP3-8B, overlay assays and docking simulations were performed. Both results indicate that TAT-BNIP3-8B binds to BNIP3 and BAX (data not shown).

[0131] It is noteworthy that in the myocardial infarction model ( Fig. 9 In A), endogenous BNIP3 and BAX monomers, homo- and heterodimers were co-immunoprecipitated with fluorescently labeled TAT-BNIP3-8B 5 min after reperfusion ( Fig. 9 B). Furthermore, reperfusion drives BNIP3 and BAX oligomerization, as evidenced by the large-scale formation of oligomers consisting of BNIP3 and 3BAX ( Fig. 9 C). SDS-PAGE and co-immunoprecipitation experiments revealed a BNIP3 / BAX hetero-interaction in a higher-order oligomeric complex, which was strongly inhibited by TAT-BNIP3-8B treatment ( Fig. 9 C).

[0132] Example 8: TAT-BNIP3-8B reduces myocardial infarction size

[0133] We then investigated the efficacy and effects of TAT-BNIP3-8B treatment given 5 min before the start of reperfusion in an appropriate myocardial infarction model ( Fig. 9 A). Notably, TAT-BNIP3-8B reduced infarct size by up to 40% in a dose-dependent manner compared with vehicle and TAT-β-Gal treatment ( Fig. 9 D and E). In contrast to TAT-BNIP-8B, TAT-BNIP3-8C treatment did not significantly affect infarct size, pointing to the importance of the phenylalanine residue ( Fig. 9 D).

[0134] Example 9: TAT-BNIP3-8B reduces mitochondrial perturbations and cell death cascade

[0135] Mitochondrial damage can occur through perturbations of the mitochondrial inner membrane (MIM) and MOM. The key mitochondrial event in necrosis is the early opening of the mitochondrial permeability transition pore (mPTP) in the MIM. 50 , which leads to a time-dependent dissipation of the potential difference across the MIM, followed by mitochondrial swelling and cell rupture. The key mitochondrial event in apoptosis is BAX activation, which induces exposure of its transmembrane region and MOM permeabilization, allowing the release of apoptotic factors such as cytochrome c, and subsequent caspase activation. 51 In the given in vivo I / R model, TAT-BNIP3-8B injected into the left ventricle 5 min before reperfusion prevented the translocation of BNIP3 and BAX to mitochondria, which led to the inhibition of downstream cell death mechanisms including mitochondrial swelling, BAX activation, cytochrome c release, and caspase-3 activity.

[0136] Example 10: TAT-BNIP3-8B inhibits apoptotic and necrotic cardiomyocyte death

[0137] We tested whether TAT-BNIP3-8B could inhibit cell death in human CMs. Human CMs were exposed to reoxygenation for 2 h after hypoxia ( Fig.11 A). BNIP3-8B significantly inhibits necrotic and apoptotic cell death ( Fig.11 B) and loss of mitochondrial inner membrane potential ( Fig.11 C).

[0138] Example 11: TAT-BNIP3-8B reduces cerebral infarction area

[0139] Since BNIP3 is believed to play a key role in cerebral ischemia 38We also evaluated the effect of the BNIP3-8B peptide on clinical outcomes in a mouse model of focal cerebral reperfusion. We subjected mice to transient middle cerebral artery occlusion (tMCAO) for 30 minutes followed by 24 hours of reperfusion. Immediate BNIP3-8B treatment after tMCAO significantly reduced infarct size by 52% ( Fig.12 ).

[0140] Example 12: TAT-BNIP3-8B reduces myocardial infarction size in pigs

[0141] We then investigated the efficacy and effects of TAT-BNIP3-8B treatment given 5 minutes before the start of reperfusion in a porcine myocardial infarction model. Pigs underwent 60 minutes of left anterior descending coronary artery occlusion followed by 4 hours of reperfusion. Notably, TAT-BNIP3-8B significantly reduced infarct size by 56% (95% CI, 2.3-1.4) compared to vehicle. Fig.13 ).

[0142] Example 13: TAT-BNIP3-8B has a protective effect on doxorubicin-induced mitochondrial damage.

[0143] We evaluated the effect of the 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, where an increase in mitochondrial volume due to swelling results in a decrease in optical density. TAT-BNIP3-8B administered simultaneously with doxorubicin prevented mitochondrial swelling, as measured by OD 540 Increase the indicated ( Fig.14 These data suggest that TAT-BNIP3-8B can protect mitochondria from damage mediated by chemotherapy drugs such as anthracyclines.

[0144] Example 14: Method

[0145] 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.

[0146] Animals. Male mice of similar age (12±3 weeks) and average body weight of 30 g were used. C57BL / 6 wild-type mice were obtained from Jackson Laboratories (Bar Harbor, ME, USA) and kept in a local animal house for one week to acclimate to the new environment.

[0147] C57BL / 6J-TgH(Bnip3 - / -) mice were obtained from Professor Gerald W. Dorn, Center for Molecular Cardiovascular Research and Department of Pediatrics, University of Cincinnati, Cincinnati, Ohio, USA. Mice were generated by replacing exons 2 and 3 with a neomycin resistance cassette 33 The mice were bred and maintained in the local animal house at University Hospital Essen. All experiments were approved by the local ethics committee in accordance with the European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Directive 2010 / 63 / EU).

[0148] In vivo myocardial infarction model in mice. Wild-type and Bnip3-deficient (Bnip3) mice were anesthetized by ip injection of ketamine (100 mg / kg) and xylazine (10 mg / kg). - / - ) mice and intubated. Using a mouse microventilator, mechanical ventilation parameters were set to a tidal volume of 2.1 to 2.5 ml and a respiratory rate of 140 breaths per minute. Deep anesthesia was maintained by adding 2 vol% isoflurane to the ventilation gas. The chest was opened by a lateral thoracotomy (1 cm left incision between the 3rd and 4th ribs). A 6-0 prolene suture was placed around the left coronary artery (LCA) and a soft silicon tube was placed on the artery. Coronary artery occlusion was achieved by tightening the suture and tying it. After 30 minutes of occlusion, the silicon tube was removed and the suture was left in place. For longer reperfusion times, the chest cavity was closed using 4-0 prolene. 5 minutes before vascular occlusion, 2, 6 and 9 nmol (in 50 μl 0.9% sodium chloride) BNIP3 was injected into the left ventricular cavity. 5 minutes before reperfusion, a peptide with 20 amino acids (2 nmol / 50 μl) and a peptide with 8 amino acids (8 nmol / 50 μl) were injected in NaCl. Sodium chloride injection (50 μl) served as a control treatment.

[0149] In vivo porcine myocardial infarction model. After induction of anesthesia, a small incision is made in the femoral artery and femoral vein; the vessels are isolated. A small hole is made in the artery and a sheath is introduced. In addition, a sheath is placed in the femoral vein or other suitable vein to allow emergency administration if necessary. A blood sample is collected before the administration of heparin and used to establish a baseline activated clotting time (ACT), which is recorded. Subsequently, under the guidance of the surgeon, heparin (250 to 350 IU / kg) is administered as needed to achieve and maintain an ACT level twice the baseline ACT. After the initial injection of heparin, the ACT is recorded again and monitored at least every 60 minutes thereafter until the completion of the surgery.

[0150] Using the visual guidance provided by fluoroscopy, a suitable guide catheter is advanced into the opening of the left anterior descending artery (LAD). Non-ionic contrast agents are used for all surgeries. The balloon catheter is introduced by advancing the balloon catheter into the left anterior descending coronary artery (LAD) through the guide catheter. The balloon is advanced into the coronary artery by the guide catheter to a suitable position above the first diagonal branch of the LAD. The balloon is then inflated to a pressure sufficient to ensure complete occlusion of the artery. Fluoroscopy is used to confirm arterial occlusion. After confirming occlusion, the balloon is allowed to expand in the artery for 60 minutes. Incidence supportive drugs and cardiac defibrillation are recorded in the study records. Peptides and vehicles are administered by intravenous injection 5 minutes before reperfusion. At the end of the vascular occlusion period, the balloon is deflated and the ischemic area is allowed to reperfuse. Fluoroscopy is used to confirm complete balloon deflation. At the end of the operation, all catheters are removed, the artery and vein are ligated, and the incision is closed in a standard manner. After reperfusion for 4 hours, the animals are euthanized.

[0151] Measurement of infarct size. To analyze the infarct size, mice were euthanized 24 hours after reperfusion; the hearts were excised and perfused with PBS for more than 5 minutes. After perfusion, the LCA was re-ligated at the same position as described above. Evans blue dye (1 ml of a 1% solution) was injected into the aorta and coronary arteries to demarcate ischemic AAR from non-ischemic areas. The tissue was wrapped in transparent food wrap and stored in a refrigerator at -20°C for one hour. The heart was then cut into 1 mm slices perpendicular to the long axis and each slice was weighed. The slices were incubated in 1% TTC at 37°C for 5 minutes to distinguish viable from non-viable myocardium in the risk zone. Infarction, AAR, and non-ischemic left ventricle were evaluated by computer-assisted planimetry by an observer who was unaware of the identity of the samples. The area of ​​myocardial infarction is expressed as a percentage of the AAR.

[0152] Immunoblotting. Tissues and human CM cells were lysed in RIPS buffer (50mM Tris-HCl, 150mM NaCl, 0.5mM EDTA, 1% NP-40 and protease inhibitors and phosphatase inhibitors, pH 7.4). Isolation of mitochondria was lysed in Mito-lysis buffer (200mM sucrose, 10mM HEPES, 1mM EGTA, 1% Triton-X100 and protease inhibitors and phosphatase inhibitors, pH 7.4). Lysates were cleared by centrifugation (20.000xg, 15min, 4°C). Protein concentration in the supernatant was measured using the DC protein assay (Bio-Rad). Samples were diluted into 4x LDS sample buffer and 10x reducing agent (Invitrogen) and prepared for SDS-PAGE by heating to 95°C for 5 minutes. 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 goat anti-mouse or anti-rabbit IgG conjugated with horseradish peroxidase (Invitrogen). Immunoblots were detected by ECL (Thermo Scientific) and imaged on an Imager 600 (Amersham).

[0153] 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 in which the wild-type sequence of BNIP3 1-20 was C-terminally, N-terminally, or C / N-terminally truncated; and (iii) BNIP3 / BNIP3 interaction studies in which a single residue of the wild-type sequence of BNIP3 1-20 was exchanged for 18 neutral amino acids.

[0154] Microarray. For protein-peptide binding studies, recombinant BNIP3 (cusabio) and BAX (MyBioSource) were used at concentrations of 10 μg / ml and 1 μm / ml, respectively. As a labeling kit, a DyLight Microscale Antibody Labeling Kit (Thermo) with the label Dylight 650 was used. The assay was performed using an automated TECAN HS4800 microarray processing station. The microarray was incubated with customer-provided samples (diluted in encapsulation buffer) at 30 ° C for 2 h. Before each step, the microarray was washed with wash buffer. The microarray was scanned using a high-resolution fluorescence scanner. The laser settings and the resolution applied were the same for all measurements performed. The resulting images were analyzed and quantified using the spot recognition software GenePix (Molecular Devices). For each point, the average signal intensity (between 0 and 65535 arbitrary units) was extracted. For further data evaluation, the so-called MMC2 value was determined. MMC2 is equal to the average of all three instances on the microarray, unless the coefficient of variation (CV)-standard deviation divided by the mean-is greater than 0.5. In this case, the average of the two closest values ​​(MC2) was assigned to MMC2. All procedures were performed by JPT Peptide Technologies (Berlin, Germany).

[0155] In silico three-dimensional (3D) structure modeling of BNIP3 The predicted in silico 3D structure of BNIP3 was obtained by homology modeling using Modeller 9.15, and the created model was energy minimized using NAMD 2.9 and CHARMM 36 force fields.

[0156] Circular Dichroism (CD) Spectra. CD spectra of BNIP3 protein and BNIP3-8B peptide were recorded on a Jasco J-715 spectropolarimeter at 37°C, pH = 7.4, 1 x PBS.

[0157] Docking simulation. Using Autodock Vina 52 and HADDOCK 53 The structure of BAX (pdb-ID: 4S00) was taken from the Protein Data Bank, while the structures of BNIP3 and peptide BNIP3-8B were obtained using Modeller 9.15. 47 Modeled. The template structures correspond to PDB codes 2k7w and 2ka1. The models were created using NAMD2.9 54 and CHARMM36 force field for energy minimization.

[0158] Immunoprecipitation. Immunoprecipitation was performed using protein G-coupled Dynabeads (Invitrogen). 500 μg of cleaved protein was incubated with 2 μg of antibody at 4°C overnight with shaking in PBS buffer containing 1 mM DTT, 0.005% Brij35 and protease-phosphatase inhibitors. 20 μl of Dynabeads were added the next day and the solution was incubated again for 1 hour. The precipitated immune complexes were washed twice, 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 minutes. After removing the Dynabeads, the eluate was analyzed by immunoblotting.

[0159] Caspase 3 activity was measured using the Caspase 3 Assay Kit from Abcam (#ab39401). Mouse hearts were harvested after 30 minutes of ischemia and 4 hours of reperfusion, and the risk area was isolated, lysed in a solution containing buffer, and assayed according to the manufacturer's instructions.

[0160] Cell culture. Human iPSC-derived ventricular cardiomyocytes (human CMs) were obtained from (axol) and cultured according to the manufacturer's instructions.

[0161] To simulate vascular occlusion, cells were incubated in buffer (113 mM NaCl, 4.7 mM KCl, 12 mM HEPES, 1.2 mM MgSO4, 30 mM taurine, 1.3 mM CaCl2, pH 7.4) at 1% O2 at 37°C. Reoxygenation was performed at 21% O2 at 37°C in buffer supplemented with 5.5 mM glucose.

[0162] 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.

[0163] JC-1 assay. To determine the mitochondrial inner membrane potential in human CM cells, human CM cells were stained with 5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanide iodide (JC-1). Cells were incubated with 6 μM JC-1 in culture medium at 37°C for 30 minutes. After washing the cells with PBS buffer at 37°C, they were fixed with 4% PFA for 15 minutes at room temperature. DAPI staining was performed and cells were analyzed using EVOS FL (life Technologies).

[0164] 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 (containing 0.5 mg / ml mitochondria in a buffer containing 250 mM sucrose, 110 mM HEPES, 1 m EGTA, pH 7.4).

[0165] Peptides. Peptides were produced by a resin synthesis method (JPT International, Berlin, Germany). Their N-termini were capped with acetyl groups and their C-termini were capped with amides. For delivery, the peptides were covalently attached to the TAT sequence GRKKRRQRRRPQ (SEQ ID NO: 31). For uptake and binding studies, the peptides were labeled with fluorophores. A complete list of peptides is attached in Appendix 1.

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Claims

1. A peptide comprising (i) a cellular uptake signal; and (ii) a BNIP3 fragment comprising positions 13 to 20 of BNIP3 or an amino acid sequence derived therefrom; wherein the peptide is 50 amino acids or less in length.

2. The peptide according to claim 1, wherein the BNIP3 fragment has a length of 12 amino acids or less, in particular 10 amino acids or less, especially 8 amino acids.

3. The peptide according to claim 1 or 2, wherein the BNIP3 fragment comprises positions 13 to 20 of BNIP3, optionally comprising 1, 2 or 3 amino acid substitutions compared to positions 13 to 20 of BNIP3.

4. The peptide according to claim 3, wherein the amino acid substitution is present at one or more of positions corresponding to positions 15, 17 and 19 of BNIP3.

5. The peptide according to claim 3 or 4, wherein the amino acid substitution is selected from (i) the glutamic acid at position 15 of BNIP3 is substituted with phenylalanine, isoleucine, leucine, valine, tyrosine, cysteine, histidine, arginine or threonine, and (ii) the histidine at position 17 of BNIP3 is substituted with valine, and (iii) The serine at position 19 of BNIP3 is substituted with tyrosine, cysteine, phenylalanine or histidine. 6 . The peptide according to claim 1 , wherein the BNIP3 fragment comprises a phenylalanine residue at a position corresponding to position 19 of BNIP3.

7. The peptide according to any one of claims 1 to 6, wherein the BNIP3 fragment (i) comprises positions 12 to 20 of BNIP3, optionally comprising 1, 2, 3 or 4 amino acid substitutions compared to positions 12 to 20 of BNIP3; (ii) comprises positions 4 to 20 of BNIP3, optionally comprising 1, 2, 3, 4, 5 or 6 amino acid substitutions compared to positions 4 to 20 of BNIP3; or (iii) comprises positions 1 to 20 of BNIP3, optionally comprising 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 SEQ ID NOs: 3 to 30.

8. The peptide according to claim 1, wherein the BNIP3 fragment comprises a D-inverted-flip sequence of positions 13 to 20 of BNIP3 or any amino acid sequence defined in claims 2 to 7.

9. The peptide according to any one of claims 1 to 8, wherein the cellular uptake signal (i) is a peptide of 5 to 30 amino acids, in particular 8 to 20 amino acids or 10 to 16 amino acids, especially 12 amino acids in length; (ii) is the protein transduction domain of the TAT protein of HIV; and / or (iii) comprises an amino acid sequence selected from SEQ ID NOs: 31 to 50. 10 . The peptide according to claim 1 , wherein the cellular uptake signal comprises a cell penetrating peptide, in particular a D-inverted-flip sequence of a cell penetrating peptide as defined in claim 9 .

11. The peptide according to any one of claims 1 to 10, consisting of the cellular uptake signal and the BNIP3 fragment, optionally comprising a linker between the cellular uptake signal and the BNIP3 fragment.

12. The peptide according to any one of claims 1 to 11, which has a length of 40 amino acids or less, in particular 35 amino acids or less, especially 30 amino acids or less.

13. A pharmaceutical composition comprising the peptide according to any one of claims 1 to 12.

14. The peptide according to any one of claims 1 to 12 for use in the treatment of reperfusion-related and / or mitochondrial-related disorders and cancer therapy-induced cardiotoxicity.

15. The peptide for use according to claim 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 blood flow arrest during organ transplantation.

16. The peptide for use according to claim 14 or 15, wherein the peptide is administered to the patient during or after an impairment of blood supply has occurred, in particular prior to reperfusion of tissue affected by ischemia.

17. The peptide according to claim 16, wherein the peptide is administered to the patient within 2 hours, in particular within 30 minutes, before reperfusion.

18. The peptide according to any one of claims 1 to 12, for use in treating or preventing tissue damage caused by mitochondrial-induced apoptosis or necrosis.

19. A method for screening compounds suitable for preventing reperfusion injury and / or mitochondrial-induced disorders and / or cancer therapy-induced cardiotoxicity, comprising (i) providing one or more candidate compounds; (ii) determine the ability of candidate compounds to interfere with the binding of BNIP3 to BAX; (iii) selecting candidate compounds that interfere with the binding of BNIP3 to BAX.

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